Ethiopian Veterinary Journal________________________
Year: 2020 Volume: 24, No. 2
Ethiop. Vet. J. is the Official Scientific Organ of the Ethiopian Veterinary Association
2020ISSN:1683-6324 (print) ISSN:2221-5034 (online)
Ethiopian Veterinary Journal (Ethiop. Vet. J.)Objectives and Scope
The Ethiopian Veterinary Journal (Ethiop. Vet. J.) is a multidisciplinary peer-reviewed journal intended to promote animal health and produc-tion of national and regional/international importance. The journal publishes review articles, original research articles, short communication as well as technical notes in English. Under special circumstances, articles in Amharic may be considered for publication.
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edu.et
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[email protected] Gijs van’t Klooster, Food and Agriculture Organization of the United Nations, E-mail: [email protected] J. B. Malone, Louisiana State University, Baton Rouge, Louisiana, USA Kurt Peters, Humboldt University, Berlin, Germany Markos Tibbo, Food and Agriculture Organization of the United Nations, Cairo, Egypt, E-mail: [email protected] Million Mulugeta, CURE/UCLA School of Medicine, Los Angeles, CA, USA Moges Woldemeskel, University of Tennessee, 2407, River Drive, Knoxville, Tennessee 37919, USA P. Dorchies, Ecole Nationale Veterinaire de Toulouse, Toulouse, France Solomon Hailemariam, African Union, Addis Ababa, Ethiopia Takele Argaw, FDA, Washington D.C., USA Teshome Mebatsion Merial & Sanofi Inc. [email protected] Teshome Yehualashet, Tuskegee University, Alabama, USA Yilma Jobre, Food and Agriculture Organization of the United Nations, Sub-Regional Office for Eastern Africa, Addis Ababa,
Ethiopia, Email: [email protected]
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Ethiop. Vet. J., is published by the Ethiopian Veterinary Association (EVA)
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Ethiop. Vet. J., is the Official Scientific Organ of the Ethiopian Veterinary Association
Table of ContentsPrevalence of wound, its associated risk factors and wound management practices in carthorses of ten selected towns of Ethiopia
Mihret Teferi, Reta Tesfaye, Hanna Zewdu, Gizaw Gemechu, Gebre Tefera, Tibebu Ashine...........................................................................................................1 https://dx.doi.org/10.4314/evj.v24i2.1
Prevalence and risk factors of swine tuberculosis in central EthiopiaKassa Demissie, Jirata Shiferaw, Girmay Medhin, Aboma Zewude, Asegedech Sirak, Takele Abayneh, Gezahegne Mamo, Gobena Ameni.........................16 https://dx.doi.org/10.4314/evj.v24i2.2
Effects of skin and hide defects on quality grades and physical characteristics of crust leather
Tewelde Tsigab, Abrha Bsrat, Redae Alemayohu, Menghistu Ashebir, Niraj Kumar, Birhanu Hadush.......................................................................................35 https://dx.doi.org/10.4314/evj.v24i2.3
Ovarian follicular dynamics in Boran and Crossbred heifers in Ethiopia: Implications for assisted reproductive techniques
Jeilu Jemal, Tamrat Degefa, Tefera Yilma, Sayid Ali, Alemayehu Lemma..................................54 https://dx.doi.org/10.4314/evj.v24i2.4
Prevalence, organ distribution and antimicrobial susceptibility profile of Salmonella isolated from chickens purchased from markets in selected districts of West Shoa, Ethiopia
Edilu Jorga Sarba, Kebene Kudama, Morka Dandecha, Lencho Megersa, Bizunesh Mideksa Borena, Endrias Zewdu Gebremdhin.............................................................73 https://dx.doi.org/10.4314/evj.v24i2.5
Trypanocidal drug utilization practices in tsetse suppression and non-suppression areas of South Omo Zone, Southwestern Ethiopia
Tegegn Tesfaye, Tekle Olbamo, Hagos Ashenafi..........................................................................90 https://dx.doi.org/10.4314/evj.v24i2.6
Fore-Stomach Foreign Bodies: prevalence, associated risk factors and types affecting cattle slaughtered at Gondar ELFORA abattoir, northwest Ethiopia
Amare Bihon, Teketaye Bayeleyegn, Ayalew Assefa and Yimer Muktar.....................................112 https://dx.doi.org/10.4314/evj.v24i2.7
Immunogenicity and protective efficacy of irradiated Salmonella Gallinarum against homologous challenge infection in Bovans brown chickens
Solomon Lulie, Haile Alemayehu, Anwar Nuru, Takele Abayneh Tadesse Eguale....................123 https://dx.doi.org/10.4314/evj.v24i2.8
Unilateral testicular degeneration in dogs: Effects on spermatozoal characteristics, testis and cauda epididymis
Chike Fidelis Oguejiofor, Kenneth Orji Anya and Nnaemeka Kingsley Ogbanya....................139 https://dx.doi.org/10.4314/evj.v24i2.9
Guidelines for authors......................................................................................................................155
Teferi et al., Ethiop. Vet. J., 2020, 24 (2), 1-15 DOI https://dx.doi.org/10.4314/evj.v24i2.1 Ethiopian Veterinary Journal
Ethiop. Vet. J., 2020, 24 (2), 1-15 1
Prevalence of wound, its associated risk factors and wound management practices in carthorses of ten selected towns of Ethiopia
Mihret Teferi, Reta Tesfaye*, Hanna Zewdu, Gizaw Gemechu, Gebre Tefera, Tibebu Ashine
Addis Ababa University, College of Veterinary Medicine and Agriculture, the Society for Protec-tion of Animals Abroad (SPANA) Ethiopia project
*corresponding author: Reta Tesfaye, email: [email protected]
Abstract A cross-sectional study was conducted from November 2017 to April 2018 to estimate the prevalence of wound and associated risk factors in carthorses, and to assess wound management practices in 10 selected towns of Ethiopia. The study was conducted through personal interview and observation of car-thorses. Randomly selected 390 drivers and their carthorses were included in this study. The study revealed that, the overall prevalence of wound was 51.5% (n=201). The prevalence of wound significantly varied among the study towns. It was high (70%, n =28) in Shashemene and low (30%, n=9) in Sheno. More than one type of wound was the most common (21%, n=82) type to occur. Harness related wounds such as girth sore (20.8%, n=81), chest sore (11.8%, n=46), bit sore (11.8, n=46), saddle sore (7.9%, 31), tail sore (3.1%, n=12) and collar sore (0.5%, n=2) were observed. The non-harness related wounds identi-fied were fetlock sore (12.3%, n=48), knee sore (3.3%, n=13), whip sore (3.1%, n=12) and other sores (1.5%, n=6). Generally, wound related with harnesses were more frequent. The prevalence of wound was observed significantly dif-ferent between body condition scores. Horses with body condition score 2 were 2 times more likely to be wounded than horses with better body condition score (BCS=3) [OR: 2.4 95%CI (1.36-4.25)]. Wound prevalence was low in horses where owner own only one horse compared to owners keeping two and more horses [OR: 1.56, 95% CI (1.01-2.33)]. Bits with smooth surface were used in less than half (44.4%, n=173) of the observed animals. Variation in frequency of bit sore was observed based on bit types used. The majority of bit sores were caused by metal bits with rough surface (χ2 =10.52, p= 0.03). Majority (83.3%, n=325) of the owners were aware of proper wound management prac-tices. Carthorses often transport 3 to 4 people at a time, the average working
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hours being 6.22 hours per day. In conclusion, the study has indicated wound as highly prevalent health and welfare problem of carthorses in the study area. Therefore, intervention should be in place to reduce the prevalence of wound. Cart horses should be fed well to keep them in a good body condition. Proper type of bits, harnessing materials and cart design should be used. Non-harness related wound should also be avoided.
Key words: Carthorse; Ethiopia; Prevalence; Risk factors; Wound
Introduction Equine are important working animals for the resource-poor communities of rural and urban areas of Ethiopia (Wilson, 1991; Starkey and Fielding, 2000; Mengistu, 2003). They are used for various purposes. In rural areas, equines are used to transport people and items from one place to another. They carry water for home consumption and transport goods to and from market. They are also involved in agricultural activities such as transport of agricultural products from field to home. Their contribution in social events such as holi-days, wedding and funeral ceremonies is also significant (SPANA, 2019, un-published). In urban and peri-urban areas, horses are mainly used in pull-ing carts to transport people and materials such as water, crop, vegetable, construction materials, firewood and other goods to market (Mekuriaw, 2019). Equine owners rely on the direct or indirect income generated from these ani-mals and life would be very difficult without them (Mekuriaw, 2019; SPANA, 2019, unpublished).
Wound is one of the most common equine health problems in daily veterinary practice. Wound beneath overlying tack or equipment is the most frequent type of wound to occur in equine (Pritchard et al., 2005). Wound compromise the welfare of equine, reduces work efficiency and predispose the animal to diseases. Wound can involve superficial layer, deep skin underlying tissue or it may involve vital structures such as joints (Knottenbelt, 2003; Stashak and Theoret, 2008).
There are few studies conducted on equine wound in the study area. Under-standing the status of wound and owners’ practice with regards to wound man-agement is crucial for prevention and control of wound in equine. Therefore, the present study aims to estimate the prevalence of wound and identify its
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associated risk factors, and assess owners’ wound management practice in car-thorses.
Materials and methodsStudy area
The study was conducted in 10 towns, namely, Adama, Akaki, Bishoftu, Debre-Brihan, Hawassa, Holeta, Modjo, Sebeta, Shashemene and Sheno. They are located within about 275 kilometers radius from Addis Ababa, capital of Ethio-pia. These sites have different agro ecology ranging from midland with an al-titude range of 2200-2600 meters above sea level to highland with an altitude of above 2600 meters. Debre-Brihan and Sheno belong to highland category while the remaining towns are in an altitude range of midland. Horses in these towns were used for pulling carts to transport people, agricultural products and inputs, and construction materials among others. They provide service within the towns and to the nearby peri-urban areas.
Study design
A cross sectional study was conducted from November 2017 to Aril 2018 in 10 selected towns. Personal interview was conducted on randomly selected 390 carthorse drivers and physical examination was conducted on carthorses of the same drivers. The questionnaire included information on socio-demography of carthorse drivers such as sex, age, level of education, marital status, cart-horses’ driving experience, horse ownership and duration of relationship with the horse. Besides, number of people transported per trip, working hours per day, time taken per trip and resting time between trips were recorded by ask-ing the owners.
Practices of carthorse drivers on wound management were also assessed dur-ing the interview. The practices were categorized as modern, traditional and unacceptable treatments. Modern treatment refers to treating animal with scientifically accepted wound management practices. Traditional treatment included the use of medicine such as herbs known for wound treatment by the community or traditional healers. Unacceptable treatment practices refer to application of agents that are known to hinder wound healing such as bat-tery acids, mud, engine oil and caustic chemicals. The use of the unacceptable
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wound treatment in combination with any other one was considered as unac-ceptable treatment approach.
During physical examination of the selected horses, age, body condition score, location of the wound and types of bits used were recorded. Age of the horses was determined based on dental eruption pattern. Horses were grouped as young and adult. Those horses who fully replaced all incisor milk teeth were grouped as adult and others were categorized as young. Scoring of the body condition was based on 1 to 5 scales (AWIN, 2015). External injuries were considered in this study. Scars, alopecia, specific disease conditions such as epizootic lymphangitis and sarcoid were not included as wound.
Sampling technique and sample size
To determine the sample size, the expected prevalence in the study area was assumed 42% at confidence interval of 95% (Chala et al., 2017). The formula given by Thrusfield (2007) for simple random sampling method was used.
N= 1.962 *pexp *(1-pexp)/d2
Where: N=required sample size, Pexp=expected prevalence, d=desired abso-lute precision. Accordingly, the calculated sample size was 374 and 390 horses were sampled.
Ten towns were selected from central part of Ethiopia. Almost equal number of carthorses was selected from each town. Accordingly, forty horses were select-ed from the nine towns and in one town 30 horses were selected. Systematic random sampling was used in selecting individual horses from their stations where they line to wait for their turn to transport people and goods.
Data analysis
Data collected from the study sites were coded and entered to Microsoft excel spreadsheet program for analysis. Statistical analysis was done by Statisti-cal Package for Social Sciences (SPSS) software version 20. Descriptive sta-tistics, percentage, was used to express prevalence and socio-demography of the participants. Chi-square (χ2) test was used to check which variables have
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association with the dependent variable individually. Variables found to have a P-value of <0.2 in χ2 test was entered into multivariable logistic regression model for controlling the possible effect of confounders and finally variables which had significant association were identified based on 95% confidence in-terval. Odds Ratio (OR) was used in assessing the strength of association.
ResultsDemography of the respondents
Almost all carthorse drivers interviewed were male. The proportion of young age group was very high compared to other age groups. Different level of edu-cation was recorded with most people being grade 1-8. More than half of the drivers had been involved as a carthorse driver for less than two years. The majority of the respondents drive their own horse while small proportion were recruited as a driver or rent horses from others. Most of the respondents own two horses followed by those who own one horse. Limited number owns three or more horses. Nearly 90% of the horses had been working for up to only 2 years. The demographic characteristics of the respondents are summarized in table 1. The mean family size of the respondents was 4.3, ranging from the carthorse driver himself alone to 14 families supported by the income gener-ated from carting.
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Table 1: Demographic characteristics of the respondents (n=390) Characteristics Sample size %
Sex Male 389 99.7Female 1 0.3
Age [18-28] 263 67.4(28-38] 86 22.1(38-48] 25 6.4>48 16 4.1
Education Can’t read and write 30 7.7Can only read and write 15 3.8Grade 1-8 259 66.4Grade 9 and 10 73 18.7Above grade 10 13 3.4
Marriage Married 240 61.5Single 145 37.2Divorced 5 1.3
Owners driving experience
Up to 1 year 77 19.7(1-2] years 138 35.4Above 2 years 175 44.9
Ownership Owner and driver 333 85.4Driver only 57 14.6
Number of horses owned
One 168 43.1Two 204 52.3Three 11 2.8Four 4 1Five and above 3 0.8
Horse working experience
Up to 1 year 202 51.8(1, 2] years 137 35.1(2-3] years 35 9.0> 3 years 16 4.1
Prevalence and types of wound
The overall prevalence of wound in the carthorses was 51.5% (201/390). The prevalence was significantly different among the study sites (Table 2).
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Table 2: Prevalence of wound in carthorses in selected towns of central Ethio-pia (n=390)Study towns Number of
horses with wound
Prevalence (%)
χ2 p-value
Sheno 9 30.0
21.23 0.01
Debre-Brihan 13 32.5Akaki 18 45.0Bishoftu 19 47.7Modjo 21 52.5Sebeta 22 55.0Holeta 22 55.0Hawassa 24 60.0Adama 25 62.5Shashamene 28 70.0
Wound was observed on various body parts of the animals due to various causes. Wound on more than one body parts accounted 21%. Girth sore was also almost equal to this; 20.8%. Generally, several harness related wounds with high proportion were found in the study area. Fetlock sore is most com-mon from the non-harness related wounds and others occur less frequently (Table 3).
Table 3: Types of wound based on its location on animals’ body or its causes (n=390)Types of wound Number of animals with wound Prevalence (%)More than one type of wound 82 21Girth sore 81 20.8Fetlock sore 48 12.3Chest sore 46 11.8Bit sore 46 11.8Saddle sore 31 7.9Knee sore 13 3.3Whip sore 12 3.1Tail sore 12 3.1other wounds 6 1.5Collar sore 2 0.5
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Types of bit and associated wound
From 390 horses examined 173 (44.4%) were using imported bit made of metal which do not rust and has smooth surface. The remaining were using bits lo-cally made of metal having different size and surface texture. The difference in prevalence of bit sore in relation to bit types used was significant. Majority of bit sores were caused by bits made of metal with rough surface (Table 4).
Table 4: Wound associated with different types of bits (n=46)Type of bit Frequency
(n)Proportion (%)
P value
Metal bit with smooth surface 11 6.4 0.003Metal bit with different size and texture 35 16.1
χ2 =10.52
Factors associated with wound occurrence
Chi-square test indicated that, educational status, number of horses owned and body condition score of the horses were a possible risk factors associated with the occurrence of wound (p<0.5). Other factors including age of the driv-ers, owners’ driving experience, marriage status, ownership of the carthorse, horses’ work experience, working hours per day and age of the horses were not statistically associated with the occurrence of wound. After the multivariable logistic regression analysis was applied, it was identified that animals’ body condition score and number of horses owned had significant associations with the occurrence of the wound. Those horses who had body condition score 2 has shown about 2 times more likely to be wounded than horses scoring 3 [OR: 2.37, 95% CI (1.34-4.20)]. Wound was 1.5 times higher in horses where owners owned two or more horses compared to horses where owners owned only one (Table 5).
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Table 5: Factors associated with wound occurrenceVariables Wound
Yes NoOR (95%) CI
Body condition score 2 179 143 2.4 (1.36-4.25)
3 22 46
Number of horses owned 1 75 93
More than 1 126 96 1.56 (1.01-2.33)
Age of the driver 18-28 149 114 0.76(0.23-2.54)
29-39 38 48 1.09(0.32-3.73)
40-49 9 16 1.29 (0.32-5.27)
>50 6 10 1.00
Educational status Cannot read and write
15 15 2.79 (0.69- 11.38)
Can only read and write
4 11 1.05 (0.47-2.36)
Grade 1-8 134 125 0.81(0.32-2.03)
Grade 9 and 10 45 28 3.48 (0.76-15.89)
Above 10 3 10 1.00
Number of people transported per trip
Carthorses in the study area transport 3 to 10 people at a time. Most of them transport 3 to 4 people (Table 6).
Table 6: Number of people transported per tripNumber of people transported Frequency (n) %
3 - 4 335 89.85 -6 35 9.47 -10 3 0.8
Working hours
Average working time per day stated was 6.22 hours, with maximum of 12 hours and minimum of 2 hours. Most of the horses work for 2-6 hours per day (Table 7).
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Table 7: working hours per day Working hours per day Frequency (n) Percent
1-6 273 70.4>7 - 12 115 29.6
Break time between trips
The traveling time taken per trip and break time in between trips are sum-marized in table 8.
Table 8: Traveling and break time Time Mean (minutes) Minimum (minutes) Maximum (hours)Traveling time 25 5 3 Break time 42 1 4
Wound management practice
Majority (83.3%) of the owners know how to properly treat or what to do when their horses are wounded. They treat with the acceptable treatment approach like washing with homemade saline water and or look for veterinary care. The remaining owners/ drivers used or believe to use traditional treatment as well as other unacceptable treatment options (Table 9).
Table 9: Wound management practice by carthorse owners/driversWound management Frequency (n) %
Modern treatment 325 83.3Traditional treatment 25 6.4Unacceptable treatment alone or in combination to others
38 9.7
Modern and tradition treatment 1 0.3
Discussion
The current study revealed an overall wound prevalence of about 51% in carthorses. There was significant difference in the prevalence of wound among the study towns ranging from 30% to 70%. The overall prevalence was lower than several reports from Ethiopia, which include 65.4% in Hawassa (Biffa and Woldemeskel, 2006), 64.2% in Kombolcha (Fikru et al., 2015), 65.8% in Asella
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(Abdela et al., 2017) and 68.2% in Jimma (Genetu et al., 2017). However, it is higher than the finding of Chala et al. (2017) who reported 42.5% in Bishoftu. This variation might be due to the difference in owners’/drivers’ awareness on wound control and prevention, agro-ecology, cart design or type of harnessing materials used among the study areas.
The current study indicated the occurrence of wound on various parts of the body of carthorses. In majority (21%) of the cases, the wounds were found in more than one body parts. Wounds related with harness were higher compared to human inflicted and accidental wounds. From harness related wounds the highest number of wounds was found on girth area, which accounted about 21% of the examined horses. Other harness related wounds such as bit sore, chest and saddle sores were also among frequently occurring wounds. Tail sore and sore due to collar were observed on few animals. In line with this, reports from Kombolcha and Asella towns indicated the occurrence of high proportion of harness related wounds (Fikru et al., 2015; Abdela et al., 2017). Harnesses have a close relationship to the body of the animal. The skin of equine is sen-sitive to rubbing, and rough materials or lack of enough padding can cause injury (Starkey, 1998). A properly designed, well-fitted and comfortable har-ness allows the working animal to pull the equipment to the best of its ability without risk of injuries (Pearson et al., 2006).
Among the non-harness related wounds, fetlock sore relatively accounted the largest proportion occurring in about 12% of the examined horses. Knee and whip sores were observed in about 3% of the horses and other non-harness related injuries was observed in 1.5% of the horses. Wound on fetlock might be due to problem in hoof conformation, improper shoeing practice and overwork-ing of the horses, which might lead to fatigue, and kicking of foot together. Knee sore may be due to high speed during driving and overworking which could cause the animals to fall down. Whip sore is human made which might be done deliberately to force an animal to work or the horses might be beaten when they resist the order given by the drivers.
Higher proportion of bit sore was observed due to bits which were made of metal with rough surface. This result is in agreement with Mekuriaw (2019). These types of bits are not standardized to fit to the horses as this are locally
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produced by people with low knowledge about its fitness and comfort. This implies use of proper bit reduces the occurrence of bit sore significantly, and hence reduces animal suffering.
In this study, body condition score and number of horses owned were identified as risk factors. Wound was more common in horses having a body condition score of 2 compared to horses having better body condition (BCS = 3). This find-ing is similar with report by several authors: Biffa and Woldemeskel (2006), Herago et al. (2015), Abdela et al. (2017), Chala et al. (2017) and Fsahaye et al. (2018). Horses with good body condition are strong enough to pull loads to the best of their ability without risk of injury. Horses with poor condition are inca-pable of pulling equipment/humans due to less transferred power from them to attached implement, this leads to fatigue as well as injury (Starkey, 1998). Besides, equids with low body condition score have reduced body fat and con-sequently, may have less natural padding protecting them from wound caused by harness (Pritchard et al., 2005).
Majority (83%) of the owners treated their horses or believed that the wound should be treated with acceptable type of treatment like washing with home-made saline water and seeking for veterinary care. Others used traditional as well as other unacceptable treatment options. This result is in contrary with the result obtained in Kombolcha where majority (79.4%) of the owners did not provide any treatment to their animal for wound. This might be due to varia-tion in awareness among the carthorse owners/drivers about wound treatment.
Horses pulling carts transport commonly 3 to 4 people at a time. They work in average for about 6 hours a day. This is in line with report by Bifa and Woldemeskel (2006) who reported that pulling carts often work continuously for 6 to 7 hours/day, carrying 3 to 4 persons in a single trip.
Conclusion
Wound was highly prevalent health and welfare problem in the study area. Variation in the occurrence of wound was observed among the study sites. Har-ness related wound was more common to occur compared to the non-harness
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related wound. From the several potential risk factors assessed only body con-dition score and number of horses owned were found to have significant as-sociations with the occurrence of wound. Some carthorse owners’ apply agents that are known to hinder wound healing. Therefore, intervention should be implemented to reduce wound in the carthorses. Carthorses should be fed well to keep them in a good body condition. The harness and cart in use should fit the animal properly. Community education should be enhanced focusing on the negative impacts of wound, proper harnessing and wound management practices. Research should be conducted to identify the contribution of local cart design, harness materials in use and its fitness for wound occurrence in carthorses.
Acknowledgements The authors acknowledge the Society for Protection of Animals Abroad (SPANA) Ethiopia Project for its financial support in this study. We also ac-knowledge the cooperation and support provided by the carthorse drivers dur-ing the interview and examination of the animals.
Limitation of the study Harness materials used, cart design and fitness of the harnesses were not in-cluded in the current study.
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Demissie et al., Ethiop. Vet. J., 2020, 24 (2), 16-34DOI https://dx.doi.org/10.4314/evj.v24i2.2 Ethiopian Veterinary Journal
Prevalence and risk factors of swine tuberculosis in central Ethiopia
Kassa Demissie*1, Jirata Shiferaw2, Girmay Medhin3, Aboma Zewude4, Asegedech Sirak5, Takele Abayneh6, Gezahegne Mamo7, Gobena Ameni3 1Department of Animal Sciences, College of Agriculture and Natural Resource Sciences, Debre Berhan University, Debre Berhan, Ethiopia, P.O. Box 445
2 College of Veterinary Medicine and Agriculture, P.O. Box 34, Addis Ababa University, Bishoftu, Ethiopia
3 Aklilu Lemma Institute of Pathobiology, Addis Ababa University, P.O. Box 1176, Addis Ababa, Ethiopia
4 Ethiopian Public Health Institute (EPHI), P.O. Box 1242, Addis Ababa, Ethiopia
5 National Animal Health Diagnostic and Investigation Center (NAHDIC), P. O. Box 04, Sebeta, Ethiopia
6 National Veterinary Institute, P.O. Box 19, Bishoftu, Ethiopia
7 College of Veterinary Medicine and Agriculture, Addis Ababa University, P.O.Box 34, Bishoftu, Ethiopia
*Corresponding author: Email: [email protected]; Tel.: +251-911-993242, (K. Demissie)
Abstract There is paucity of information in the epidemiology and pathology of tuber-culosis in swine though the endemic occurrence of bovine tuberculosis was elucidated in Ethiopia. A cross-sectional study was employed to investigate the epidemiology of Mycobacterium tuberculosis complex and Mycobacterium avium complex in swine. The study was conducted from September 2016 to December 2017 using single intradermal comparative tuberculin test, gross pathology and histopathology. Tuberculin test was carried out in farmed swine of central Ethiopia while the necropsy at Addis Ababa Abattoirs Enterprise, Ethiopia. Of the total 329 heads of swine tuberculin tested by bovine purified protein derivative antigen, an animal level prevalence of 3% (95% CI: 2-6) and a herd level prevalence of 11% (95% CI: 1-49) were observed at a cut-off value of >2mm. Multivariable logistic regression analysis revealed the protective ex-posure effect of body condition to tuberculin test positivity in swine (Adjusted OR: 0.06; 95% CI: 0.0-1.1; P: 0.055). Swine were predominantly in close prox-imity with dairy cattle which is an important risk factor for the transmission of bovine tuberculosis from cattle to swine. Necropsy study of tuberculosis-like le-
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sions in 640 heads of swine revealed an overall lesion prevalence of 4.1% (95% CI: 2.8-6.0). In conclusion, the present study demonstrated the occurrence of swine tuberculosis in low prevalence in farmed swine and swine slaughtered at Addis Ababa Abattoirs Enterprise in central Ethiopia. Further in-depth study covering larger sample size and wider areas is warranted so as to identify the prevalent species and their zoonotic importance.
Keywords: Abattoir; Epidemiology; Ethiopia; Pathology; Swine; Tuberculosis
IntroductionTuberculosis (TB) in animals is one of the most widespread infectious diseas-es in the world that can be described as a chronic granulomatous lethal dis-ease caused by tubercle bacilli. It is widely distributed in developing countries where surveillance and control activities are often inadequate or unavailable. Risk factors for Mycobacterium bovis (M. bovis) in both animals and humans are present in the tropics (Cosivi et al., 1998). M. bovis infection has been re-ported in a wide range of animal species and is endemic in Africa (Cosivi et al., 1995). The course of TB is slow taking months or years to kill an infected animal and an animal can spread the disease to many other herd mates before it begins to manifest clinical signs (Elias et al., 2008; OIE, 2019).
The endemic nature of bovine TB (bTB) has long been reported in Ethiopia (Ameni et al., 2003; Shitaye et al., 2006; Sibhat et al., 2017). Swine are suscep-tible to Mycobacterium tuberculosis complex: MTBC (M. tuberculosis, M. bovis) and M. avium complex (MAC). Most of the TB compatible lesions in swine are localized along the digestive tract indicating the mode of infection is predomi-nantly through ingestion of untreated milk from TB infected dairy cattle which suggests transmission between species (Barandiaran et al., 2011).
The MTBC can cause similar lesions with that of MAC (Agdestein et al., 2011). Based on molecular biological studies, swine were found to be a potential source of MAC infection for humans. Based on IS1245 restriction fragment length polymorphism (RFLP) analysis, at least 75% similarity of IS1245 RFLP types was detected among 61% human and 59% swine isolates (Matlova et al., 2004). Serovars of MAC recovered from swine lymph nodes have been isolated from many environmental sources including soil, dust, water, feed, pig-compost and bedding (Gardener and Hird, 1989). Skin test by purified protein derivative (PPD) antigen is a good tool and feasible to detect TB infected herds. Abattoir surveillance of animal TB is also a rou-
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tine procedure and cost-effective method of diagnosis (Radostits et al., 2007). The single intradermal comparative tuberculin test (SICTT) with bovine and avian PPDs is used mainly to differentiate between animals infected with M. bovis and those sensitized to tuberculin due to exposure to other mycobacteria or related genera. Due to their higher specificity and easier standardization, PPD products have replaced heat-concentrated synthetic medium tuberculins (OIE, 2009).
Retrospective pork inspection data analysis for the years 1996-2005 (Shitaye et al., 2006) at Addis Ababa Abattoirs Enterprise (AAAE) and necropsy study (Arega et al., 2013) at both AAAE and ALEMA Farms PLC (Bishoftu) were the only studies undertaken in swine in Ethiopia. Despite evidence that swine are affected by TB in Ethiopia, information on its epidemiology and pathology are still lacking. The current study of swine TB by skin test is the first of its kind executed in Ethiopia. Both skin test and necropsy studies generated useful in-formation to all interested scientific audience with regard to swine TB. There-fore, the present study was initiated with the objectives of investigating the epidemiology of swine TB in dynamic herds of farmed swine in selected sites in central Ethiopia and its pathology in slaughtered swine at AAAE.
Materials and methods
Study areas
The study was conducted in farmed swine of Alage Agricultural, Technical and Vocational Education and Training (ATVET) College and farms in Bish-oftu town and Addis Ababa from September 2016 to December 2017. Necropsy study was conducted in those swine brought to AAAE to be slaughtered for pork. Moreover, necropsy study was also carried out in those swine that react-ed to both avian and bovine PPDs during SICTT. The study sites were located in central Ethiopia (Figure 1). The husbandry practices of swine in these study farms were intensive and semi-intensive.
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Figure 1. Map of the Study Areas
Study animals and sampling
Swine for skin testing were 329 heads included in 9 clusters. There were 12 clusters in the study areas representing 464 heads of swine. Necropsy exami-nation of TB-like lesions was conducted in 640 heads of apparently healthy swine slaughtered at AAAE. Dairy cattle which were 28 in number (26 females and 2 males) herded with swine in one farm were also skin tested. A swine farm has equivalent meaning to a cluster or a herd. An individual cluster was taken as a unit of sampling. The sampling frame containing lists of clusters was established for each study site by the help of local livestock development agents. Clusters were selected by simple random sampling. Before the test, each swine was identified by ear tag or a temporary ID number using an in-delible ink to write (for those without ear tags) on the back that could not be erased at least for a week. The ID numbers and all the corresponding data were recorded in a LogBook.
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Age, sex, body condition score (BCS), physiological state (open or pregnant), lactation status (non-lactating or lactating) and parity of swine were collected from farm records/owners and registered before skin test. The ages of swine were categorized into <2 years as young and ≥ 2 years as adult based on the dentition formula adopted by USDA (2018). BCS of swine was categorized into poor, medium or good according to the guidelines given in CFSPH (2011). Sows that were more than 6 weeks of prepartum and postpartum (due to immuno-logical hyporeactivity occurred in association with farrowing) and piglets with the age of above 3 months (piglets drinking colostrum from infected sows give positive reactions for up to 3 weeks after birth even though they may not be infected) were included in the skin test (Radostits et al., 2007). All apparently healthy swine brought for slaughtering to AAAE during the study period were included in necropsy study. Age, sex, origin and body condition scores of swine were recorded.
Study design and sample size
A cross-sectional study design was employed. The sample size for swine skin testing was determined using the formula for one-stage cluster sampling (Thrusfield, 2007) by considering:
g = number of clusters to be sampled; 1.96 = multiplier of the 95% confidence interval (CI); n = the average number of swine per cluster (25); Pexp= expected prevalence = 0. 5(since this study is the first of its kind in Ethiopia); d= desired absolute precision (5%) and Vc = assumed between-cluster variance = 0.02 (previous experience in bovine TB studies of central Ethiopia). Accordingly, the calculated number of clusters to be sampled was nearly 46 representing 1150 heads of swine. However, the actual number of clusters representing the study population was 12 and the number of swine in the current study sites were small (N = 464). Hence, the sample size needed an adjustment (Adj) as follows:
Adjn = G x g/ G + g = 12 x 9/ 12+ 9 = 6 clusters
The required number of swine to be PPD tested was not restricted to 6 clus-ters. Therefore, 9 out of 12 clusters representing 329 heads of swine that ful-filled the inclusion criteria were tuberculin skin tested. Those swine (n = 135) that were under 3 months of age, late pregnant and near to farrow were not PPD tested. Five strong PPD reactors’ swine (at > 2 mm cut-off point) which
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were herded with dairy cattle (4 heads) and alone (1 head) in Bishoftu were purchased, slaughtered and inspected in detail at necropsy facility of the Col-lege of Veterinary Medicine and Agriculture of Addis Ababa University (CV-MA-AAU), Bishoftu, Ethiopia. Those 635 heads of swine which were presented for slaughter at AAAE were eligible for both detailed antemortem examination and necropsy. In total, 640 heads of swine were used for the current necropsy study. Antemortem examination and necropsy were conducted from July 2015 to March 2016 for the duration of seven months following the procedure de-scribed in FAO (1994).
Skin testing
Tuberculin testing of swine was carried out by SICTT following the protocols (Songer et al., 1980; OIE, 2009). Before injection of any PPD, the hair immedi-ately caudal to the right and left base of the ears was shaved at one site on both sides of the neck (approximately 4cm square) and the skin thickness of each shaved site was measured with 0.01mm graduated callipers. All swine in 9 se-lected clusters were PPD tested. The right side was injected with bovine PPD (0.1ml of 3000 IU/ml) while the left side was injected with avian PPD (0.1ml of 2500 IU/ml) intradermally (PRiONiCS, Lelystad, The Netherlands) with 1mL sterile insulin syringe with needle separately into the respective shaved sites.
The intradermally injected tuberculin into the raised fold was indurated so that a pea-like nodule was palpable. The tuberculin skin test result was read and the thickness of the skin at each injection site was measured again after 48 hours of post PPD injection (Songer et al., 1980; APHA, 2019). The test results were interpreted based on the guidelines provided by CFIA (2019) as follows: An increase in skin-fold thickness of more than 2mm (>2mm) and/or oedema observed after the injection of avian or bovine tuberculin was considered posi-tive while no visible or palpable change in tissue at the site of injection and an increase of 2mm or less (≤2mm) in skin thickness with no oedema indicates a negative reaction. Swine reacted to either bovine or avian PPD were obtained using the formula:
[(Bov48−Bov0) − (Av48−Av0)] > 2mm to bovine PPD and [Av48−Av0) >2mm to avian PPD. Bov0 and Av0 indicated skin thickness before injecting bovine and avian tuberculins, respectively. On the other hand, Bov48 and Av48 were the corresponding skin fold thickness 48h post injection of bovine and avian tuberculins, respectively (OIE, 2009).
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Dairy cattle herded with swine were tuberculin skin tested following OIE (2009) recommendations with the aim to hypothesize interspecies transmis-sion of bovine TB. Single intradermal comparative cervical tuberculin test (SICCTT) was used. The test results were read after 72 hrs. and interpreted using ≥4mm cut-off values. In both dairy cattle and swine, a herd was consid-ered as positive if it had at least one tuberculin reactor animal (OIE, 2009).
Necropsy examination
Whenever gross lesions suggestive of TB were detected in any of the tissues, the tissues were classified as having lesions. Hence, TB-like lesions were collected from 276 lymph nodes and tissues of swine slaughtered at AAAE. Necropsy and gross pathological examination were conducted following estab-lished protocols in FAO (1994). Histopathological examination
Fat and other tissues were trimmed from TB-like lesions collected during nec-ropsy examination. They were preserved in 10% neutral buffered formalin and transported to histopathological examination laboratory at the National Ani-mal Health Diagnostics and Investigation Center (NAHDIC), Sebeta, Ethio-pia. The tissue processing for histopathological examination was performed according to Santos et al. (2010) protocol. Briefly, the tissues were dehydrated in different grades of ethanol (70%, 95% and 100%), cleared in xylene and re-fixed with formalin in an automatic tissue processing machine. Then, the tis-sues were embedded in paraffin using an embedding machine and cut into thin sections of 4-5μm using a microtome. Subsequently, the tissue sections were stained with haematoxylin and eosin and prepared for microscopic examina-tion following the procedures described previously (Bancroft and Cook, 1994).
Ethical consideration and clearance
The ethical clearance for the study was provided by the Animal Research Eth-ics Review Committee of College of Veterinary Medicine and Agriculture, Ad-dis Ababa University (CVMA-AAU) (Ethical Clearance Certificate Ref. No.: VM/ERC/007/03/09/2016). Swine suffering during skin test and antemortem inspection were ameliorated by safe handling.
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Dataset Management and Statistical Analysis
Data were entered and stored in separate MS-Excel 2007 spread sheet, thor-oughly screened for errors, coded, imported and analyzed in Stata Version 12.0 for Windows. Descriptive statistics was used to determine the prevalence of swine TB. The associations of putative risk factors to swine TB were deter-mined using chi-square (χ2) test of independence in univariate logistic regres-sion. The strength of associations of the potential risk factors to swine TB was indicated by the odds ratio in multivariable logistic regression. The 95% CI other than a value of one and P<0.05 was set for statistical significance and effect of interactions. Swine herd was considered as a random effect and risk factors were considered as fixed effects. Variables in univariate logistic regres-sion analysis were selected for multivariable logistic regression analysis when P<0.05. Variables were fitted separately to the final multivariable logistic re-gression model. A variable was considered to be a confounder and included in the model if its inclusion altered the OR of the estimated risk by 25% or more.
Results
Prevalence and risk factors
Dairy cattle herded with swine in one farm were tuberculin skin tested by us-ing SICCTT. Of the total 28 dairy cattle tuberculin skin tested, 32% (9/28; 95% CI: 2-27) reacted to avian PPD, 86% (24/28; 95% CI: 11-44) reacted to bovine PPD and 18% (5/28; 95% CI: 0.5-22) reacted to both avian and bovine PPDs at ≥4mm cut-off value.
The SICTT was conducted on 329 heads of swine. The individual apparent prevalence of bTB in swine was 3% (10/329; 95% CI: 2-6) at >2mm cut-off val-ue. In the total 9 clusters tested, the herd prevalence was 11% (1/9; 95% CI: 1-49). In addition, 2.7% (9/329; 95% CI: 6-13) of swine reacted to avian PPD and 1.5% (5/329; 95% CI: 3-8) reacted to both avian and bovine PPDs at >2mm cut-off value (Table 1).
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Table 1. Number and percent of swine positive to tuberculin skin test at >2mm value to different antigens by host related factorsVariables No.
testedSICTT ResultsPPDApositive
% PPDBpositive
% Mixedpositive
%
SexMale
329133
91
2.70.8
10 2
31.5
51
1.50.8
Female 196 8 4.1 8 4.1 4 2Age 329 9 2.7 10 3 5 1.5<2 years 261 2 0.8 0 0 0 0≥2 years 68 7 10.3 10 14.7 5 7.4BCS≥5: Good
329105
90
2.70
10 0
3 0
50
1.50
[3-4]: Medium 178 3 1.7 2 1.1 1 0.6[1-2]: Poor 46 6 13 8 17.4 4 8.7Physiological state
196 8 4.1 8 4.1 4 2
Open 137 5 3.6 3 2.2 1 0.7Pregnant 59 3 5.1 5 8.5 3 5.1Lactation status 196 8 4.1 8 4.1 4 2Non-lactating 146 7 4.8 6 4.1 3 2.1Lactating 50 1 2 2 4 1 2Parity 196 8 4.1 8 4.1 4 2No parity 111 3 2.7 1 0.9 4 3.6[1-3] Parity 64 5 7.8 6 9.4 0 0[4-6] Parity 21 0 0 1 4.8 0 0
No.: total number of swine tuberculin skin tested; BCS: body condition score; PPDA: avian purified protein derivative antigen; PPDB: bovine purified pro-tein derivative antigen; Open: dry or non-pregnant
The gross pathological changes observed on the skin in some of test positive swine were nodular swelling characterized by firm erythematic nodules. The injection site constitutes a diffuse swelling, skin thickening, superficial necro-sis and sloughing.
Univariate logistic regression analysis of host risk factors revealed body condi-tion (P = 0.000) and parity (P = 0.026) were significantly associated with tuber-culin skin test positivity. Moreover, physiological state of swine was associated
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to skin test positivity with marginal statistical significance (P = 0.058) (Table 2). Table 2. The associations of different risk factors to swine skin test positivity post bovine PPD injection and interpreted at >2mm cut-off value
Variables No.tested
SICTT ResultsPPDBpositive
% χ2 P-value
SexMale
329133
10 2
31.5
1.96 0.199
Female 196 8 4.1
Age 329 10 3 0.00 -
<2 years 261 0 0
≥2 years 68 10 14.7
BCS≥5: Good
329105
10 0
3 0
17.29-
0.000*
[3-4]: Medium 178 2 1.1
[1-2]: Poor 46 8 17.4 -
Physiological state 196 8 4.1 3.74 0.058**
Open 137 3 2.2
Pregnant 59 5 8.5
Lactation status 196 8 4.1 0.00 0.973
Non-lactating 146 6 4.1
Lactating 50 2 4
Parity 196 8 4.1 7.57 0.026*
No parity 111 1 0.9
[1-3] Parity 64 6 9.4
[4-6] Parity 21 1 4.8
BCS: body condition score; *: statistical significance; **: statistical marginal significance; Open: dry or non-pregnant
Multivariable logistic regression analysis revealed that body condition in swine has a protective exposure effect to tuberculin skin test positivity (adjusted OR = 0.06; 95% CI: 0.0-1.1) with statistical marginal significance (P = 0.055) (Table 3).
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Table 3. Multivariable logistic regression analysis of tuberculin reactors’ swine with the associated risk factors
Variables No. tested No. positives
COR (95% CI) AOR (95% CI) P-Value to AOR
SexMale 133 2 1 1Female 196 8 0.36 (0.1-1.7) * -Age (Years)<2 261 0 1 1≥2 68 10 - * -
BCS≥5: Good 105 0 - -[3-4]: Medium 178 2 1 1[1-2]: Poor 4 6 8 0.05 (0.01-
0.26)**0.06 (0.00-
1.07)**0.055**
Physiol. stateOpen 137 3 1 1Pregnant 59 5 0.24 (0.06-
1.05)1.17 (0.06-
22.45)0.917
LactationNon-lactating 146 6 1 1Lactating 50 2 1.03 (0.2-5.27) 1.03 (0.04-
25.63)0.987
ParityNo parity 111 1 1 1[1-3] parity 64 6 11.38 (1.3-
96.79)3.80 (0.08-
185.02)0.501
[4-6] parity 21 1 5.5 (0.3-91.58) 1.54 (0.05-45.23)
0.803
*: Sex and age were not considered in multivariable model due to collinearity issues thus not presented in the table; BCS: body condition score; COR: Crude Odds Ratio; AOR: Adjusted Odds Ratio; Number 1: refers to reference; **: statistical significance; Physiol. state: Physiological state; Open: dry or non-pregnant
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Abattoir lesion prevalence
The abattoir lesion prevalence in swine was 4.1% (26/640; 95% CI: 2.8-6.0) (Table 4).
Table 4. Association of host related risk factors in relation to TB-like-lesions in swineVariables No.
inspdNo. positive
Prevalence(%)
χ2 OR 95% CI for OR
P-value
Sex 640 26 4.1 0.85 1.46 0. 64-3.33 0.365Female 277 9 3.2Male 363 17 4.7Age 640 26 4.1 2.21 1.81 0.83-4.00 0.137<2 years 386 12 3.1≥2 years 254 4 1.6BCS 640 26 4.1 0.82 1.16 0.45-3.00 0.753Poor 206 7 3.4Medium 305 12 3.9Good 129 7 5.4Origin 640 26 4.1 0.51 1.29 0.55-3.03 0.558AA 262 9 3.4Bishoftu 319 14 4.4Others 59 3 5.1
No. inspd: total number of swine inspected; OR: Odds Ratio; AA: Addis Ababa; Others: refer to names of regional towns like Bahr Dar, Debre Berhan, Dessie and Gondar.
Gross pathology and histopathology
Gross pathological characterization of TB-like lesions revealed caseous, en-larged and cheesy in submandibular lymph nodes. Lesions in the retropharyn-geal lymph nodes were characterized as purulent, early caseation and caseous. The mesenteric lymph nodes were enlarged. Caseous lesions were observed in the spleen. Focal calcification was observed in the liver. White spots, focal cal-cification and caseous lesions were seen in the lungs. Gross TB-like lesions in the present study were repeatedly encountered in retropharyngeal (27%: 7/26), submandibular (12%: 3/26) and mesenteric (15%: 4/26) lymph nodes as well as 4% (1/26) in each of the lungs, spleen and liver.
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Histopathological analysis of TB-like lesions revealed granulomas with cen-tral necrosis and calcification. The histological arrangement from the center to outer was made up of lymphocytes, macrophages and epithelioid cells dis-tributed under connective tissue layers. Moreover, the central area is made of necrotic cellular debris, calcium deposits and connective tissue capsule walled off the granulomas from the surrounding tissue. The presence of concomitant pyogranulomatous and granulomatous lesions in different organs were also observed. Some granulomas were characterized by necrotic foci, intense calcifi-cation and fibrosis with absence of epithelioid cells. Multiple small granulomas in the lymph node with less dense lymphocyte at periphery and epithelioid cells surrounding the deep outer lymphatic layer of the granulomas were observed.
DiscussionThe current study of swine TB by making use of SICTT is the first of its kind executed in Ethiopia. Tuberculin skin test and necropsy were employed to investigate the epidemiology of swine TB in central Ethiopia. Skin test (3%) and necropsy (4.1%) studies have confirmed the occurrence of swine TB at low prevalence in central Ethiopia. The lower relative prevalence of TB in swine is due to the tendency of the disease to remain localized in this species and the early age of slaughter (Radostits et al., 2007).
The greater of the reactions to either avian PPD or bovine PPD indicates the organism responsible for sensitization. There exists no clear evidence on the immunological relationship between these two but studies have shown that co-infection with MAC compromises bTB skin test results by negatively influ-encing the sensitivity of the tuberculin test. Some decrease in skin sensitivity after parturition occurs in sows infected with M. bovis but may not occur when the infection is associated with M. avium (Radostits et al., 2007). Cattle sen-sitized by MAC might conceal M. bovis for a period of time. However, it is not clear to what extent this disease could jeopardize the detection of bTB with skin test thus requiring further research (Mekonnen et al., 2019).
SICCTT in this study revealed 86% of dairy cattle herded with swine reacted to bovine PPD. Cattle served as sources of infection to swine in bTB endemic farms (Arega et al., 2013). When the disease is common among dairy cattle in an area, 10-20% of the local swine are likely to be infected due to interspecies transmission from bovine to swine. Uninfected swine can easily get bacilli from their contaminated bedding, feed and drinking water (Radostits et al., 2007). Inhalation of infected aerosols or feeding on contaminated feed or water might
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be hypothesized to be sources of mycobacterial infection to swine in the cur-rent study. The predominant reaction of swine and bovine to bovine PPD in the current study has indicated its zoonotic risk to human beings in the commonly experienced shelter sharing of swine with human beings and herding together different species of livestock in Ethiopia.
The tuberculin skin test was evaluated in Arizona as an epidemiological tool for measuring prevalence of TB infection in swine herd and 63.3% of swine revealed positive responses. At slaughter, 61.5% were shown to have TB-like lesions in one or more lymph nodes of the digestive tract. A good correlation (97.1%) exists between skin test reactors and swine with lesions (Matlova et al., 2004).
Female swine in this study were more reactive to bovine PPD than males due to their longer duration in the farms. Adult female breeding animals had lon-ger and repeated chance of exposure to mycobacterial infection due to factors such as lactation, pregnancy and parturition which all are physiological stress-ors that suppress the immune responsiveness (Ameni et al., 2007; Elias et al., 2008; Mamo et al., 2013; Terefe, 2014; Bekele et al., 2016) and might cause in-fection via endogenous reactivation of baciili. The immunological hyporeactiv-ity that occurs in association with pregnancy and parturition generally makes pregnant sows susceptible to the disease (Radostits et al., 2007).
Swine with poor body condition were more affected in this study and field observation revealed poor feeding regimen. Stress caused by poor feeding in animals could subject to severe diseases like TB (Ameni et al., 2006). Higher prevalence of bTB was reported in animals with poor body condition which con-forms well to the established fact that animals’ resistance to TB is reduced by a shortage of feed and/or unbalanced diet attributable to a deficiency of proteins, minerals and vitamins in the diet (Elias et al., 2008). In addition, poor condi-tioned animals were susceptible to bTB infection due to weak immunological responses (Nuru et al., 2015).
Necropsy revealed low prevalence of TB-like lesions in slaughtered swine in the current study. Necropsy has low sensitivity of lesion detection (Shitaye et al., 2007) due to lack of visible lesions in tuberculin reactors in the early stage of infection (Tsegaye et al., 2010). However, some dairy cattle without visible lesions were culture positive which may be due to prior spread of the bacterium in the tissues during an early stage of infection but yet to result in lesion (Berg et al., 2009). The lesion prevalence in the present study is higher than 0.009%
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(Shitaye et al., 2006), 0.02–1.83% (Shitaye et al., 2007) and 1.48% (Bogale et al., 2004) but slightly lower than 5.16% (Ameni and Wudie, 2003) and 5.8% (Arega et al., 2013). Arega et al. (2013) experienced 67% (563/841) of the swine brought for slaughter were less than one year of age. Experience indicated that swine are slaughtered at their early age for digestible pork. Significantly higher number of swine >1year of age were found to be lesioned than those of ≤1 year (Arega et al., 2013) which supported the frequent obser-vation of gross TB-like lesions in swine of ≥2years of age in this study. Gross TB-like lesions in the present study were repeatedly encountered in lymph nodes of the gastrointestinal tract. The percentage of gross TB-like lesions was the highest (29%) in submandibular lymph node and the lowest (6%) in me-diastinal lymph node (Arega et al., 2013) which is congruent to this finding. Majority of TB-like lesions were detected in swine where localization as non-progressive abscesses occurs in the lymph nodes of the head and neck (Ra-dostits et al., 2007; Ameni et al., 2013).
The study in the Czech Republic detected TB-like lesions in 3.6% of the slaugh-tered swine (Matlova et al., 2004). Among the predominant localization of TB-like lesions in mesenteric and cervical lymph nodes in slaughtered swine, it could be suspected that animals were primarily infected by the ingestion of mycobacteria (Songer et al., 1980; Matlova et al., 2004). Study on environmen-tal source of mycobacteriosis in swine which were exposed to dirt pens and then slaughtered in California revealed an abattoir TB-like lesion prevalence of 9.4% (Gardener and Hird, 1989).
Haematoxylin-eosin (H-E) stained TB-like lesions in histological analysis in this study revealed the presence of lymphocytes, epithelioid cells, necrotic cel-lular debris, fibrosis, calcification and necrosis. Parallel to this, Ameni et al. (2000) indicated the presence of cellular infiltration, macrophages, giant cells, necrosis or calcification in H-E stained tissues in dairy cattle. Moreover, soft foci of caseous necrosis were usually present upon gross and microscopic ex-amination (Songer et al., 1980) which is in line with the present finding.
ConclusionTuberculosis in swine in the study area occurred in low prevalence.
31 Ethiop. Vet. J., 2020, 24 (2), 16-34
Demissie et al.,
Limitation of the studySwine farm owners were not voluntary for their swine to be PPD tested and hence small sample size was one of the limitations. In addition, the mycobacte-rial species in swine were not identified and characterized due to time limita-tion.
Conflict of interestThe authors declared there is no conflict of interest exists.
AcknowledgementsThe authors would like to acknowledge the TB laboratory of Aklilu Lemma In-stitute of Pathobiology (ALIPB), participated professionals from CVMA-AAU, animal health and production staffs of Oromia, owners of swine farms and field staffs for their time. Ali Gebeyehu and Dr. Estifanos Lemma are acknowledged for development of the GIS map to indicate the study sites.
References
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Tsigab et al., Ethiop. Vet. J., 2020, 24 (2), 35-53DOI https://dx.doi.org/10.4314/evj.v24i2.3 Ethiopian Veterinary Journal
Ethiop. Vet. J., 2020, 24 (2), 35-53
Effects of skin and hide defects on quality grades and physical characteristics of crust leather
Tewelde Tsigab1, Abrha Bsrat1, Redae Alemayohu2, Mengstu Ashebre Arefe2, Niraj Kumar1, Birhanu Hadush1*1College of Veterinary Sciences, Mekelle University, Mekelle, Tigray, Ethiopia
2Department of Manufacturing Engineering, Ethiopian Institute of Technology, Mekelle Univer-sity, Mekelle, Tigray, Ethiopia
*Corresponding author: [email protected], ORCID, 0000-0003-3614-595X, Tel: +251 919 366578
Abstract Leather is one of the important export commodities in Ethiopia. However, its quality and physical characteristics are affected by diseases, handling and storage problems. A cross-sectional study was conducted to identify major hide and skin defects and assess their effect on quality grades and physical characteristics of crust leather. A total of 6530 hides and skins were inspect-ed at Sheba Tannery and Leather Industry Private Limited Company (PLC) through standard operative procedures. For tests on physical characteristics of crust leather, top 6 defects of skin and hide were identified thereby each 5-crust leather per defect were evaluated by standard experiments for deter-mining testing tensile strength, tear strength and percentage elongation at break. The overall hide and skin rejection was 32.7%. The rejection in wet blue hide (23.5%) was higher than for wet blue salted and dry goatskins (5.1%) and pickled sheepskins (4.1%). Scratch (20.5%), Wound (14.6%) and Cockle (12.7%), were the most common pre-slaughter skin and hide defects found in wet blue hide, goatskin and pickled sheepskin, respectively. Knife cut with proportion of 21.1%, 17.5% and 4.5% respectively in wet blue hide, goatskin and pickled sheepskin was the major slaughter defect. Putrefaction was of the most common post-slaughter defect in wet blue hide (5.3 %), pickled sheepskin (1.6%) and goatskin (2.7%). Among the identified major defects, statistically significant reduction (p<0.05) were noted in tensile strength, tear strength and percent elongation of the crust leather. Major skin and hide defects lead to considerable economical losses through reducing quality and physical perfor-mance characteristics of crust leather. Hence, innovative leather grade correc-tion technologies are timely important.
Keywords: Defects; hide; physical characteristics; quality; skin
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IntroductionLeather industry is one of the oldest and largest industries in the global econ-omy through its massive potential for employment, growth and exports earn-ings. Leather is an important input to footwear, gloves, belts, apparels and others. In the 2001, world demand for the leather and leather products was USD 24.3 billion (Adem, 2019) and it escalated to USD 347.50 billion in 2010 (TDAP, 2011). Leather upper footwear market is the biggest market amongst all the leather sub-sectors, accounting for more than 70% of the global leather consumption (TDAP, 2011) and 58.5% of the global footwear types (IBISWorld, 2010; TDAP, 2011). Footwear consumption has been rapidly increasing world-wide from year to year; from 11 billion pairs in 1999 (Ashebre, 2014) to more than 20 billion pairs of shoes in 2005 (Shahin et al., 2007) and then estimated to be 25 billion pairs in 2018 (IBISWorld, 2010; TDAP, 2011). About 83% of the global footwear products are manufactured in Asian countries, where china shares 70% (TDAP, 2011).
Ethiopia has the largest livestock population in Africa. The country is 10th larg-est in the world with an estimated population of 60.3 million heads of cattle, 31.3 million sheep and 32.7 million goats (CSA, 2017). The animal resources gave the country a comparative advantage in raw materials needed for the leather sector. The country has a long tradition in processing and export of leather and its products. The modern leather goods industry dates back to the time when the modern tanning industry was established in mid 1920s by the Ethiopian Investment Agency (Asegedom et al., 2019). Next to coffee, leather and leather products contribute a lot for Ethiopian export earnings ( Fereja et al., 2017; Asegedom et al., 2019). However, the leather sector in Ethiopia still suffers with tremendous challenges along its value chain. Traditional hus-bandry management, extensive farming system, prevalence of rampant ani-mal diseases, backyard slaughtering habit, lack of modern facilities i.e. slaugh-tering, preservation and transport, weak marketing system and information and lack of awareness of actors on the value chain are the major obstacles in this industry (Mesele et al., 2015; Fereja et al., 2017). Estimates of losses to the Ethiopian economy due to such problems reached US$ 14 million per year (Solomon, 2011). Hence, this study had identified major defects of hide and skin (pre-slaughter, slaughter and post-slaughter) and determined their effect on quality and physical characteristics of crust leather.
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Materials and Methods Description of the study area
The study conducted in Sheba Tannery and Leather Industry Private Limited Company (PLC). It is located in Wukro town in eastern zone of the Tigray Regional State, Northern Ethiopia between longitudes and latitudes of 13° 47’ 59.99” N 39° 35’ 59.99” E. It is 47 km north from Mekelle, the capital city of the region. The tannery obtains rawhide and skin mainly from Tigray region, Addis Ababa, Gondar and Wollo areas of Amhara region. The company is one of the largest tanneries in the country that exports most of its products to Italy, China, India, Pakistan, the Netherlands, Turkey, Thailand, Malaysia and other countries. Besides, it supplies finished products like shoes and other leather articles to domestic markets (Kahsay et al., 2015; Kuria et al., 2016).
Study design and sampling
A cross-sectional study design was employed on wet blue hide, pickled sheep-skin, and air-dried and salted goatskin collections of the tannery. One batch, a total of 6,530 skins and hides, of which 1,950 pickled sheep skin, 1,800 air dry goat skin, 1,780 wet salted goat skin and 1,000 wet blue hide received from the beam house operation were examined for defect type at crust leather stage. Moreover, 9,500 hides and skin (3,900 sheep skin at pickled stage, 3,600 goats at wet blue stage and 2,000 hides at wet blue) used for further analysis of de-fects and quality grading.
Defect assessment and quality grading
Each selected skin or hide was examined for defects (pre-slaughter, slaughter and post-slaughter) in natural light by trained skin selectors of the company and the research groups. Criteria indicated in Quality Standard Authority of Ethiopia as described in the International Organization for Standardization (Muralidharan et al., 1999) were used to identify defects and graded leather quality.
Determination of effect of defects on physical characteristics of crust leather
Crust leather is the term applied to leather, which is dried after tanning but has not yet been dyed. After descriptive analysis of data on defects observed
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(Table 1-3), top six major defects for each of hide, sheep skin and goat skin (wound, scratch, brand mark, putrefaction, knife cut and cockle) were selected for determining the effect of those defects on physical characteristics of leather (tensile strength, tear strength and percent elongation). For each defect, 5 each for wet blue goat skin, wet blue sheep skin and wet blue cattle hide were se-lected. Considering five skin/hide per an identified major defect, 90 samples (5 skin/hide*6 defects = 30 hide, 30 sheep skin and 30 goat skin) were purposively selected at crust level. All samples were collected by cutting in accordance with official sampling position as per the Society of Leather Technologist and Chem-ists methods IUP 2 (Kuria et al., 2016). Tensile-strength, percent elongation and tearing strength evaluation were conducted in accordance with the official method of analysis adopted by the Society of the Leather Technologists and Chemists as it has been stated by Kuria et al., (2016). Then the specimens were conditioned by keeping them in standard environment, i.e., temperature 20 ± 2 °C and relative humidity 65 ± 2% for 48 hours before testing as it has been de-scribed in the International Organization for Standardization (Muralidharan et al., 1999).
Tensile strength test (TNS): Tensile strength indicates the overall strength of the leather. Thicknesses of the specimens were measured according to IUP/6 testing method, using a standard measuring instrument as described by Kuria et al., (2016). The cross-sectional area of each specimen calculated by multiply-ing its width by its thickness, and values expressed in square centimeters. The span length of the tensile test is 100 mm apart and the crust leather specimen clamped in the jaws so that the edges of the jaw lie along lines AB, CD (Figure. 1). The machine then run until the specimen is broken and the highest load reached taken at the breaking load. The formula is Tensile strength (kg/cm2) = Load at breaking (kg)/Cross sectional area (cm2) (Kuria et al., 2016).
Figure 1: Schematic illustration of a standard tensile sample -Dumbbell shape (ISO 2589:2016 [IULTCS/IUP 4]).
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Tear strength (TRS): Tearing strength is the load required to continue a tear in a crust leather sample, once load is applied. There are different ways to measure tearing strength. Most common double edge tear method used in this study using the standard IUP/8 method as described by Kuria et al., (2016). The samples were punched out using a steel die of standard dimension, and taken for thickness measurement and the specimens were conditioned by keep-ing them in standard environment, i.e., temperature 20 ± 2 °C and relative humidity 65 ± 2% for 48 hours before testing (Muralidharan et al., 1999). The prepared samples were mounted in the universal tensile measuring machine (UTMM), and then the load was applied at a constant rate of 100 mm/min, till break (Kuria et al., 2016).
Data analysis
Data entry made through Excel spreadsheet version 2010 and analyzed using STATA12 statistical software. Descriptive statistics used to report percentages and counts. One sample t-test used to compare deviation of mean of physi-cal characteristics of the selected defects against their standard. At all levels, p<0.05 was considered statistically significant.
Ethical approval and consent to participate
A letter of request to work in the tannery was prepared and approved by the management body of the tannery. This study was conducted once a consent was obtained and necessary inputs was permitted. Examination of skins and hide for defects and physical quality performance characteristics made by will-ing and allowed expertise.
ResultsDefects of skins and hides
In this study, wet blue hide had the highest percentage of defects. The major pre-slaughter, slaughter and post-slaughter defects were scratch (20.5%), knife cut (21.1%), and putrefaction (5.3%), respectively (Table 1). On pickled sheep-skin, the prevalence of pre-slaughter defect (27%) was higher than at slaugh-ter (5.6%) and at post-slaughter (4.2%). The major pre-slaughter, slaughter and post-slaughter defects on pickled sheepskin were cockle (12.7%), knife cut (4.5%), and putrefaction (1.6%) respectively (Table 2). Major pre-slaughter de-fect of both wet blue salted and air-dried goat skin were presence of wound (14.6 %) followed by scratch marks (9.2 %) while knife cut (18 %) was the high-
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est slaughter defect (Table 3). Their effects on the quality grading and physical performance of leather were as follows.
Table 1: Pre-slaughter, slaughter and post-slaughter defects on wet blue hideDefects Pre-slaughter
(N=1000)Slaughter (N=1000) Post-slaughter (N=1000)
N % n % N %Brand mark 29 2.9Scratch 205 20.5LSD 27 2.7Wound 17 1.7Cockle 61 6.1Wart 3 0.3Poor pattern 4 0.4Hole 19 1.9Knife cut 211 21.1Machine defect 1 0.1Putrefaction 53 5.3Heat 29 2.9
N=total sampled hide/skin; n=samples with defects, LSD= Lumpy skin disease
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Table 2: Pre-slaughter, slaughter and post-slaughter defects on pickled sheepskinDefects Pre-slaughter
(N=1950)Slaughter (N=1950) Post-slaughter
(N=1950)N % n % n %
Brand mark 0 0Scratch 202 10.4Pox 19 0.9Wound 132 6.7Cockle 248 12.7Wart 0 0Poor pattern 9 0.5Hole 12 0.6Knife cut 87 4.5Machine defect 29 1.4Putrefaction 32 1.6Heat 24 1.2
N=total sampled hide/skin; n=samples with defects
Table 3: Pre-slaughter, slaughter and post-slaughter defects on air dried and salted goatskinDefects Pre-slaughter (3580) Slaughter (3580) Post-slaughter (3580)
Dry skin (N=1800)
Salted skin (N=1780)
Dry skin (N=1800)
Salted skin (N=1780)
Dry skin (N=1800)
Salted skin (N=1780)
n % n % n % n % n % n %
Brand mark 3 0.2 8 0.4
Scratch 87 4.8 82 4.6
Pox 65 3.6 71 4
Wound 163 9 100 5.6
Cockle 19 1 22 1.2
Wart 0 0 0 0
Poor pattern 12 0.7 8 0.4
Hole 9 0.5 7 0.4
Knife cut 100 5.5 221 12.4
Machine defect 13 0.7 11 0.6
Putrefaction 15 0.8 35 1.9
Heat 4 0.2 8 0.4N=total sampled hide/skin; n=samples with defects
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Quality grading of skins and hides
The overall rejection of skins and hides was 32.7% of which it was 23.5% for wet blue hide, 5.1% for wet blue salted and air-dried goat skin, and 4.1% for pickled sheepskins. None of the skin and hide scored grade I-III. Only 3% of wet blue salted and air-dried goatskin, 1.1% of pickled sheepskin and 0.07% of wet blue hide were of grade IV. The majority of the skin and hide were in grade VI and VII (Table 4). The distribution of defects in the quality grades of skins and hides was as shown in Tables 5 -7.
In pickled sheepskin, most defects, particularly cockle/ekek and scratch were highly distributed in grade IV-VII. The defects responsible for rejection of pickled sheepskin were cockle (18.7%), putrefaction (15.2%), flay cut (10.3%), scratch (9.5%), scar (9.2%), poor pattern (8.5%), pox (7.1%), machine defect (6.4%), poor substance (4.3%), vein mark (4.9%), and crack (4.6%) (Table 5). On wet blue goatskin, most defects distributed in grade IV-VII. The defects respon-sible for rejection of wet blue goatskin were crack (18.9%), cockle (18.6%), scar (17.4%), scratch (15.9%), vein mark (11%), flay cut (5.4%), brand mark (3.4), poor pattern (2.3%), putrefaction (1.5%), pox (1.5%), machine defect (0.4%), and wound (0.4%) (Table 6). On wet blue cattle hides; brand mark, knife cut, putrefaction and scratch marks were most important defects observed in hide quality grades of IV-VII. Defects responsible for rejection of wet blue hide were knife cut (25.6%); putrefaction (20.2%), scratch (15.7%), scar (11.5%), cockle (7.4%), brand mark (6.4%), wound (3.8%), machine defect (3.5%), poor pattern (1.6%), tick damage (1.6%), crack (1.3%), lumpy skin disease (1%), and vein mark (0.3%) (Table 7).
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Table 4: Grading proportion of hide and skin in Sheba tannery and leather industry Input type IS IQ Grades
I II III IV V VI VII Rejected
Wet blue hide Small 516 - - - 17 79 217 203
Medium 720 - - - 2 28 315 195 180
Large 638 - - - 103 201 189 145
Extra large
978 - - - 73 718 51 136
Total 2852 - - - 2 (0.07)
221 (7.4)
1313 (46)
652 (23)
664 (23.5)
Wet blue salted and dry goatskin
Small 945 - - - 18 64 492 319 52
Medium 766 - - - 17 308 261 126 54
Large 789 - - - 39 323 271 119 37
Extra large
1003 - - - 25 412 270 257 39
Total 3503 - - - 99 (3) 1107 (31.6)
1294 (37)
821 (23.4)
182 (5.1)
pickled sheepskin
Small 945 - - - 12 58 500 325 50
Medium 766 - - - 5 363 249 103 46
Large 789 - - - 391 298 93 7
Extra large
1003 - - - 23 412 267 260 41
Total 3503 - - - 40 (1.1)
1224 (35)
1314 (37.5)
781 (22.2)
144 (4.1)
IS = Input size; IQ = Input quantity
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Table 5: Distribution of defects on sheep skin at pickled stage in different quality grades (N=3900)Type of defects Grade
IV V VI VII/rejected Total
n % n % n % n %
Cockle/ekek 59 19.5 43 14.5 38 16.7 53 18.7 193
Scratch 47 15.6 54 18.2 35 15.4 27 9.5 163
Flay cut 29 9.6 38 12.8 20 8.8 30 10.6 113
Scar 22 7.3 26 8.8 23 10.1 26 9.2 97
Crack 24 7.9 22 7.4 18 7.9 13 4.6 77
Veins 34 11.3 24 8.1 13 5.7 14 4.9 85
Putrefaction 20 6.6 21 7.1 31 13.6 43 15.2 115
Poor pattern 23 7.6 20 6.8 15 6.6 24 8.5 82
Pox 19 6.3 15 5.1 12 5.3 20 7.1 66
Machine defect 13 4.3 22 7.4 12 5.3 18 6.4 65
Poor substance 12 4.0 11 3.7 11 4.8 15 5.3 49
Total defects 302 296 228 283 1105NB: n=samples with defects
45
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Ethiop. Vet. J., 2020, 24 (2), 35-53
Table 6: Distribution of defects on goat wet blue stage in different quality grades (N=3600)
Defects Grade
IV V VI VII/reject Totaln % n % n % N %
Cockle/ekek 19 15.4 66 21.6 59 19.8 49 18.6 193
Scratch 11 8.9 42 13.8 35 11.7 42 15.9 130
Flay cut 6 4.9 18 5.9 9 9.7 43 16.3 76
Scar 12 9.8 53 17.4 55 18.5 46 17.4 166
Crack 13 10.6 45 14.8 52 17.4 50 18.9 160
Veins 49 39.8 56 18.4 47 15.8 29 11.0 181
Putrefaction 3 2.4 5 1.6 6 2.0 4 1.5 18
Poor pattern 3 2.4 7 2.3 5 1.7 6 2.3 21
Pox 2 1.6 2 0.7 2 0.7 4 1.5 10
Brand mark 0 - 3 1.0 2 0.7 9 3.4 17
Machine defect
2 1.6 2 0.7 0 - 1 0.4 5
Wound 2 1.6 2 0.7 1 0.3 1 0.4 6
Hole 1 0.8 2 0.7 2 0.7 0 - 5
Total grade 123 305 298 264 988
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Table 7: Distributions of defects on wet blue hide stage in different grades (N=2000)Defects Grade
I-III IV V VI VII/rejected Total
n % n % n % n % N %
Cockle/ekek 0 0 1 1.6 2 1.7 25 9.7 23 7.4 51
Scratch 0 0 13 20.6 17 14.3 32 12.4 49 15.7 111
Knife cut 0 0 6 9.5 53 44.5 87 33.7 80 25.6 226
Scar 0 0 4 6.3 8 6.7 31 12.0 36 11.5 79
Crack 0 0 5 7.9 1 0.8 3 1.2 4 1.3 13
Veins 0 0 0 - 0 - 6 2.3 1 0.3 7
Purification 0 0 6 9.5 14 11.8 47 18.2 63 20.2 130
LSD 0 0 2 3.2 3 2.5 0 - 3 1.0 8
Machine defect 0 0 1 1.6 3 2.5 6 2.3 11 3.5 21
Brand mark 0 0 22 34.9 5 4.2 7 2.7 20 6.4 34
Poor pattern 0 0 0 - 5 4.2 3 1.2 5 1.6 13
Wound 0 0 3 4.8 6 5.0 8 3.1 12 3.8 29
Tick hole 0 0 0 - 2 1.7 3 1.2 5 1.6 10
Total defects 0 0 63 119 258 312 732
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Tabl
e 8:
T te
st R
esul
t for
phy
sica
l cha
ract
eris
tics
of g
oat a
nd s
heep
skin
cru
st le
athe
r an
d cr
ust h
ide
leat
her
Var
iabl
esP
hysi
cal t
est
OB
SC
rust
goa
t lea
ther
Cru
st s
heep
leat
her
Cru
st h
ide
leat
her
Mea
nSD
CI
(95%
)M
ean
SDC
I (9
5%)
Mea
nSD
CI
(95%
)
Wou
ndTe
nsile
str
engt
h5
131.
1420
.96
105.
10-
157.
1719
0.02
6.91
181.
43-1
98.6
018
4.12
4.83
178.
17-1
90.1
2
Tear
str
engt
h5
88.2
410
.71
74.9
3-10
1.54
81.0
610
.32
36.3
1-16
5.8
90.5
42.
8087
.06-
94.0
1
Elon
gatio
n5
53.3
25.
6746
.27-
60.3
661
.42
1246
.50-
76.3
364
.32.
760
.8-6
7.7
Bran
d m
ark
Tens
ile s
tren
gth
518
3.8
3.38
179.
59-1
8817
8.64
5.47
171.
84-1
85.4
317
3.66
0.09
164.
85-1
82.4
6
Tear
str
engt
h5
92.2
4.46
86.6
5-97
.74
89.7
81.
188
.40-
91.1
588
.06
2.43
85.0
4-91
.07
Elon
gatio
n5
61.2
2.01
58.6
9-63
.70
62.1
42.
8558
.59-
65.6
862
.92
4.67
57.1
1-68
.72
Scra
tch
Tens
ile s
tren
gth
517
8.62
4.62
172.
87-
184.
3617
5.46
8.67
164.
69-1
86.2
218
0.44
10.9
316
6.8-
194.
01
Tear
str
engt
h5
90.2
42.
7386
.84-
93.6
388
.26
5.06
81.9
6-94
.55
90.3
41.
1988
.85-
91.8
2
Elon
gatio
n5
63.5
43.
9358
.65-
68.4
269
.19.
0757
.83-
80.3
660
.74
2.25
57.9
3-63
.54
Kni
fe cu
tTe
nsile
str
engt
h5
182.
143.
7717
7.45
-18
6.82
128.
32.
7812
4.84
-131
.75
138.
222.
4313
5.19
-141
.24
Tear
str
engt
h5
77.6
28.
5866
.95-
88.2
881
.56
4.91
67.9
2-95
.19
95.0
42.
292
.30-
97.7
7
Elon
gatio
n5
58.2
46.
8649
.71-
66.7
649
.34
1.33
47.6
8-50
.99
60.8
1.73
58.6
4-62
.95
Putr
efac
tion
Tens
ile s
tren
gth
513
8.9
7.71
129.
31-
148.
4812
9.58
8.3
119.
26-1
39.8
914
6.1
4.64
140.
33-1
51.8
6
Tear
str
engt
h5
91.4
1.23
89.8
6-92
.93
89.1
29
77.9
3-10
0.3
79.4
21.
7977
.19-
81.6
4
Elon
gatio
n5
43.5
2.59
40.2
8-46
.71
45.3
23.
2641
.26-
49.3
64.1
82.
7560
.76-
67.5
9
Cock
le/e
kek
Tens
ile s
tren
gth
518
9.96
2.27
187.
13-
192.
7817
8.1
3.51
173.
73-1
82.4
618
3.62
1017
1.19
-196
.04
Tear
str
engt
h5
83.6
64.
1278
.54-
88.7
779
.46
5.63
72.4
5-86
.46
92.2
43.
4487
.96-
96.5
Elon
gatio
n5
51.2
6.89
42.6
4-59
.75
42.5
45.
6535
.51-
49.5
668
.24.
8662
.15-
74.2
4N
B: T
ensi
le s
tren
gth
(kg/
cm2 =
200)
; Tea
r str
engt
h (k
g/cm
2 =20
0); E
long
atio
n (%
70-
80);
OBS
=obs
erva
tion;
SD
=sta
ndar
d de
viat
ion;
CI=
confi
denc
e in
terv
al
48
Tsigab et al.,
Ethiop. Vet. J., 2020, 24 (2), 35-53
Effect of skin and hide defects on physical characteristics of crust leather
Normal standards values of TNS (200 kg/cm2); TRS (200 kg/cm2); PEL (70-80%) were compared with measurements of crust leather of skins and hides affected by either of wound, scratch, brand mark, putrefaction, knife cut or cockle. The difference in mean of the five measurements of each defect com-pared to the standard of the TNS, TRS and PEL was statistically significant (p< 0.05) (Tables 8).
Discussion The quality of the rawhide and skin plays a decisive role on the quality of the leather and its’ constituents. Quality of rawhide and skin is largely dependent on the extent of ante-mortem and postmortem defects. Slaughter and postmor-tem defects are controllable to certain extent, while ante-mortem defects pose serious challenges to tanneries (Habib et al., 2015). Scratch was the most fre-quent pre-slaughter defect of hides. Kahsay et al., (Kahsay et al., 2015) had a similar observation in Sheba Tannery. However, Urgessa (2013) had reported cockle as most frequent pre-slaughter defect of hide from Addis Ababa and Modjo tanneries. These variations might be due to the differences in agro-ecology, deworming activities husbandry managements and the season that samples had collected. Agroecology that affects quality of feed and texture of the skin and hide reflects the substance or quality nature of the skin and hide whereas poor husbandry practices downgrade it. Cockle was the most frequent pre-slaughter defects on pickled sheepskin. The finding was in agreement with reports of Ashenafi et al., (2013) in Sheba Tannery and in Addis Ababa and Modjo (Urgessa, 2013). In wet blue goat skin, wound was most frequent pre-slaughter defect observed in the current study although cockle (Urgessa, 2013) and scratch (Sertse and Wossene, 2007) were reported from Addis Ababa and Modjo, and Sebeta, respectively.
Of the slaughter defects, knife cut was most frequently observed slaughter defect on hide and skin which was in agreement with Berhe (2009) from Sheba tannery and leather industry. However, poor pattern was reported with high-
49
Tsigab et al.,
Ethiop. Vet. J., 2020, 24 (2), 35-53
est frequency in Bahirdar (Zembaba et al., 2012). Likewise, Hailu, (2013) had also reported poor pattern as a slaughter defect of sheep and goats skin in many of the municipal abattoirs in the country. It might be due to skill and awareness of butchers. Commonly practiced backyard slaughtering habit of the community may also have great role for both defects. Among the post-slaughter defects, putrefaction was in highest percentage on hide and skin. This finding was in agreement with those reported by Behailu (2015) from Colba (Ashenafi et al., 2013) and Sheba tanneries (Ahenafi et al., 2013). The prevalence of putrefaction was at higher rate on wet blue hide as compared to wet blue skin. This finding was in agreement with those reported by Kahsay et al., (2015) from Sheba tannery and Yacob et al., (2008). Most of the post-slaughter defects are due to poor management or improper preserva-tion and storage of skin and hide.
The observed defects have reduced the quality grading and resulted in signifi-cant reduction. Larger proportion of wet blue hide rejected, followed by wet blue goatskin. (Ashenafi et al., 2013) had also reported financial loss of around USD 800, 000.00 and USD 250, 000.00 due to cockle defect-based rejection of pickled sheep and wet blue goatskin, respectively. The dominant defects were cockle, which were higher in both sheep and goatskin than hide. This was in line with the result observed by Berhanu et al., (2011) and Asefa et al., (2012)who had reported cockle as leading defects on sheep skin rejection. However, Kahsay et al., (2015) had reported scratch as the dominant defect of sheep skin, goat skin and hide.
During the current study, none of the skin and hides scored I-III quality grade which was unlike to the observations of (Behailu, 2015) who had reported only 8.8% from Colba tannery. Most of the skins and hides of this study scored quality grades of VI-VII which was in line with reports of 69% by Behailu from Colba tannery and 69.6% by Urgessa (2013) from tanneries in Addis Ababa and Modjo. The overall hide and skin rejection in this study was 32.7% of which 23.5% was wet blue hide, 5.1% for wet blue salted and dry goatskin and 4.1% for pickled sheepskin. Degrading and rejection of skin and hide after beam house operation incurs loss of cost of purchase and processing and sub-sequently incurring extra cost for correction during finishing or total rejection (Solomon, 2011 and Teklay et al., 2019).
Measurable properties of leather are physical properties such as tensile prop-erties that determines the structural resistance of leather to tensile forces,
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hence its state and usability (Habib et al., 2015). Understanding the changes made on physical properties due to the various defects is a prerequisite for identification and development of leather defect correction technology through synthesis and application of selected tanning agent or filler material. Percentage Elongation determines the elasticity of the material especially upper leather and footwear upper should possess high flexibility to prevent the appearance of cracks and tears in the ball area. High elasticity allows the material to withstand the elongation stresses to which it is subjected during footwear lasting, especially on the toe area (Nalyanya et al., 2013). In this study, compared to standards, there was reduction in the mean differences of Tensile strength, Tear strength and percent elongation measurements of the skins and hides with any of the major defects (wound, scratch, brand mark, putrefaction, knife cut or cockle). This signifies serious damages attributed to poor quality leather, characterized by reduced percent elongation, elasticity, and tear strength. This seriously reduces quality of the raw materials required for finished products, thereby, a drop in the national economy.
ConclusionVarious skin and hide defects were identified among which pre-slaughter de-fects took higher proportion followed by slaughter defects. The defects serious-ly affected quality grading and significantly reduced tensile strength, percent elongation and tear strength. Rejection of the defective skin and hides incurs serious financial loss across the raw skin and hides marketing value chain and beam house operations. Therefore, we recommend further research on novel grade correction technologies of leather. Moreover, policies and actions on im-proved animal husbandry and locally feasible animal diseases control policies should be established in the country.
Competing InterestsThe authors declare that they have no competing interests.
AcknowledgmentsThe staffs of the Sheba Tannery and Leather Industry are acknowledged for their technical support during the study. The study received financial support from large and medium scale project schemes of Mekelle University.
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constraints: A review paper. Cogent Food & Agri., 5, 1-9.Asegedom, B.H., Mezgebe, T.T., Desta, A., 2019. Analyzing the causes of rejection and
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Jemal et al., Ethiop. Vet. J., 2020, 24 (1), 54-72 DOI https://dx.doi.org/10.4314/evj.v24i2.4 Ethiopian Veterinary Journal
Ethiop. Vet. J., 2020, 24 (2), 54-72
Ovarian follicular dynamics in Boran and Crossbred heifers in Ethiopia: Implications for assisted repro-ductive techniques
Jeilu Jemal1*, Tamrat Degefa2, Tefera Yilma3, Sayid Ali2, Alemayehu Lemma3
1National Agricultural Biotechnology Research Center, Animal Biotechnology Research
P O Box 249 Holeta, Ethiopia
2 Debre zeit Agricultural Research Center, Animal Biotechnology Research
3 Addis Ababa University, College of Veterinary Medicine and Agriculture, Department of Clinical Studies
*Corresponding author: - Tel-+251911549606, E-mail: - [email protected]
AbstractThe study was conducted to characterize the follicular dynamics of purebred Boran (Bos indicus) and Boran * Holstein Friesian crossbred heifers during estrous cycles; for use in ovum pick up and in-vitro embryo production. Insight of reproductive physiology of cattle would help to understand and exploit the reproductive potential of elite animals for breed improvement. Follicular de-velopment, growth and atresia during estrous cycles were evaluated using a trans-rectal real-time B-mode ultrasound system for three consecutive estrus cycles. Luteal activity was evaluated by serum progesterone level. Follicular aspirations were done to investigate the potential of Boran cattle and their crosses for transvaginal oocyte production; using a vacuum pressure pump and Aloka SSD Prosound-2 ultrasound device. Boran heifers (n=15) manifested two (n = 6, 40%), three (n = 5, 33%), four (n= 3, 20%) and five (n=1, 6.7%) follicular waves. Crossbred heifers (n=14) showed one (n= 2, 14%), two (n= 6, 43%) and three (n= 6, 43%) follicular waves. Interovulatory interval was 21.1 ± 3.4 and 21.4 ± 2.7 days for Boran (n=45) and crossbred (n=42) heifers, respectively. The progesterone level in Boran and Crossbred heifers during diestrus was 9.5 ± 11.0 and 4.6 ± 8.8 ng/ml, respectively. The maximum diameter of the ovulatory follicle for crossbred heifers was higher (15.4 ± 1.6 mm) than the diameter of the Boran (14.0 ± 1.9 mm) heifers (p<0.005). Differences (p<0.005) were observed in the size of both right (26.6 ± 5.14 and 28.6 ± 5.1 mm) and left (21.7 ± 4.85 and 24.1 ± 5.07 mm) ovaries of Boran and Crossbred heifers, respectively. Difference (p<0.005) was also observed in follicular count of the
55
Jemal et al.,
Ethiop. Vet. J., 2020, 24 (2), 54-72
right ovaries of Boran (4.84 ± 1.96) and Crossbred (5.13 ± 2.05) heifers. Oocyte recovery rate in once weekly collection scheme for Boran and their crossbred heifers was (n=19, 42.08%) and (n=17, 42.55%), respectively. The recovery rate for twice weekly collection scheme was (n=24, 34.53%) and (n=23, 40.44%) for Boran and crossbred heifers, respectively. Follicular dynamics in Boran heifers is characterized by a higher incidence of cycles with two, three and four waves, associated with a low persistence of the dominant follicles; and smaller size of ovulatory follicles and less intense heat signs from their crossbred counterpart. Boran heifers proved to have potential for comparable number of follicular population and ease of aspiration procedures that can be tapped for advanced reproductive techniques.
Keywords: Estrus cycle; follicular wave; ovarian follicle; ovum pickup; ultra-sound.
IntroductionIncreasing knowledge of ovarian physiology in cattle has provided opportuni-ties for the improvements of assisted reproductive techniques over the years. The manipulation of ovarian function had great contribution to design proto-cols to control luteal and follicular function of ovaries in cattle. Cows can be classified as low, intermediate or high antral follicle count (AFC), according to the number of antral follicles detected via ovarian ultrasonography (Zan-girolamo et al., 2018). A sound understanding of the processes involved in the growth and differentiation of antral follicles destined for ovulation is es-sential for artificial maturation of bovine oocytes and in developing improved hormonal regimens to control estrus in the cow (Gordon, 2003). Further, it is also important in improving techniques such as estrus synchronization, in vivo and in vitro embryo production and transfer. The maturation of bovine oocyte technique helps breeders to exploit the genetic potential of elite animals since young age. This would accelerate the genetic advancement rate of genetically superior animals (Baldassarre et al., 2018). Ovarian physiological characteris-tics can directly influence the number and quality of oocytes available for ovum pick up procedures (OPU) and in-vitro embryo production (IVEP) Lonergan et al (1994); which have great contribution on selection of elite females to be the parent of the next generation through assisted reproductive techniques.
Evaluation of antral follicles by ultrasound is considered the most practical strategy to recruit a female for reproductive improvement; and hence ultra-
56
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sound scanning is quite useful for studying follicular and luteal dynamics (Mo-rotti et al., 2017). Follicles in cattle grow in wave-like pattern (Driancourt, 2001) and follicular population is related to the stage of the estrous cycle. Ev-ans et al (1994) reported that follicular development in calves or heifers less than 36 weeks of age occurred in a wave-like pattern as early as 2 weeks. This has practical implications for OPU, IVEP and estrus synchronization work. OPU technique enables to aspirate follicles from immature oocytes and from animals that are not showing regular estrous cycles (Gordon, 2003).
Understanding the differences between breeds of cattle is also important for the establishment of correct reproductive management procedures (Viana et al., 2000). Reproductive physiology of zebu cattle is not identical to the Europe-an breeds of cows, and differences are known to exist in characteristics such as diameter of dominant follicle (Figueiredo et al., 1997), ovulation moment and a higher number of follicular waves during the estrus cycle (Viana et al., 2000).
Ethiopian Boran cattle are a fast growing, fertile and good milk producer com-pared to other indigenous cattle breeds in Ethiopia (Haile et al., 2011). The growth, reproduction and milk production performance of Boran has been im-proved in different parts of the world including, South Africa, Kenya, Austra-lia and USA (Haile et al., 2011). The breed has huge potential that could be tapped with appropriate reproductive tools in selection scheme breeding pro-grams to improve its beef and dairy attributes. However, there is inadequate information regarding follicular population and transvaginal oocyte aspira-tions in zebu breeds in general. Hence, the mechanisms that controls follicular dynamics during estrous cycles in Boran and their crossbred cattle needs to be understood to optimize reproductive management techniques and make the best use of reproductive biotechnology tools. The aim of this study was to char-acterize ovarian follicular dynamics and determine oocyte production potential of Boran and its Holstein crosses for the purpose of recruiting these breeds for advanced assisted reproductive techniques.
Materials and MethodsLocation
Experiments were conducted at Holeta Agricultural Research and National Agricultural Biotechnology Research Centers, Holeta, Ethiopia; from February to April and November to December 2019 and January 2020. Holeta is located
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about 29 km west of the capital city, Addis Ababa, with Longitudes 38º38’ east’ Latitude 09º04’ north and with Altitude of 2,390 meters above sea level. The minimum and maximum temperatures at Holeta are 6.13°C and 22.8°C, re-spectively. The rainfall pattern is bimodal with short rains from March to May and long rains from June to August. The mean annual rainfall is about 1,243.7 mm with peak rain in August.
Animal
The study animals constituted indigenous purebred Boran (n=15) and Boran * Holstein Friesian (HF) Crossbred heifers (n=14) that were used for follicular dynamics experiment. All the Crossbred heifers used for this experiment have 75% exotic blood level. The heifers were between 1.5 and 3 years of age. The mean (±SD) body condition score (scale 1-5) of Boran and Crossbred heifers were 3.3 ± 0.5 and 3.4 ± 0.5, respectively. The mean (±SD) body weight of Boran and Crossbred heifers were 266 ± 27.7 kg and 297 ± 25.4 kg during and few weeks before the start of the experiment, respectively.
All animals were allowed to graze on natural pasture. Hay made from grass (Pennisetum, Festuca, Eragrostis and Hyparrhenia) constituted the major pro-portion of the roughage supply. Concentrate composed of wheat by-products or maize (28-30%); Noug seed cake (68-70%; Guizotia abyssinica) and 1% salt were used as supplementary feed for heifers every morning before the start of the experiment. Water was provided ad-libitum.
Procedure for characterizing follicular dynamics
Estrus was synchronized to bring experimental animals on similar reproduc-tive stage, using an intramuscular injection of 2ml PGF2α (Estrumate, Germa-ny) hormone. Date of ovulation (considered as day 0) was determined ultrason-ically after animals showed behavioral estrus signs and thereafter, monitoring follicular dynamics was commenced. The nonidentity (mathematical) method was used to manage ultrasonic follicular data (Ginther, 1993). Estrus detec-tion was scheduled twice a day and reported by assistants. Trans-rectal real-time B-mode ultrasound system with a 7.5 MHz linear array rectal transducer (SIUI, CTS-3300V; Shantou, China) was used for serial scanning of the ova-ries. Each animal was scanned at least for two consecutive cycles. Follicular diameter was measured by the internal electronic calipers and later grouped as small (3-5mm), medium (6-8mm) and large (≥9 mm). Each time, the three
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largest follicles were measured and the total number of visible follicles counted in each ovary. At each scan, ovary size was measured (taking the dimension of the longest axis) in both right and left ovaries. Inter ovulatory interval (IOI) was defined as the length of time between successive ovulations. Date of wave Emergence was determined retrospectively from the date of ovulation (Day 0). Date of divergence (DD) was defined as the date at which the dominant fol-licle (DF) of the wave dramatically diverged in growth rate from the rest of its subordinate follicles (SF). Luteal activity was evaluated by serum progester-one concentrations. Blood samples were collected every other day using plain vacutainer tubes. The serum was separated and stored at -20oC until analysis. Progesterone analyses were performed by serum progesterone analysis device (eProCheck® 2.0 - 2400), using a commercial kit (Mini tube, Germany). Num-ber of follicular waves, length of IOI, DD, growth and atresia rate, and total number of follicles in each ovary, follicular size and group, diameter of the larg-est follicle, diameter of ovulatory follicles and ovary size were used to compare the breeds.
Procedure for follicular aspirations
At the end of studying the follicular dynamics, follicles were aspirated from both breeds (n=12) on once and twice (three days interval) weekly collection scheme for three months. The OPU set-up had a real-time B-mode ultrasound system (Aloka SSD Pro Sound 2, Japan) with a 6.5-MHz convex sector probe transducer (Hitachi Medical Co., Tokyo, Japan) and a needle guide system (Mini tube, GmbH, Germany). Follicles were aspirated at 70-80 mmHg vac-uum pressure at a flow rate of 15–20 ml ⁄ min. Oocytes were collected into a PBS media supplemented with heparin 20µg/ml, 2% FCS, 50µg/ml gentamicin, and 25mM HEPS and maintained at 370C. Each heifer had received 2-3 ml of epidural anesthesia (2% lidocaine) before follicular aspiration procedures. Ethical clearance for animal research was granted by research ethical commit-tee of AAU CVMA. Aspirated COCs were searched and categorized according to Seneda et al (2001) with some modifications as; Grade I= COCs with an even cytoplasm and three or more cumulus-cell-layers; Grade II= COCs with an even cytoplasm and less than three cumulus-cell-layers; Grade III= COCs at least with one layer of cumulus cells or partially denuded and Grade IV= Denuded oocytes.
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Statistical analysis
Data were grouped according to breed of animals and follicular characteristics in each estrous cycle. Descriptive statistics were used to determine the propor-tion of animals that exhibited different number of follicular waves. Frequency analysis and chi-square test were used to determine occurrence of ovulation. Differences between breeds on follicular population and among animals with the same number of follicular waves per cycle were evaluated by t-test, repeat-ed measures and PROC-Mixed ANOVA. Differences in follicular growth and atresia characteristics among the breeds with similar follicular wave number were evaluated by F test. The relationship between the number of waves and estrous cycle length was estimated by Pearson’s correlation method. Graphical analyses were performed using Microsoft offices excel. Results are shown as mean ± standard deviation (SD) and level of significance was held at P<0.05.
Results All except one heifer exhibited heat within 2-5 days after 2ml PGF2α hormone injection. Boran heifers (n=15, 100%) and crossbred heifers (n=14, 93.3%) ex-hibited behavioral heat in five consecutive days after 48 hours of PGF2α in-jection. Prominent behavioral and physiological heat signs observed during heat manifestation period were mounting each other, vaginal discharge and bleeding.
The three estrous cycles evaluated for 69 days on purebred Boran and Boran * HF crossbred heifers manifested characteristic pattern of follicular waves, with the recruitment of a group of 3 to 5 mm follicles, followed by the selec-tion, development and atresia of a dominant follicle. The Boran heifers (n=15) manifested two (40%), three (33%), four (20%) and five (6.7%) follicular waves whereas the crossbred heifers (n=14) showed one (14%), two (43%) and three (43%) follicular waves.
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Figure 1. Two wave follicular development in Boran heifers (left panel) and crossbred heifers (right panel).
A Boran heifer (6.7%) showed two follicular waves in the first and second es-trus cycles and three follicular waves during the third cycle. A double ovulation was observed in two Boran heifers (13.3%) and one crossbred heifer (7.1%). Two Boran heifers (13.3%) and one (7.1%) crossbred heifer exhibited prolonged estrus signs (three days); and the size of ovulatory follicle remained constant or slightly reduced in size (1 mm) until they eventually ovulated and the be-havioral estrus signs vanished.
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Figure 2. Three wave follicular development in a Boran heifer (left panel) and Cross bred heifer (right panel).
The majority of the Boran and crossbred heifers (n=11, 73% and n=12, 85%) respectively, exhibited estrus cycles with two and three follicular waves. The main characteristics of the follicular dynamics in these two breeds with two and three follicular waves are shown in Tables 1 and 2. There was no differ-ence (p>0.05) between Boran (n=45) and crossbred (n=42) heifers in IOI (21.1 ± 3.4 and 21.4 ± 2.7 days) and serum progesterone level (9.5 ± 11.0 and 4.7 ± 8.8 ng/ml) during diestrus, respectively.
There was, however, a difference (p<0.05) in IOI between the heifer with five follicular waves and other heifers with one, two, three and four follicular waves. There was a positive correlation (p<0.05) between the number of fol-licular waves in a cycle and IOI (r = 0.27, p = 0.05). There was a tendency of increase in the number of follicular waves with the prolongation of inter ovu-latory interval. The mean IOI was 27.0 ± 0.0 days, for a heifer with five-wave cycles, 22.6 ± 2.9 days, for four-wave cycles, 20.8 ± 2.9 days for three-wave cycles and 19.5 ± 3 days for two-wave cycles, and 18.8 ± 2.4 days for one-wave cycle.
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The mean maximum diameter of the ovulatory follicle for crossbred heifers (15.4 ± 1.6 mm) was higher than the diameter of the Boran heifers (14 ± 1.9 mm) (p<0.005).
4
6
8
10
12
14
16
0 2 4 6 8 10 12 14 16 18 20 22 24
Folli
cle
diam
eter
in [m
m]
IOI (Days)
1st wave 2nd wave 3rd wave 4th wave (Ovulatory)
Figure 3. Four wave follicular development in a Boran heifer. The dynamics and size of the DF of the first, second, third and fourth (ovulatory) waves are indicated by solid lines with different symbols.
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Table 1. Characteristics of follicular waves, growth and atresia rate of domi-nant follicle in Boran and Crossbred heifers with two follicular waves during estrus cycleCharacteristics Follicular Waves (mean ±SD)* First SecondBoranWave onset (day) 0.59 ± 0.71 10.7 ± 3.53Wave length (days) 15.1 ± 2.40 10.0 ± 2.26Day of maximum diameter (day) 8.47 ± 2.38 19.5 ± 2.98Maximum diameter (mm) 12.1 ±1.56a 14.2 ±1.63b
Growth rate (mm/day) 0.97 ± 0.58a 0.91 ± 0.26 a
Divergence day (day) 4.41 ± 1.37 13.8 ± 3.50Length of growth phase (days) 8.35 ± 2.29 8.82 ± 4.02Atresia rate (mm/day) 1.25 ± 0.76 -CrossbredWave onset (day) 0.83 ± 0.62 12.9 ± 3.29Wave length (days) 16.0± 3.36 10.9 ± 2.19Day of maximum diameter 9.50 ± 2.60 22.2 ± 2.38Maximum diameter (mm) 14.0± 2.47a 15.8 ± 1.51b
Growth rate (mm/day) 0.93 ± 0.28a 1.00 ± 0.28a
Divergence day 3.67 ± 1.41 16.4 ± 3.54Length of growth phase (days) 9.50 ± 2.60 10.8 ± 2.13Atresia rate (mm/day) 1.41 ± 0.69 -* Means followed by different letters within rows significantly differ at p< 0.05.
The growth pattern for the first dominant follicle of Boran and crossbred heifers with two waves emerged on day (0.6 ± 0.7 and 0.8 ± 0.6) and its regression occurred on day (9.4 ± 2.3 and 10.5 ± 2.6) of the estrus cycles, respectively. The dominant follicle reached its maximum diameter on day 8.5 ± 2.4 for pure Bo-ran and 9.5 ± 2.6 for crossbred heifers. The second wave emerged on day 10.7 ± 3.5 for Boran and 12.9 ± 3.3 for crossbred heifers, the dominant (ovulatory) follicle selected on day 13.8 ± 3.5 for pure Boran, and 16.4 ± 3.5 for crossbred heifers. The maximum diameter of the dominant follicle for the first wave was smaller (p<0.001) than the diameter of the second wave dominant (ovulatory) follicle in both breeds.
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Table 2. Characteristics of follicular waves, growth and atresia rate of dominant follicles in Boran and crossbred heifers with three follicular waves during the estrus cycle Characteristics Follicular Waves(mean ± SD)* First Second Third BoranWave onset (day) 0.69 ± 0.79 7.06 ± 2.18 14.0 ± 2.45Wave length (days) 11.4 ± 2.68a 11.8 ± 2.62a 8.25 ± 1.57b
Day of maximum diameter 6.63 ± 1.20 11.8 ± 1.95 20.8 ± 2.91Maximum diameter (mm) 11.9 ± 2.46a 11.5 ± 1.21a 14.6 ± 2.06b
Growth rate (mm/day) 1.09 ± 0.31a 1.25 ± 0.47a 1.23 ± 0.30a
Divergence day (DD) 3.94 ± 0.99 10.2 ± 1.97 16.7 ± 2.41Length of growth phase (days) 6.63 ± 1.20 6.06 ± 3.02 7.94 ± 0.93Atresia rate (mm/day) 1.56 ± 1.09 1.29 ± 0.61 - CrossbredWave onset (day) 0.83 ± 0.71 8.22 ± 2.92 14.1 ± 3.07Wave length (days) 11.7 ± 3.68a 11.0 ± 3.01a 8.17 ± 1.72b
Day of maximum diameter 6.17 ± 2.53 12.9 ± 2.99 21.50 ± 2.71Maximum diameter (mm)Growth rate (mm/day)Divergence day (DD)
12.5 ± 2.09a
1.23 ± 0.49a
3.61 ± 1.38
12.2 ± 1.62a
1.11 ± 0.46 a
10.7 ± 3.29
15.17± 1.86b
1.22 ± 0.32a
16.8 ± 3.09
Atresia rate (mm/day) 1.36 ± 0.56 1.75 ± 0.64 -* Means followed by different letters within rows significantly differ at p< 0.05.
The first wave in Boran and crossbred heifers with three follicular waves emerged on day (0.7 ± 0.8 and 0.8 ± 0.7) of the estrous cycle, respectively. The dominant follicle growth pattern of the first follicular wave for Boran and crossbred heifers with three follicular waves reached maximum diameter on day 6.6 ± 1.2 and 6.2 ± 2.5 and began atresia on day 7.6 ± 1.2 and 7.2 ± 2.5 re-spectively, showing a quite short period of stabilization or apex plateau.
The second wave showed up on day 7.1 ± 2.2 for Boran and 8.2 ± 2.9 for cross-bred heifers, and the dominant follicle had selected on day (10.2 ± 1.97 and 10.7 ± 3.3) reaching maximum diameter on day (11.8 ± 2.0 and 12.9 ± 3.0) and beginning atresia on day (13.7 ± 2.2 and 14.7 ± 3.1) respectively, for pure Boran and Boran * HF cross heifers.
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The third wave (ovulatory) emerged on day (14.0 ± 2.5 and 14.1 ± 3.1), with the dominant follicle being selected on day (16.7 ± 2.4 and 16.8 ± 3.1) and reaching its maximum diameter on day (20.8 ± 2.9 and 21.5 ± 2.7) of the estrus cycle, respectively.
The ovulatory follicle developed in the right ovary in proportion of (62%, n=45) in Boran heifers and (60%, n=42) in crossbred heifers.
Table 3. Comparison of the size of ovaries in Boran and Crossbred heifers Ovary size Breed N Mean ± SD
Size of ovary [mm] t P
Right ovary Boran 820 26.6 ± 5.14a -7.92 0.000Cross 815 28.6 ± 5.10b
Left ovary Boran 817 21.7 ± 4.85a -9.84 0.000Cross 808 24.1 ± 5.07b
There was difference (p<0.001) between the size (both right and left) of pure-bred Boran and crossbred heifers ovaries and follicular count of the right ova-ries. However, there was no difference (p>0.05) in the total follicular count of the left ovaries. The mean number of follicular populations per animal was 9.4 ± 3.0 for Boran heifers and 9.6 ± 3.3 for crossbred heifers. Regarding the size, the mean number of small, medium and large follicles was (5.6 ± 3.0, 2.5 ± 1.3, and 1.3 ± 0.9) respectively for Boran heifers and the mean number of small, medium and large follicles for Crossbred heifers was (5.5 ± 3.3, 2.4 ± 1.3 and 1.7 ± 1.0) respectively. A repeated measures ANOVA, with Greenhouse-Geisser correction was conducted to assess differences between the means of the three groups of follicles. Results indicated that there was difference F (1.5, 45005) = 3514, p<0.001 between the populations of the three follicular groups in both breeds. Examination of these means indicated that small and medium follicles combined were found more abundantly (84%, n= 1732) in both breeds than the large follicles and with the mean number of (8.16 ± 3.13, n=871 and 7.89 ± 3.50, n=859) for Boran and Crossbred heifers, respectively, during the three estrus cycles.
A mixed ANOVA was conducted to assess the follicular population difference between Boran and Crossbred heifers. Results indicated there was no differ-ence between the breeds in the mean count of the follicular groups, F (1, 1724) = 0.005, p = 0.94.
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The mean number of right ovaries follicular count for Crossbred heifers is higher (p<0.003) than Boran heifers. However, there is no difference in total number of follicular populations between the breeds.
Table 4. Comparison of follicular counts in the right and left ovaries of Boran and crossbred heifers Follicular counts Breed N Mean (± SD)
No of follicles t p
Right ovary Boran 870 4.84 ± 1.96 a -2.97 0.003 Cross 856 5.13 ± 2.05b
Left ovary Boran 871 4.56 ± 1.96a 0.34 0.735 Cross 855 4.53 ± 1.95a
Oocyte recovery rate for the once weekly and twice weekly collections scheme was 42.3% (n=36) and 37.4% (n=47), respectively. Oocyte recovery rate for Bo-ran and Crossbred heifers in once weekly collection scheme was 42.1% (n=19) and 42.6% (n=17), respectively. The recovery rate in twice weekly collection scheme was 34.5% (n=24) and 40.4% (n=23) for Boran and Crossbred heifers, respectively. The mean number of oocyte collected in a once weekly scheme was (5.6 ± 2.2 and 5.9 ± 2.1) for purebred Boran and crossbred heifers, respec-tively. The mean number of aspirated oocyte in twice weekly collection scheme was (2.8 ± 1.7 and 3.1 ± 2.3) for Boran and their crosses, respectively. There was no difference (p>0.05) between purebred Boran and crossbred heifers in total number of oocyte aspirated in the once and twice weekly OPU session.
Table 5. Number of COC obtained in Boran and Crossbred heifers by once and twice weekly follicular aspirations scheme
Follicular Aspiration
COC QualityBreed N Mean (± SD)*
No of COCGI GII GIII GIV
Once weekly Boran Cross
1917
5.58 ± 2.16a
5.88 ± 2.05 a 1.00 1.18
1.58 1.41
1.791.88
1.211.41
Twice weekly Boran Cross
2423
2.79 ± 1.69a
3.13 ± 2.30 a 0.58 0.78
1.13 1.13
0.670.91
0.420.30
* Means followed by different letters within column differ (p < 0.005).
There was difference (p<0.001) in mean number of oocyte collected per session between once (5.72 ± 2.09) and twice (2.96 ± 2.0) a week collection scheme.
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There was no difference between the breeds regarding the number of quality oocyte collected by both (once and twice weekly) collections.
DiscussionAll animals except one heifer exhibited heat within five consecutive days after estrus synchronization. Behavioral signs of heat were weaker in Boran heifers as compared to crossbred heifers. During pro-estrus, estrus and met-estrus the Crossbred heifers manifested a good deal of behavioral estrus signs (mount-ing each other, vaginal discharge and bleeding) and have higher interaction among them. Estrus signs in Boran heifers were mostly observed during rec-tal palpation (vaginal discharge) and when the animals mixed with Crossbred heifers (mounting). Bleeding at the end of estrus period never manifested in Boran heifers. The practical implication of this phenomenon during estrus syn-chronization work should be emphasized. Local zebu breeds require intensive follow up for proper heat detection and timely insemination.
The three estrous cycles evaluated for 69 days on both breed of heifers mani-fested characteristic pattern of follicular waves. Two- and three- wave cycles occurred in the majority of animals (n=23, 79.3 %) during the estrus cycles. The incidence of higher estrus cycles with two and three follicular waves was also observed in European Taurus breeds (Savio et al., 1988) and Zebu cows (Gambini et al., 1998). Zebu breeds tend to have more follicular waves com-pared to B. Taurus breeds (Viana et al., 2000). The length of the luteal phase is considered as the main factor determining the number of follicular waves (Figueiredo et al., 1997). The length of inter ovulatory interval had shown no difference between the breeds. This result is in agreement with previous work (Degefa et al., 2016) in Ethiopian Boran and their crossbred cows’ estrous cycle length. Long luteal phase maintains a high serum progesterone concentration that suppresses ovulation of the dominant follicle hence, inducing their atresia and onset of a new follicular wave (Gong et al., 1995).
The marked decline in diameter of the second dominant (ovulatory) follicle (in-dicated in the right panel of figure: 1) of Boran heifer with two follicular waves could be due to abrupt energy imbalance. Nutrition has effect on animal repro-duction and follicular development. A report by Rhodes et al (1995) on Bos-in-dics cows that had negative energy balance indicated a reduction in dominant follicle maximum diameter, growth rate and persistence of dominant follicle. Follicular dynamics, behavioral heat manifestation and oocyte quality can be
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impacted by dietary energy and protein intake that related with the function of the ovarian insulin-like growth factor system. Crossbred heifers had higher diameter of ovulatory follicle than Boran heifers. Muraya, (2013) reported that the maximum diameter of ovulatory follicle in Kenyan Boran cows’ was small-er than those reported in European cows by Ginther et al (1989). But compa-rable in size with other Bos indicus cows (Figueiredo et al., 1997). There was no difference in dominant or subordinate follicles growth or atresia rates between the breeds and between animals with different follicular waves. Follicular dy-namics in Boran heifers is characterized by a higher incidence of cycles with two, three and four waves, associated with a low persistence of the dominant follicle, however, in this current observation in few animals (n=2, 13.3%) the dominant follicle remained for longer times (three days) without ovulation. The failure of the dominant follicles to ovulate could be due to lack of LH serge. LH pulse frequency is the key determinant of the fate of the dominant follicle (Duffy et al., 2000).
Occurrence of double ovulation was manifested in Boran heifers. This phenom-enon could either be a defect in the deviation mechanism or a case in which twining would normally occur (Fricke and Wiltbank, 1999). Wiltbank et al (2000) reported the occurrence of more than one dominant follicle during a follicular wave in monovular species. The dominant follicles in both breeds developed in the right ovaries in higher proportion than the left ovaries. This incidence is attributed to high frequency of corpora lutea present in the right ovaries (Viana et al., 2000) and to the fact that these ovaries receive more blood supply compared to the left ones (Muraya, 2013).
There was no difference in total number of follicular populations between the breeds. However, there was difference in mean number of follicular counts of the right ovaries between the breeds. Boran heifers have a fairly compara-ble follicular population with Boran*HF crosses. In recent study, Degefa et al (2016) reported the total number of follicles greater than 4 mm in diameter for Ethiopian Boran cows is similar to the number reported for Bos taurus breeds (Bastos et al., 2010) and lower than the number of follicles reported for Nelore. The greater number of growing ovarian follicles in Ethiopian Boran cattle would help for OPU and subsequent IVEP (Degefa et al., 2016). As it was the case in this study and previous reports, different cattle breeds present different follicle numbers on the ovaries in a follicular wave cycle. The number of antral follicles is highly repeatable in the same animal over several evalu-ations even though there is considerable variability among individual cows
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(Morotti et al., 2017) and improved reproductive parameters and response to reproductive technologies in cows with higher antral follicle also count (Silva-Santos et al., 2014).
The mean number of oocyte retrieved in once weekly collection scheme from Boran heifers was comparable with Crossbred heifers. There was no differ-ence in mean number of oocyte retrieved in twice weekly collection scheme (which is done in three days interval) between Boran and crossbred heifers. There was difference (p<0.001) in mean number of oocyte collected per session between once and twice a week collection scheme. The recovery rate in both breeds was less than 50 %. This could be due to technician skill on aspiration and searching, vacuum pressure and also animal handling facility. Boran heif-ers had ease of anatomical structure for OPU work compared to their cross-bred counterpart. The size of the probe holder that meant for European breed (mini-tube, Germany) had no negative effect on the ease of oocyte collection process in Boran heifers. Once weekly follicular aspiration scheme was more convenient than twice weekly collection scheme for coordination of field and lab works and for longer time repeated collections.
Introduction of advanced reproductive biotechnology techniques such as IVEP in Ethiopian cattle production systems believed to hasten the process of breed improvement activities. It offers an opportunity for cattle breeding and im-provement centers to deliver embryo as a genetic improvement and multiplica-tion tool beside semen. To our knowledge, this is the first study in Ethiopia that focused on the investigation of the potential of zebu breed for oocyte retrieval. Boran breed proved to be capable of producing oocyte in comparable quan-tity and quality with Boran*HF crosses. IVEP like other assisted reproductive techniques could help to enhance genetic gain from these cattle by shortening the generation interval and by increasing accuracy of selection. Knowledge of follicular dynamics help in tackling some underling infertility problems such as delayed ovulation and silent heat manifestation (which is prominent in lo-cal cattle breeds), and may increase efficiency of AI by predicting exact ovula-tion time for proper insemination. The breeding program should give focus for these techniques to exploit the genetic potential of local zebu breed for milk and meat production.
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ConclusionThere are marked differences in the follicular dynamics between purebred Bo-ran heifers and their Holstein Frisian crosses. In the Boran, it is characterized by a higher number of follicular waves, smaller size of ovulatory follicles and less intense heat signs. Behavioral signs of heat were weaker in Boran heifers as compared to crossbred heifers. Boran heifers proved to have potential for comparable number of follicular population and ease of aspiration procedures that can be tapped for advanced reproductive techniques. For longer time re-peated collections and coordination of field and lab work, once weekly follicular aspiration had advantage over twice weekly aspirations.
Further improvement of the procedures such as, optimizing vacuum pres-sure, needle operation and aspiration time, hygienic oocyte collection environ-ment, COCs transportation equipment and proper temperature maintenance for laboratory equipment would go long ways in enhancing local and national capacity to run a full-fledged bovine IVF laboratory. Boran cattle could be a good candidate for implementation of OPU-IVP embryo production program in Ethiopia.
AcknowledgementsThe authors gratefully acknowledged the financial and research inputs sup-port of this study by the Ethiopian Institute of Agricultural Research (EIAR), National Biotechnology Research Center (NABRC). The financial support of Agricultural Growth Project (AGP II) of the (EIAR).The provision of experi-mental animals by Holeta Agricultural Research Center (HARC). And, the as-sistance of Tariku Bekele, Teshome Asfaw and Teshome Nigusse is gratefully acknowledged.
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Sarba et al., Ethiop. Vet. J., 2020, 24 (2), 73-89 DOI https://dx.doi.org/10.4314/evj.v24i2.5 Ethiopian Veterinary Journal
Ethiop. Vet. J., 2020, 24 (2), 73-89
Prevalence, organ distribution and antimicrobial susceptibility profile of Salmonella isolated from chickens purchased from markets in selected dis-tricts of West Shoa, Ethiopia
Edilu Jorga Sarba1, Kebene Kudama2, Morka Dandecha3, Lencho Megersa3, Bizunesh Mideksa Borena1, Endrias Zewdu Gebremdhin1 1Ambo University, College of Agriculture and Veterinary Sciences, Department of Veterinary Sci-ence, P. O. Box 19, Ambo, Ethiopia
2Adaberga District Agriculture Office, Livestock and Fishery Agency, West Shewa Zone, Oromia region, Ethiopia
3Ambo University, College of Agriculture and Veterinary Sciences, Department of Veterinary Laboratory Technology, P. O. Box 19, Ambo, Ethiopia
*Corresponding author, Email: [email protected], Tel.: +251911957036
Abstract Salmonella is one of the major causes of heavy losses in chicken and food-borne diseases worldwide. The current study was conducted from November 2015 to May 2016 to estimate the prevalence of Salmonella and determine the antimicrobial susceptibility of isolates in chickens. Chickens (n=205) were pur-chased from local markets of five selected districts of West Shoa Zone, Central Ethiopia. Following clinical examination, chicken were euthanized and 2-3 ml of blood sample was collected immediately. Then after postmortem examina-tion, samples were collected from the liver, kidney, ovary, and spleen. The slide agglutination test was used to assess the seroprevalence of Salmonella anti-bodies. Isolation of Salmonella was performed according to the ISO-6579 pro-cedure. The isolates were subjected to antimicrobial susceptibility testing (us-ing 13 antimicrobial drugs) following the Kirby-Bauer disc diffusion method. The seroprevalence of Salmonella antibodies was 63.5% (95% CI: 55.9-70.5). The isolation rate of Salmonella was 19.0% (95% CI: 13.9-20.1) at the chicken level and 7.3% (95% CI: 5.5-9.4) at the organ level. The detection rate was 11.2%, 7.0%, 6.1%, and 4.4% for spleen, liver, ovary, and kidney, respectively. The majority of the Salmonella isolates were susceptible to norfloxacin (97.4%) and chloramphenicol (92.3%). All the 39 isolates were resistant to amoxicillin, tetracycline, and nitrofurantoin. Three multidrug resistance patterns to six antimicrobial classes were observed. Four isolates were resistant to five anti-microbial classes. Therefore, regular surveillance of Salmonella and its antimi-
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crobial resistance is needed for a better understanding of the epidemiological dynamics. Awareness creation for chicken farmers about improving farming practices and the risks of antimicrobial resistance warrants special attention.
Keywords: Antimicrobial susceptibility; Chicken; Prevalence; Salmonella; Ethiopia
IntroductionSalmonellae are Gram-negative bacteria that are facultative intracellular hu-man and animal pathogens and members of the family Enterobacteriaceae. Salmonella Gallinarum and S. Pullorum are the highly host-adapted serovars affecting birds.S. Enteritidis and S. Typhimurium are the common serotypes causing disease in humans and animals (Gal-Mor, 2019). Salmonellosis is an infection ranging from subclinical to acute fatal septicemia or chronic diar-rhea and death (Quinn et al., 2011). In Ethiopia, salmonellosis is one of the important diseases of poultry reported from local and exotic chickens (Aragaw et al., 2010; Chaka et al., 2012; Kindu, and Addis, 2013). Fowl typhoid, which is caused by S. Gallinarum is a per-acute, acute, or chronic disease affecting mostly adult chickens, whereas pullorum disease, caused by S. Pullorum, af-fects young chickens of mostly 2−3 weeks of age (Parveen et al., 2007).
In Ethiopia, fowl typhoid and pullorum diseases are mentioned to cause heavy economic losses through mortality, morbidity, and reduced productiv-ity (Chanie et al., 2009). Poultry salmonellosis has the ability of both verti-cal and horizontal transmission, which complicates the spread and control of the disease. As a result, layer chickens can become sub-clinically infected carriers and pass the infection to their embryo in the egg (Muktaruzzaman et al., 2010). Other Salmonella serovars such as S. Typhimurium might also colonize the gastrointestinal tract of adult poultry asymptomatically (Gal-Mor, 2019). There are several reports on the detection of non-typhoidal Salmonella serovars from chicken samples (Molla et al., 2003; Eguale, 2018), as poultry is the major source of foodborne salmonellosis for humans (Sanchez et al., 2002).
On the other hand, Salmonella species could acquire antimicrobial resistance (AMR). The resistant strains are non-treatable with currently available anti-microbials, thus are serious global public health concerns both in humans and food animals (Parveen et al., 2007). In Ethiopia, the veterinary drug regulation and guidelines are not well developed and there is no enforcing the standard to
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practice responsible and prudent use of antimicrobials. As a result, indiscrimi-nate use of antimicrobials, the sale of counterfeit drugs, the involvement of untrained personnel, and illegal drug market are big challenges (DACA, 2009). Moreover, as the veterinary service is not well developed, chicken owners have a practice of treating sick chickens using antimicrobials either from veterinary or medical pharmacies (Sambo et al., 2015; Tufa et al., 2018).
Culture-based and serological studies detected Salmonella Gallinarum, S. Pul-lorum, and other serovars from chicken in Ethiopia previously (Aragaw et al., 2010; Berhe et al., 2012; Dagnew et al., 2020). There is also a report on the existence of multidrug-resistant Salmonella strains in chicken farms (Bekele and Ashenafi, 2010). However, there is little information about the prevalence and antimicrobial resistance of Salmonella species from chicken in Ethiopia (Molla et al., 2003, Eguale, 2018) and no data is available in the study area. Thus, for a better understanding of the situation, and to make an evidence-based decision, investigation in this aspect will have a significant contribution. Therefore, the objectives of the present study were to estimate the prevalence of Salmonella and to determine antimicrobial susceptibility of the isolates in selected districts of West Shoa, Central Ethiopia.
Materials and methods
Description of the Study Area
Chickens were purchased from local markets of Ambo, Holeta, Guder, Ijaji, and Dire Inchini districts of the West Shoa Zone, Oromia, Ethiopia. Ambo is an administrative center of the Ambo district and center of West Shoa Zone lo-cated 114 Km West of Addis Ababa. It has a midland altitude. Holeta is located 40 Km west of Addis Ababa and Dire Inchini is located 70 Km southwest of Ambo, both are in highland altitude range. Guder and Ijaji are located 15 Km and 80 Km West of Ambo, respectively, and both are in the tropical climate. All the study districts experience a bimodal rainfall pattern. The chicken popula-tion of each district is approximately between 350, 000 to 500,000 (Etefa and Dibaba, 2011).
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Study animalsChickens of both sexes and local and hybrid breeds managed under the back-yard or small-scale management system were included. The chickens in this system, scavenge their feed with a limited supplement and often share the same house with humans or other livestock. Chickens were categorized as young (≤6 months) and adult (>6 months) based on their age (Calnek et al., 1991). Chickens with clinical signs of diseases such as decreased appetite, de-pression, weight loss, ruffled feathers, and watery to mucoid diarrhea, dehy-dration, and anemia were categorized as clinically sick and otherwise appar-ently healthy.
Study design and sample size determination
A cross-sectional study was conducted from November 2015 to May 2016. The sample size was determined using Thrusfield (2007) formula with an expected prevalence of 8% (Aragaw et al., 2010) and with 0.05 precision. Accordingly, the calculated sample size was 113 chickens, but 205 chickens were considered for this study. Eighty-four clinically sick were purchased purposely and 121 apparently healthy chickens were randomly selected and purchased from local markets. Sick and healthy chickens were separately transported in a cage with adequate space and ventilation. Chickens were slaughtered immediately upon arrival, but whenever there is a delay they were kept for a maximum of one day in Ambo University in a room with adequate living space, ventilation, and feed and clean water in ad libitum.
Postmortem examination
Post mortem examination was performed according to procedures by Chauhan and Roy (2007). The cervical dislocation was used to euthanize chickens. Fol-lowing slaughtering, the gross lesions on the visceral organs were recorded.
Sample collection
Approximately 2-3 ml of the blood sample was collected immediately following eutha-nasia from each chicken in a new plain vacutainer tube. The blood samples were kept in a slant position at room temperature overnight. After 24 hrs. of clotting, the sera were subjected to centrifugation at 1000 RPM for 10 minutes and sera were collected using sterile Pasteur pipettes in cryovial tubes, labeled and stored frozen at –20°C until tested. A total of 741 samples comprising liver (n=205),
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spleen (n=205), kidney (n=205) and ovaries (n=131) was sampled. About 25g of each organ was collected from the internal portion aseptically in a sterile polyethylene zipped plastic bag (Falconpack, UAE). The samples were kept at +4°C for a maximum of 24 hrs until culturing.
Serological examination
A serum slide agglutination test was performed following the test procedure described by OIE (2012). The sera and the reagent (crystal violet stained Sal-monella Gallinarum antigens) were brought to room temperature (22 ± 5 °C) before use. Thirty microliters of the reagent and equal volume of the serum were gently mixed on a sterile slide by rocking and rotating for about 2-3 min-utes. Any degree of agglutination was taken as evidence of seropositivity. The serological test was performed at the National Veterinary Institute, Bishoftu, Ethiopia.
Isolation and identification of Salmonella
Isolation of Salmonella was performed as recommended by ISO 6579 (2002) Amendl: 2007. Tissue samples were crushed by gentle maceration in polyeth-ylene zipper bag mixed with buffered peptone water (BPW) in a ratio of 1: 9 and is incubated at 37 oC for 24 hrs. Then, 0.1 ml aliquots were inoculated into tubes containing 10 ml Rappaport Vassiliadis (RV) broth (HiMedia, Pvt. Ltd., India) and are incubated for 24 hrs at 42 oC. Xylose Lysine Deoxycholate Agar (XLD) and Brilliant Green Agar (BGA) (HiMedia, Pvt. Ltd., India) plates were inoculated in parallel from the RV broths and incubated for 24-48 h at 37 °C. Up to five suspect colonies with typical Salmonella morphology on XLD (red colonies with/without black spots) and BGA (pink colonies) were streaked onto nutrient agar plates and incubated at 37 oC for 24-48 hrs. Finally, colonies from nutrient agar were confirmed biochemically by inoculating into Lysine Iron Agar (LIA), Triple Sugar Iron (TSI) Agar, Simmon Citrate (SC) agar Urea agar test, and motility test.
Antimicrobial susceptibility testing
All Salmonella isolates were tested for susceptibility to 13 different antimi-crobial drugs using Kirby Bauer disc diffusion method as described by CLSI (2014). The antimicrobial impregnated discs (Oxoid Ltd, Hampshire, Eng-land) used were streptomycin, amikacin, gentamicin, amoxicillin, cefotaxime, ceftazidime, cefuroxime, chloramphenicol, ciprofloxacin, nitrofurantoin, tetra-
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cycline, trimethoprim-sulfamethoxazole and norfloxacin with appropriate con-centration. Two to three pure colonies were picked up and emulsified in 3 to 4 ml of sterile normal saline. Dilution of the suspended colonies was performed until the turbidity matches the 0.5 McFarland Standards and transferred to Mueller-Hinton agar (HiMedia, Pvt. Ltd., India) using a sterile cotton swab. Following air drying the plates were incubated aerobically at 37 °C for 18 to 24 hrs. The diameters of the zone of inhibitions were measured with a caliper and the susceptibility of the isolates was recorded as susceptible, intermediate, and resistant. Escherichia coli ATCC 35218 was used as quality control.
Data analysis
Data were entered into an MS Excel Spreadsheet and descriptive statistics were used to summarize the data. SATA version 11 (Stata Corp, 2009) for Windows was used for analysis. Prevalence was sorted by district, sex, age, and season, health status, diarrhea status, and organ. The association of the studied variables with the outcome variables was analyzed using logistic re-gression. Variables that were non-collinear and with P-value less than 0.25 during the univariable analysis were selected for multivariable logistic regres-sion analysis. The odds ratio was used to compare the degree of association and the difference was considered significant when the P-value was less than 0.05. The percentages of antimicrobial resistance (AMR) were calculated as suscep-tible, intermediate, and resistant.
Ethical consideration
Permission for this study was obtained from the Ambo University Animal Re-search Ethics Review Committee (ARERC) and conducted under the approved protocol Ref. No. RD/AREC/003/2015.
ResultsClinical and postmortem findings
The clinical signs recorded in sick chickens in this study were decreased ap-petite, depression, dehydration, weight loss, ruffled feathers, and watery to mucoid diarrhea, anemia with pale, and shrunken combs. Accordingly, out of the 205 chickens, 84 (44.12%) and 121 (55.88%) were clinically sick and ap-parently healthy, respectively. Salmonella culture positive spleen, liver, and kidney showed one or more of the pathological signs such as enlargement,
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congestion, white necrotic foci, and mottling. Ovarian follicles were regressed, misshapen, and discolored (Fig. 1). Out of 40 lesions, almost all were recorded from adult chickens.
Figure 1. Gross lesions on Salmonella positive organs: enlarged and mottled spleen (A), discoloration and enlargement of the liver (B), enlargement and white spot appearance of kidney (C) discoloration and misshaping of ovarian follicles (D)
Serological result
Seroprevalence of S. Gallinarum was 63.5% (95% CI: 55.9-70.5). According to the multivariable logistic regression analysis, the study site showed significant variation in seroprevalence (p<0.05), whereas other variables did not show sig-nificant association (p>0.05). Hence, chickens from the Guder area were with greater Odds of seropositivity (OR=3.76, 95% CI: 1.09,12.98, p= 0.036) as com-pared to those from Dire Inchini (Table 1).
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Table 1. Logistic regression analysis of factors for seroprevalence of S. Gal-linarum in poultry Factors Category No.
positive (%)
Univariable Multivariable OR (95% CI) p-value OR (95%
CI)p-value
Breed Local 95 (62.9) 1Cross 18 (66.7) 1.18 (0.50,2.80) 0.709
Age Young 60.6 (60.6) 1Adult 70 (65.4) 1.23 (0.66,2.29) 0.510
Sex Male 38 (60.3) 1Female 75 (65.2) 1.23 (0.65,2.32) 0.516
Study site Dire Inchini 17 (41.5) 1 - -Ijiaji 11 (44.0) 1.11 (0.41,3.03) 0.840 0.84
(0.29,2.45)0.754
Ambo 34 (66.7) 2.82 (1.20,6.61) 0.017 1.82 (0.59,5.60)
0.295
Guder 33 (82.5) 6.65 (2.39,18.55) 0.000 3.76 (1.09,12.98)
0.036
Holeta 18(85.7) 8.47 (2.15,33.37) 0.002 4.82 (0.88,26.28)
0.069
Season Wet 38 (46.3) 1 - -Dry 75 (78.1) 4.13 (2.16,7.92) 0.000 2.15
(0.91,5.14)0.082
Health status
App. Healthy
59 (57.8) 1 - -
Sick 54 (71.0) 1.79 (0.95,3.367) 0.070 0.64 (.20,2.02)
0.448
Diarrhea Absent 73 (58.9) 1 - -Present 40 (74.1) 2.00 (0.98,4.04) 0.055 2.00
(0.62,6.48)0.247
Total 113(63.5%)CI= Confidence interval
Culture-based prevalence
From 205 chickens examined, 19.0% (95% CI: 13.9-20.1) were positive for Sal-monella spp. About 38.5% (15/36) were non-motile, while 61.5% (24/39) of the isolates were motile. Of the 741 organs sampled, 7.3% (95% CI: 5.5-9.4) were Salmonella positive. The isolation rates from the spleen, liver, ovaries, and kidneys were 11.2%, 7.0%, 6.1%, and 4.4%, respectively (Table 2). Not all the studied variables showed a significant difference (p> 0.05). However, a rel-
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atively higher isolation rate was observed in chickens of the Guder district (29.0%) as compared to Ijaji (8.0%), and adults (25%) as compared to young chickens (23.1%)(Table 3).
Table 2. The isolation rate of Salmonella from chicken organsOrgan No. of organs No. positive Isolation rate (%)Spleen 205 23 11.2Liver 200 14 7.0Ovary 131 8 6.1Kidney 205 9 4.4Total 741 54 7.3
Chi-square value =3.47, p-value >0.05
Table 3. Prevalence of Salmonella compared with the breed, sex, age, study sites, season, health and status of diarrheaFactors Category No. of
chicken No. positive (%)
Odds ratio
95% CI p-value
Breed Cross 30 4 (13.3)Local 175 35 (20.0) 1.62 0.53, 4.96 0.390
Sex Female 131 22 (16.8)Male 74 17 (22.9) 1.47 0.73, 3.00 0.281
Age Young 82 21 (17.1)Adult 123 18 (21.9) 1.37 0.68, 2.76 0.384
Site Ijaji 25 2 (8.0)Dire Inchini 41 4 (9.8) 1.24 0.21, 7.34 0.810Ambo 68 14 (20.6) 2.98 0.63, 14.18 0.170Holeta 40 10 (25.0) 3.83 0.76, 19.22 0.102Guder 31 9 (29.0) 4.70 0.91, 24.24 0.120
Season Wet 98 15 (15.3)Dry 107 24 (22.4) 1.60 0.78, 3.26 0.197
Health status Healthy 121 21 (17.4)Sick 84 18 (21.4) 1.30 0.64, 2.62 0.466
Diarrheal status Diarrheic 57 9 (15.8)Non-diarrheic 148 30 (20.3) 1.35 0.60, 3.07 0.465
Total 205 39 (19.0)CI= Confidence interval
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Antimicrobial susceptibility
Salmonella isolates showed higher susceptibility to norfloxacin (97.4%), chlor-amphenicol (92.3%), trimethoprim-sulfamethoxazole (89.7%) and streptomycin (84.6%). All the isolates were resistant to amoxicillin, tetracycline, and nitro-furantoin, thus 100% multidrug-resistant (MDR) to these three antimicrobial classes (Table 4). Besides, three different MDR patterns to six antimicrobial classes were observed. Four isolates were multidrug-resistant to five antimi-crobial classes (Table 5).
Table 4. Antimicrobial susceptibility test of Salmonella isolatesAntimicrobial class
Antimicrobial Agent
Disc Code
Potency (μg)
No. (%) resistant
No. (%) interm ediate
No. (%) suscep tible
Aminoglycosides Streptomycin S 10 2 (5.1) 4 (10.3) 33 (84.6)
Amikacin AMK 30 0 10 (25.6) 29 (74.4)
Gentamycin GEN 10 0 15 (38.5) 24 (61.5)β-lactams Amoxicillin AMX 25 39 (100) 0 0
Cephems Cefotaxime CTX 30 0 10 (25.6) 29 (74.3)Ceftazidime CAZ 30 5 (12.8) 5 (12.8) 29 (74.4)
Cefuroxime CRX 30 3 (7.7) 21 (53.8) 15 (38.5)
Phenicols Chloramphenicol CHL 30 3 (7.7) 0 36 (92.3)Quinolones Ciprofloxacin CPR 5 0 20 (51.3) 19 (48,7)
Nitrofurans Nitrofurantoin NIT 300 39 (100) 0 0
Tetracyclines Tetracycline TET 30 39 (100) 0 0
Folate pathway inhibitors
Trimethoprim-sulfamethoxazole
SXT 23.75 0 4 (10.3) 35 (89.7)
Fluoroquinolones Norfloxacin NOR 10 µg 0 1(2.6) 38 (97.4)
Table 5. Resistance patterns in Salmonella isolated from chicken (N=39)Number Antimicrobial resistance pattern No. of resistant isolates (%)Three AMX TET NIT 39 (100)Four AMX TET NIT CRX 20 (51.3)Five AMX TET NIT SXT CHL 4 (10.3)
AMX Amoxicillin, TET Tetracycline, NIT Nitrofurantoin, CRX Cefuroxime, SXT Trimethoprim-sulfamethoxa-zole, CHL Chloramphenicol.
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DiscussionSalmonella in poultry causes heavy losses through mortality and reduced pro-duction (Jordan and Pattison, 1996). The current study revealed 63.5% se-ropositivity to chicken salmonellosis, which is in agreement with 61.7% re-port of Sundar et al. (2007) and 64.2% Ashenafi et al. (2003). However, the present finding was higher than the 35.9% reported by Afera et al. (2012) from Ethiopia and 45.9% seroprevalence reported by Ahmed et al. (2008) from Bangladesh. The possible causes of the high prevalence recorded in this study could be the reason that salmonellosis in chicken is complicated by vertical and horizontal transmission. Subclinical carrier layer chickens might pass the infections to their embryos in the egg and to the rest of the flock, as there is no practice of culling carrier chickens in most of the chicken keeping communities in Ethiopia. The higher odds of seropositivity in Guder as compared to chick-ens from Dire Inchini could be due to possible contamination of chicken in the nearby village chickens from sick chicken brought to the big livestock market at Guder.
The 19.0% culture-based prevalence of Salmonella in the current study was comparable to a 16.7% reported by Abdi et al. (2017) but much higher than the 8% reported by Aragaw et al. (2010) using direct cloacal swab plating tech-nique. This high prevalence in the present study could be related to the higher probability of Salmonella detection from spleen, liver, ovary, and kidney of carrier chickens. In this study, 38.5% of the isolates were non-motile and are either S. Gallinarum or S. Pullorum which are responsible for active clinical cases or chronic carrier state.
The organ level isolation rate was 7.3%, which is lower than 19.7% in China (Li et al., 2013). Higher prevalence such as 47% (Uyttendaele et al., 1999), and 23% were also reported(Alali et al., 2012). In these studies, Salmonella con-tamination from the surface of the retail chicken carcass was the target, which could result in a higher prevalence rate compared to chicken organs that are free of any external contamination. The higher detection rate of Salmonella spp. in the spleen (11.2%) and liver (7.0%) compared to ovaries (4.4%) in the current study is nearly in line with the report of Rahimi (2012) from Iran who reported 14% (spleen), 6% (liver), and 4% (ovaries). Nazir et al. (2014) reported that the liver and spleen were the primary target organs involved in S. Gal-linarum infection, irrespective of the route of infection. The infected liver and spleen were characterized by lesions such as hepatomegaly, discoloration of
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liver, splenomegaly, congestion, and necrotic foci (Nazir et al., 2012). This is in line with the current finding of lesions in the positive liver and spleen.
The lower detection rate of Salmonella spp. in young (17.1%) than adult (21.9%) chickens in the present study are in agreement with the reports of Temelli et al. (2012). According to Zewdu and Cornelius (2009), the prevalence of Salmo-nella increased with the increasing age of chickens. Generally, Salmonella in adult chickens might be attributed to the physiological stress of layers during egg-laying and molting, which depress their immune response and increase the susceptibility as compared to young chickens (Landers et al., 2005). In this study, there was a lower detection rate of salmonella from exotic than local chickens. This could be because most of the exotic chickens sampled in the present study were growers recently distributed to farmers from the growing center, which are not expected to be the carrier. Moreover, the sample size of exotic chickens is small. The absence of a significant association of Salmonella detection rate and seropositivity with sick chicken could be because the clini-cal signs considered were not specific and some of the isolates might not be pathogenic to chicken.
Antimicrobial resistance in Salmonella spp. has been a global problem, with the rates increased to as high as 70% in some countries (Su et al., 2004). In the present study, 100% resistance to amoxicillin, tetracycline, and nitrofu-rantoin are in accordance with the 100% resistance to amoxicillin and tetra-cycline (Hassan et al., 2014), 100% to nitrofurantoin (Rampling et al., 1990), and 97.8% to tetracycline (Abdi et al., 2017). In contrast to the present find-ing, a lower resistance rate of Salmonella from poultry farms was reported to the aforementioned antimicrobials (Eguale, 2018; Dagnew et al., 2020). This variation could be due to the different samples considered which in turn might also be associated with different serovars.The other probable reason could be that most chickens in this study were obtained from local chicken traders who use different antimicrobials unprofessionally for prophylaxis in healthy and treatmentof sick chickens, that might result in possible contamination with resistant strains. The MDR at least to three antimicrobials used which is simi-lar to the pevious report (Yildirim et al., 2011). The absence of resistance to norfloxacin indicates that the drug is a reserve drug as reported by Lee et al. (2005). These reports indicate that the utilization of antimicrobials is different from region to region and between antimicrobials themselves.
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The finding of MDR to six antimicrobial classes in the present study supports the report of Abdi et al. (2017) who reported resistance isolates from chicken to 8 antimicrobials. The current finding supports the idea that Salmonellae are among the most known bacteria to carry plasmids, which encode for drug resistance (Poppe et al., 2002). The widespread use of antimicrobials in ani-mals/humans might cause an increase in the frequency of bacteria resistant to other antimicrobials as the R plasmid may encode resistance to additional antimicrobials.
Generally, poor hygiene and biosecurity, undeveloped diagnostic capabilities, lack a strong system for notification of pathogens, and antimicrobial resistance could contribute to higher disease burden in Ethiopia (Pagani and Wossene, 2008). However, the limitation of genotype data and the inability to get enough information from chicken owners in the present study made it difficult to ana-lyze their role on pathogenesis and risk factor for drug resistance, respectively. According to ISO 2002, parallel selective enrichment- RP and Tetrathionate are recommended, but as the latter doesn’t support the growth of S. Gallina-rum and S. Pullorum we didn’t use it, which might have affected the isolation rate of another Salmonella serotypes.
Conclusion The present study revealed Salmonella is prevalent in chickens in the study areas. A higher isolation rate of Salmonella was recorded in the spleen and liver. A considerable number of Salmonella isolates from chickens were multi-drug-resistant to antimicrobial drugs commonly used for animals and humans. Therefore, there is a need for regular surveillance and monitoring of salmo-nellosis and antimicrobial resistance. Awareness creation for chicken farmers about improving farming practices and the risks of antimicrobial resistance warrants special attention.
AcknowledgmentsEthiopian Institute of Public Health is acknowledged for giving us antimicro-bial discs and reference organisms and Ambo University for funding this re-search.
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Trypanocidal drug utilization practices in tsetse sup-pression and non-suppression areas of South Omo Zone, Southwestern Ethiopia
Tegegn Tesfaye1*, Tekle Olbamo2, Hagos Ashenafi3
1 Jinka Agricultural Research Center, SNNPR, Jinka, Ethiopia, P.O. Box 96
2 Jinka University, Department of Animal Science, Jinka, Ethiopia
3 Addis Ababa University, College of Veterinary Medicine and Agriculture, Bishoftu, Ethiopia
* Corresponding author, e-mail: [email protected]
AbstractTrypanosomosis control in Ethiopia is largely rely on use of available trypano-cidal drugs although there are other options such as vector control and use of trypanotolerant hosts. A cross-sectional survey aimed at assessing the knowl-edge, attitude and practices of trypanocidal drug utilization and constraints of trypanosome infection conducted in tsetse suppression and non-suppression areas of South Omo Zone, Ethiopia. The questionnaire based survey was con-ducted from November 2018 to May 2019. Descriptive statistics was used to summarize the field data obtained from 184 cattle owners. Sixty (60) of the cattle owners were from suppression area and 124 from tsetse non-suppression area. Accordingly, draft oxen and milking cows respectively from tsetse sup-pression and non-suppression areas were classes of animals which were given priority in trypanocidal drug treatment. About 79.03% and 81.7% of cattle own-ers respectively from tsetse suppression and non-suppression areas witnessed that they treat their sick animals by themselves; indicating that veterinarians and other animal health experts have very little role in medication of sick ani-mals. Diminazine aceturate (DA) was the main trypanocidal drug preferred by cattle owners in tsetse suppression area while both DA and Isometamidium chloride (ISM) were used in non-suppression areas. About 83.1% of the re-spondents from tsetse suppression areas and 86.7% from non-suppression area reported treatment failures following the use of trypanocidal drugs. Moreover, about 79.61% and 86.53% of respondents respectively from tsetse suppression and non-suppression areas observed that drugs obtained from private drug stores were less effective compared to drugs obtained from governmental vet-erinary clinics. Furthermore, the respondents disclosed that DA was the most
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horrible trypanocidal drug in showing treatment failures despite high prefer-ence by cattle owners. It was also noted that treatment frequency was higher in tsetse suppression areas than non-suppression areas regardless of vector suppression campaign. In conclusion, higher dependency of cattle owners on trypanocidal drugs, limited trypanocidal drug availability in the veterinary pharmaceutical market, frequent trypanocidal drug usage and injection by un-skilled herdsmen and owners report on trypanocidal drug treatment failures may point out the issue of trypanocidal drug resistance in the area. Therefore, awareness creation to livestock owners on the effect of misuse of trypanocidal drugs and safe trypanocidal drug usage policy should be put into effect to up-hold the effectiveness of currently available trypanocidal drugs.
Keywords: Trypanosomosis; Trypanocidal Drugs; Tsetse suppression; South Omo Zone; Ethiopia
Introduction Trypanosomosis is a complex debilitating and often fatal disease, caused by species of unicellular parasite (trypanosome), which is found in the blood and tissues of vertebrate including livestock, wild life and people (Claes et al., 2005). Absence of effective vaccine against trypanosome and absence of coher-ent environmentally friendly and sustainable vector control strategies makes the disease very complex (Claes et al., 2005; Chitanga et al., 2011; Assefa and Shibeshi, 2018). This disease is controlled by using trypanotolerant host, by controlling of the vector and parasite by using different approaches or a combi-nation of three. However, in poor rural communities, which are mostly affect-ed by the disease, control is mainly relying on the use of trypanocidal drugs. The main drugs used by livestock keepers are Isometamidium chloride (ISM), which has both curative and prophylactic effects, Diminazine Aceturate (DA), which has only curative properties, and homidium salts (chloride and bromide) which has curative and limited prophylactic effect (Van den Bossche, 2000; Giordani et al., 2016).
About 35 million doses of drugs are used in Sub- Saharan Africa (SSA) each year, with ISM, homidium/ethidium bromide and DA representing 40%, 26% and 33% respectively and with about 50–70 million animals at risk from try-panosomosis. All effective trypanocidal drugs developed between 1940 and 1960 have been extensively used for more than half a century (Chitanga et al.,
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2011). Due to this extensive use, they can cause the appearance of the drug resistant strains of trypanosomes and it is of utmost importance that measures are taken to avoid or delay the development of resistance and to maintain the efficacy of the currently available drugs (Claes et al., 2005; Chitanga et al., 2011).
Despite the high usage of these veterinary trypanocides and increasing drug resistance problems, the interest of pharmaceutical industries to invest in re-search for developing new products remains low, leaving farmers to rely on the existing drugs (Cross, 2001; Cossic et al., 2017). Due to the privatization of veterinary services in most parts of Africa, farmers have easy access to try-panocides and this has resulted in rampant misuse and under-dosage of the medications, actions that have been blamed for the emergence of trypanocidal drug resistance. Currently, trypanocidal drug resistance has been reported in 18 Sub-Saharan African countries. However, most of these reports seem to be confined to areas where the disease is endemic (Delespaux et al., 2008).
In Ethiopia, bovine trypanosomosis and its cyclical vectors were widely dis-tributed in Western and Southwestern part of the country with annual es-timated losses of $200 million, in terms of mortality and morbidity losses in livestock and the costs of controlling the disease (IAEA, 1996). Similar to other SSA countries, trypanocidal drug resistance is reported from different parts of Ethiopia by using either a mice experiment model or block treatment methods in field conditions. Accordingly, multiple drug resistant T. congolense popula-tions (resistant to DA and ISM) in naturally infected cattle of Metekel district (Afewerk et al., 2000) and T. congolense populations resistant to DA, ISM and homidium chloride were reported in cattle from Ghibe (Mulugeta et al., 1997). Moreover, T. congolense isolates resistant to DA and ISM were found in Ghibe valley Moti et al. (2012) and Bedelle and Sodo (Chaka and Abebe, 2003). Simi-larly, Tewelde et al. (2004) reported ISM resistant T. congolense, T. vivax and T. brucie in cattle from upper Didessa valley of Western Ethiopia. Addition-ally, ISM and homidium resistant T. evansi was reported from Tigray and Afar regions (Mekonnen et al., 2018). Despite of these numerous researches on trypanocidal drug resistance, some parts of the country, which are endemic to trypanosomosis and its vectors, are devoid of information about trypanocidal drug resistance and drug utilization practice.
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South Omo zone is one of pastoral areas in Ethiopia, which owns huge live-stock resources. Although there is scarce data on trypanosomosis, trypanoci-dal drug resistance and its utilization practice among livestock owners, it is suspected that South Omo zone is one of endemic area, which shares favorable conditions for the occurrence of bovine trypanosome infection, and its control depends mainly on use of available trypanocidal drugs. Furthermore, demand and supply of trypanocidal drugs was unbalanced due to huge cattle popula-tion and limited veterinary pharmaceuticals in the area. This in turn exposes the herdsmen to purchase poor quality trypanocides from unauthorized routes (personal communication). Moreover, extensive treatment of sick animals by untrained livestock owners and use of poor quality trypanocides might aggra-vate the extent of trypanocidal drug resistance and its associated problems. Therefore, the objective of this survey was to assess the knowledge, attitudes and trypanocidal drug utilization practices of livestock owners in South Omo Zone, as initial step for further study of trypanocidal drug resistance.
Materials and methodsDescription of study area
South Omo zone is situated in the Southern Nations, Nationalities and Peoples Region (SNNPR) of Ethiopia. The zone is bordered on the South by Kenya, on the Southwest by the Ilemi Triangle, on the West by Bench Maji, on the Northwest by Keffa, on the North by Konta, Gofa and Basketo on the Northeast by the Dirashe and Konso Special Dis-tricts, and on the East by the Oromia Region. The administrative center of South Omo zone (Jinka city) is located at 750 Km and 525 Km from South of Addis Ababa and Hawassa respectively (SOZLFD, 2018).
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Figure 1. Map showing study zone on regional map (left bottom) and four study districts (right) (Designed by using ArcGIS Version 10.2.2 by taking GPS points of each study sites).
According to population projections of Central Statistical Agency of Ethiopia (CSA, 2013), more than 700,000 people were projected to be living in the South Omo zone which has an area coverage of 24,249 Km2. Similarly, agricultural sample survey indicated the total cattle, sheep and goat population of South Omo zone (in million) as 1.75, 1.55 and 2.88 respectively (CSA 2017). There are eight (8) districts representing South Omo zone, which have diverse ethnic groups. Out of 56 ethnic groups found in the SNNPR, 16 ethnic groups were from South Omo zone.
The zone is located between 40 43’ North to 60 46’ North latitude & 350 79’ East to 360 06’ East longitude, and found between 376 m and 3,500 m. a. s. l. with mean annual rainfall ranging from 400 mm to 1,600 mm. It has a diverse agro-ecological zone ranging from hot arid to the tropical humid with average temperature ranging from minimum of 10.10C to maximum 27.50C (SOFED, 2012). In the study area, rain is erratic and usually bimodal occurring from
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September to December and from March to May (NMSA, 2005; FEWSNET, 2012).
Mixed crop-livestock farming is the main source of livelihoods in agro-pastoral districts of the zone (Debub Ari, Semen Ari, Male and few areas of Benatse-may district) where maize, sorghum and legumes are the major subsistence crops, and onion, cabbage and cardamom are the cash crops. Other crops in-clude root crops (yams, sweet and Irish potatoes, cassava, enset, “godere”); fruit trees and small-scale horticulture (vegetables) are also available in these agro-pastoral districts. Out of eight districts in the zone, four districts namely Hammer, Dasenech, Gnangatom and Selamago are occupied largely by pasto-ralists even though there are few pastoralists practicing crop cultivation near Omo River after the river get out from the farming land following heavy rain which is locally known as ‘Omo shish’. The livelihood of these pastoral districts depends on livestock rearing which are mainly local breeds of both large and small ruminants.
Regarding livestock health infrastructures, the zone has seven (7) veterinary clinics, six of which are found in six different districts under the zone and one in the zonal center (Jinka). The zone also has 86 animal health posts, 9 veteri-narians, 301 animal health experts and 364 community animal health workers (CAHWs). According to SOZLFD (2018), there are fifteen (15) veterinary drug shops situated in different districts of the zone.
Glossina vector (tsetse fly) suppression activities in South Omo zone was start-ed in 2013 by Southern Tsetse Eradication Program (STEP) based on initial entomological survey conducted on 2012 by the program office. According to the initial survey, the vector density of South Omo zone was found to be 51.5 F/T/D; dominantly of G. pallidipes and few undefined species (STEP, 2012). After this initial survey, four districts namely Debub Ari, Benatsemay, Male and Sela-mago, which were suspected to be highly infested by the Glossina vector were selected and vector suppression activity was initiated by using deltamethrin (1%) pour-on technique with regular vector survey between each suppression campaign. For the sake of this study, the above mentioned four districts were taken as tsetse suppression areas. Whereas, the remaining districts (Hamer,
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Dasenech, Semen Ari and Gnangatom), which were not included in vector sup-pression campaign of STEP were classified as tsetse non-suppression areas.
Study design and methods of sampling
Cross-sectional questionnaire survey was used to undertaken this study. Rep-resentative study districts and Kebeles were purposively selected based on ease of access and sake of convenience. From selected Kebeles, representative respondents (livestock owners) were selected by simple random method by us-ing their lists from Kebeles administration.
Sample size determination
The sample size for trypanocidal drug utilization practice survey was calcu-lated by the formula given below to calculate survey sample size in social re-search according to Taherdoost (2017).
z2*p(1-p)__________
e2
________________________________
1+ [z2*p(1-p)]
e2NWhere:N= total population size of study districts which was obtained from (CSA, 2007)
E= margin of error (equal to 0.05 or 1/20)
z= z-score indicating the number of standard deviations a given proportion is away from the mean whose value is equal to 1.96 for 95% confidence level
p=percentage of my sample that picks a particular answer. Accordingly, 184 agro-pastoralists/pastoralists, 124 and 60 respectively from tsetse suppressing and non-suppressing districts were participated in questionnaire survey.
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Data collection
A structured questionnaire was administered to agro-pastoralists/pastoral-ists to collect data on problems of trypanosomosis and trypanocidal drug uti-lization. The questionnaire comprised of respondent’s background, knowledge about trypanosomosis, information about trypanocidal drugs (source, drug preference, frequency of treatment, treatment failure, and cost/year) and any death occurrence due to trypanosomosis. Prior to dissemination of question-naire to respective participants, the questionnaire was translated in to Amhar-ic version for easy translation to local languages and then to the three major local languages (Ari, Bena and Hammer). Moreover, for compliance of ethical standard, participants were briefed about the objectives of the survey and con-fidentiality of information they provide and all participants gave their oral informed consent before the interview. In addition to primary data sources, secondary information from recorded data at study zonal, district and Kebele level were also used.
Data analysis
Data recorded during questionnaire survey was coded and entered into Micro-soft Excel Spread Sheet to create a database and were imported to SPSS ver-sion 20 for descriptive analysis. Descriptive statistics like means, frequencies and percentages were calculated for different parameters.
Results
The findings of questionnaire survey declared that majority of the respondents were male and illiterate. Accordingly, 84.67% and 74.19% of the respondents in tsetse suppression area were male and illiterate respectively. Likewise, 86.7% and 88.3% of respondents from tsetse non-suppression areas were male and illiterate respectively.
About 63.7% of the respondents from tsetse-suppression area gave priority to draft oxen in trypanosomosis treatment. However, 58.3% of interviewee from non-tsetse suppression areas mentioned that they give priority to milking cow in trypanosomosis treatment. Moreover, about 63.70% and 81.7% of respon-dents respectively from tsetse suppression area and non-suppression areas ob-tain trypanocidal drugs from private veterinary drug shops. Veterinarians and
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other animal health experts have very little role in treatment of animals with trypanocidal drugs and other medications in the study area as 79.03% and 81.7% of respondents respectively from tsetse suppression area and non-sup-pression areas witnessed that treatment was given by themselves (Figure 2).
Figure 2. Source of trypanocidal drugs and respondent’s access to veterinary service in tsetse suppression and non-suppression areas of South Omo Zone
Diminazine aceturate (DA) is the most preferred drug by herd owners of tsetse non-suppression areas as affirmed by 70% of respondents which is followed by both DA and ISM (16.7%) (Figure 3 B). However, in tsetse suppression area both DA and ISM (62.1%) occupy the first place in their preference followed by DA (18.7%) and both DA and homidium (5.6%) (Figure 3 A).
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Figure 3. Trypanocidal drug preference by respondents
Respondents from both tsetse suppression area (81.5%) and non-suppression areas (73.3%) replied that their animals show frequent symptom/sign of try-panosome infection such as erection of hair, erection of hair and depression and erection of hair and dried feces (Table 2) during wet season as compared to dry season. With occurrence of disease symptom, the owners treat their animals with their own preferred trypanocidal drugs. However, if the animals continue showing the disease symptoms following treatment, it is perceived as failure
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of the trypanocidal drug to cure the patient. This condition was witnessed by 83.1% and 86.7% of the respondents respectively from tsetse suppression and non-suppression areas. These failures of trypanocidals to cure the patient were most common when the origin of the drug is from private drug shops compared to drugs from governmental veterinary clinic as the cattle owners’ response indicated (Table 1).
Table 1. Season of trypanosomosis occurrence, occurrence of treatment fail-ure and source of trypanocidals with failure reportDescription of interview Response Response rate (%)
TSA TNSASeason of frequent trypanosomosis occurrence
Dry 12.1 18.3wet 81.5 73.3Equal in both seasons 6.5 8.3
Encountering failure of trypanocidal drug
Yes 83.1 86.7No 16.9 13.3
Source of failure trypanocidal Gov. veterinary clinic 16.5 9.6Private drug stores 79.6 86.5Both sources 3.9 3.8
TSA= Tsetse suppression area, TNSA= Tsetse Non- suppression area
Diminazine aceturate is the most dominant trypanocidal showing failures af-ter treatment which was respectively acknowledged by 54% and 70% of respon-dents from tsetse suppression and non-suppression areas districts followed by both DA and ISM in both areas (Figure 4). The frequency of trypanocidal treatment per year is higher in tsetse suppression area as half of the respon-dents replied that they treat their animals four times a year followed by three times a year (25.8%). However, in tsetse non-suppression areas, trypanocidal medication of two times a year (60%) is maximal followed by three times a year (16.7%) as interviewee confirmed (Figure 4).
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Figure 4. Trypanocidal drug treatment response and frequency of treatment per year
Erection of hair (47.58% and 65% respectively in tsetse suppression area and non-suppression areas) was the dominant symptom manifested by cattle own-ers as indicative of trypanosomosis, which was followed by concurrent occur-rence of erection of hair and depression in both study areas. In tsetse suppres-sion area, although the cattle owners invest more than 500 ETB to purchase trypanocidal in a year, about 39.51% ascertained that they repeatedly encoun-tered death due to the disease in the past five years. The cattle owners response indicated less average cost on trypanocidal purchase in tsetse non-suppression area which ranged between 100-200 ETB per year as compared to >500 ETB per year in tsetse suppression area (Table 2). The respondents from tsetse non-suppression area also affirmed that they encountered less death due to trypanosomosis in past five years as witnessed by 73.3% of the respondents (Table 2).
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Table 2. Interviewee response on drug costs per year, cattle deaths and ob-served symptoms of trypanosomosisDescription of interview
Categories Frequency Percentage (%)TSA TNSA TSA TNSA
Cost for trypanocidal per year
50-100 ETB 21 13 16.93 21.7100-200 ETB 20 20 16.12 33.3200-500 ETB 31 9 25.00 15.0> 500 ETB 32 9 25.80 15.0I don’t know 20 9 16.12 15.0Total 124 60 100.0 100.0
Death due to Trypanosomosis in past 5 years
Yes 49 16 39.51 26.7No 75 44 60.48 73.3
Total 124 60 100.0 100.0
Indicative signs/symptoms of trypanosomosis
Erection of hair and diarrhea 12 0 9.67 0.00
Erection of hair 59 39 47.58 65.0Erection of hair and depression 45 13 36.29 21.7
Erection of hair and drying of faces 8 8 6.45 13.3
Total 124 60 100.0 100.0TSA= Tsetse Suppressing Area, TNSA= Tsetse Non- Suppressing Area, ETB= Ethiopian Birr
Discussion The current study revealed that trypanosomosis (locally known as “Kusu-pho”, meaning the disease caused by flies) is one of the major bottlenecks for livestock production in both tsetse-suppressing and non-suppressing areas of South Omo Zone. All the respondents (100%) from both study areas witnessed that their animals had encountered the disease for decades. Similar reports about trypanosomosis was revealed from tsetse infested and non-tsetse infest-ed areas of Northwest Ethiopia (Dagnachew et al., 2017), Abbay basin areas of Northwest Ethiopia (Dagnachew, 2004), Tselemti Woreda of Tigray region (W/yohannes et al., 2010), Western Ethiopia (Tewelde et al., 2004), South Western Ethiopia (Tekle et al., 2018), and Northern Ethiopia (Birhanu et al., 2015).
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This study has also indicated that cattle owners had different approach in giving priority to their animals from treatment point of view. Animal owners from tsetse suppression areas give priority to draft oxen in treatment of try-panosome infection and reasoned that oxen are the most valuable class of ani-mals in agricultural land preparations since they are practicing mixed farming system and the market fetch value of oxen is higher than other class of ani-mals even after long term serving on traction. Previous report of Seyoum et al. (2013) indicated similar condition of bovine trypanosome infection severity and more investment of owners to treat draft oxen than other class of animals in Baro-Akobo and Gojeb river basins in SNNPR, Southwestern Ethiopia. On the contrary, to tsetse suppression areas, respondents from tsetse non-suppression areas of current study gave priority to milking cow in treatment of trypano-somosis and stated that milk is their primary base of livelihood since most of respondents from tsetse non-suppression areas were pastoralists who rear livestock for their livelihood.
Private drug shops were the major suppliers of trypanocidal drugs for the residents of South Omo Zone (for both tsetse suppression and tsetse non-sup-pression areas) followed by both sources together (government vet clinic and private drug shop) and government veterinary clinics. Due to strict control made on unauthorized drug sellers (personal communication with each study district’s livestock and fishery department head), the vet drug from this route is negligible in the study area. Moreover, community awareness creation on disadvantage of unauthorized drug usage and strong participation of autho-rized drug sellers play key role in minimizing unauthorized veterinary drug sources in the area. Consistent report was declared from tsetse- infested ar-eas of Jabitehenan by Dagnachew et al. ( 2017) where 48% of his interviewee responded private drug shops as their primary drug route. However, reports disagree with present study was declared from Guraghe zone of SNNPRS and Jimma zone of Oromia region by Tekle et al. (2018) and Chewaka settlement station and Bikiltu Didessa peasant associations of South West Ethiopia by Anberber et al. (2014) which stated that unauthorized route as primary source of trypanocidal drug.
Assessment on person responsible for injection of sick animals with trypano-cidal drugs indicated that 79.03% and 81.7% of respondent; respectively from tsetse suppression and tsetse non-suppression areas treat their animals when
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sick. Only 8.87% and 6.7% of the respondents respectively from tsetse suppres-sion and tsetse non-suppression areas send their sick animals to veterinary clinic or animal health post to be treated with veterinarian or other trained personnel. The cattle owners reasoned out that they were far away from ani-mal health post and veterinary clinics so that their only chance to save sick animal is treating by themselves. According to respondents, one animal health expert or community animal health worker (CAHW) was assigned for clusters of Kebeles (one cluster containing 3-4 Kebeles) (SOZLFD, 2018). Therefore, waiting animal health expert’s service is very difficult to save diseased ani-mals. Person responsible for trypanocidal injection in the current study was found in agreement with previous studies of Denu et al. (2012); Hadush et al. (2014); Tsegaye (2014) and Tekle et al. (2018).
Diminazine aceturate (DA), locally known as “Kacho” was the major drug of choice for treatment of animals with trypanosomosis symptom in tsetse non-suppression areas as DA is cheaper than ISM (“Singula”) in the private drug stores. However, it was both DA and ISM in tsetse suppression areas according to current survey report. DA preference by cattle owners of tsetse non-suppres-sion areas agree with reports of Van den Bossche et al. (2000) and Tewelde et al., (2004) who stated that most farmers prefer to use DA to ISM. Simi-larly, DA was the drug of choice for non-tsetse infested area residents of North West Ethiopia (Tsegaye 2014). Tsetse suppression areas drug preference of the present study was similar to Tsegaye (2014) report from tsetse-infested areas of North West Ethiopia who reported that majority (68%) of the respondents prefer both ISMM and DA. Both DA and ISM preference in tsetse suppres-sion area of South Omo zone might be associated with the cattle owners’ belief in which they perceive treating the two drugs interchangeably brings better achievement in the control of trypanosomosis. Moreover, easy availability of both drugs in all available sources of veterinary pharmaceuticals of tsetse sup-pression area may increase their preference by cattle owners.
Trypanocidal treatment frequency of 4 times per year (50%) and two times per year (60%) was reported respectively from tsetse suppression areas and tsetse non-suppression areas of current study area. Comparable treatment frequen-cies were reported from tsetse free areas of North West Ethiopia (1-3 times per year) by Tsegaye (2014) and Metekel district of Benshangul Gumuz region (three times per year) by Afewerk and collegues (2000). On the contrary, very
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higher treatment frequency reports were disclosed by Dagnachew (2004) in the lowland (3-4 times per month) and midland (1-2 times per month) areas of Abbay basin, Tewelde et al., (2004) in Western Ethiopia (2.5-3.5 times per month), Tsegaye (2014) in tsetse-infested areas of North West Ethiopia (> 10 times per year) and Seyoum et al. (2013) in Baro-Akobo and Gojeb River basins (6 times per year).
Higher treatment frequency in tsetse suppression areas compared to tsetse non-suppression areas in current study might indicated higher challenge of the disease due to non-sustainability and less participation of community on vector suppression activities. Close interaction of wild life conservation areas with tsetse suppression areas may increase the chance of animals to be infect-ed since wild animals in conserved areas may act as source of infection. This in turn increases the frequency of treatment in the area as compared to tse-tse non-suppression areas. Moreover, easy access to trypanocidal drug sources may also play great role in increased frequency of treatment since most of the owners in tsetse suppression areas were close to zonal and district cities where all sources (veterinary clinic and private drug vendor shops) of trypanocidal drugs were located.
High trypanosomosis occurrence during wet season according to respondents of current study was similar with the report of Abebe, (2018). The respondents of this study associated with high wet season occurrence of the disease with increased fly number in the area following rain since they associate the dis-ease with fly population (“Kusupho” in local language meaning disease caused by flies). They also explained that there was a tree called “Gadaq” in local language whose flower can highly attract the flies (including tsetse) during wet season so that their animals were easily infected by the disease caused by flies (“kusupho”). On the contrary, equal occurrence of trypanosomosis in both dry and wet seasons was reported previously by Tewelde et al., (2004) and Afewerk and collegues (2000). from Ethiopia and Ngare and Mwendia (2000) from Kenya.
Treatment failure was witnessed by 83.1% and 86.7% of the interviewee from tsetse suppression areas and tsetse non-suppression areas respectively. High-er treatment failure of present study area might be hugely associated with injection of trypanocidal drugs by unskilled herdsmen and erroneous dilution
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of trypanocidals. Additionally, it also might be associated with under dosage and use of poor quality of trypanocidals to treat the animals. Similarly, Tekle et al. (2018) survey report indicated that all of his respondents perceived the occurrence of treatment failure after medication. The current survey indicated that drugs from authorized private drug stores show more failure than those drugs from government veterinary clinics. However, reports of Tekle et al. (2018) pointed that trypanosomosis treatments were more likely to be success-ful when the drugs are sourced from both government veterinary clinics and authorized private sources, although he and his co-workers found 27.3% DA and 29.4% ISM sampled from both authorized and unauthorized drug sources were non-compliant due to insufficient active ingredients.
DA is the most horrible trypanocidal drug that exhibit treatment failure in current study areas of South Omo Zone as approved by 54% and 70% partici-pant respondents, respectively from tsetse suppression areas and tsetse non-suppression areas. Different reports from various angles of Ethiopia as well as other Sub-Saharan African countries revealed this situation through assess-ment as well as via experimental studies by using laboratory animals. Experi-mentally Moti et al. (2012) in Gibe valley, Chaka and Abebe (2003) in Gibe val-ley, Bedelle and Sodo, Mekonnen et al. (2018) in Tigray and Afar and Afewerk et al. (2000) in Metekel reported single as well as multiple trypanocidal drug resistant occurrence. Low market price of DA when compared with ISM, its high preference by cattle owners of the study area (especially of tsetse non-suppression areas) and lack of dosage knowledge as well as administration by unskilled personnel may predispose this trypanocide for treatment failure. Despite of numerous reports on resistant development against DA by trypano-some species, Mekonnen and colleagues reported comparative advantage of DA over ISM by completely curing mice infected with T. evansi isolated from dromedary camel in Tigray and Afar, Northern Ethiopia which makes DA as drug of choice for treatment of Surra (Mekonnen et al., 2018).
Higher treatment cost in tsetse suppression areas as compared to tsetse non-suppression areas in this study may indicate high challenge of the disease to the residents of tsetse suppression area although there were vector control activities and it shows more reliance of herdsmen on trypanocidal drug. More-over, it may indicate better awareness of cattle owners in prophylactic treat-ment of non-sick animals in tsetse suppression areas, which may increase an-nual cost for trypanocidal. Furthermore, increased access of veterinary health
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provision centers such as veterinary clinic, animal health post and veterinary drug shops in tsetse suppression areas may increase the frequency of trypano-cidal drug purchase per year. Similar assumption was reported by Uilenberg and Boyt (1998) who said that the number of treatments over a year reflects the magnitude of trypanosomosis challenge in an area. Mean annual treat-ment cost report for trypanosomosis from Gimbo and Guraferda of Baro-Akobo and Gojeb river basins in SNNPRS by Seyoum et al. (2013) was almost similar with the current treatment cost report from tsetse non-suppression areas but it was lower than annual treatment cost of respondents from tsetse suppres-sion areas.
ConclusionThis study result leads to the conclusion of higher dependency of cattle owners on trypanocidal drugs and frequent trypanocidal drug usage and injection by unskilled herdsmen was common in South Omo Zone. Furthermore, limited trypanocidal drug availability in the market in conjunction with owners report on trypanocidal drug treatment failures may point out the issue of trypano-cidal drug resistance in the area. Therefore, awareness creation to livestock owners on the effect of misuse of trypanocidal drugs and safe trypanocidal drug usage policy should be engaged to uphold the effectiveness of currently available trypanocidal drugs. Moreover, participatory vector control activities and further trypanocidal drug resistance and efficacy investigation should be conducted in the area to discern the status of available trypanocidal drugs.
Limitations of the studyFurther information might be generated especially from tsetse non-suppression areas if the number of participants (herd owners) in the survey were higher than those used in our study. Additionally, brand identification of circulating trypanocidals in the area, especially trypanocidal drugs that shown treatment failure was not covered by this study due to the interviewee knowledge hin-drance to identify each brand through our questionnaire survey, which needs further exploration along with their efficacy study.
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Acknowledgements The authors would like to acknowledge Southern Agricultural Research In-stitute (SARI) for funding this research and Livestock Research Directorate of Jinka Agricultural Research Center (JARC), Jinka University (JKU) and Jinka Veterinary laboratory for further logistic contribution. The contribution by the study participants is highly acknowledged, without their collaborative effect the study would not be possible.
Conflict of interestThe authors declare that there is no conflict of interest.
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Bihon et al., Ethiop. Vet. J., 2020, 24 (2), 112-122 DOI https://dx.doi.org/10.4314/evj.v24i2.7 Ethiopian Veterinary Journal
Ethiop. Vet. J., 2020, 24 (2), 112-122
Fore-Stomach Foreign Bodies: prevalence, associat-ed risk factors and types affecting cattle slaughtered at Gondar ELFORA abattoir, northwest Ethiopia
Amare Bihon1*, Teketaye Bayeleyegn1, Ayalew Assefa2 and Yimer Muktar1
1College of Agriculture, Woldia University, Woldia, Ethiopia
2Sekota Dryland Agricultural Research Center, Sekota, Ethiopia
*Corresponding author: Email: [email protected]; Tel: +251-(0)9-45143238 and ORCID: https://orcid.org/0000-0003-1766-9889
AbstractIn Ethiopia, recurrent drought and feed shortage coupling with high level of environmental pollution predispose the animals to foreign body ingestion. A cross-sectional study with systematic random sampling approach was em-ployed from November 2018 to April, 2019 with the objectives of estimating the prevalence; identify associated risk factors and to categorize the types of foreign body in cattle slaughtered at Gondar ELFORA Abattoir. Ante-mortem and postmortem examinations were used to collect the data. From the total of 384 animals examined, the overall prevalence of foreign body was 83(21.61%). Adult and old animals were 4.33 (95% CI=0.98, 19.00, p=0.052) and 4.54 (95% CI=1.03, 19.96, p=0.045) times more likely to have a chance of getting foreign body than young animals by keeping another factors constant, respectively. However, the difference is not statistically significant for adult cattle. More-over, poor and medium body conditioned animals were 2.19 (95% CI=1.04, 4.56, p=0.037) and 1.51 (95% CI=0.72, 3.13, p=0.273) times more likely to acquire foreign bodies than good body conditioned animals by keeping another factors constant. In the positive cases (N=83), 41(49.40%), 34(40.96%), 8(9.64%) and 0(0%) of the foreign bodies were found in the rumen, reticulum, both rumen and reticulum, and omasum, respectively. Majority of foreign bodies identified (79.51%) were non-metallic in nature, including clothes, plastics, rope, sand and stone. Designing and implementation of appropriate solid waste disposal and management practices are strongly recommended to reduce the risk of ingestion of indigestible foreign bodies.
Keyword: Abattoir; Cattle; Foreign body; Fore stomach; Prevalence
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IntroductionA lot of documents witnessed huge livestock resource of Ethiopia by describing its priority rank in Africa and 5th in the world (FAO, 2015). According to CSA, (2015) report, the country hosts 56.71 million cattle, 29.33 million sheep and 29.11 million of goats almost all of which are local breed animals. In Ethiopia, like other developing countries, domestic animals contribute in every aspect of household’s survival (Kassahu and Wale, 2017). Among domestic animals, cattle take the leading advantage, but still, its contribution is below its ex-pected potential due to different hindering factors like disease (Jemberu et al., 2018). Disease, by its nature, could be infectious and non-infectious in origin (Radostits, 2007). Gastrointestinal foreign bodies are among non-infectious diseases and one of surgical emergencies in veterinary medicine. Cattle are the most susceptible species because of its indiscriminate feeding habit (Tesfaye and Chanie, 2012).
Recurrent drought and feed shortage in Ethiopia coupling with high level of environmental pollution predisposes animals for foreign body ingestion (Tes-faye et al., 2012). Nature and way of entrance of foreign bodies to tissues de-termine the level and type of complication from glossitis to traumatic reticulo-peritonitis (TRP). The ingested foreign bodies may be metallic or non-metallic objects. Metallic foreign bodies mostly reside in the reticulum and may cause perforation of the reticulum resulting in peritonitis, pericarditis or both (Ra-dostitis, 2007) while non-metallic objects may cause ruminal impaction, which hampers the absorption of volatile fatty acids and reduces the level of animal fattening (Vanitha et al., 2010). Foreign body ingestion also called hardware disease and can be diagnosed by ultrasonography, radiology, wither pinch test and rumenotomy. Rumenotomy is both diagnostic and treatment method (Sharma and Pankaj, 2006).
According to Ramaswamy et al, (2011), the presence of extensive construction sites and improper waste disposal can cause the death of beef and dairy cattle due to foreign body ingestion that resulting in a huge economic loss. However, the problem is left unnoticed while it is life threatening and economically dev-astating. Moreover, its diagnosis, treatment and control in live animals are costly and difficult. Thus, it can be adequately studied in abattoirs. Therefore, the objectives of the present study were assessing the prevalence, identifying risk factors and types of fore-stomach foreign bodies in cattle slaughtered at Gondar ELFORA Abattoir.
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Materials and methodsStudy Area
The study was conducted at Gondar ELFORA Abattoir, North Gondar zone, Amhara National Regional State. Gondar town is located at 750Km from Ad-dis Ababa at an elevation of 2200 m above sea level. The city situated be-tween a latitude and longitude of 12°36′N and 33°28′E. Rain fall varies from 880-1172mm with the average annual temperature of 19.7°C. The abattoir has the capacity of slaughtering up to 250 animals per day. Its daily service ranges from 6-10 animals per day during Christian fasting season and the maximum up to 150 in Christian holydays. Generally, it serves Gondar univer-sity student’s café which presented 6-10 animals per day except Tuesday and Thursday and some other customers in the town. The university’s agent has focal points in Chilga, Metema, Fogera, Debretabor and Gondar town, so the animals are mainly presented from these areas.
Study Animals
Three hundred eighty four (384) apparently healthy animals presented to Gondar ELFORA abattoir from Gondar town, Debretabour, Fogera, Metema, Chilga and Kolladeba were included in the study. All animals were males and both local and cross breed cattle were included. Age category was done based on dentition eruption described by Pace and Wakeman (2003) as young (≤5 years), adult (5-10) years) and old (≥10 years). Moreover, Body condition of cat-tle was recorded based on the appearance of the animal and manual palpation of the spinus and transverse processes of the lumbar vertebrae as described by Nicholson and Butterworth (1986) as poor, medium and good body conditions.
Study design and sampling technique
A cross sectional study with systematic random sampling technique with the inclusion of every 5th animal from the start was included in the sample until the required sample size was fulfilled. Because of the variability of animals presented in the abattoir every day, its service is limited in Christian fasting days (minimum of 6) and higher in the holydays (maximum of 150) from No-vember, 2018 to April, 2019.
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Sample size determination
Thrusfield (2005) formula was used to calculate the number of animals includ-ed in the study. Considering the absence or limitation of previous works, 50% expected prevalence with 5% desired level of precision and 95% of confidence interval, 384 animals were taken.
Where: n = required sample size, 1.96=the value of Z at 95%confidence interval,
Prexp = expected prevalence and d = desired absolute precision
Data collection method
Ante mortem examination: This was done according to FAO, (2009) describ-ing a specific range of procedures by considering the behavior, appearance and signs of disease. Animals were examined at rest and in motion during their stay in the lairage for the general status. A unique identification code was given. Then, age, breed and their origin was recorded.
Postmortem examination: palpation, incision and visual inspection were done on Rumen, Reticulum and Omasum immediately after slaughter. Short-ly, in the evisceration stages the stomach were carefully removed from the abdominal cavity and incision was given to remove the ingesta and foreign bodies were taken out after visual inspection. Then it was washed with water to remove adhering feed material and to identify types of foreign bodies. When the finding is positive, the location and type of the foreign bodies were recorded and photographed.
Data management and statistical analysis
The collected data were entered and scored in Microsoft excel worksheet. Before subjected to statistical analysis, the data were thoroughly screened for errors, adequately coded and summarized using descriptive statistics. For analysis, STATA 13 (Stata Corp, TX, USA) was used. Descriptive analysis (frequency and Pearson chi-square (χ2) test) and inferential statistics (multiple logistic regressions) were employed to measure the risk caused by associated factors. For all comparisons, P-values less than 0.05 were considered as significant.
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ResultsRisk factors associated with foreign body
The overall prevalence of fore-stomach foreign body was 83(21.61%) from a to-tal of 384 animals examined. Adult and old animals were 4.33 and 4.54 times more likely to have a chance of getting foreign body than young animals by keeping other factors constant and the association between age groups were significant in old animals (p<0.05). Moreover, poor and medium body condi-tioned animals were 2.19 and 1.51 times more likely to acquire foreign bodies than good body conditioned animals by keeping other factors constant. The association between body condition is significant among poor and good body conditioned animals (p>0.05) (Table 1)
Table 1: Logistic regression analysis of associated risk factors with foreign bodyVariable No.
examinedPositive (%) AOR (95%CI) p-value
Age Young 39 2(5.12) Reference ReferenceAdult 173 40(23.12) 4.33 (0.98-19.00) 0.052Old 172 41(23.84) 4.54 (1.03- 19.96) 0.045
BCS Good 92 12(13.04) Reference Reference
Medium 163 34(20.85) 1.51 (0.72 - 3.13) 0.273
Poor 129 37(28.68) 2.19 (1.04- 4.56) 0.037
Breed Local 364 75(20.6) Reference ReferenceCross 20 8(40.0 ) 2.10 (0.78-5.61) 0.140
Origin Gondar 67 21 (31.34) Reference Reference Debretabour 61 12(19.67) 2.10 (0.89 – 4.94) 0.090Fogera 70 14(20) 2.03 (0.88 – 4.65) 0.093Metema 80 14(17.5) 2.16 (0.97- 4.82) 0.058Chilga 42 13(30.95) 1.04 (0.43 - 2.53) 0.916Kolladeba 64 9(14.06) 2.78 ( 1.10 - 6.98) 0.029
Total 384 83 (21.61) - -BSC = Body condition score
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Foreign body location, type and penetration
From the positive cases (N=83), 41(49.40%), 34(40.96%), 8(9.64%) and 0(0%) was found in the rumen, reticulum, both rumen and reticulum, and omasum respectively. Regarding types of foreign bodies, 66 (79.51%), 12(14.46%), and 5(6.06%) were non-metal, metal and mixed respectively. Among the positive cases there were 3 (3.61%) cases of penetration on the fore-stomach itself and neighboring organs (Table 2).
Table 2: Foreign body location, type and penetration Variable Category Frequency (%)FB location Rumen
ReticulumBoth rumen and reticulum Omasum
41(49.40)34(49.96)8(9.64)
-Type of FB Metal
Non metalMixed
12(14.46)66(79.51)5(6.06)
Penetration PresentAbsence
3(3.61)80(0)
Total 83(100)
Distribution of foreign body type due to associated risk factors
From 384 examined animals, a total of 83 metallic and non-metallic foreign bodies were identified. Non-metallic foreign bodies comprise clothes, plastics, stone, rope and sand whereas, metallic foreign bodies comprise wire, nail, blade and needle. The distribution of foreign body type showed significant dif-ference among breeds (Table 4).
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Tabl
e 4:
Dis
trib
utio
n of
fore
ign
body
type
by
asso
ciat
ed r
isk
fact
ors
Var
iabl
eC
ate
gori
esN
egat
i ve
Met
allic
Non
-met
allic
Mix
edFi
sher
s ex
act
test
P-v
alue
Wir
ena
ilB
lade
Nee
dle
Clo
the
Pla
stic
Ston
ero
pesa
nd
Age
Youn
g37
--
--
--
--
2-
29.6
532
0.07
6
Adul
t13
31
11
18
11-
104
3
Old
131
32
-3
615
3-
63
BCS
Goo
d80
-1
--
14
1-
41
17.5
499
0.61
7
Med
ium
129
11
12
610
16
42
Poor
923
1-
27
121
44
3
Bree
dLo
cal
289
42
14
1324
39
123
32.3
137
0.00
0
Cro
ss12
-
1-
-1
2-
1-
3
Ori
gin
Gon
dar
to
wn
46-
--
23
52
26
165
.926
90.
065
Deb
reta
bo
ur49
--
--
-5
33
12
Foge
ra56
21
--
16
11
3-
Met
ema
66-
2-
15
41
1-
1
Chilg
a29
2-
--
44
--
21
Kol
a de
ba55
--
11
12
33
-2
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DiscussionFore-stomach foreign body is an important potential health problem in Gondar area. The finding of the present study demonstrated that one in fifth (21.61%, 83/384) examined cattle were affected in the study area. This finding concurs with the findings of Tesfaye et al (2012) who reported 23.9% from Hirna mu-nicipal abattoir and 23.4% published from Addis Ababa municipality abattoir by Tiruneh and Yesuwork (2010). It was slightly higher than the reports of Rahel (2011), Kassahun et al. (2015), Tesfaye and Chanie (2012), who reported 17.07% in Hawassa Municipal Abattoir, 14.8% in Gondar ELFORA abattoir and 13.22 % in Jimma Municipal Abattoir, respectively. In the contrary, Is-mael et al. (2007) reported higher overall prevalence of foreign body (77.41%) in Jordan. The variation could be due to differences in the waste management systems in different study areas, sex of animals and season which study was conducted. Moreover, it might be due to indiscriminate feeding habit of cattle.
The higher prevalence rate was found in older animals (23.84%) than adult and young animals. This finding is close to the reported 25.6% prevalence in older animals by Kassahun et al (2015). But it is lower than the results of Tesfaye and Chanie (2012), Rahel (2011), Radostitis et al. (2007) and Ismael et al. (2007) who reported 81.25%, 63.3%, 56.6% and 32.25% in older animals respectively. This might be associated with extended time of exposure which increases with the age of the animal and positivity of chronically affected ani-mals.
Poor body conditioned animals were more affected than medium and good body conditioned animals. The present finding was in agreement with Tesfaye et al. (2012) who reported 26%, lower than the results of Tesfaye and Chanie (2012) who reported 72.72% and was higher than the findings of Rahel (2011) who reported 15% in poor body conditioned animals. Poor body condition by itself might be due to the contribution of the foreign body, or it might be due to the interference of the foreign body with the absorption of volatile fatty acid and thus causes reduced weight gain.
Our study demonstrated that forestomach foreign body prevalence was higher in crossbreed animals (40.0%) than local breeds (20.5%). The result is in agree-ment with Tesfaye and Chanie (2012) as 70%, Nugusu et al. (2013) as 62.2% and Rahel (2011), who reported 58.82% prevalence of forestomach foreign body in crossbreeds. This might be associated with body size and higher productiv-
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ity which requires high demand of nutrition and hence increase exposure for foreign bodies.
Animals originated from Kolladeba were 2.78 times more likely to have risk of acquiring foreign body than animals originated from Gondar town. This might be due to animals in Gondar town are reared in small feedlots that restrict their movement, whereas, in Kolladeba most of the farmers practiced free grazing. From the positive cases (N=83), higher proportion of fore stomach foreign body is recovered from rumen (49.40%) and zero from omasum. This finding is higher than the findings of Bassa and Tesfaye (2017) who reported 10.83% in rumen at Wolaita Sodo municipal Abattoir and lower than the reports of Ushula and Nana (2017) in the rumen at Hawassa municipal abattoir (87%). This might be associated with the larger rumen volume, the cumulative size and material composition of the foreign bodies and the types of materials, with metals and sharp objects tending to localize preferentially in reticulum (Radostits, 2007). Besides, the present study revealed zero prevalence from omasum which might be associated with the anatomical position of omasum and structure of reticu-lo-omasal orifices which is elevated above the floor to retain heavy object in the reticulum and the material that can pass omasal compartment is very small in size (less than1.2mm). Hence, such size of material was not possible to visual-ized with naked eye.
The types of foreign bodies detected were plastics, cloths, sands, ropes, nee-dles, wires, nails, stones, blades and mixed. From those, the most frequently observed fore-stomach foreign bodies in cattle slaughtered at Gondar ELFO-RA abattoir were plastics 26 (31.33%). This finding agreed with various re-ports from different areas of Ethiopia 21(41.2%) by Kassahun et al., 2015 and 22(44.89%) by Tesfaye and Chanie (2012). The highest prevalence of plastics might be due to lack of good waste management, lack of awareness in the society about the side effect of plastic material in livestock production and improper disposal of plastics.
Conclusion
The present study revealed an overall prevalence of 21.61% fore-stomach for-eign body in cattle in the study area. Adult and old animals were 4.33 and 4.54 times more likely to have a chance of getting foreign body than young ani-mals, respectively. Rumen harbored more foreign bodies (49.40%) than other compartments of the fore-stomach. Therefore, proper waste management, an
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adequate mineral supplement for animals and increasing awareness about the side-effects of plastics should be considered in the study area.
Declaration of conflict of interest Authors declare, there is no conflicts interest.
AcknowledgmentThanks to Gondar ELFORA abattoir for their unreserved support and volun-tariness to perform this study.
References
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Lulie et al., Ethiop. Vet. J., 2020, 24 (2), 123-138 DOI https://dx.doi.org/10.4314/evj.v24i2.8 Ethiopian Veterinary Journal
Ethiop. Vet. J., 2020, 24 (2), 123-138
Immunogenicity and protective efficacy of irradiated Salmonella Gallinarum against homologous chal-lenge infection in Bovans brown chickens
Solomon Lulie1, Haile Alemayehu2, Anwar Nuru1, Takele Abayneh3 Tadesse Eguale2*
1College of Veterinary Medicine and Animal Sciences, University of Gondar, P.O. Box 346
Gondar, Ethiopia.
2Aklilu Lemma Institute of Pathobiology, Addis Ababa University, P.O. Box 1176, Addis Ababa, Ethiopia
3National Veterinary Institute, P.O. Box 19, Bishoftu, Ethiopia
* Corresponding author email address: [email protected]
AbstractFowl typhoid is a systemic poultry disease caused by Salmonella Gallinarum (SG). It is responsible for significant economic loss, due to its severe morbid-ity and mortality. An irradiated vaccine is one of the possible alternatives to prevent and control fowl typhoid. This study aimed to evaluate the safety, immunogenicity, and protective efficacy of irradiated SG using a randomized control trial in chicken. A field strain of SG was exposed to different doses of gamma irradiation to determine its effect on the viability of SG. Safety and im-munogenicity were assessed by administering irradiated SG orally to 3 groups (5 each) of 5 weeks old Bovans brown chickens at 2400, 2500, and 2600 gray (Gy). The protective efficacy of 108colony forming units (CFU) of SG irradi-ated at 2400 Gy administered orally and subcutaneously was then evaluated using homologous challenge infection and compared with SG 9R commercial vaccine using 40, 5-week old Bovans brown chickens where the chickens were randomly assigned to 4 groups. Chickens in Group 1were exposed to 108 CFU of irradiated SG orally; Group 2 to the same dose subcutaneously; Group 3 to SG 9R strain commercial vaccine subcutaneously, Group 4 to phosphate-buffered saline (PBS) orally. Data related to survival, antibody response, and patho-logical lesions were recorded. Mann-Whitney U-test, Kruskal-Wallis test, and Fisher’s exact tests were used to examine for statistical significance. Irradia-tion at 2600 Gy caused complete inactivation of SG whereas SG exposed to 2400 Gy showed better immunogenicity and was safe for chickens. Antibody response in a group of chickens vaccinated with irradiated SG administered
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subcutaneously (SC) was significantly higher than those vaccinated with the SG 9R vaccine on day 7 (p=0.003) and day 14 (p=0.002) post-immunization. Comparative evaluation of the protective efficacy based on the mortality rate of chickens after challenge showed that 2400 Gy irradiated SG vaccine ad-ministered SC and SG 9R vaccine-induced equal protection of 50% while the irradiated vaccine administered orally protected only 10% of chickens against homologous challenge infection. SG was not isolated from the liver, spleen, and feces of chickens that survived challenge infection until the end of the experiment. Irradiated SG administered SC is shown to be a promising vac-cine against fowl typhoid. Further studies using a large sample size involving tuning of irradiation dose to improve immunogenicity and use of booster vac-cination are recommended.
Keywords: Chicken; Fowl typhoid; Gamma irradiation; Salmonella Gallina-rum; Vaccine
IntroductionFowl typhoid (FT) is a systemic disease of poultry caused by Salmonella Gal-linarum (SG), which results in septicemia, high morbidity, and mortality as well as severe inflammation of internal organs such as liver and spleen. Chick-ens are the natural hosts of SG which is responsible for significant economic losses to the poultry industry worldwide (Barrow and Freitas, 2011). Although FT has been eradicated from commercial poultry farms in many developed countries through the isolation, removal of contaminated flocks, implementa-tion of biosecurity, and hygienic practices; it is still a major problem of poultry production in many developing countries (Desin et al., 2013; Revolledo, 2018). Previous studies conducted in different areas of Ethiopia showed that the over-all prevalence of SG among chicken flocks ranges from 0.8% to 44.8% (Aragaw et al., 2010; Alebachew and Mekonnen, 2013; Kumar et al., 2014). Vaccina-tion has been the most practical and effective strategy for the control of fowl typhoid in developing countries (Laniewski et al., 2014).The most commonly used vaccine against FT is a commercial live vaccine derived from the stable rough strain of S. Gallinarum (SG 9R)(Revolledo, 2018). However, this vac-cine has several drawbacks such as the presence of residual virulence, which is particularly serious in some strains of chickens, prolonged survival in feces, litter or dust, and its persistence in immunized chickens leading to transmis-sion through eggs (Kwon and Cho, 2011). Therefore, there is a need for novel, safe and effective vaccines for prevention and control of FT.
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Inactivated vaccines produced by killing the pathogens with chemicals, heat, or radiation are believed to be more stable and safe compared to live vaccines (Rosenthal and Zimmerman, 2006). Vaccines developed by irradiating patho-gens have been reported as strong inducers of cellular and humoral immune responses (Eberl et al., 2001; Zorgi et al., 2011). Gamma irradiation is capable of destroying nucleic acids without damaging the pathogen surface antigens and can also eradicate chemical contaminants (Seo, 2015). A previous study on the possible use of gamma-irradiated rotavirus showed a lack of infectivity of virus inactivated at 50 Kilo Gray(KGy) and induction of strong neutralizing antibody responses in mice (Shahrudin et al., 2018). This study aimed to evalu-ate the safety, immunogenicity, and protective efficacy of irradiated SG and compare it with the existing commercial SG 9R vaccine.
Materials and methodsExperimental animals and experimental design
A total of 60 female Bovans brown breed of chickens at the age of 5 weeks were purchased from a small-scale poultry farm in Bishoftu town, located about 40 Km South of Addis Ababa. The farm receives and grows day-old chickens from a branch of EthioChicken Private Limited Company, one of the large chicken multiplication farms in Ethiopia located in Adama Town. The chickens were purchased twice, first 20 of them for investigation of safety and immunogenic-ity of irradiated SG, and on the second occasion, the other 40 chickens for as-sessment of protective efficacy of irradiated bacteria. The chickens were kept in cages with area of 1 meter by 1 meter and height of 90 cm. Five chickens were kept per cage in the animal house facility of Aklilu Lemma Institute of Pathobiology, Addis Ababa University. They were provided with water and feed free of antibiotics. All chickens used in the current experiment were vac-cinated against Marek’s, Gumboro, and Newcastle disease while they were on multiplication farm. The complete randomized experimental design was used to group experimental animals into different treatment groups.
Bacterial strain and vaccineA field strain of SG originally isolated from a poultry farm in Ethiopia during the previous epidemiological study of Salmonella in a poultry farm was used to assess the effect of different irradiation doses and to evaluate the protective efficacy of the irradiated vaccine. The isolate was confirmed with biochemical tests and serotype-specific PCR as described previously (Yang, et al., 2014).
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Live attenuated rough strain of SG (SG 9R) strain vaccine, produced at Na-tional Veterinary Institute, Bishoftu, Ethiopia (Batch no. Ft 02-18) containing 5x107CFU per dose was employed as a control vaccine.
Determination of optimum bacterial titer and irradiation process
Three uniform isolated colonies of SG grown for 18 hrs. on Tryptone Soya Agar (Oxoid, UK) were inoculated into 5ml nutrient broth (Oxoid, UK) and cultured at 37°C for 6 hrs. One ml of the suspension was withdrawn every hour for five hrs. and serial dilution ranging from 10-1 to 10-6 was performed in 9 ml phosphate-buffered saline (PBS). Hundred µl of each dilution was inoculated to plate count agar (Oxoid, UK) using the spread plate technique. The number of CFU was counted after 24 hours of incubation at 37°C to determine the time required to reach 108 CFU/ml.
After determination of the time required to reach 108 CFU/ml, which was 5 hrs., 3 uniform colonies were inoculated into the nutrient broth and grown for 5 hrs. at 37°C. Then culture suspension was centrifuged at 6000 revolutions per minute at 4°C for 15 min. The supernatant was discarded and the pellet of cells was washed twice with PBS and re-suspended in 5ml PBS in 15 ml falcon tubes. It was then transported to the National Institute for Control and Eradi-cation of Tsetse and Trypanosome (NICETT) at Kaliti, Addis Ababa, in an ice-box where irradiation was conducted. Suspended bacterial cells were exposed to gamma irradiation with doses ranging from 500-3000 Gy using a cobalt 60 irradiation machine (MDS NORDION, Canada).
Viability, safety, and immunogenicity of irradiated SG
Viability of SG exposed to different irradiation doses was assessed by inoculat-ing serial dilution of irradiated SG in PBS on plate count agar and incubating for 24-48 hrs. at 37°C. Then the number of CFU/ml of suspension and lethal irradiation dose of the bacteria were determined through counting visible colo-nies. Various irradiation doses were examined to find the lowest optimum ir-radiation at the margin of the lethal dose. Twenty, 5 weeks old Bovans brown chickens were acclimatized to the laboratory condition for 1 week before initia-tion of the experiment. During this time, the chickens were screened for the antibody of SG using slide agglutination test (SAT) according to Quinn et al (1999) and none of the experimental chickens were seropositive.
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Chickens were randomly assigned to four groups each containing 5 chickens. The first 3 groups G1, G2, and G3 were administered subcutaneously (SC) with 108 CFU of SG irradiated with 2400, 2500, 2600 Gy in 0.5 ml of PBS, respec-tively. The fourth group served as a negative control and administered with 0.5ml of PBS SC. Following vaccination, chickens were monitored daily for any unwanted reactions such as anorexia, depression, diarrhea, and death if any for 21 days. The liver and spleen of dead chickens were examined for the pres-ence of SG by plating on general and selective media. For the detection of SG antibody using the SAT test, blood samples were collected from wing vein at day 7 and day 14 post vaccination (Quinn et al., 1999). Safety was measured by injection site reactions such as pain and swelling, systemic reactions like fever and anorexia, and lesion in the liver and spleen.
At the end of the experiment, all chickens were sacrificed and a postmortem examination was conducted to assess gross pathology in each group. Chickens were euthanized by cervical dislocation. It was carried out by grasping the leg of the chicken and stretching the neck and pulling on the head through apply-ing a ventrodorsal rotational force to the skull (AVMA, 2013). Gross lesions on the liver and spleen were given scores of “0” when there is no lesion, “1” for mild few necrotic foci in the liver “2” for enlarged liver, but gizzard not covered, or “3” when there is a severe lesion that covered the gizzard as described previ-ously (Matsuda et al., 2011; Adamu et al., 2017). Samples of liver and spleen were ground and cultured for isolation of Salmonella strains from chickens sacrificed for postmortem examination at the end of the experiment.
Vaccination and challenge infection
Once the best immunization irradiation dose was established, i.e. 2400 Gy, forty, 5 weeks old chickens confirmed negative for the antibody of SG using SAT were randomly assigned into 4 groups, containing 10 chickens per group. Group 1 and Group 2 received 108 CFU of SG irradiated at 2400 Gy in 0.5 ml of PBS orally and subcutaneously, respectively. Group 3 received 0.2 ml of commercial SG 9R strain vaccine containing 107 CFU and Group 4 received 0.5 ml of PBS orally. Twenty-one days post-immunization; all chickens were chal-lenged with 108 CFU of field strain SG suspended in 1 ml of PBS orally.
Chickens were observed daily for any clinical signs such as depression, loss of appetite, and diarrhea, and the number of sick and healthy chickens per each
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group was recorded daily. Blood samples were collected from each chicken af-ter immunization, at day 7, and 14 post-vaccination to determine the presence of specific antibody using SAT (OIE, 2018). Agglutination reaction was given scores of “0” for no agglutination, “1” for weak agglutination, “2” for moderate agglutination; and “3” for strong agglutination. Inventory of healthy, sick and dead chickens was recorded every day. Postmortem examination was conduct-ed in all dead chickens during the challenge period and for all survivors, at the end of the study on day 21 post-challenge. The scoring of gross lesions observed was conducted as shown above. Liver and spleen samples were collected asep-tically into a sterile plastic container for isolation of Salmonella from chickens that were sacrificed for postmortem examination at the end of the experiment (day 21). Vaccine protective efficacy was calculated using the formula devel-oped previously (Orenstein et al., 1985). Briefly, EV = [(ARU-ARV)/ARU]*100; where EV= Efficacy of vaccine; ARU=Attack rate (morbidity and/or mortality) of the unvaccinated group; and ARV= Attack rate (morbidity and/or mortality) of the vaccinated group.
Ethics consideration
This study was reviewed and approved by the Institutional Review Board of Aklilu Lemma Institute of Pathobiology, Addis Ababa University (Ref. No.: ALIPB/IRB/008/2015/16) prior to initiation of the experiment. Chickens were handled following the guiding principles for biomedical research involving animals and complies with the ARRIVE guideline of National Centre for Re-placement, Refinement, and Reduction of Animals in Research (Kilkenny et al., 2010). A trained veterinarian performed chicken handling and sample col-lection humanely.
Statistical analysis
Analysis of data was performed using Stata software version 14. Descriptive statistics were used to summarize clinical data (lesion score and mortality). Results were expressed as mean ± standard deviation and Mann-Whitney U-test and Kruskal-Wallis test were employed to examine for the significant dif-ference in lesion scores and the immune responses among groups. The differ-ence in mortality of chickens between the immunized and control groups was analyzed using the Fisher exact test. At all levels, p-value of less than 0.05 was considered as statistically significant.
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ResultsViability of S. Gallinarum exposed to different irradiation doses
An exponential decrease in viability of SG was observed while increasing the dose of gamma irradiation (Figure 1). Salmonella Gallinarum exposed to doses greater than or equal to 2600 Gy of irradiation was completely inactivated and no growth was observed after culturing for 48 hrs. Therefore, the lethal dose (2600 Gy) and two doses close to lethal dose 2400, 2500 Gy were selected to as-sess the safety and immunogenicity of irradiated SG in chicken.
Figure 1: Effect of different irradiation doses on the viability of S. Gallinarum Immunogenicity and safety of irradiated bacteria
The proportion of SG seropositive chickens 7-14 days after immunization with irradiated SG at 2400 Gy and 2500 Gy was 100%. In chickens immunized with irradiated SG at 2600 Gy, the percentage of seropositive chickens was 80% on day 7 and 100% on day 14, while all chickens in G 4 (negative control) were seronegative both on 7thand 14th days. According to the SAT score, chickens im-munized with SG irradiated with 2400 Gy showed a better antibody response compared to the other two higher doses. Overall, it appears that increasing irradiation dose from 2400 Gy to higher doses is associated with a decrease in the level of mean antibody production (Table 1). All vaccinated chickens did
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not show any clinical signs of disease, injection site reactions, and pain. No lesion was observed in the liver and spleen of chickens in all groups. Based on the immunogenicity and safety data, irradiation at 2400 Gy was selected for further investigation.
Table 1: Antibody response of chickens after immunization with Salmonella Gallinarum irradiated at various dosesGroup (n=5)
Irradiation dose (Gy)
Immunization route
Weeks after immunization
Positive after vaccination (%)
Mean agglut ination score ± SD
Group1 2400 SC 1 5(100) 2.8±0.22 5(100) 3.0±0
Group2 2500 SC 1 5(100) 2.4±0.42 5(100) 2.8±0.2
Group3 2600 SC 1 4(80) 1.8±0.62 5(100) 2.4±0.4
Group4 PBS SC 1 0(0) 02 0(0) 0
SC=Subcutaneous; PBS=Phosphate buffered saline
Immunogenicity of 2400 Gy irradiated SG and SG 9R commercial vaccine
The proportion of seropositive chickens after vaccination was 100% in the ir-radiated SG immunized orally and subcutaneously on day 7 and day 14 post-vaccination. On the other hand, the rate of seropositivity in those chickens vaccinated with SG 9R was 80% on day 7 and 90% on day 14 post-vaccination. The highest mean SAT score was recorded in chickens immunized subcutane-ously at day 14 post-vaccination and the second mean score was also recorded in this group at day 7 of vaccination. The mean SAT score for chickens im-munized with irradiated SG subcutaneously and orally was higher than those immunized with commercial SG 9R vaccine. None of the chickens in the unim-munized group were positive for the SG antibody on day 7 and day 14 post-immunization (Table 2).
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Table 2: Antibody response of chickens immunized with S.Gallinarum irradi-ated at 2400 Gy
Group Type of vaccine
Route of admini stration
Week Post-vaccination
No. positive (%)
Mean aggl utination + SD
G1 Irradiated SG Oral 1 10(100) 2.0+0.26
2 10(100) 2.3+0.21 G2 Irradiated SG SC 1 10(100) 2.7+0.15
2 10(100) 3.0+0.0G3 9R strain SG SC 1 8(80) 1.4+0.31
2 9(90) 1.8+0.33
G4 PBS Oral 1 0
SC= subcutaneous, PBS= Phosphate-buffered saline
Analysis of the level of antibody production using the Kruskal-Wallis test be-tween the three vaccine groups revealed that the mean agglutination value of the three groups of vaccinated chickens was significantly different at day 7 (p=0.007) and day 14 (p= 0.005) post-vaccination. Mann-Whitney U-test analy-sis showed that there was no significant difference between the mean agglu-tination value of groups of chickens vaccinated orally with the irradiated vac-cine (G1) and those vaccinated with commercial SG 9R (G3) vaccine at day 7 and 14 post-vaccination. On the other hand, there was a significant difference between those immunized with irradiated vaccine orally (G1) and subcutane-ously (G2) on day 7 (p=0.042) and day 14 (p=0.005). Similarly, there was a sig-nificantly higher mean serum agglutination value of chickens in G2 compared to those chickens in G3 at both day 7 (p=0.003) and day 14 (p=0.002) (Table 3).
Table 3: Comparative serum antibody response among chickens immunized with irradiated S. Gallinarum and SG9R vaccine using Kruskal-Wallis and Mann-Whitney U-tests Among group Type of test p-value
First week Second weekG1 Vs G2 Vs G3 Kruskal-Wallis test 0.007 0.005
G1 Vs G2 Mann-Whitney U-test 0.042 0.005
G1Vs G3 Mann-Whitney U-test 0.176 0.261G2 Vs G3 Mann-Whitney U-test 0.003 0.002
G1= Immunized with irradiated SG orally, G2= Immunized with irradiated SG subcutaneously, G3= Immu-nized with SG9R vaccine.
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Morbidity and mortality of chickenspost-challenge infection
The number of apparently healthy, sick, and dead chickens during 21 days fol-lowing challenge infection for vaccinated and unvaccinated control groups is presented in Figure 2. Typical signs of morbidity like depression, loss of appe-tite, and yellowish diarrhea were seen as of day 3 post-challenge in the unvac-cinated group (Group 4), and on day 5 in chickens vaccinated with irradiated SG orally (Group 1). The course of disease establishment in the unvaccinated group was acute and all the chickens died within 7 days of the challenge with gross lesions on liver and spleen at postmortem examination. In chickens vac-cinated with irradiated SG orally (Group 1), 5 (50%) of them got sick and died in the first 6 days of infection and on day 10, 9 (90%) died from the group with gross pathology in internal organs. Morbidity and mortality rates in groups of chickens vaccinated with irradiated SG subcutaneously (Group 2) and SG 9R commercial vaccine (Group 3) were similar. In both groups, 10 to 20% of chickens were found sick on days 5, 6, and 7 post-challenge, and a total of 5 (50%) of chickens died on day 9 and day 10 from Group 2 and Group 3, respec-tively. All the remaining 50% of chickens from both groups were apparently healthy until the end of the experiment. There was no statistically significant difference in the mortality rate of chickens vaccinated with irradiated vaccine orally and unvaccinated group (p > 0.05). However, more chickens survived in those vaccinated with the irradiated vaccine administered SC and SG 9R vac-cine compared to the unvaccinated group (p <0.05). Both of them induced 50% protection compared to mortality recorded in the unvaccinated control group (Table 4).
Figure 2: Inventory of apparently healthy, sick and dead chickens immunized with irradiated SG at 2400 Gy and SG
9R followed by homologous challenge infection compared to the unvaccinated control group (G1=vaccinated with
irradiated SG orally, G2 = vaccinated with irradiated SG subcutaneously, G3 vaccinated with SG 9R vaccine; G4
unvaccinated control group). All chickens were challenged with 108 CFU of SG 21 days post-vaccination orally and
were followed for the next 21 days.
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Salmonella Gallinarum was isolated from liver and spleen of 6 (66.7%) of 9 chickens that died from G1, 3(60%) of 5 chickens from G2, 4 (80%) of 5 chickens from G3 and 9 (90%) of 10 chickens from G4. On the other hand, SG was not iso-lated from the intestinal contents of dead chickens from all groups. Salmonella Gallinarum was not isolated from the liver, spleen, and intestinal contents of all vaccinated chickens that survived the challenge infection upon postmortem examination at the end of the experiment on day 21. The mean lesion score of all chickens in the immunized group was significantly lower compared to those in the unimmunized group (Table 4).
Table 4: Protection efficacy of S. Gallinarum irradiated at 2400 Gy, lesion score and recovery of bacteria after oral homologous challenge with 108CFU of S. GallinarumGroup (n=10)
No. dead (%)
P-valueb Efficacy (%)
Bacteria recovery from liver and spleen (%) a
Mean lesion score ±SD
p-valuec
G1 9(90) 1 10 6(66.7) 1.6±0.31 0.002G2 5(50) 0.033 50 3(60) 1.1±0.38 0.001G3 5(50) 0.033 50 4(80) 1±0.33 0.000G4 10(100) - 9(90) 2.9±0.1
G1 = vaccinated with irradiated S. Gallinarum orally, G2 = vaccinated with irradiated S. Gallinarum subcuta-neously, G3 = irradiated with SG9R vaccine; G4 = unvaccinated but challenged with 108 CFU of S. Gallinarum, bFischer’s exact test; cMann-Whitney U-test, aonly for those chickens that died during the experiment
DiscussionCommercial poultry farming is one of the fastest-growing sectors in most devel-oping countries. However, it has several challenges including poultry diseases of which fowl typhoid is known to cause heavy economic loss through mortal-ity and reduced productivity (Shivaprasad, 2000). Vaccination is the best way of protecting poultry from this disease. Adequate balance between safety and immunogenicity is crucial for designing effective vaccines (OIE, 2018). Howev-er, safety is particularly a major concern when using live attenuated vaccines due to their residual virulence and long persistence in the internal organs of immunized hosts, which may cause adverse effects in immunized hosts and contaminate the environment via shedding of live attenuated strains through feces (Silva et al., 1981; Kwon and Cho, 2011).
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The least lethal dose of 108 CFU/ml of SG (suspended in 5ml of PBS) recorded in the current study was 2600 Gy of gamma irradiation. In a previous study, it was shown that a dose of 3000 Gy was sufficient to inactivate Salmonella serotypes and Vibrio parahaemolyticus (Jakabi et al., 2003). The minimum effective dose of irradiation required for SG inactivation varies depending on the volume of liquid in which bacteria are suspended and the concentration of bacteria. Therefore, optimization of irradiation dose is required under different conditions.
Although the SG 9R vaccine was also administered subcutaneously, the inten-sity and proportion of antibody production were less than the one produced by irradiated vaccine administered subcutaneously. The percentage of chickens detected seropositive after vaccination reached 100% in those vaccinated with irradiated SG while in chickens vaccinated with SG 9R, it reached only 90% at day 14 post-vaccination. This could be due to the high immunogenicity of irra-diated vaccine than the live rough avirulent strain of SG. The avirulent strain may lack some of the antigens present in the virulent strain of SG and could still be retained in irradiated vaccines. Kwon and Cho (2011) previously dem-onstrated that SG 9R has rough lipopolysaccharide (LPS) with defective O-side chain repeats. Further investigation is required in this area. In the present study, antibody production by chickens inoculated with irradiated SG at 2400 Gy subcutaneously was higher compared to those inoculated with the same doses orally. This could be due to the fact that orally administered vaccines need to pass through different barriers of the gastrointestinal tract, which delay absorption as compared to subcutaneous administration and enzymatic degradation in the gastrointestinal tract (Davitt and Lavelle, 2015). Previous work also demonstrated less protection of the SG 9R strain vaccine through oral than subcutaneous vaccination (Silva et al., 1981).
The observed seroconversion and protection against challenge infection in chickens vaccinated with irradiated SG is in agreement with the previous re-port where irradiated Listeria monocytogenes efficiently activated dendritic cells via Toll-like receptors and was capable of inducing protective T-cell re-sponses in mice(Datta et al., 2006). A previous study showed that gamma-irradiated toxid produced from Salmonella Typhimurium to be effective in protecting poultry against challenge with S. Typhimurium and S. Gallinarum (Begum et al., 2011).Another study also showed that irradiated Mannheimia hemolytica protected rabbits from challenge infection (Ahmed et al., 2016).
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Similarly irradiated Brucella abortus was shown to induce better protective efficacy compared to strain 19 B. abortus vaccine in mice (Mahmoud et al., 2016). Despite eliciting different immune responses compared to heat-killed or chemically killed vaccines, the efforts to gamma irradiation technology for production of vaccine is increasing because of its ability to penetrate different cells and can damage both double and single-stranded DNA without affecting surface antigens (Seo, 2015).
Irradiated SG vaccine administered subcutaneously showed comparable pro-tection with the SG 9R vaccine against homologous challenge infection. Despite the high rate of antibody production demonstrated by strong slide agglutina-tion reaction, only 50% protection from the mortality induced by homologous challenge infection was recorded in chickens vaccinated with irradiated SG subcutaneously. In the current study, the SG 9R vaccine also showed only 50% protection against challenge infection in Bovans brown breed of chickens. This is closely in line with the report of Silva et al (1981) where SG 9R vaccine gave 64.3% protection in the immunized group against SG wild type challenge infec-tion at the dose of 108CFU/ml in meat-breed of chickens. However, the current finding is contrary to the report of Adamu et al (2017) where SG 9R vaccine showed 100% protection in the immunized group and 53.4% mortality rate was recorded in the unvaccinated control group in White leghorn breed of chickens unlike that of 100% mortality in the unvaccinated challenged group in the current study. This disparity may be due to differences in the level of suscepti-bility of breed of chickens used in the two experiments. It has been previously shown that level of protection of SG 9R varies with types of breed of chickens involved (Kwon and Cho, 2011). Besides, the difference in the dose of challenge bacteria used could have also contributed to the observed variation.
ConclusionSG Irradiated at 2400 Gy administered subcutaneously induced good antibody response and provided comparable protection with SG 9R strain commercial vaccine against homologous challenge infection indicating its potential of be-ing an effective vaccine against fowl typhoid. A further detailed study involv-ing optimization of irradiation dose, quantification, and characterization of im-munological responses, assessment of the effect of booster vaccination on the protective efficacy using a large sample size is recommended.
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Conflict of interestThe authors declare that they have no conflict of interest.
AcknowledgmentsThis study was supported by International Atomic Energy Agency under the project CRP-D32033; Irradiation of Transboundary Animal Diseases (TAD) Pathogens as Vaccine and Immune Inducers. The authors are grateful for Na-tional Tsetse and Trypanosomiasis Control and Eradication Institute for al-lowing us to use the irradiation facility. The technical assistance of Mr. Moges Hiddoto from the National Institute for Control and Eradication of Tsetse and Trypanosome and Ms. Azeb Teklu from Aklilu Lemma Institute of Pathobiol-ogy is highly appreciated. We also thank Drs. Hermann Unger, Viscam Wijew-ardana, and Richard Kangethe from IAEA for giving constructive suggestions during the study period.
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Oguejiofor et al., Ethiop. Vet. J., 2020, 24 (2), 139-154 DOI https://dx.doi.org/10.4314/evj.v24i2.9 Ethiopian Veterinary Journal
Unilateral testicular degeneration in dogs: Effects on spermatozoal characteristics, testis and cauda epi-didymis
Chike Fidelis Oguejiofor*, Kenneth Orji Anya and Nnaemeka Kingsley Ogbanya
Department of Veterinary Obstetrics and Reproductive Diseases, Faculty of Veterinary Medicine, University of Nigeria, Nsukka 410001, Nigeria
*Corresponding author: Chike Fidelis Oguejiofor, Email: [email protected]
AbstractTesticular degeneration is an important cause of poor fertility in dogs, but there is little knowledge on its effects on spermatozoa in affected dogs. The study in-vestigated the specific effects of unilateral testicular degeneration (UTD) on spermatozoal characteristics in the testis and epididymis. Ten sexually mature Nigerian indigenous breed of dogs, comprising 5 normal dogs and 5 dogs with UTD were used for the study. The testis and epididymis were removed via or-chidectomy for morphological and histopathological evaluation. Sperm in the testis and cauda epididymis were analysed. The samples were grouped into four as N1 (normal right testis), N2 (normal left testis), ND (non-degenerated testis in UTD dogs), and D (degenerated testis in UTD dogs). Data were ana-lysed using one-way ANOVA. There were significant decreases (p<0.001) in testes weight, length, width and volume, and the gonado-somatic index in the D testes compared to the contralateral ND and the N groups. The D group also had significantly lowered epididymal sperm total and progressive motility, vi-ability and concentration (p<0.001), and a decreased testicular sperm concen-tration (p<0.01). Moreover, there was a decreased percentage (p<0.001) of mor-phologically normal sperm, with increased prevalence of sperm abnormalities in the D group compared to the other groups. In comparison with the N groups, the ND group had a significantly lowered (p<0.05) epididymal sperm progres-sive motility, with increased percentage (p<0.01) of sperm with proximal cyto-plasmic droplets and looped tails. The findings demonstrated that UTD in dogs adversely affected spermatozoa in the testis and cauda epididymis. There was also evidence of compromised spermatozoa in the epididymis contralateral to the degenerated testis.
Keywords: Canine; Epididymis; Spermatozoa; Testicular atrophy; Testicular degeneration
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Oguejiofor et al.,
Introduction Dogs are mainly bred for companionship, showmanship, security, hunting, and to provide valuable source of income for breeders. In addition, the slaughter and consumption of dog meat is popular in some countries (Podberscek, 2009; Ehimiyein et al., 2014). Therefore, infertility is likely to cause significant eco-nomic losses in canine breeding.
The testis is the primary organ of reproduction in male animals and func-tions in spermatogenesis (sperm production) and steroidogenesis (testoster-one production). Testicular degeneration (TD) is considered as one of the most common causes of poor semen quality and acquired infertility in male dogs (Fontbonne, 2011; Domingos and Salomão, 2011) with a prevalence of 15-58% (Ortega-Pacheco et al., 2006; Câmara et al., 2014). Testicular degeneration in-volves deterioration in the structure of the testis with a consequent loss of testicular function (Turner, 2007). It may affect one testis (unilateral) or both testes (bilateral), involving parts of the testis or the whole testis. The seminif-erous epithelium of the testis is highly susceptible to damage, with a wide vari-ety of agents causing reversible or irreversible degeneration (Parkinson, 2001). Acute TD can result secondary to a known insult such as exposure of the testes to toxins, radiation, extreme scrotal temperature, scrotal trauma, autoimmune disease, certain nutritional deficiencies, and infection with pathogenic organ-isms (Turner, 2007; Obi et al., 2013). Idiopathic TD has no identifiable underly-ing cause and has also been reported in dogs (Rehm, 2000; Fontbonne, 2011).
Spermatozoa can be recovered from the cauda epididymis of dogs by percuta-neous epididymal aspiration, following castration, or at post mortem; for use in the investigation of sperm quality and spermatozoal characteristics (Varesi et al, 2013; Chima et al., 2017; Bhanmeechao et al., 2018), and for artificial breeding of bitches (Wydooghe et al., 2016). Ejaculated semen comprises sperm from both testes as well as secretions of the accessory sex glands. Thus, while evaluation of the ejaculate is used to assess semen for fertility potential, it precludes the investigation of specific testis/epididymis-derived effects of tes-ticular disorders, and their impact on spermatozoa.
A few studies reported adverse effects of various testicular disorders, including TD on testicular and sperm morphology (Ortega-Pacheco et al., 2006; Câmara et al., 2014). In addition to sperm morphology, several other sperm character-
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istics and parameters (e.g. motility, viability, concentration) are known to have major influence on the integrity, functionality and fertility potential of sper-matozoa (Johnston et al., 2001; Robert et al., 2016; Kolster, 2018). However, there is little knowledge on the effect of TD on these sperm characteristics in the testis or epididymis of affected dogs. Therefore, the aim of the study was to investigate the effects of unilateral testicular degeneration (UTD) on sperma-tozoal characteristics in canine testis and epididymis.
Materials and methodsAnimals
Ten sexually mature (9-12 months old) apparently healthy Nigerian indige-nous breed of dogs comprising 5 normal dogs: N (both testes normal and pres-ent in the scrotum) and 5 dogs with UTD: one non-degenerated (ND) and one degenerated (D) scrotal testes, were used for the study. Only dogs with no other observed reproductive pathology during clinical examination, but with a presentation and history of progressive unilateral scrotal testicular atrophy were selected. Animal welfare was observed in accordance with the National Institute of Health’s Guide for the Care and Use of Laboratory Animals (Na-tional Research Council, 2011), and the protocol was approved by the Research Ethics Committee of the Faculty of Veterinary Medicine (2016/10-173537). The animals had body weights ranging from 7.8-9.7 kg. Following an elective re-quest for castration, orchidectomy (open castration) was performed as previ-ously described (Hassan and Hassan, 2003). The surgery was performed follow-ing premedication with atropine sulphate (Pauco Atropine®, Jiangsu Huayang Pharmaceutical, Jiangsu, China) at 0.04 mg/kg b.w., IM and xylazine hydro-chloride (AnaSed®, Akorn, Lake Forest, IL, USA) at 0.5 mg/kg b.w., IM., and under general anaesthesia using ketamine hydrochloride (Ketmin®, Laborate, Panipat, India) at 5 mg/kg b.w., IM. Post-surgical pain was alleviated with tramadol hydrochloride (Tradyl®, PT Interbat, East Java, Indonesia) at 2.5 mg/kg b.w., IM twice daily for four days. The recovered testis and epididymis were dissected, and then utilized for gross morphometry, histopathology and sperm analysis. Each testis/epididymis was considered as the experimental unit. Thus, the samples were grouped into four as N1 (normal right testis), N2 (normal left testis), ND (non-degenerated testis in UTD dogs), and D (degener-ated testis in UTD dogs).
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Morphological and histopathological evaluation
The weights of testis and epididymis were measured using an electronic weighing scale (Ohaus, Pine Brook, NJ, USA). Testis length and width, and epididymal length were measured with a vernier calliper. Testes volume was measured by the water displacement method (Sakamoto et al., 2007). The go-nado-somatic index (GSI, g/kg) was calculated by dividing the testis weight (g) by the body weight (kg). Likewise, the epididymo-somatic index (ESI, g/kg) was calculated by dividing the epididymis weight (g) by the body weight (kg) (Omari et al., 2018).
Small portions of cauda epididymal and testicular tissues were immersed in Bouin’s fixative solution for 24 h and then transferred to 70% ethanol until processing. Histopathological sections were prepared and stained with haema-toxylin and eosin as previously described (Slaoui and Fiette, 2011). Sections were evaluated for histopathology under light microscopy (Figure 1A-D). Sam-ples were designated as normal (N) in the absence of testicular or epididymal histologic abnormalities. Testicular degeneration was confirmed based on the presence of characteristic histopathological features as previously described (Yuan and McEntee, 1987; McGavin and Zachary, 2007; Câmara et al., 2014). Samples with absence of spermatozoa (azoospermia) in the epididymides indi-cated severe testicular degeneration and were excluded from the study, as the aim was to investigate spermatozoal characteristics in the different groups.
Epididymal and testicular sperm analyses
Sperm evaluation was performed as previously described (Seed et al., 1996; World Health Organization, 2010). Sperm motility was determined using sperm diffusion in phosphate buffered saline (PBS; pH 7.4, 37 °C) from sec-tioned cauda epididymis. Sperm motility (%) was determined at ×400 using a phase-contrast microscope (Motic B3; Motic, Carlsbad, CA, USA) equipped with a stage slide warmer (TCS-100; Amscope, Ivrine, CA, USA) set at 37 °C. Sperm viability (%) was evaluated using eosin-nigrosin vital staining, and sperm were categorized as live (unstained head) or dead (marked pink-stained head) under light microscopy at ×1000 magnification. Sperm morphological abnormalities were evaluated using phase-contrast microscopy and eosin-nigrosin staining. All values in percentage were determined by examining 200 sperm cells across different fields in duplicates. Cauda epididymal and testicular sperm concen-
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trations were determined following cauda epididymal/testicular tissue homog-enization in PBS, and counting of sperm cells using a haemocytometer (Weber, England). Total sperm count was expressed as the number of sperm per gram cauda epididymis, and per gram testis, respectively (Seed et al., 1996).
Statistical analysis
Data were analysed with the one-way analysis of variance (ANOVA) tool us-ing GraphPad Prism version 6.01 (GraphPad Software, Inc.), and the results presented as Mean ± standard deviation (SD). Significant differences between means were confirmed using Tukey’s honestly significant difference post hoc test. Results were considered statistically significant when p<0.05.
ResultsMorphological evaluation of testis and epididymis
The results of gross morphological evaluation of the testis and epididymis in normal dogs and UTD dogs were as shown in Table 1. UTD affected the right testis in two dogs and the left testis in three dogs. There were significant de-creases (p<0.001) in testes weight, length, width and volume, and the GSI in the D testes compared to the contralateral ND testes in UTD dogs, and also compared to the N testes in the normal dogs. However, these parameters did not differ (p>0.05) between the ND and the N testes. In addition, there were no significant differences in epididymal weight and length, and the ESI in all the four groups.
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Table 1. Testis and epididymis morphological characteristics in dogs with normal testes and unilateral testicular degeneration (UTD).
Normal UTDParameter N1 N2 ND DTestis weight (g) 7.74 ± 0.34a 7.80 ± 0.28a 7.92 ± 0.31a 5.12 ± 0.64b
Testis length (cm) 2.78 ± 0.04a 2.76 ± 0.04a 2.83 ± 0.09a 2.01 ± 0.15b
Testis width (cm) 2.26 ± 0.04a 2.27 ± 0.03a 2.29 ± 0.05a 1.57 ± 0.10b
Testis volume (cm3) 7.13 ± 0.18a 7.14 ± 0.19a 7.21 ± 0.21a 4.47 ± 0.53b
Epididymis weight (g) 1.25 ± 0.11 1.23 ± 0.08 1.25 ± 0.12 1.19 ± 0.09Epididymis length (cm) 4.81 ± 0.05 4.83 ± 0.04 4.81 ± 0.06 4.72 ± 0.09Gonado-somatic index (g/kg) 0.89 ± 0.01a 0.90 ± 0.02a 0.88 ± 0.05a 0.56 ± 0.02b
Epididymo -somatic index (g/kg) 0.14 ± 0.01 0.14 ± 0.00 0.14 ± 0.00 0.13 ± 0.01
N1 (normal right testis); N2 (normal left testis); ND (non-degenerated testis in UTD dogs); D (degenerated testis in UTD dogs). Values represent mean ± SD, n = 5. Rows with different superscript letters indicate sig-nificant differences between groups (p<0.05).
Histopathology of the testis and cauda epididymis
Evidences of TD were observed (Figure 1B) and these included the presence of decreased seminiferous tubular diameter, reduced thickness of the seminif-erous epithelium, and scanty presence of spermatogenic cells. The cauda epi-didymis of the degenerated testis (Figure 1D) showed evidence of regression of epididymal tubules with reduced tubular diameter, scanty or no presence of spermatozoa in the tubular lumen and increased inter-tubular connective tissue.
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Figure 1. Histomicrographs of the testis and cauda epididymis in dogs with normal testes and unilateral testicular degeneration (UTD). A: Normal testis showing large diameter of seminiferous tubules (ST), thick multi-layered and active seminif-erous epithelium (SE) comprising numerous cells of the spermatogenic lineage. B: testis showing evidence of degeneration including decreased seminiferous tubular diameter, reduced thickness of the SE, and scanty pres-ence of spermatogenic cells. C: Normal cauda epididymis showing large diameter of epididymal tubules (ET), presence of abundant spermatozoa (SP) in the tubular lumen (TL), scanty inter-tubular connective tissue (CT). D: cauda epididymis of degenerated testis showing regression of ET with reduced tubular diameter, scanty or no presence of SP in the TL and increased inter-tubular CT. (H & E stain; scale bar: 50 μm, 100 μm).
Cauda epididymal and testicular sperm analysis
The results of the cauda epididymal and testicular sperm analysis in normal dogs and UTD dogs are shown in Table 2 and Figure 2A-F. There were no significant differences (p>0.05) in all the sperm parameters between the two N (N1 and N2) groups in the normal dogs. In comparison with the N groups, the ND group in dogs with UTD did not differ with respect to epididymal sperm total motility, viability and concentration, and testicular sperm concentration. However, they had a significantly lowered (p<0.05) epididymal sperm progres-sive motility. The percentage of sperm with normal morphology and the preva-lence of detached head, abnormal head, coiled midpiece (Dag defect) and coiled tail did not differ between the ND and N groups. However, there was increased
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percentage (p<0.01) of sperm with proximal cytoplasmic droplets (PCD) and looped tails in ND compared to the N groups.
Table 2. Cauda epididymis and testis spermatozoal characteristics in dogs with normal testes and unilateral testicular degeneration (UTD).Testis/Epididymis Normal UTD
N1 N2 ND DEpididymal sperm parameter (%)Total motility 83.6 ± 2.7a 84.8 ± 3.0a 81.3 ± 5.7a 11.5 ± 9.1b
Progressive motility 74.5 ± 4.7a 72.9 ± 3.8a 59.6 ± 7.9b 4.4 ± 8.3c
Viability 94.6 ± 2.3a 94.2 ± 2.8a 87.6 ± 4.8a 27.2 ± 8.3b
Epididymal sperm morphology (%)Normal sperm 61.0 ± 4.6a 63.4 ± 3.9a 55.2 ± 7.2a 23.7 ± 11.3b
Detached head 4.1 ± 1.5a 3.7 ± 1.1a 4.8 ± 2.8a 13.7 ± 4.2b
Abnormal head 1.2 ± 0.8a 0.9 ± 0.8a 1.4 ± 0.9a 5.2 ± 1.8b
Bent midpiece 2.9 ± 1.3 3.2 ± 0.9 3.0 ± 1.7 6.5 ± 4.1Coiled midpiece/Dag defect 0.8 ± 0.8a 0.6 ± 0.9a 0.8 ± 1.1a 4.8 ± 2.5b
Proximal cytoplasmic droplets 0.2 ± 0.4a 0.4 ± 0.5a 2.8 ± 1.1b 0.6 ± 0.5a
Distal cytoplasmic droplets 21.4 ± 9.9 19.6 ± 10.8 22.6 ± 11.6 27.4 ± 16.5Looped tail 6.6 ± 1.5a 7.0 ± 1.2a 10.8 ± 2.5b 13.1 ± 3.9b
Bent tail 4.5 ± 1.8 4.1 ± 1.6 4.4 ± 2.1 4.6 ± 2.5Coiled tail 3.4 ± 1.1a 4.0 ± 1.0a 4.5 ± 1.5a 8.6 ± 2.6b
Sperm concentration Epididymis (x106 sperm/g) 926.0 ± 23.9a 912.4 ± 19.1a 874.0 ± 78.7a 3.29 ± 2.1b
Testis (x106 sperm/g) 31.6 ± 5.7a 29.3 ± 4.8a 23.2 ± 9.3a 5.81 ± 2.9b
N1 (normal right testis); N2 (normal left testis); ND (non-degenerated testis in UTD dogs); D (degenerated testis in UTD dogs). Values represent mean ± SD, n = 5. Rows with different superscript letters indicate sig-nificant differences between groups (P<0.05).
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Figure 2. Micrographs of cauda epididymal sperm characteristics in dogs with normal testes and unilateral testicular degeneration (UTD). A and B: Evaluation of sperm morphology using phase-contrast microscopy. Note the normal sperm (NS) and the presence of coiled midpiece (CM) or Dag defect and looped tail (LT) abnormalities. C-F: Evaluation of sperm viability and morphology using eosin-nigrosin staining. Note the live sperm (LS) and dead sperm (DS), and sperm with bent tail (BT), bent midpiece (BM), LT, pear-shaped abnormal head (AH), detached head (DH), looped tail enclosing a distal cytoplasmic droplet (LT-DCD), and distal cytoplasmic droplet (DCD) abnormali-ties. Scale bar (10 µm).
On the other hand, there were significant differences in most of the observed sperm parameters in the D group in dogs with UTD, compared to the contralat-eral ND and the N groups. The D group had a significantly decreased (p<0.001)
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epididymal sperm total and progressive motility, viability and concentration, compared to the other groups. Testicular sperm concentration was also sig-nificantly decreased in the D group compared to the contralateral ND (p<0.01) and the N groups (p<0.001). There was decreased percentage (p<0.001) of nor-mal sperm, and increased percentage of sperm with detached head (p<0.001), abnormal head (p<0.05), and coiled midpiece and coiled tail (p<0.01) in the D group compared to the other groups. The percentage of sperm with looped tail was also increased in the D group (p<0.01), but did not differ from the contra-lateral ND group. Out of all the observed sperm abnormalities, the percentage of sperm with bent midpiece, bent tail and distal cytoplasmic droplets (DCD) did not differ across all the groups.
DiscussionThe study involved only dogs with a history of progressive unilateral testicular atrophy, and with histological evidence of TD and the presence of spermato-zoa in the cauda epididymis. This differentiated the dogs from animals with testicular hypoplasia. Therefore, the selected animals were considered to be in a state of progressed TD but not in the advanced or terminal stage charac-terized by total loss of seminiferous epithelia and the absence of spermatozoa (azoospermia). We applied the term ‘non-degenerated’ rather than ‘normal’ in the contralateral ND group for two reasons. First, there were no specific infor-mation on the aetiologies of the TD, and to what extent they may have affected the contralateral testes. Second, we observed some sperm morphological ab-normalities in the contralateral ND group compared to the normal dogs.
The results showed evidence of adverse effects of UTD in canine testes and sper-matozoa. All the testicular gross morphological parameters (weight, length, width, volume and GSI) were markedly reduced following TD. This is consis-tent with other reports of a decline in testis size and weight in severe cases of TD or testicular atrophy in male animals (Parkinson, 2001; Ortega-Pacheco et al., 2006; Teankum et al., 2013). In contrast, epididymal weight and length and the ESI were not significantly altered by TD. This observation is also in agree-ment with previous reports of unaltered epididymal size or weight, concurrent with testicular atrophy in cases of TD or testicular atrophy in the stallion and boar (Blanchard and Varner, 1993; Teankum et al., 2013). Ortega-Pacheco et al. (2006) reported a decreased epididymal weight only in dogs with advanced TD but not partial TD, compared to the contralateral normal testes.
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The percentage motility and viability of cauda epididymal spermatozoa ob-served in this study for the N groups were comparable to the values reported previously in normal dogs (Chima et al., 2017; Bhanmeechao et al., 2018). Tes-ticular degeneration decreased the epididymal sperm total and progressive motility in the D group. A low percentage of progressively motile sperm was also reported in the semen of stallions with TD (Blanchard et al., 2000). De-creased sperm motility may be related to abnormal sperm formation, and the observed increase in the proportion of sperm with morphologic abnormalities. Testicular degeneration also caused an increase in the percentage of dead epi-didymal sperm (decreased sperm viability) in the D group. This may be related to a disruption of the normal processes of spermatogenesis and sperm epididy-mal maturation, as a consequence of testicular insult and degeneration. Sperm motility is a critical indicator of normal structural and functional competence of spermatozoa, and there is positive correlation between the proportion of progressively motile sperm and sperm with normal morphology (Robert et al., 2016). Lowered fertility has been associated with <70% total and progressive sperm motility and with increased proportion of dead sperm in the semen of dogs (Johnston et al., 2001; Oguejiofor, 2018).
The mean proportion of morphologically normal cauda epididymal sperm in the N groups in the study (62%) was lower than the previous reports of 80% in epididymal sperm (Ortega-Pacheco et al., 2006) and 81% in vas deferens sperm (Câmara et al., 2014). This difference was attributed to the exclusion of epididymal sperm with DCD from the proportion of morphologically nor-mal sperm in the study. However, Ortega-Pacheco et al. (2006) considered the presence of DCD as normal for sperm harvested from the cauda epididymis; a site for sperm maturation and storage. Although the proportion of sperm with DCD did not vary across the different groups, we observed a lower mean prevalence (21%) in the N groups, in contrast to the 49% reported by that study (Ortega-Pacheco et al., 2006). A high prevalence (76%) of total morpho-logically abnormal sperm was observed in the D group. Similarly, more than 80% cauda epididymal sperm abnormality was reported in cases of partial TD with oligospermia (Ortega-Pacheco et al., 2006). TD reduced the proportion of normal sperm in the study from 62% to 24%. A decrease in normal sperm from 81% to 55% was also observed in vas deferens sperm of dogs with severe TD (Câmara et al., 2014). In addition, the findings here are consistent with the previous reports of a high prevalence of sperm with detached head abnormal-ity (Ortega-Pacheco et al., 2006; Câmara et al., 2014), and other abnormalities
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including acrosomal defects, abnormal midpiece, and bent tails in severe TD (Ortega-Pacheco et al., 2006). Remarkably, these sperm defects are considered to have significant negative effect on canine fertility (Kolster, 2018).
Furthermore, TD decreased the testicular and cauda epididymal sperm con-centrations. This may be a consequence of atrophy of the testicular parenchy-ma, degeneration of seminiferous epithelia, and decreased spermatogenesis. Since the cauda epididymis is the site of sperm storage, the lowered epididy-mal sperm concentration reflected a decrease in sperm production in the testis. Similarly, there was lowered daily sperm production per millilitre of testis and decreased sperm concentration in stallions with TD (Blanchard et al., 2001). Oligospermia and azoospermia may result in dogs with TD depending on the severity of the condition (Ortega-Pacheco et al., 2006; Fontbonne, 2011; Câma-ra et al., 2014). Lowered sperm numbers can lead to subfertility or infertility in affected dogs (Robert et al., 2016).
Interestingly, the findings suggest that TD in one testis may impair spermato-zoa in the contralateral epididymis. In comparison with the N groups, the ND group had decreased sperm progressive motility and increased prevalence of PCD and looped tail sperm abnormalities. However, the mechanism for this impairment (whether direct or indirect) was unclear due to the unknown un-derlying cause of UTD in the affected dogs. Most of the sperm parameters were not significantly different between the N testes in normal dogs and the contralateral ND testis in UTD dogs. However, it is likely that the overall fer-tility outcome in dogs with UTD will depend on the progression and severity of TD. Extragonadal (epididymal) sperm reserve have been reported to positively correlate with gonadal sperm reserve and testicular weight (Ajani et al., 2015), and these in addition to testicular volume, were all significantly decreased by TD. Lowered testicular volume has also been associated with oligospermia and subfertility in males (Tijani et al., 2014). Taken together, TD caused decreased testicular and epididymal sperm concentration, decreased epididymal sperm motility, viability and concentration, and increased epididymal sperm abnor-malities, and these defects can adversely affect fertility in dogs (Johnston et al., 2001; Robert et al., 2016; Kolster, 2018; Oguejiofor, 2018).
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ConclusionThe study demonstrated that UTD in dogs decreased testicular sperm concen-tration. It also adversely affected epididymal spermatozoal motility, viability, morphology, and sperm concentration in the testis and epididymis. Moreover, there was evidence of compromised spermatozoa (decreased sperm motility and increased sperm abnormalities) in the epididymis contralateral to the de-generated testis. These findings provide more information on alterations in sperm quality in relation to UTD. Although sperm cells may be recovered from the cauda epididymis of affected dogs for application in assisted reproductive technologies, further studies could investigate the functional capacity and fer-tilizing potential of such recovered spermatozoa.
Conflict of interestThe authors declare that there is no conflict of interest.
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Copyright © Ethiopian Veterinary Association (EVA)Ethiop. Vet. J.,ISSN: 1683-6324
All articles as well as the editorials published in the Ethiopian Veterinary Journal represent the opinion of the author(s) and do not necessarily reflect the official view of the Ethiopian Veterinary Association, the Editorial Board or the institution within which the author(s) is/are affiliated unless this is clearly stated. Furthermore, the author(s) is/are fully responsible for the contents of the manuscript and for any claim or disclaim therein.
No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or any means, electronic, mechanical, photocopying, recording or otherwise, without the prior permission of the Association.
Ethiop. Vet. J., is the Official Scientific Organ of the Ethiopian Veterinary Association
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ETHIOPIAN VETERINARY JOURNAL
(Ethiop. Vet. J.)
Objectives • To stand as the official scientific organ of the Ethiopian Veterinary Associa-tion and serve as medium of communication with professionals in the fields of animal health and production and other related disciplines.
• To effectively disseminate research outputs in all spheres of veterinary sci-ence, veterinary public health, animal production and other related fields of study.
• To help promote agricultural development in Ethiopia and the subregion as a whole through generation via research of technologies, methods etc and dissemination of scientific knowledge to stakeholders and ultimately to end users.
Type of papers
• Original research papers • Review articles • Short communications
Original articles
This includes original articles presenting outputs from basic, applied and adaptive research activities related to animal and public health and diverse aspects of animal production in Ethiopia, and from regional and international sources. The material must not have been previously submitted or published elsewhere. The research article should not contain more than 6000 words.
Review articles
These include papers covering baseline data/information as well as advances in the field of veterinary medicine, public health and related fields of research and development. Often review articles will be prepared by specialist or re-searchers actively engaged in particular area of study for a considerable period of time with proven success. This must be supported by a strong track record
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of publications in the area of the proposed review. Before submitting a review, authors must first contact the Editor with an outline of a proposed review. Ac-quisition of such papers my also be done through invitation. The review article should not contain more than 9000 words
Short communications
This is devoted to entertain results of original research, selected clinical case reports, brief scientific notes including preliminary results, scientific obser-vation, experimental techniques, recent technological advances and news of interest in the field. Short communication must be written in a synthesis form where all necessary information are presented in a condensed manner. The material must not have been previously published elsewhere. Short communi-cation should not contain more than 3000 words
Guidelines for authors
The Ethiopian Veterinary Journal (Ethiop. Vet. J.) is a multi-disciplinary and peer-reviewed publication intended to promote animal health and production of national, regional and international importance. All submitted manuscripts should be in English. SubmissionsElectronic copy in Word file should be addressed to the Editorial-Office. All manuscripts should be accompanied with a letter (or a filled Manuscript Sub-mission and Copyright Transfer Form) signed by all authors, who clearly state that the paper, other than oral presentation or abstract, has not been submit-ted for publication elsewhere in any form. The Editorial Board reserves the right to accept or reject any paper submitted.
ManuscriptsIn general, all submitted manuscripts must conform to the requirements set by the Journal. Failure to follow instruction in the preparation of manuscripts may result in total rejection or delay in publication. It should be prepared with Times New Roman 12 pt, double-spaced, minimum 1.5 cm margins. To facili-
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tate the review process give line numbers in Arabic numerals. Give also page number in the lower middle of each page, starting from the title page.
Title pageThe title page should consist of:
a) Title of the article, which should be concise and descriptive enough; should be in sentence case, and justified left; avoid abbreviations;b) Name(s) of author (s), institutional affiliation, email addresses of all au-thors in italics (s); andc) Name and address of the corresponding author, in case of more than one authors.
Abstract
The second page should provide an abstract of not more than 300 words sum-marizing the background, objective, materials and methods, major findings and their significance, and conclusions. Avoid the use of undefined abbrevia-tions.
KeywordsBelow the abstract, in a separate line, keywords, up to six in number should be given in alphabetical order separated by “;”.
IntroductionGive sufficient information on the associated background zooming in to the subject in question; articulate the specific research question, and the contri-bution of your current research in bridging the gap, followed by statement of objective(s) is in order.
Materials and methodsUnder this section, briefly state the materials used, the selection of study ani-mals, subjects, herds or materials, and the design for observational or exper-imental study and the nature of controls. Briefly describe the methods and procedures followed in sufficient detail and give reference; if new or modified methods are used, give justification for using them. Precisely specify appara-tus, instrument or chemicals used in terms of manufacturer’s name and ad-
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dress in parentheses. Similarly, drugs, biologicals, medicinal plants or others used should be sufficiently described in terms of generic name(s) or scientifi-cally accepted identifications, dose(s), and route(s) of administration. Describe sufficiently the statistical methods used and how the magnitude or precision of analysis results reached.
ResultsQuantify qualitative findings and present them with appropriate indicators of measurement of error or uncertainty, such as by using confidence intervals. Likewise, when reporting quantitative data, authors must indicate the extent of variability by either using standard deviation or standard error. Present the results in logical sequence in the text, tables, or illustrations. The results have to be presented in the same order as the questions raised in the objective(s) and methods sections. Results should be concise and no need of interpretation. In clinical or therapeutic trials, report complications or losses or even dropouts of such observations giving numbers. Support all findings by using appropriate statistical analysis.
Tables should be complete enough to be informative. They should be presented in separate pages, numbered consecutively using Arabic numerals and provid-ed with captions. Avoid using vertical lines to separate columns in Tables. De-tails essential to further explain specific aspects of the Table should be given as a footnote below the table, by using appropriate symbols or lower case letters as superscripts.
Figures and the captions should also be placed in separate sheets, numbered in Arabic numerals according to the sequence of their appearance in the text. Lengthy and complicated Tables and Figures are discouraged. When data are reported, avoid also duplications in presenting them. In other words, the same data should either be presented in a Table or Figure or text but not more than one of these options. All Tables and illustrations must be cited in the text. Pho-tographs of only good quality with TIFF, PDF, JPEG format with minimum dpi of 300 are accepted. Colored illustrations are considered if the costs are covered by the author.
DiscussionUnder this heading you should explain the findings and the associated inter-pretations. Relate the results with the objective of the study and with previ-
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ously published work. Here emphasize the new and important aspects of the study and not repeat the results. In the discussion, clearly indicate the signifi-cance of the work and implications of the findings for future research.
ConclusionThis section should be separately presented with supporting evidences based on the major findings of the study. Appropriate recommendations can be made if necessary
AcknowledgementsAcknowledgments should be briefly stated after the Conclusions. Under this, technical, financial and material support can be mentioned.
Conflict of interestAuthors should declare that they have no conflict of interest.
ReferencesAll citations of publications in the Text, Tables and illustrations must follow the requirements. Show such references by author’s name (without initials) followed by year of publication in Arabic numerals and all that in parentheses. When reference is made to a publication written by more than two authors, then indicate the first author’s name (without initials) followed by “et al.,”. When you want to use name of author out of paranthesis in case of more than two authors use last name of first author et al (year). More than one refer-ences cited together should follow a chronological order and separated by “;” in a parenthesis. Personal communications and unpublished work must not be included in the reference list. They should be mentioned in the text only. Ex-amples: (Dagnachew, 2004, personal communication). (Gopelo, 2004, unpub-lished).
All cited publications must be presented in the list of references. In such a list, the authors’ names are presented in alphabetical order and then chrono-logically by author. All publications by the single author precede those multi-authored publications. Works of the same author should be arranged according to publication dates. Publications by the same author(s) with the same year should be listed, using ‘a’, ‘b’, ‘c’, etc after the year, eg. 2004a, 2004b, etc. If
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the number of authors is more than six, ‘et al.,’ should be used after the sixth author. Titles of references should be given in the original language, except for instances involving non-Latin alphabets, in which case the title should be transliterated and a notation identifying the language, for example ‘in Am-haric’, be added. Abbreviations of the titles of periodicals mentioned in the reference list must be according to the International List of Periodical Title Word abbreviations.Use the following method for presenting references:
A) Article in journals:
Ameni, G., Miorner, H., Roger, F. and Tibbo, M., 2000. Comparison between compara-tive tuberculin and gamma-interferon tests for the diagnosis of bovine tuberculosis in Ethiopia. Trop. Anim. Hlth. Prod., 32, 267-267.
B) Accepted Articles in press with doi.no.
Jobre, Y., Malone, J. B., McCarroll, J. C., Erko, B., Mukaratirwa, S. and Xinyu, Z., 2001. Satellite climatology and the environmental risk of Schistosoma mansoni in Ethiopia and east Africa. Acta Trop., (In press).
C) Papers in proceedings
Tegegne, A., Wirtu, G., Mukasa-Mugerwa, E. and Kassa, T., 1994. Oestrus phenom-enon and oestrus detection efficiency using androgenized cows and entire bulls in Boran and Boran x Friesian crossbred cows. In: Proceedings, Advances in Tropical Agriculture in the 20th Century and Prospectus for the 21st: TA 2000, 4-9 Septem-ber 1994, Port-of- Spain, Trinidad.
D) Books or chapters in books:
Thrusfield, M., 1995. Veterinary Epidemiology, 2nd edition, Blackwell Science Ltd, Ox-ford, UK.
Malone, J. B and Jobre, Y., 1999. Predicting outbreaks of fasciolosis from Ollernshaw to satellites. In: Dalton, J.P. (Ed.), Fasciolosis. CBA International Publications, Cambridge, Pp. 151-183.
E) Organizations as author:
OIE, 1992. Bovine tuberculosis. OIE manual for diagnostic techniques of livestock dis-eases. Office International des Epizooties (OIE), Paris, France. Pp. 287-296.
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NomenclatureAuthors, reviewers as well as editors should follow the rules governing bio-logical nomenclature, as indicated in the International Code of Botanical No-menclature, the International Code of Nomenclature of Bacteria, the Interna-tional Code of Zoological Nomenclature and the Standardized Nomenclature of animal Parasitic Diseases. All biotica (crops, plants, insects, birds, mammals, etc.), with the exception of common domestic animals, must be identified by scientific names when such terms are used first.
All biocides and other organic compounds should be identified by their Generic names when first used in the text; likewise, the active ingredients of all for-mulations should be identified. For chemical nomenclature, the conventions of the International Union of Pure and Applied Chemistry and the official recom-mendations of the IUPAC-IUB Combined Commission on Biochemical Nomen-clature should be observed.
EthicsWhen reporting experiments involving animals, authors are expected to have observed all ethical standards on the care and use of animals or any pertinent national law. The Editorial Board reserves the right to reject papers that have been judged to have subjected animals to unnecessary handling or exposure to unacceptable pain or detention. Experimental studies should be accompanied by institutional ethical clearance.
Units of measurementAll measurements should be reported in SI units; examples are meter, kilo-gram, liter and degree Celsius. All dates in manuscripts should be based on the Gregorian Calendar. When reporting financial matters on data collected within Ethiopia, the preferred currency to use is Birr, with exchange rates indicated in US Dollar.
AbbreviationsUse standard abbreviations only. The full terms for which an abbreviation stands should precede its first use in the text. Abbreviations of the titles of periodicals mentioned in the reference list must be according to the Interna-tional List of Periodical Title Word Abbreviations. Such a list can be avail-
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able from the Editorial Office upon request. E.g ‘Veterinary Pathology’ as ‘Vet. Pathol.,’, ‘Veterinary Parasitology’ as ‘Vet. Parasitol.,’, ‘Tropical Animal Health and Production’ as ‘Trop. Anim. Hlth. Prod.,’, ‘Preventive Veterinary Medicine’ as ‘Prev. Vet. Med.,’, ‘Revue Medecine Veterinaire’ as ‘Revue Med. Vet.,’, ‘Acta Tropica’ as ‘Acta Trop.,’, ‘Journal of Helminthology’ as ‘J. Helminthol.,’ ‘East African Medical Journal’ as ‘E. Afr. Med. J.,’, etc… NB: All journal citations in reference list must be written in ITALICS.
CopyrightAuthors are expected to observe all copyright related matters. It has to be noted that opinions expressed by the author(s) are not necessarily the views of the Journal or the Association. Authors assume full responsibility for the con-tents of the manuscript and for any claim or disclaim therein. All submissions are also with the understanding, knowledge and consent of copyright transfer to the Association.
Proofs and reprintsProofs will be sent to the corresponding author. They should be corrected and returned within 48 hrs. If this is not done in the specified period, for timely publication of the Journal, the editorial staff will have it proofed and published without the author’s correction.
Mailing addressAll contributions must be addressed to:
The Editorial OfficeEthiopian Veterinary Journal (Ethiop. Vet. J.) by email. Email: [email protected]
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Manuscript submission and copyright transfer formAll EVA publication submissions require completion of the following form addressed to:
Ethiopian Veterinary JournalP.O. Box 2462, Addis Ababa, EthiopiaTel. 251-118697868Email: [email protected]
1. Manuscript title: ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------2. Name of author(s): -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------3. Corresponding author:
Name: -------------------------------------------------------------------------------------------------------------
Institution: -------------------------------------------------------------------------------------------------------
P.O. Box: --------------------------------------------------------------------------------------------------------
City/Town: ------------------------------------------------------------------------------------------------------
Telephone: ------------------------------------------------------------------------------------------------------
Fax: --------------------------------------------------------------------------------------------------------------
Email: -----------------------------------------------------------------------------------------------------------4. The above indicated manuscript is submitted to appear in: (check only one)
_____________ The proceedings_____________ The journal (Ethiop. Vet. J.)
5. If the manuscript is submitted to the Journal, the column proposed is: (check only one)
_________ Original articles_________ Review and feature articles_________ Short communications, clinical/case Reports
6. The author(s) hereby:
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6.1. Agree to assume full responsibility for the contents of the manuscript and for any claim or disclaim therein.6.2. If the submission is to the Journal, that no similar paper other than oral presentation or the abstract has been or will be submitted for publication elsewhere prior to the written decision of the Editor-in-Chief.
6.3. Agree to the transfer of the copyright to the Association.
Name ____________________ Signature__________________ Date___________
