RNI No. RAJ ENG/2000/3243 ISSN 0972-4036 VETERINARY ... Veterinary Practitioner Volume 18(1) 2017...

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Veterinary Practitioner Vol. 18 No. 1 June 2017

RNI No. RAJ ENG/2000/3243 ISSN 0972-4036

Volume 18 No. 1 June 2017

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CONTENTS Titles Page No.

Genetic diversity analysis of Marwadi sheep breed of Rajasthan using microsatellites markers 1 - 4Kritika Gahlot, Mukul Purva, Rahul Yadav, Anupama Deora, Sunil Maherchandani, G.C. Gahlot andSudhir Kumar Kashyap

Detection of Theileria annulata infection in cattle-calves using nested PCR for targeting the 5 - 6small sub-unit ribosomal RNA (SSU r-RNA) geneP. Goyal, A. Chahar, R.K. Tanwar, Fakhruddin and A.P. Singh

Evaluation of the cytotoxic activity of crude aqueous extracts of medicinal plants of Kashmir 7 - 9I. A. Reshi, T.K. Sarkar, H.U. Malik, A. Muhee and Ashiq Hussain Bhat

Cytological and bacteriological evaluation of tracheal aspirates for the diagnosis of lung 10 - 14affections in horsesA.K. Sharma, Charanjit Singh Randhawa, Asmita Narang, Naresh Kumar Sood and Tejinder Singh Rai

Study of the potential of Ocimum sanctum in sub clinical mastitis in Rathi cattle 15 - 17Aprajita Johri, A.P. Singh, A. Gaur, J.P. Kachhawa, Ankita Sharma and P. Sharma and R.K. Joshi

Therapeutic efficacy of parenteral administration of gentamicin and enrofloxacin along with 18 - 21parenteral fluid therapy in the treatment of neonatal diarrhoeic calves - a comparative studyHimanshu and B. Pal

Oxidative stress and hemato-biochemical indices in Marwari horses during moderate intensity 22 - 26field exerciseR.K. Dedar, Vijay Kumar, P.A. Bala, R.A. Legha and B.N. Tripathi

Effect of enrofloxacin and enrofolxacin in combination with fish oil on haematobiochemical 27 - 30and otic cytological parameters in canine bacterial otitis externaSandeep Kumar, Kafil Hussain, Azhar Shuaib Batoo, Sunil Chaudhary, Afaq Amin Najar and Sumreen Kour

Management of thrombocytopenia in canine Ehrlichiosis by herbal therapy 31Kapil Kumar Gupta, Alok Singh, Hemant Kumar Shah, Ankur Upadhaya, Amanjeet Parashar and Sujeet Kumar

Efficacy of various hormonal protocols for improvement of fertility in post-partum anoestrus 32 - 34buffaloes during breeding seasonA.D. Patil, S.K. Sahatpure, P.T. Jadhao, J.P. Korde, M.S. Patil and D.V. Patil

Comparative study of haemoprotozoan and haematological examination in cyclic and 35 - 36non-cyclic Murrah buffaloes at organised dairy farmGyan Singh, R.K. Chandolia, Ravi Dutt and R.K. Malik

Per-vaginal delivery of conjoined twin monster by obstetrical intervention in a Murrah buffalo 37 - 38Anil Saini, Gyan Singh and Ravi Dutt

Schistosomus reflexus in a kid 39Manjusha Patil and P.B. Hase

Dystocia due to lateral devation of head in mare -a case report 40Gyan Singh, Devender, Jasmer Dalal and Ravi Dutt

Assisted delivery of mummified foetus in primipara cow 41A.K. Pandey, S. Agarwal, S.K. Gupta and S.E.H. Andrabi

Validation of sericin concentration in semen freezing media and its effect on post-thaw 42- 44quality of buffalo bull semenRidhima Demra, V.K. Gandotra, A.K. Singh and Ajeet Kumar

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Effect of soya-milk extender on release of phosphatases from buffalo bull spermatozoa 45 - 46during cryopreservationSajad Hussain Wani, Sudershan Kumar, M. Mutha Rao, A.K. Pandey, S.K. Gupta, Anavil Bhardwazand Sudhir Kumar

Study of certain physical parameters of fresh semen in Marwari horse 47 - 51Tejpal, J.S. Mehta, S.K. Ravi, Swati Ruhil, T.R. Talluri, Ashok Kumar and Diler Singh

Haematobochemical evaluation of levobupivacaine with and without fentanyl citrate in cow calves 52 - 53G. Roonwal, B.P. Shukla, S.Shukla, R. Jain and A. Jaiswal

Comparative evaluation of haemato-biochemical changes during infected wound healing in 54 - 55cow calves with the use of silver nano particle gel and povidone iodineYogendra Singh, B.P. Shukla, Reshma Jain and Aditya Jaiswal

Surgical excision of lipoma in a dog: a case report 56 - 57Munna Lal, S.K. Jhirwal, Satyaveer Singh and P. Bishnoi

Prepucial prolapse in Rathi bull- a case report 58N.P. Singh, S.K. Jhirwal, A. Sangwan and T.K. Gahlot

Cypermethrin and sodium fluoride induced haematological studies on male Wistar rats 59 - 62Renu Singh, A. K. Srivastava and Neeraj Kumar Gangwar

Histopathological observations of hydronephrosis in sheep (Ovis aries) 63 - 64Sarita, H. Dadhich , M. Mathur, V.K. Dhaka and A. K. Limba

Pathomorphological and histopathological observations of fibroma of cervix in sheep (Ovis aries) 65P.C. Kankhedia, Manish Agrawal, A. Dadhich, M. Mathur, H. Dadhich and A.P. Singh

Pathological observations of retention cyst in caprine kidney 66 - 67V.K. Dhaka, Sarita, H. Dadhich, A. Kumar, M. Mathur and I. Vyas

Radiographic anatomy of the wing of small Indian kite (Milvus migrans govinda) 68 - 69Aarti Sharma and S.C. Dubal

Age related histomorphological studies of small intestine of Uttara fowl 70 -73Jigyasa Rana, B. S. Dhote, I. Singh and Meena Mrigesh

Postnatal age related histochemical studies on the testes in Assam goat (Capra hircus) 74 - 76Kamal Sarma, S.N. Kalita and J. Devi

Gross, histomorphology of thyroid gland and thyroid hormonal changes in Pati ducks 77 - 79Anas platyrhynchos domesticus) of Assam with ageSnehangsu Sinha, Munmun Sarma and K.B. Dev Choudhury

Gross structural studies on spleen of large White Yorkshire pig 80 - 81Nikhil Shringi, Rakesh Mathur, Sanjeev Joshi and Kavita Rohlan

Studies on Tetrameres mohtedai v: pathology of spontaneous infection in local poultry 82 - 86Pallabi Pathak, Saidul Islam, Sushanta Goswami and Gouranga Das

Effects of ketosis on complete lipid profile in buffaloes 87 - 88Usha Choudhary, Meenaxi Sareen, Anil Moolchandani and N.S. Rathore

Effects of direct sunshine and melatonin on physiological responses and haematological 89 - 93profiles in Beetal and Assam hill goat in assamD. Hasin, A. Bora, J. Goswami, A. Barua, A. Saleque and B.K. Sarmah

Effect of treadmill exercise and heat on hormonal profile level in male buffalo calves 94 - 96J.P. Meharda, V.P. Varshney and Govind Ram Meghwal

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Role of aldosterone in hot ambience associated oxidative stress in Murrah heifers 97 - 101Ashish Joshi, N. Kataria, S. Asopa and A. K. Kataria

Epidemiological study on prevalence of Cysticercus cellulosae in pigs of Jammu region 102 - 105Rashmi Sharma, Harsh Kumar Sharma, Rohini Sharma, Rajesh Katoch, Rabjot Kaur, Mohd. Rashidand Alveena Ganai

A study on isolation of common pathogenic bacteria from bio-medical wastes 106 - 107Krupa K. Soni, Rakesh Rao, Rajani Joshi, Munna Lal, S. Maherchandani, Pankaj Mangal and Balram

Comparison of different methods of sire evaluation for first lactation milk yield in Rathi cattle 108 - 110K. S. Nehra, N. K. Sharma R.K. Joshi M.Sain and G. K. Dakhera

Comparative performance of Marwari and Karakul sheep in arid zone: growth 111 - 112N. K. Sharma, M.K. Sharma and G.C. Gahlot

Effect of feeding rosemary and fenugreek alone and in combination on the dry matter 113 -114digestibility and nitrogen balance of broiler chicksM. Meena, T. Sharma, R. Nehra, Nirmala Kumari and J.Saini

Effect of feeding detoxified Karanj seed cake (Pongamia glabra vent) based diets on intake of 115 - 117nutrients in goat kidsSonal Thakur, B.S.V. Reddy, T.M. Prabhu, Vijay Kumar Agrawal and Minali Jain

Antioxidant and antibacterial potential of condensed tannins containing tree leaves extract 118 - 121Mohd. Iqbal Daing, A. K. Pathak, Md. Altaf Bhat and Mohd. Aqib Zargar

Effect of feeding mushroom (Agaricus bisporus)as feed additive on the performance of broiler chicks 122 - 123Nirmala Kumari, R.K. Dhuria, T. Sharma, J. Saini, M.P. Kajla and M. Meena

Effect of feeding Withania somnifera and enzyme alone and in combination on dry matter 124 - 125digestibility and nitrogen balance of broiler chicksJ. Saini, T. Sharma, R.K. Dhuria, Nirmala Kumari, N. Mitharwal A.K. Limba and M. Meena

Effect of herbal products on in vitro fermentation of wheat straw based complete feed 126 - 129A.K. Jain, R.K. Dhuria, T. Sharma and M. Jain

Effect of various levels of Methi straw in complete feed on rumen fermentation pattern and 130 - 132haemato-biochemical parameters of sheepR.K. Saini, T. Sharma, R.K. Dhuria and L.N. Sankhala

Effect of feeding Aloe vera alone and combination with feed additive like enzyme on the 133 - 135dry matter metabolizability, n-balance and carcass traits of broiler chicksS.K. Saini, R.K. Dhuria, T. Sharma, M.S. Meel, Manish Bhati and M.P. Kajla

Development and quality assessment of fibre enriched functional Carabeef cookies 136 - 140Meena Goswami, B.D. Sharma, S.K. Mendiratta and Vikas Pathak

Effect of season of first calving on productive herdlife, longevity and life time calf production 141 - 143in Kankrej cow at organised farmAbhishek Joshi, V.K. Patel, R.P. Kalma, A.K. Srivastava and J.B. Patel

Effect of different litter materials on carcass attributes of broiler chicken 144 - 146Rahul Sigroha, Devender Singh Bidhan, Dipin Chander Yadav, Sajjan Sihag, Sandeep Dhillodhand Ravi Kumar

Comparative study of synthetic vitamin ‘C’ vis-a-vis herbal vitamin ‘C’ on performance of Giriraja 147 - 148(cross bred) chicksP.K. Kaushik, R.S. Choudhary, A. Singh, S. Meel and R.S. Godara

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Effect of feeding of multi-enzymes on performance of broiler chicks in hot arid zone of Rajasthan 149 - 151S. Kumar, R.S. Choudhary, S.C. Goswami, S.Meel, R.S. Gadhwal, D.S. Manohar, J. Saini andN. Mitharwal

Impact of mass media and extension worker contact on degree of adoption of improved animal 152 - 153husbandry practices by livestock farmers of Tarai area of district U.S. Nagar (Uttarakhand)A.S. Arora, Vasundhara Dawra, Avadhesh Kumar and Y.P.S. Dabas

Knowledge of tribal livestock owners about livestock health care practices in Banswara 154 - 155district of RajasthanPankaj Mishra, Devi Singh Rajput and Mohan Lal Yadav

A comparative study of knowledge about improved scientific management practices of members 156 - 159and non-members of dairy cooperative societyO.P. Meena, N.K. Sharma, N.K. Jeph, A.K. Jain and R.K. Dhuria

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#1Part of Ph.D. Thesis and corresponding author email: [email protected]; 2 SRF, Dept. of Vet. Micro. and Biotech; 3Ph.D. student, Dept. ofVet. Micro. and Biotech ; 4Teaching Associate, Dept. of Micro. and Biotech. , RAJUVAS, Bikaner, Rajasthan

GENETIC DIVERSITY ANALYSIS OF MARWADI SHEEP BREED OFRAJASTHAN USING MICROSATELLITES MARKERS

Kritika Gahlot1, Mukul Purva2, Rahul Yadav3, Anupama Deora4, Sunil Maherchandani, G.C. Gahlotand Sudhir Kumar Kashyap

Department of Veterinary Microbiology and Biotechnology, College of Veterinary and Animal Science Rajasthan University of Veterinary and Animal Sciences, Bikaner- 334 001, Rajasthan, India

ABSTRACT

The Genetic variability of Marwadi sheep was studied using 18 microsatellite markers, proposed by The Food and AgricultureOrganization and the International Society for Animal Genetics (FAOISAG). A total of 158 alleles ranging between 4 to 15 were foundacross 18 microsatellite loci. The most polymorphic marker was OarFCB48 with a total of 15 alleles and least polymorphic loci wasTGLA377 with 4 alleles were detected. The high genetic variation (MNA=8.7) was observed in Marwadi sheep compare to otherreported breed in Rajasthan. All studied loci contained high PIC value (>0.5) in the present study, where mean PIC value (0.751) ishigher than earlier reported study on sheep in India, which indicates highly informativeness of markers to characterize Marwadi sheepbreed population and showed abundant genetic diversity in this population. Further, the expected gene diversity (Hexp) ranged from0.637 (OarHH35) to 0.900 (OarFCB48) with an overall mean of 0.797 and observed heterozygosity (Hobs) across 18 loci rangedbetween 0.300 (TGLA137) and 0.900 (OarVH72, BM757) with a mean of 0.602 were estimated. The present study has shown a highlevel of genetic variability in Marwadi sheep, suggesting good breeding practices were carried out in the farms and other placeswhere samples were collected. Similarly, all the loci were detected highly polymorphic with 100% value of polymorphism observed inMarwadi sheep breed. The high polymorphic information content (PIC) value indicating informativeness of markers to characterizeMarwadi sheep population. Hence, these markers could be used in various applications like conservation programmes, biodiversitystudies and disease diagnosis.

Key words: Genetic diversity, microsatellite markers, polymorphic information content, heterozygosity, allelic diversity

IntroductionSheep are one of the earliest ruminants to be tamed by

human at Fertile Crescent, 9000 years back (Peter et al., 2007;Tapio et al., 2006; Zeder et al., 2006). It plays a vital role in thelivelihood of alarge proportion of small farmers and indigenouspeople. It considers as one of the most significant smallruminants of India as the native sheep breeds are distributedthroughout the country. According to Indian Council forAgricultural Research (ICAR), there are nearly 40 sheep breedsin India. The methodology utilized to categorize a large numberof sheep and goat breeds in India were relied upondistinguishable morphological characteristics that weredistinctive from other populations in the locality, mainly thosewith a local name (Acharya, 1982; Shrestha, 2005). MarwadiSheep, the name derives from the home tract of the breed:Marwad, is one of the 8 sheep breeds in Rajasthan and deliverlustrous carpet wool. The breed resembles black-headedPersian sheep, yet is smaller in size and produce great amountof fleece. This significant breed is predominantly found in theJodhpur, Jalore, Nagour, Pali and Barmer districts, spreadingup to Ajmer and Udaipur districts of Rajasthan and the Jeoriaregion of Gujarat. Studies on genetic diversity of smallruminants have been exaggeratedly hastened over the pastdecades based on microsatellite markers (Bhatia and Arora,2005). Microsatellite markers have been recognized to bevaluable for genetic diversity studies, parentage test, linkageanalysis and population genetic studies, because of theirsuperior features over the other molecular markers (Bruford

and Wayne, 1993). This technique represents the arrays ofrepeat sequences that display length variations, differentalleles containing different number of repeat units (Purohit etal., 2016). Evaluation of genetic diversity is the key step towardsconservation and viable utilization of genetic resources (Dalvitet al., 2008; Glowatzki-Mullis et al., 2008; Kevorkian et al., 2010),and could prove to be an important technique to conserve thebreeds.

Materials and MethodsSampling and DNA isolation

A total of 40 blood samples of genetically unrelated Marwarisheep were randomly collected from across their breedingtract and from the flock maintained on the farm at Central Sheepand Wool Institute, Bikaner and Kodamdesar farm, BikanerRajasthan. Genomic DNA was extracted from whole bloodusing the QIAamp® DNA Mini Kit with slight modification. AllDNA samples were analyzed on 1.5% agarose gel throughhorizontal electrophoresis. Furthermore p36 Nanophotometerwas used to measure the concentration and purity of the DNAsample.

Microsatellite DNA typingA total of 18 microsatellite marker loci (Table 1) was

selected from the list as suggested by International Society forAnimal Genetics and FAO’s (1996) (MoDAD) for ovine, basedon their allele size, level of polymorphism and authenticity ofthe allele to characterize and uncover the level of genetic diversityin Marwadi sheep breed. PCR was carried out in 25 ìl reaction

Received on: 29.01.2017Accepted on: 12.03.2017

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volume containing 1.5 mM MgCl2, 200 μMdNTPs, 0.75 μl ofeach primer, 3.5 μl of template DNA and 0.20 μl of Taqpolymerase (Promega, Madison, USA) using Eppendorfmastercycler gradient. PCR cycling conditions were 5 min at95°C, after that 30 cycles of 1 min at 94°C, 1 min of annealingtemperature (55-58°C) of each primer, 1 min at 72°C and finalextension of 7 min at 72°C. The exact annealing temperaturewas determined by Gradient PCR. A constant concentration of1.5 mM MgCl2 was used for all amplification reactions whichwere already present in the reaction buffer because there wasno significant change was observed by varying MgCl2concentration. The polymorphic typing of microsatellite markerwas done on 8% native polyacrylamide gel electrophoresis(PAGE) at 80 V (Hoefer SE 600 series electrophoresis unit)and visualized by ethidium bromide staining. Allele sizes wereestimated utilizing a 100 bp ladder (Promega, USA). Thegenotype of each individual animal at 18 different loci wasrecorded by manual counting and data was further analyzedusing different statistical tools.

Statistical analysisAllele frequency, Observed number of alleles (Na), Effective

number of alleles (Ne), Observed heterozygosity (Ho) andExpected heterozygosity (He) were calculated by GenAlEx 6.5version(Peakall and Smouse, 2006, 2012). Hardy-Weinbergequilibrium (HWE) was estimated using GENEPOP 1.2 version(Raymond and Rousset, 1995). Fixation Index (Fis) wasestimated using FSTAT version 2.9.3 (Goudet, 2001).Polymorphic Information content (PIC) for each locus wasestimated according to (Botstein et al., 1980).

Results and DiscussionThe Numerous genetic diversity measures viz., allele

frequency, the observed number of alleles, effective number ofalleles, observed heterozygosity, expected heterozygosityassessed across 18 microsatellite loci in Marwadi sheep isshown in Table 2. All 18 microsatellite markers weresuccessfully amplified and produced clear banding patterns.A total of 158 alleles ranging between 4 to 15 were found across18 microsatellite loci. The most polymorphic marker was

Table 1: Details of microsatellite markers used in sheep of Marwari breed (Crawford et al., 1995).

Locus Primer sequence Allele Size Chr No.

Ann. Temp. (in°C)

BM6526 F-CAT GCC AAA CAA TAT CCA GC R-TGA AGG TAG AGA GCA AGC AGC

146-172 26 60

BM757 F-TGG AAA CAA TGT AAA CCT GGG R- TTG AGC CAC CAA GGA ACC

178-198 9 55

BM8125 F- CTC TAT CTG TGG AAA AGG TGG G R- GGG GGT TAG ACT TCA ACA TAC G

107-135 17 55

BM827 F- GGG CTG GTC GTA TGC TGA G R- GTT GGA CTT GCT GAA GTG ACC

210-222 3 55

CSSM31 F- CCA AGT TTA GTA CTT GTA AGT AGA R- GAC TCT CTA GCA CTT TAT CTG TGT

130-170 23 55

OarAE129 F- AAT CCA GTG TGT GAA AGA CTA ATC CAG R- GTA GAT CAA GAT ATA GAA TAT TTT TCA ACA CC

140-164 5 60

OarCP34 F- GCT GAA CAA TGT GAT ATG TTC AGG R- GGG ACA ATA CTG TCT TAG ATG CTG C

110-128 3 63

OarFCB128 F- CAG CTG AGC AAC TAA GAC ATA CAT GCG R- ATT AAA GCA TCT TCT CTT TAT TTC CTC GC

108-134 2 63

OarFCB48 F- GAG TTA TGT ACA AGG ATG ACA AGA GGC AC R- GAC TCT AGA GGA TCG CAA AGA ACC AG

138-166 17 55

OarHH35 F- AAT TGC ATT CAG TAT CTT TAA CAT CTG GC R- ATG AAA ATA TAA AGA GAA TGA ACC ACA CGG

128-160 4 63

OarHH41 F- TCC ACA GGC TTA AAT CTA TAT AGC AAC C R- CCA GCT AAA GAT AAA AGA TGA TGT GGG AG

96-120 6 63

OarHH64 F- CGT TCC CTC ACT ATG GAA AGT TAT ATA TGC R- CAC TCT ATT GTA AGA ATT TGA ATG AGA GC

116-132 4 55

OarJMP29 F- GTA TAC ACG TGG ACA CCG CTT TGT AC R- GAA GTG GCA AGA TTC AGA GGG GAA G

130-150 24 55

OarJMP8 F- CGG GAT GAT CTT CTG TCC AAA TAT GC R- CAT TTG CTT TGG CTT CAG AAC CAG AG

119-131 6 63

OarVH72 F- CTC TAG AGG ATC TGG AAT GCA AAG CTC R- GGC CTC TCA AGG GGC AAG AGC AGG

113-137 25 63

RM4 F- CAG CAA AAT ATC AGC AAA CCT R- CCA CCT GGG AAG GCC TTT A

135-143 15 55

TGLA137 F- GTT GAC TTG TTA ATC ACT GAC AGC C R- CCT TAG ACA CAC GTG AAG TCC AC

119-161 5 55

TGLA377 F- GAC TGT CAT TAT CTT CCA GCG GAG R- GAT CTC TGG TTG AAA TGG CCA GCA G

86-122 2 55

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OarFCB48 with a total of 15 alleles and least polymorphic lociwas TGLA377 with 4 alleles. The allele diversity, considered tobe a reasonable indicator of genetic variation within thepopulation, displayed high genetic variation (8.7) in Marwadisheep. The average number of observed alleles detected inMarwadi sheep was comparable with three Iranian pelt sheepbreed, namely: Gray (8.1), Zandi (8.0) and Karakul (8.1)(Nanekarani et al., 2010). The effective number of alleles (Ne)ranged from 2.757 (OarHH35) to 10.0 (OarFCB48) with a meanvalue of 5.5. According to data shown in the present studyallelic diversity found in greater extent in comparison to earlierreported study by (Sodhi et al., 2005; Mukesh et al., 2006; Sodhiet al., 2003; Sodhi et al., 2004; Sharma et al., 2016). Thus, itcan be concluded that sufficient genetic variability exists inMarwadi sheep despite its declining population.

All loci found highly polymorphic with a 100% value ofpolymorphism in Marwadi breed population. Polymorphicinformation content (PIC) value ranged between 0.589 (HH35)and 0.856 (OarVH72, RM4) with a mean value of 0.763. Themean PIC value (0.763) in the present study is higher thanearlier reported in Chokla, 0.605 (Sodhi et al., 2005; Mukeshet al., 2006), Magra, 0.648 (Arora and Bhatia, 2006),Tibetan 0.632 (Sharma et al., 2016), Nali, 0.613 (Sodhi etal., 2005; Mukesh et al., 2006) sheep breed. Accordingly,all the markers considered for this study are highlyinformative (PIC>0.5) to characterize Marwadi sheep breedpopulation and showed abundant genetic diversity in thispopulation.

The heterozygosity calculates genetic variab ilitywithin a population in a significant manner. Observed andexpected heterozygosity at individual loci are presentedin Table 2. The expected heterozygosity (Hexp) rangedfrom 0.637 (OarHH35) to 0.900 (OarFCB48) with an overallmean of 0.797. Observed heterozygosity (Hobs) across

18 loci ranged between 0.300 (TGLA137) and 0.900(OarVH72, BM757) with a mean of 0.602. The averageobserved heterozygosity (Ho=0. 602) detected in thepresent study is higher than previously reported Chokla(0.47), Garole (0.44), Nali (0.47) sheep breed (Mukesh etal., 2006) and few breeds from northern temperate region,namely: Rampur bushair, 0.515 (Pandey et al., 2008),Gurej, 0.490 (Gupta et al., 2007), Karnah, 0.530 (Guptaand Gannai, 2007) and Tibetan, 0.473 (Sharma et al.,2016). Similarly, much higher heterozygosity was detectedin Magra sheep, 0.597 (Arora et al., 2006), six Indiansheep breed of the north western arid region (Arora et al.,2011), Gray (0.984), Zandi (0.985) and Karakul (0.988)pelt sheep (Nanekarani et al., 2010). The expectedheterozygosity (0.797) obtained in the present study ishigher than the earlier reported study by (Mukesh et al.,2006; Sodhi et al., 2005; Arora et al., 2011; Sharma et al.,2016) but lower than that of Pelt sheep (Nanekarani etal., 2010). Therefore, in the present study data has showna high level of heterozygosity suggesting good breedingpractices were carried out in the farms and other placeswhere samples were collected.

ReferencesAcharya RM (1982) Sheep and goat breeds of India. FAO Animal

production and Health Paper. 30. FAO of United Nations,Rome, Italy.

Arora R and Bhatia S (2006) Genetic diversity of Magra sheepfrom India using microsatellite analysis. Asi. Aus. J.Ani. Sci. 19: 938-942.

Arora R, Bhatia S, Mishra BP, Jain A and Prakash B (2011)Divers ity analys is of sheep breeds f rom Southernpeninsular and Eastern regions of India. Trop. Anim.Health Prod. 43(2): 401-408.

Bhatia S and Arora R (2005) Biodiversity and conservation of Indian

Table 2: Details of observed heterozygosity (Ho), expected heterozygosity (He),number of alleles (Na, Observed and Ne, effective), polymorphism informationcontent (PIC)

Locus Ho (Observed) He (Expected)

Na (Observed)

Ne (effective)

PIC ( overall)

BM8125 0.500 0.814 7.000 5.373 0.786 FCB48 0.467 0.900 15.000 10.000 0.8006 FCB128 0.500 0.721 9.000 3.579 0.6879 HH35 0.667 0.637 5.000 2.757 0.5898 HH41 0.667 0.840 10.000 6.250 0.8232 HH64 0.767 0.819 8.000 5.538 0.7947 JMP8 0.867 0.854 11.000 6.870 0.8222 JMP29 0.467 0.842 12.000 6.338 0.8049 TGLA137 0.300 0.851 9.000 6.691 0.8317 CP34 0.633 0.836 8.000 6.102 0.8138 AE129 0.633 0.784 6.000 4.627 0.7488 BM827 0.467 0.807 8.000 5.172 0.7776 CSSM31 0.367 0.739 5.000 3.838 0.6987 BM6526 0.533 0.802 10.000 5.042 0.783 BM757 0.900 0.688 9.000 3.209 0.6559 TGLA377 0.633 0.671 4.000 3.035 0.6048 VH72 0.900 0.873 11.000 7.895 0.8563 RM4 0.567 0.869 11.000 7.660 0.856 Mean±SE 0.602±

0.041 0.797± 0.018

8.778± 0.655

5.554± 0.450

0.763

4


sheep genetic resources-An overview. Asi. Aust. J. Anim.Sci. 18(10): 1387-1402.

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Sodhi M, Mukesh M and Bhatia S (2004) Microsatellite based geneticcharacterization of three breeds of Indian sheep. InProceedings of the 91st Indian Science Congress, 3-7January 2004, Chandigarh, India.

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5


DETECTION OF THEILERIA ANNULATA INFECTION IN CATTLE-CALVESUSING NESTED PCR FOR TARGETING THE SMALL SUB-UNIT

RIBOSOMAL RNA (SSU r-RNA) GENE#

P. Goyal1, A. Chahar, R.K. Tanwar, Fakhruddin and A.P. Singh2Department of Epidemiology and Preventive Veterinary Medicine, College of Veterinary and Animal Science

Rajasthan University of Veterinary and Animal Sciences, Bikaner-334 001, Rajasthan, India

ABSTRACT

A total of 100 blood samples were collected in ethylene diamine tetra acetic acid (EDTA @ 1 mg/ml) vacutainer from cattle-calves irrespective of their age, sex and breed brought to Teaching Veterinary Clinical Complex of College of Veterinaryand Animal Science, Bikaner for treatment. By nested polymerase chain reaction (nPCR), 41 samples were found positivefor Theileria annulata infection which produced 372-bp amplicon in 1 per cent agarose gel.

Key words: nPCR, Theileria annulata, amplicon, agarose gel.

#1Part of M.V.Sc. Thesis of first author submitted to RAJUVAS, Bikaner, Email:[email protected]; 2Professor, Department of ClinicalVeterinary Medicine, Ethics and Jurisprudence, CVAS, RAJUVAS, Bikaner.

IntroductionTheileria annulata, a blood protozoan parasite, causes

bovine tropical theileriosis and it is transmitted by tickHyalomma anatolicum anatolicum. It causes significanteconomic losses in large parts of Asia (Hasanpour et al., 2013).It is mainly seen in cattle, sheep and goat as well as in wildand captive ungulates (Radostits et al., 2007). This intracellularinfection inflicts economic burden on cattle breeders in termsof mortality and morbidity as well as expenses spent onprophylactic measures against disease and treatment (Durraniet al., 2008).

This parasite is a serious constraint to cattle productionin endemic areas, causing lethal infection in exotic cattle andconsiderable mortality in indigenous as well as crossbredstocks (Forsyth et al., 1997). The economic losses to animalsin terms of morbidity and mortality are due to their heavyincidence (Fadraga et al., 1991). Although, the sporadic casesare seen throughout the year however, the outbreak in exoticand cross-bred cattle is mostly reported during the hot andhumid months (July-September) of the year (Ashfaq et al., 1983and Zahid et al., 2005).Tropical theileriosis caused by Theileriaannulata may result in 80 per cent mortality in susceptibleanimals (Gill et al., 1977). Diagnosis of the disease is basedon clinical findings and microscopic examination of blood andlymph node aspirate smears stained with Giemsa anddetection of macroschizonts in acute cases. Smear method isassociated with technical problems and even wrong diagnosisand has low sensitivity in diagnosis of carrier cattle (Nayel etal., 2012). Recently, diagnostic methods like PCR have beendeveloped for the rapid and accurate detection of Theileriaspp. (Zaeemi et al., 2011 and Ghaemi et al., 2012). This studywas carried out to diagnose the Theileria annulata infection inblood samples obtained from cattle-calves in Bikaner,Rajasthan using PCR for amplification of SSU r-RNA gene ofT. annulata.

Materials and MethodsA total of 100 blood samples were collected in ethylene

diamine tetra acetic acid (EDTA @ 1 mg/ml) vacutainer fromcattle-calves irrespective of their age, sex and breed brought toTeaching Veterinary Clinical Complex of College of Veterinaryand Animal Science, Bikaner for treatment. Genomic DNA wasextracted using QIAamp® DNA blood mini kit (QIAGEN, GmbH,Germany) as per procedure. Aliquots of extracted DNA werekept at -20°C. PCR was carried out using primers as reportedby D’Oliveira et al. (1995).

Primary PCR assay was performed using the Theileriagenus specific primers (989 and 990) for targeting the smallsub-unit ribosomal RNA (SSU r-RNA) gene in a final reactionvolume (master mix) of 25 µl containing:

989 (F): 5’ AGT TTC TGA CCT ATC AG 3’990 (R): 5’ TTG CCT TAA ACT TCC TTG 3’The thermo cycle profile was as described below:1) Initial denaturation at 94°C for 3 minutes.2) Then denaturation at 94°C for 1 minute for 30 cycles.3) Annealing at 60°C for 1 minute for 30 cycles.4) Extension at 72°C for 1 minute for 30 cycles.5) Final extension at 72°C for 3 minutes.6) Holding at 4°C until the samples were taken out from thermalcycler.

Then nested PCR assay was carried out in a final reactionvolume of 25 µl as described above but instead of templateDNA, 3 µl of the primary PCR product was used and amplified

Reagent Volume CoralLoad PCR buffer 5.00 µl 25 mM MgCl2 3.00 µl 10 mMdNTP mix 1.00 µl Taq DNA polymerase 0.25 µl Forward primer (989) 0.75 µl Reverse primer (990) 0.75 µl Template DNA isolated from the blood samples

3.00 µl

Nuclease free water 11.25 µl


mailto:Email:[email protected];

6


Fig.1: Photograph showing 372-bp amplicon (Theileria annulataspecies specific) which subjected to electrophoresis on 1% agarosegeland stained with ethidiumbromideLane M: 100-bp GenerulerTMDNA ladderLane 1: Negative controlLane 2: Positive controlLane 3-12: Clinically suspected samplesLanes 6-10: Positive samples

with 0.75 µl (20 pmol) each of forward primer (989) and reverseprimer (1347) for targeting the SSU r-RNA gene and the thermocycle profile was same as above.

1347(R): 5’ TGC ACA GAC CCC AGA GG 3’The PCR products were checked by electrophoresis on 1

per cent agarose gel and visualized using a gel documentationsystem (Syngene, UK).

Results and DiscussionOut of 100 DNA extracted from blood samples of cattle-

calves, only 41 samples were found positive for Theileriaannulata which produced 372-bp amplicon in 1 per centagarose gel (Fig.1).

Microscopic detection of the piroplasms and schizonts inthe blood smears and/or lymph node aspirate smears fromthe suspected host is the true ‘gold standard’ diagnostic testavailable for theileriosis, along with the clinical signs of thedisease, such as high fever and enlargement of lymph nodesalso help in diagnosis. These tests are robust, but have poorsensitivity and higher dependency on the qualified laboratorytechnicians. The low level of parasitemia in the case of carrieranimals and chronic cases holds importance as they makethe animal a reservoir of infection for the whole herd. PCRassays permit identification of parasite at levels far below thedetection limit of the commonly used parasitologicaltechniques and has superiority in separating clinical andsubclinical forms of parasitic infection.

Thus, PCR is preferred method for diagnosis oftheileriosis because this method is more sensitive and specific

than other conventional methods (Alhassan et al., 2005; Altayet al., 2005 and Nagore et al., 2004).

AcknowledgementsThe authors are thankful to Dr. S.K. Kashyap, Professor

and Head, Department of Microbiology and Biotechnology andDr. G.C. Gahlot, Professor and Head, Department of AnimalGenetics and Breeding, College of Veterinary and AnimalScience, Bikaner, Rajasthan for providing necessary facilitiesto carry out the present investigation.

ReferencesAshfaq M, Ajmal M and Ahmad S (1983) An outbreak of theileriosis in

crossbred neonate calves. Pakistan Vet. J. 3: 44-46.D’Oliveira C, Vandermerve M, Habela M, Jacquiet P and Jongejan F

(1995) Detection of Theileria annulata in blood samples ofcarrier cattle by PCR. J. Clin. Microbiol. 33(10): 2665-2669.

Durrani AZ, Shakoori AR and Kamal N (2008) Bionomics of Hyalommaticks in three districts of Punjab. Pakistan J. Anim. PlantSci. 18(1): 20-23.

Fadraga M, Cordoves and Puentes T (1991) Circulation of antibodiesto haemoparasites in cattle (Bos taurus) of high geneticvalue in Cuba. Rev. Cub-de-Cien. Veterinarias. 22: 249-255.

Forsyth LMG, Jackson LA, Wilkie G, Sanderson A, Brown CGD andPreston PM (1997) Bovine cells infected in vivo with Theileriaannulata express CD11b, the C3bi complement receptor.Vet. Res. Commun. 21: 249-263.

Ghaemi P, Hoghooghi-Rad N, Shayan P and Eckert B (2012) Detectionof Theileria orientalis in Iran by semi-nested PCR. ParasitolRes. 110: 527-531.

Gill BS, Bhattacharyulu Y and Kaur D (1977) Symptoms and pathologyof experimental bovine tropical theileriosis (Theileriaannulata infection). Annales de Parasitologie Humaine etComparee. 52: 597-608.

Hasanpour A, Sabegh YG and Sadeghi-nasab A (2013) Assessmentof serum antioxidant enzymes activity in cattle sufferingfrom theileriosis. European Journal of Experimental Biology.3(1): 493-496.

Nagore D, Gracia-Sanmartin J, Gracia-Perez AL, Juste RA and HurtadoA (2004) Identification, genetic diversity and prevalence ofTheileria and Babesia species in a sheep population fromNorthern Spain. Int. J. Parasitol. 34: 1059-1067.

Nayel M, El-Dakhly KM, Aboulaila M, Elsify A, Hassan H and Ibrahim E(2012) The use of different diagnostic tools for Babesiaand Theileria parasites in cattle in Menofia. Egypt Parasitol.Res. 111(3): 1019-1024.

Radostits OM, Gay CC, Hinchcliff KW and Constable PD (2007)Veterinary Medicine, 10th Ed., W.B. Saunders Company Ltd.Philadelphia.

Zaeemi M, Haddadzadeh H, Khazraiinia P, Kazemi B and BandehpourM (2011) Identif ication of different Theileria species(Theileria lestoquardi, Theileria ovis and Theileriaannulata) in naturally infected sheep using nested PCR-RFLP. Parasitol. Res. 108: 837-843.

Zahid IA, Latif M and Baloch KB (2005) Incidence and treatment oftheileriosis and babesiosis. Pakistan Vet. J. 25(3): 137-139.

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1Corresponding author: e-mail: [email protected] , Mobile: 7006888290; 2Department of Veterinary Gynaecology and Obstetrics, KCVAS, Amritsar.

EVALUATION OF THE CYTOTOXIC ACTIVITY OF CRUDE AQUEOUSEXTRACTS OF MEDICINAL PLANTS OF KASHMIR

I. A. Reshi1, T.K. Sarkar, H.U. Malik, A. Muhee and Ashiq Hussain Bhat2Division of Clinical Veterinary Medicine, Ethics and Jurisprudence, Faculty of Veterinary and Animal ScienceSher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir Jammu and Kashmir, India

ABSTRACT

The objective of this study was to evaluate the cytotoxicity potential of aqueous extracts of Nepeta cataria, Fumaria indica, Boragoofficinalis, Adiantum capillus, Levandula stoeches on HeLacell line. Plant material was washed with distilled water, dried in shade,grinded to fine powder and stored in airtight container at room temperature in the dark until used. The powdered samples weresubjected to extraction using distilled water. Different concentrations of the extracts of herbals were prepared. Cytotoxicity studywas carried out by The MTT Cell Proliferation Assay on HeLa cell line. The MTT Cell Proliferation Assay measures the cell proliferationrate and conversely, when metabolic events lead to apoptosis or necrosis, there is reduction in cell viability. Fumaria indica exhibitedcell viability ranging from 89.60 to 100 per cent against the extract concentration of 5 to 100 μg/ml with IC50 (µg/ml) on HeLA cell line as-581.79 Adiantum capillus extract revealed cell viability from 81.91 to 100 per cent against the extract concentration of 5 to 100 μg/ml with IC50 (µg/ml) on HeLA cell line as 36.96. Nepata cataria extract showed cell viability ranging from 84.36 to 96.09 per cent againstthe extract concentration of 5 to 100 μg/ml with IC50 (µg/ml) on HeLA cell line as -112.07. In case of Levandula stoeches extract percent cell toxicity ranged from 0 to 41.77 against the extract concentration of 5 to 100 μg/ml. Borago officinalis extract exhibited per centcell toxicity ranging from 4.27 to 43.17 against the extract concentration of 5 to100 μg/ml with IC50 (µg/ml) on HeLA cell line as 26.91.

Key words: Herbal, extracts, cell line, cytotoxicity.

IntroductionPlants are used medicinally in different countries and are

a source of many potent and powerful drugs (Srivastava et al.,1996). A wide range of medicinal plant parts are used for extractof raw drugs and they possess varied medicinal properties.The use of conventional plant products described in ancientliterature in modern medicine suffers from the fact that scientificevidence and explanation are lacking in most cases.Considering the vast potentiality of plants as sources forantimicrobial and anti-inflammatory drugs, the present studywas undertaken to screen the cytotoxic potential of crudeaqueous extracts of some plants. Cytotoxicity testing isimportant for the purpose of determining the potential toxicityof the compounds being studied. Cytotoxicity studies of localplant extracts or folk medicinal plant extracts has not beenstudied extensively and this is vital for the safety evaluation ofany herbal preparations.Thus the objective of this study was toevaluate the potential cytotoxicity of aqueous extracts of Nepetacataria, Fumaria indica, Borago officinalis, Adiantum capillus,Levandula stoeches on HeLacell line.

Materials and MethodsFollowing herbs (crude aqueous extract) were evaluated

for their in-vitro cyto-toxicity trial on cell line (HeLa cell lines):

Plant materialThe selected herbs (leaves) were purchased from

registered herbal shops from local market Srinagar, J & K.Plant material was washed with distilled water, dried in shade,grinded to fine powder and stored in airtight container at roomtemperature in the dark until used. The powdered sampleswere subjected to extraction using distilled water following themethod of Nair et al. (2005). Different concentrations of theextracts of herbals were prepared.

In vitro toxicity study on cell lineMeasurement of cell viability and proliferation forms the

basis for numerous in vitro assays of a cell population’sresponse to external factors. The MTT Cell Proliferation Assaymeasures the cell proliferation rate and conversely, whenmetabolic events lead to apoptosis or necrosis, there isreduction in cell viability.

DMEM (Dulbecco’s modified Eagles medium), MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide],trypsin, EDTA Phosphate Buffered Saline (PBS) and werepurchased from Sigma Chemicals Co. (St. Louis, MO) andFetal Bovine Serum (FBS) were purchased from Gibco. 25cm2 and 75 cm2 flask and 96 well plated purchased fromEppendorf India.

Maintenance of cell lineThe Hela Cervical cancer cell line were purchased from

NCCS, Pune and the cells were maintained in DMEMsupplemented with 10% FBS and the antibiotics penicillin/streptomycin (0.5 mL-1), in atmosphere of 5% CO2/95% air at37 oC.

Preparation of test compoundFor MTT assay, each test compounds were weighed

separately and dissolved in DMSO. With media make up the

S. No. Name of the herb

Part used

1 Nepata cataria Leaves 2 Levandula stoeches Leaves 3 Fumaria indica Leaves 4 Adianthum capillus Leaves 5 Borago officinalis Leaves



8


final concentration to 1 mg/ml and the cells were treated withseries of concentrations from 10 to 100 µg/ml.

HeLa cell viability by MTT AssayPrinciple: MTT Assay is a colorimetric assay that measures

the reduction of yellow 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by mitochondrial succinatedehydrogenase. The assay depends both on the number ofcells present and on the assumption that dead cells or theirproducts do not reduce tetrazolium. The MTT enters the cellsand passes into the mitochondria where it is reduced to aninsoluble, dark purple coloured formazan crystals. The cellsare then solubilized with DMSO and the released, solubilizedformbazan reagent is measured spectrophotometrically at 570nm.

Procedure: Cell viability was evaluated by the MTT Assaywith three independent experiments with six concentrations ofcompounds in triplicates. Hela cells were trypsinized andperform the trypan blue assay to know viable cells in cellsuspension. Cells were counted by haemocytometer andseeded at density of 5.0 x103 cells/well in 100 ìl media in 96well plate culture medium and incubated overnight at 37oC.After incubation, remove the old media and add fresh media100 µl with different concentrations of test compound inrepresentative wells in 96 plate. After 48 hrs discard the drugsolution and add the fresh medic with MTT solution (0.5 mg/mL-1) was added to each well and plates were incubated at37oC for 3 hrs. At the end of incubation time, precipitates areformed as a result of the reduction of the MTT salt tochromophore formazan crystals by the cells with metabolicallyactive mitochondria. The optical density of solubilized crystalsin DMSO was measured at 570 nm on a microplate reader.The percentage growth inhibition was calculated using thefollowing formula and concentration of test drug needed toinhibit cell growth by 50% values is generated from the dose-response curves for each cell line using with origin software.

Per cent inhibition =

Result and Discussion

In vitro cytotoxicity of aqueous herbal leaf extractsAdiantum capillus extract revealed cell viability which

ranged from 81.91 to 100 per cent against the extractconcentration of 5 to 100 ìg/ml with IC50 (µg/ml) 36.96 on HeLacell line. Gaikwad et al. (2013) also reported that the histologicaldamages in Adiantum capillus extract treated rats withthe doserate of 500 mg/kg b.wt. for 14 days were minimal in contrastwith the toxic drug cisplatin on rat. Ethanolic extract Adiantumcapillus-veneris showed good antioxidant activity and itexhibited low IC50 value (0.3986 mg/gm) for DPPH assay and0.695 mg/gm for ABTS assay and results obtained indicatedthat Adiantum capillus-veneris leaves are endowed with freeradical scavenging molecules and it can be used as a potentialsource of natural antioxidants and nutrients (Rajurkar andGaikwad, 2012). Paul et al. (2012.) revealed that 2000 mg/kgof Adiantum philippense on mice for 14 days was safe andconcluded that 500 mg and 250 mg/kg b.wt of Adiantumphilippense was chosen for subsequent experimentation.

Fumaria indica exhibited cell viability from 89.60 to 100

per cent against the extract concentration of 5 to 100 ìg/ml withIC50 (µg/ml) on HeLA cell line as - 581.79, while as per cent celltoxicity of Fumaria indica ranged from 0 to 12.53 against theextract concentration of 5 to 100 ìg/ml. Fumaria indica wasfound to be safe in cytotoxic test and devoid of toxicmanifestations during chronic administration (Singh et al.,2011). Erdagon (2009) also reported that the water extract ofFumaria indica showed no cytotoxicity activity.

Borago officinalis extract exhibited cell viability which rangedfrom 47.24 to 71.6 per cent against the extract concentration of5 to 100 µg/ml with IC50 (µg/ml) on HeLA cell line as 26.91.Lozano-Baena et al. (2016) also reported cytotoxic effect ofBorago officinalis with IC50 values of 0.28 mg/mL. Hamed andWahid (2015) also revealed that 150 mg/kg b.wt. of Boragoofficinalis ethanolic extract is a safe dose.

Nepata cataria extract showed cell viability ranging from34.36 to 83.67 per cent against the extract concentration of 5 to100 ìg/ml with IC50 (µg/ml) on HeLa cell line as-112.07. Percent cell toxicity ranged from 16.33 to 65.64 against the extractconcentration of 5 to100 µg/ml. Nepata cataria aqueous extractshowed low cytotoxic effect and the study corroborates withHussain et al. (2009), who revealed that LD50 (µg/ml) ofaqueous Napeta juncea equal to 88.1. Similarly aqueousfraction (AQF) of Nepeta praetervisa leaves extract has showedlowest anti-leishmanial activity with IC50 value

9


and antimicrobial activit ies of extracts of Nepetajuncea. African J. Biotechnol. 8(6): 935-940.

Nair R, Kalariya T, Chanda S (2005) Antibacterial activity of someselected Indian medicinal flora. Turk. J. Biol. 29: 41 47

Paul T, Das B, Apte KG, Banerjee S and Saxena RC (2012)Evaluation of anti-hyperglycemic activity of Adiantumphilippense Linn, A pteridophyte in Alloxan inducedDiabetic Rats . Journal of Diabêtes and Metabolism.3:226

Rajurkar NS and Gaikwad K (2012) Evaluation of phytochemicals,

antioxidant activity and elemental content of Adiantumcapillus veneris leaves. J. Chemical andPharmaceutical Res. 4(1): 365-374.

Singh GK, Chauhan SK, Rai G and Kumar V (2011) Fumariaindica is safe during chronic toxicity and cytotoxicity.A preclinical s tudy. J. Pharmacol. Pharmacother.2(3):191-192.

Srivastava J, Lambert J and Vietmeyer N (1996) Medicinal plants:An expanding role in development . World BankTechnical Paper. p. 320.

Table1: In vitro cytotoxicity study of herbal extracts on HelA cell line

Test extract Parameters

Test concentration (µg/ml) 5 10 25 50 75 100

Fumaria indica

Per cent cell viability 100 87.47 100 92.25 89.60 100 Per cent cell cytotoxicity 0 12.53 0 7.75 10.40 0 IC50 (µg/ml) -581.79

Adiantum capillus

Per cent cell viability 92.28 100 93.6 100 81.91 84.54 Per cent cell cytotoxicity 7.72 0 6.4 0 18.09 15.46 IC50 (µg/ml) 36.96

Napeta cataria

Per cent cell viability 84.36 96.09 93.29 91.67 85.99 91.69 Per cent cell cytotoxicity 15.64 3.91 6.71 6.33 14.01 8.31 IC50 (µg/ml) -112.07

Levandula stoeches

Per cent cell viability 100 82.30 100 57.44 58.33 69.3 Per cent cell cytotoxicity 0 11.70 0 32.56 41.77 30.70 IC50 (µg/ml) 34.17

Borago officinalis

Per cent cell viability 95.63 83.20 84.27 71.12 46.83 71.21 Per cent cell cytotoxicity 4.27 16.80 15.73 28.88 43.17 `28.79 IC50 (µg/ml) 19.62

10


*Correspondence: [email protected]; 1,2Professor, Department of Veterinary Medicine, C.V.Sc., GADVASU, Ludhiana, Punjab, India;3PhD scholar, Department of Veterinary Medicine, CVSc., GADVASU, Ludhiana, Punjab, India; 4Senior Veterinary Pathologist, Teaching VeterinaryClinical Complex, C.V.Sc., GADVASU, Ludhiana, Punjab, India; 5Professor, Department of Veterinary Microbiology, CVSc., GADVASU, Ludhiana,Punjab, India.

CYTOLOGICAL AND BACTERIOLOGICAL EVALUATION OF TRACHEALASPIRATES FOR THE DIAGNOSIS OF LUNG AFFECTIONS IN HORSES

A.K. Sharma1, Charanjit Singh Randhawa2, Asmita Narang3*, Naresh Kumar Sood4 and TejinderSingh Rai5

Department of Veterinary Medicine, College of Veterinary and Animal Sciences,Guru Angad Dev Veterinary Sciences University, Ludhiana-141 004, Punjab, India

ABSTRACT

In present study four horses (two foals and two adult horses) with frank respiratory signs, inflammatory leukogram and radiologicfinding suggestive of respiratory involvement, and unresponsive to routine antibiotic treatment in field settings were subjected totranstracheal wash collection. Cytology of the stained smears and culture examination revealed Rhodococcus equi in two foalswhereas in other two horses, Staphylococcus spp. was isolated. Isolates of Rhodococcus equi were sensitive to erythromycin,amoxicillin, streptomycin, neomycin, norfloxacin and sulfadiazine, whereas Staphylococcus isolates were found sensitive to amoxicillin,ampicillin, doxycycline, gentamicin, neomycin, erythromycin, oxytetracycline, penicillin and streptomycin and resistant to ciprofloxacinand norfloxacin. Two horses (treated with amikacin and penicillin) and one foal (treated with erythromycin and rifampicin) respondedto recommended doses of antibiotic therapy and recovered smoothly. Therefore, transtracheal wash is a reliable technique fordiagnosis of lower respiratory diseases in horse. R. equi can be cultured from TTW of foals undergoing antibiotic treatment, and thuscould be of help in evaluating microbiological recovery in refractory animals.

Key words: Transtracheal wash, pneumonia, Rhodococcus equi, cytology, mirobiological examination.

IntroductionDisorders of respiratory system, particularly the lower

airways, are frequently diagnosed conditions in horses. Theseconditions have been identified as one of the main causes oftraining disruption and interruption of racing competitions inthoroughbred horses (Gerber et al., 2014; Wilsher et al., 2006).The disorders of the respiratory system are responsible for42% of decreasing performance of athlete horses, second todiseases of the musculoskeletal system (Hewson and Arroyo,2015; Allen et al., 2006).

Transtracheal wash (TTW) is a reliable technique fordiagnosis of lower respiratory tract affections in animals. It is acommonly employed procedure for culture of pathogens oflower respiratory tract. Bacteriological evaluation of atranstracheal aspirate may provide useful information onetiology, antimicrobial sensitivity and aid in the selection ofappropriate drugs (Ivester et al., 2014). Similarly, cytology ofTTW can be a useful diagnostic tool and help to differentiateinflammation, neoplasia and specific pathogens of lowerrespiratory tract (Hewson and Arroyo, 2015).

The present article reports the successful transtrachealwash aspiration and its utility in the diagnostic and therapeuticprotocol in lower respiratory tract affections in four horses.Inspite of its use worldwide, the technique of TTW for diagnosisof lower respiratory tract affection in animals has not beenemployed in India and the study appears to be the first of itskind.

Materials and MethodsFour horses presented to Large Animal Clinics, GADVASU

in different months of year with frank respiratory signs and

radiologic finding suggestive of respiratory involvement weresubjected to thorough clinical examination.

Samples collectionBlood samples (2 ml) were collected aseptically from

jugular vein in EDTA coated vials. Immediately after collectionwhole blood was used for determination of haemoglobin (Hb)and total leukocytes count (TLC) by ADVIA HaematologySystem. The blood smears were prepared and evaluated forDifferential Leukocytes Count (DLC) after staining the slidesby Leishman stain and compared with reference values forthe Indian equine breeds (Dedar et al., 2015).

TTW was collected percutaneously with adequate restrainand sedation, as required to immobilize the animal in astanding position. For this, about 10 cm² area was selected atthe ventral aspect of the neck where the trachea could begrasped and the rings could be easily palpated. The skin overthe selected site was clipped and surgically prepared andinfiltrated with 2-5 ml of 2% lidocaine. A small stab incisionwas given just through the skin, using the scalpel blade.

TTW was collected using Large Animal Trans-TrachealWash Kit (MILA International, Inc. USA) containing 10 gauge x2.25 inches steel introduction catheter (like 10 gauge needle)with 12 gauge x 70 cm (28 inches) flexible plastic flushingcatheter.

Following stab incision on the skin the steel introductioncatheter was inserted on the ventral midline, while stabilizingthe trachea with the other hand, and passed into the tracheallumen between two tracheal rings. Through this catheter,flexible flushing catheter was passed towards the lungs so asto reach thoracic inlet. For washing, the sterile saline (50 ml in

Received: 15.03.2017Accepted: 18.04.2017

mailto:[email protected];

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adult horses and 20-30 ml in foals) was injected through theflushing catheter into the trachea. After injecting, it was aspiratedimmediately, in an attempt to recover at least one third of thevolume of fluid administered (Fig. 1). If recovery wasunsuccessful, an additional amount of saline equal to the firstvolume was injected to obtain fluid adequate for all diagnosticprocedures. The same volume of fluid can be administeredthree times to receive adequate fluid for proper diagnosis andwithout any side effects to the animal. After the sample wasobtained, the catheter was withdrawn and the site was dressedwith pressure bandage for 24 hours.

The aspirated fluid or the transtracheal washings weretransferred into EDTA vials and sterile containers for cytologyand bacteriology respectively. For cytologic analysis, the TTWsamples were centrifuged (1000 rpm for 5 minutes)immediately and smears were prepared from the sediment bydiscarding the supernatant and stained with Leishman’s stain.

An aliquot (100 ìl) of TW fluid was cultured on Blood Agarand MacConkey Lactose Agar. Plates were incubated in aerobicconditions at 37°C for 18 h. The isolates were identified byGram staining, growth on specific media and biochemical tests.Antibiotic sensitivity testing was done by disk diffusion method,measuring zones of inhibition.

Results and Discussions

Case 1A 3-month- old filly, was presented with a history of fever

104-106° F, bilateral purulent nasal discharge, oculardischarge, coughing and inappetance for past 15 days. Theanimal was treated with broad spectrum antibiotics for oneweek by referring veterinarian with mild remission of clinicalsigns. On clinical examination, the foal had normal rectaltemperature (101.6°F), tachycardia (124 beats/ minute),tachypnoea (75 breaths/minute) and congested mucousmembrane. Auscultation of the lungs revealed wheezes incaudal left lung lobe, and harsh lung sounds in both the lungs.Haemogram revealed neutrophilic leukocytosis with toxicchanges in most of the neutrophils, few immature neutrophilsand microcytic hypochromic anaemia (Table 1). Thoracicradiography showed fluid density in cranial chest and moderateinterstitial pattern in caudal lung lobe. Thick purulenttranstracheal wash was obtained from the foal (Fig. 2a).Cytology of the stained smear of transtracheal wash revealedmassive number of degenerated neutrophils suggestive offrank suppurative pneumonia (Fig. 4). The diagnosis wasconfirmed 48 hours later when culture results were available.Bacteriological evaluation of the transtracheal wash samplerevealed Rhodococcus equi infection, sensitive to erythromycin,amoxicillin, neomycin, streptomycin and norfloxacin; andresistant to ampicillin, doxycycline, oxytetracycline and penicillin.The foal was treated with erythromycin @ 25 mg/kg b.wt. andrifampicin @ 5 mg/kg b.wt. and responded to the antibiotictherapy initally. But, it showed relapse after 15 days anddeveloped diarrhoea. Animal was then put on gentamicin andamoxicillin, selecting least toxic among the tested antibioticsirrespective of their sensitivity pattern. Animal made goodrecovery with resolution of pulmonary abscessations on repeatradiograph. It however, again developed fever, was positive forR. equi on cultural examination of washings and succumbed

to infection following prolonged therapy with different antibioticfor 5 months

Case 2A six and half-month-old colt, had a history of fever (106-

107°F) from past one month and productive coughing since 20days. There was no nasal discharge and appetite was normal.On clinical examination, the foal was bright, its rectaltemperature was 102.6°F, heart rate was 90 beats/ minute,and respiratory rate was 40 breaths/minute. Soft moist coughwas evident and lung auscultation revealed crackles in leftlung; and crackles and wheezes on right side with thecranioventral lung most severely affected. Haemogramrevealed macrocytic hypochromic anaemia and neutrophilicleukocytosis with toxic changes (Table 1). On thoracicradiography, there was severe nodular interstitial pattern inlungs with nodules up to 3 cm in diameter indicating lungabscessation (Fig. 3). Radiographic impression suspected R.equi infection. Thick purulent transtracheal wash was obtainedfrom the foal, and cytological and cultural isolation was done.The transtracheal wash sediment smear showed relativeneutrophilia besides some intracellular, pleomorphic rodscharacteristic of Rhodococcus equi in neutrophils and presenceof alveolar macrophages along with fibrin strands (Fig. 5a andFig. 5b). Bacteriological isolation from the transtracheal washsample yielded Rhodococcus equi infection. The isolate werefound to be sensitive to erythromycin, amoxicillin, neomycin,streptomycin and norfloxacin; and resistant to ampicillin,doxycycline, oxytetracycline and penicillin. The foal was treatedwith erythromycin @ 25 mg/kg b.wt. and rifampicin @ 5 mg/kgb.wt. and responded well to the antibiotic therapy and recoveredsmoothly.

Case 3A 15 years old male horse was presented with a history of

dyspnoea, fever and coughing for past 4 months. There was ahistory of long journey following racing and jumping eventleading to onset of dyspnoea. Feed and water intake, urinationand defaecation were normal. Vitals included 100.3°Ftemperature, 60 bpm heart rate, 62/ min respiration rate andmucus membrane was slightly congested. On auscultation oflungs, there were severe harsh lung sounds. Inspiratory andexpiratory dyspnoea was recorded. Thoracic radiographyrevealed moderate interstitial pattern in chest. Grossly thewashing appeared cloudy with thick strands of mucus in it (Fig.2b). Cytologic examination of the transtracheal wash smearsshowed chronic active inflammation overlapped by markedsuppurative inflammation. Bacteriological evaluation of atranstracheal aspirate yielded growth of Staphylococcus spp.,sensitive to amoxicillin, ampicillin, doxycycline, gentamycin,neomycin, erythromycin, oxytetracycline, penicillin,streptomycin, ciprofloxacin and norfloxacin. The animal wastreated with amikacin @ 25 mg/kg b.wt. and procaine penicillin@ 20,000 IU/kg b.wt. and showed marked improvement inabout 7-10 days. Antibiotics were continued for two weeks andanimal made successful recovery.

Case 4A 6-year-old mare had cough since two weeks. There was

normal feed and water intake by the animal. Urination and

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Table 1: Haematological alterations in affected horses on first day ofpresentation

defaecation was normal as well. Clinical examination of themare revealed temperature (100.5°F), heart rate (48 bpm) andcapillary refill time (

13


Fig. 2b: Transtranstracheal aspirate from a horse with pneumonia

Fig. 3: Cotton wool abscesses (nodular interstitial pattern) in lungs inRhodococcus pneumonia in second foal- case 2

Fig. 4: Stained smears from transtracheal aspirate showing massivenumber of degenerated neutrophils suggesting frank purulentpneumonia

Fig. 6 : Stained smears from transtracheal aspirate showing fibrinouspurulent pneumonia

Fig. 5b: Stained smears from transtracheal aspirate showingRhodococcus equi in neutrophils (Black arrow)

Fig. 5a: Stained smears from transtracheal aspirate showingRhodococcus equi in neutrophils (Black arrow)

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isolation. The cytologic evaluation of transtracheal aspiratehelps in the differentiation of lower respiratory tract diseasesof inflammatory and non inflammatory origin (Condasa et al.,2015). Cultures from the aspirates may provide usefulinformation about the offending organism and its culturalsensitivity which can aid in appropriate treatment (Mathilde etal., 2011). Cytological and microbiological evaluation of trachealwashes (TW) has proven to be important tool in the diagnosisof respiratory diseases in horses. In animals with lower airwaydiseases, the nasal flora may not reflect that in the lung andcultures are best taken as transtracheal aspirates of the lowerrespiratory system. Tracheal wash samples collected usingthe percutaneous transtracheal technique are preferred forbacterial culture because these are not contaminated byoropharyngeal organisms. However, care should be takenwhile interpreting microbiological cultures taken from the lowerrespiratory tract as it may be due to the normal microflora.Furthermore, possible complications like subcutaneousemphysema or infection at puncture site may be encounteredbut are usually infrequent.

ReferencesAllen KJ, Tremaine WH, Franklin SH (2006) Prevalence of inflammatory

airway disease in National Hunt horses referred forinvestigation of poor athletic performance. Equine Vet J.36: 529-534.

Condasa L, Ribeiroa, MG, Olivob G, Francoa MMJ, Gonoic T, MatsuzawacT, Yazawac K, Gonçalvesb RC and Borges AS. (2015)Nocardia nova identification in a transtracheal wash of ahorse with recurrent airway obstruction. Archivos deMedicina Veterinaria. 47: 231-235.

Dedar RK, Kumar V, Bala PA, Legha, RA, Pal Y and Gupta AK (2015)Hematology and plasma electrolyte profile of some equinesbreeds Vet. Pract. 16:212-215.

Gerber V, Tessier C and Marti E (2014) Genetics of upper and lowerairway diseases in the horse. Equine Veterinary Journal.47(4): 390-397.

Hewson J, Arroyo LG (2015) Respiratory Disease: DiagnosticApproaches in the Horse. Veterinary Clinics of NorthAmerica: Equine Practice. 31: 307-336.

Holcombe SJ, Robinson NE, Derksen FJ, Bertold B, Genovese R, MillerR, De Feiter R H, Carr EA, Eberhart SW, Boruta D, KaneeneJB (2006) Effect of tracheal mucus and tracheal cytologyon racing performance in Thoroughbred racehorses. EquineVet. J. 38: 300-304.

Ivester KM, Couetil LL, Moore GE, Zimmerman NJ, Raskin RE (2014)Environmental exposures and airway inflammation in youngthoroughbred horses. J. Vet. Internal Med. 28: 918-924.

Malikides N, Hughes KJ, Hodgson DR, Hodgson, JL (2003) Comparisonof tracheal aspirates and bronchoalveolar lavage inracehorses. 2. Evaluation of the diagnostic significance ofneutrophil percentage. Austr. Vet. J. 81: 685-687.

Mathilde LK, Magdesian G, Philip HK, Nicola P, Rhodes DM (2011)Comparison of the clinical, microbiological, radiological andhaematological features of foals with pneumonia causedby Rhodococcus equi and other bacteria. The Vet. J. 187:109-112.

Muscatello G (2012) Rhodococcus equi pneumonia in the foal - Part 2:Diagnostics, treatment and disease management. The Vet.J. 192: 27-33.

Pal M, Rahman T (2015) Rhodococcus equi: An emerging zoonoticpathogen. Annals of Vet and Anim. Sci. 2(1): 3-8.

Wilsher S, Allen WR and Wood JL (2006) Factors associated withfailure of thoroughbred horses to train and race. EquineVet. J. 38: 113-118.

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1Part of M.V.Sc. Thesis of first author; 2Co-P.I. Centre for Ethno Veterinary Practices and Alternative Medicine; 3Veterinary Officer, I/c VeterinaryHospital, Durgpura, Department of Animal Husbandry, Jhalawar, Raj.; 4Assistant Professor, Department of Veterinary Pharmacology; 5PI, LRS,Rathi, Bikaner

STUDY OF THE POTENTIAL OF OCIMUM SANCTUM IN SUB CLINICALMASTITIS IN RATHI CATTLE#

Aprajita Johri1, A.P. Singh, A. Gaur2, J.P. Kachhawa, Ankita Sharma3, P. Sharma4 and R.K. Joshi5Department of Veterinary Clinical Medicine, Ethics and Jurisprudence

College of Veterinary and Animal ScienceRajasthan University of \veterinary and Animal Sciences, Bikaner-334 001, Rajasthan, India

ABSTRACT

The present investigation was carried out at local veterinary hospitals and LRS, Rathi of College of Veterinary and AnimalScience, Bikaner where milk samples of 48 quarters from 12 apparently healthy Rathi cows were subjected to varioustests viz. modif ied California mastitis test (MCMT), total somatic cell count (TSCC), pH, electrical conductivity (EC) andcultural examination for the diagnosis of sub-clinical mastitis. The aim of present investigation was to evaluate thetherapeutic potential of Ocimum sanctum (Tulsi). The 12 Rathi cows were divided into two groups with 6 animals in eachgroup viz. Group I powdered O. sanctum @ 600 mg/kg b.wt., Group II aqueous extract of O. sanctum @ 150 mg/kg b.wt.On 8th day post-treatment, there was nonsignif icant decrease (p>0.05) in electrical conductivity (EC) in both the groups.There was signif icant change (p>0.05) in pH in Group I and Group II post-treatment. Similarly, there were significantchanges (p

16


mg/kg body weight, orally daily for 7 days. After administrationof treatment in both groups for 7 days evaluation of milksamples was done on 8th day to note the efficacy of the drug.

The data obtained in the research work undertaken werestatistically analyzed and compared using standard formulagiven for mean, standard error and analysis of variance as perthe procedures explained by Snedecor and Cochran (1994).

Results and DiscussionsModified California mastitis test (MCMT)

In the present study, 48 quarter milk samples of 12 Rathicows were screened by modified California mastitis test forsubclinical mastitis and 13 (27.08%) quarter milk were foundpositive. California mastitis test reactions were scoredaccording to Radostits et al. (2007). Similar findings in cattlewere also recorded by previous workers viz. Reddy et al. (2014)and Hanam et al. (2015).

Cultural examinationIn Group I, 7 quarters milk samples and in group II 6

quarters milk samples were subjected to culture and sensitivityaccording to results of CMT. The bacterial growth waspreliminary identified as Staphylococcus spp. E. coli, Klebsiellaspp. and on the basis of colonial characteristics, morphology,Gram’s reaction, catalase and oxidase activities.

Out of 7 quarters in group I, Staphylococcus spp. wasisolated from 3 quarters (42.85 per cent) and E. coli infectionwas isolated from 3 quarters (42.85 per cent) and 1 quartershowed (14.30 per cent) mixed infection of Klebsiella spp. andStaphylococcus spp. While Out of 6 quarters in group II,Staphylococcus spp. was isolated from 3 quarters (50.00 percent), E. coli isolated from 2 quarters (33.33 per cent) and 1quarter showed (16.67 per cent) mixed infection of both bacterialspecies viz. E. coli and Staphylococcus spp.

In vitro antibacterial activity sensitivity of herbal preparationsThe antibacterial activity of the herb against particular

mastitis pathogen was determined as proportion of zones ofgrowth inhibition formed by the herbal preparations and thestandard antibiotic i.e. cefotaxime depicted as percentage. Thezones of inhibition produced by powdered and aqueous extractO. sanctum and antibacterial activity for different bacteria interms of zones of inhibition formed by cefotaxime have beendepicted in Table 1.

In this study, the average zone of inhibition shown bypowder of O. sanctum (Tulsi) were found 12.60 mm againstStaphylococcus spp. and 12.30 against E. coli and averagezone of inhibition shown by aqueous extract of O. sanctumagainst Staphylococcus spp. was 20.00 mm and 18.00 mmagainst E. coli. The zone of inhibition shown by control antibioticcefotaxime against Staphylococcus spp. was 23.5 mm and24 mm against E. coli during course of the presentinvestigation.

The aqueous extract of O. sanctum showed higherantibacterial activity against Staphylococcus spp. 85.10 percent and against E. coli 75 per cent as compared to powder ofO. sanctum which showed antibacterial activity againstStaphylococcus spp. 53.87 per cent and against E. coli 51.37per cent.

Mukherjee (2006) also recorded the similar zones ofinhibition ranging from 12.5 to 18.3 mm for aqueous extract of

O. sanctum leaves against S. aureus, S. agalactiae and E. coli.Ali and Dixit (2012) observed the zones of inhibition forflavanoids of O. sanctum as 20.12, 20.75, 20.95, 19.55 and20.1 mm against E. coli, Proteus, S. aureus, Staphylococcuscohni and Klebsialla pneumonia, respectively. Gupta (2010)reported that at a concentration of 100 mg/ml, the hydro-alcoholic (1:1) extract of O. sanctum leaves, exhibited theantibacterial activity against S. aureus, S. agalactiae and E.coli. Prakash and Gupta (2005) found eugenol (1-hydroxy-2-methoxy-4-allylbenzene), the active constituent present in O.sanctum, to be largely responsible for its therapeutic potential.

Treatment trialThe efficacy was recorded after observing the results of

modified California mastitis test (MCMT), pH, electricconductivity (EC) test and total somatic cell count (TSCC) (Table2).

Group I animals were administered powdered Ocimumsanctum @ 600 mg/kg and Group II animals wereadministered prepared aqueous extract of Ocimum sanctum@ 150 mg/kg body weight, orally daily for 7 days. To determinethe therapeutic efficacy of prepared herbal powder andaqueous extract of leaves of Ocimum sanctum; samples werecollected on day 0 as pre-treatment and on 8th day as post-treatment to record the changes in electrical conductivity (EC),milk pH and somatic cell count (SCC).

There was significant (p

17


398.Atakisi O, Oral H, Atakisi E, Merhan O, Metin PS, Ozcan A, Marasli

S, Polat B, Colak A and Kaya S (2010) Subclinical mastitiscauses alterations in nitric oxide, total oxidant andant ioxidant capacity in cow milk . Res.Vet. Sc i.89(1):10-13.

Bauer AW, Kirby WMM, Sherris JC and Turek (1966) Antibioticssusceptibility testing by a standardized s ingle discmethod. Am. J. Cli. Pathol. 45: 493-496.

Chahar A (2001) Studies on some epidemiological and diagnosticaspects of bovine subclinical and clinical mastitis.M.V.Sc. Thesis submitted to Rajasthan AgriculturalUniversity, Bikaner, Rajasthan.

Chevallier A (1996) The Encyclopedia of Medicinal Plants. DoorlingKindersley, London, New York, Stutgart, Moscow.

Cowan ST and Steel KJ (1975) Mannual for the identification ofmedical bac teria. Cambr idge Univers ity Press,Cambridge.

Gupta DK (2010) Evaluation of immuno-therapeut ic and

ant ioxidative ef fects of some herbs in bovinesubclinical mastitis. Ph.D dissertation, Pant Universityof Agriculture and Technology, Pantnagar, India.

Hanam M, El-Hewairy S, Galal A, Hamouda RH and Dohreig RMA(2015) Immunological and bacteriological f indingsassociated with subclinical mastitis in dairy farm. LifeSci. J. 12(2):139-146.

Mukherjee R (2006) Antibacterial and therapeutic potential ofOcimum sanctum in bovine subclinical mastitis. IndianVet. J. 83(5): 522-24.

Mukherjee R, Dash PK and Ram GC (2005) Immunotherapeuticpotential of Ocimum sanctum L. in bovine subclinicalmastitis. Res. Vet. Sci. 79(1): 37-43.

Prakash P and Gupta N (2005) Therapeutic uses of Ocimumsanc tum L . wi th a note on eugenol and i tspharmacological actions: A short review. Indian J.Physiol. Pharmacol. 49(2):125-131.

Radostits OM, Gay CC, Hinchcliff KW and Constable PD (2007)Veterinary Medicine. A text book of diseases of cattle,horses, sheep, pigs and goats. 10 th Ed. Saunders,Elsevier, London.

Reddy BSS, Kumari KN, Reddy YR, Reddy MVB and Reddy BS(2014) Comparison of dif ferent diagnostic tests insubclinical mastitis in dairy cattle. Int. J. Vet. Sci. 3(4):224-228. www.ijvets.com

Richter O, Kneinschioth E, Gruber R and Hauge G (1960) TheCalifornia mastitis test for examination of can samplesin relation to cont rol udder health . Berliner undMûncheer tierÓrztliche Wochenschrift, 74(7):127-130.

Sadekar RD, Pimprikar NM, Bhandarkar AG and Barmase BS(1998) Immunopotentiating effect of Ocimum sanctumL. dry leaf powder on cell mediated immune responsein poultry, naturally infected IBD virus . Indian Vet. J.75: 168-69.

Singh E, Sharma S, Dwivedi J and Sharma S (2012) Diversifiedpotentials of Ocimum sanctum L. (Tulsi): An exhaustivesurvey. J.Natural Prod. Plant Res. 2(1):39-48.

Snedecor GW and Cochran W G (1994) Statistical Methods. 8thEd. IOWA State, University Press, Ames, IOWA, USA.

Yang F Li, and Li XS (2015) Role of antioxidant vitamins andtrace elements in mastitis in dairy cows. J. Adv. Vet.Anim. Res. 2 (1): 1-9.

Table 1: Mean ± SE values of zones of inhibition (mm) of powder of leaves of O. sanctum againstsubclinical mastitis pathogens of Group I and Group II Rathi cows

Pathogen

Group I Average Zones of Inhibition (mm) Per cent Sensitivit

y

Group II Average Zones of Inhibition

(mm) Per cent sensitivity O. sanctum Cefotaxime O. sanctum Cefotaxime

Staphylococcus spp.

12.60 23.50 53.61 20.00 23.50 85.10

E. coli 12.30 24.00 51.25 18.00 24.00 75

Table 2: Pre and post-treatment mean ±SE values of EC, pH, SCC inapparently healthy and Group I and II suffering from subclinical mastitis.

Group

Diagnostic Test

E.C. (mS) Mean ± SE

pH Mean ± SE

SCC (million/ml) Mean ± SE

Healthy 4.78a±0.105 6.43a±0.058 2.74a±0.18

I

Pre-treatment 5.73b±0.189 7.02b±0.144 5.62b±0.68 Post-

treatment 5.66b±0.177 6.95b±0.141 5.47b±0.69

II

Pre-treatment 6.16b±0.130 7.42c±0.03 6.40c±0.29 Post-

treatment 5.76b±0.09 6.84b±0.08 4.82b±0.42

Table 3: Efficacy of treatments given in various groups

Group

Pre-treatment

CMT positive quarters

Post –treatment

CMT positive quarters

Per cent Efficacy

I 7 3 42.85 II 6 4 66.66

http://www.ijvets.com

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*Part of M.V.Sc. thesis work submitted by the first author to Chaudhary Sarvwan Kumar Himachal Pradesh Krishi Vishvavidyalays, Palampur 176062 (H.P.); 1Corresponding author: Professor of Veterinary Medicine, KVK , CSK HPKV, Dhaulakuan- 173 031, Distt. Sirmour (H.P.);E-mail:[email protected]

THERAPEUTIC EFFICACY OF PARENTERAL ADMINISTRATION OFGENTAMICIN AND ENROFLOXACIN ALONG WITH PARENTERAL FLUID

THERAPY IN THE TREATMENT OF NEONATAL DIARRHOEIC CALVES – ACOMPARATIVE STUDY#

Himanshu and B. Pal1Department of Veterinary Medicine, Dr.G.C. Negi College of Veterinary and Animal Sciences

Chaudhary Sarwan Kumar Himachal Pradesh Krishi Vishvavidyalaya, Palampur-176 062, Himachal Pradesh, India

ABSTRACT

Neonatal calf diarrhoea, being the leading cause of calf mortality in dairy farm. The clinical trial was conducted in 16 neontal dirrahoeiccalves suffering from severe degree of dehydration associated with Escherichia coli. These diarrhoeic calves were divided into twoequal groups consisting of 8 in each. The group I was treated with parenteral fluid therapy (Ringer’s lactate solution) along with intramuscular administration of gentamicin @ 3 mg/kg b. wt bid x 4 days. The calves of group II were treated with Intravenous administra-tion of Ringer’s lactate solution along with subcutaneous administration of enrofloxacin @ 5 mg/kg b wt bid x 4 days. The therapeuticefficacy of group I and II were assessed and compared on the basis of restoration of clinical appraisal, haemato-biochemicals andelectrolytes changes. The results showed that in severe dehydrated calves in group II treated with parenteral fluid therapy along withsubcutaneous administration of enrofloxacin showed better improvement as compared to group I treated with parenteral fluid therapyalong with intramuscular administration of gentamicin.

Key words: Diarrhoea, Escherichia coli, enrofloxacin, gentamicin, neonatal calves.

IntroductionNeonatal calves with diarrhoea often have small intestinal

overgrowth with Escherichia coli bacteria, regardless of theinciting cause for the diarrhoea, and 30% of systemically illcalves with diarrhoea have bacteraemia, predominantlybecause of E. coli. Antimicrobial treatment of diarrhoeic calvesshould therefore be focused against E. coli in the smallintestine and blood, the 2 sites of infection. Antimicrobial efficacyis therefore, best evaluated by the clinical response as well ashaemato-biochemicals and electrolytes changes of a numberof calves to treatment, with calves assigned in two treatmentgroups. (Khan and Zaman, 2007) reported that mild dehydrationmight be corrected by oral rehydration solution while inmoderate and severe dehydration; rehydration solutionintravenous administration (i/v) along with antibiotic is bettertreatment for neonatal calf diarrhoea. Although theadministration of intravenous fluids and oral electrolytesolutions plays a central role in treatment, the efficacy ofantimicrobial agents in treating calf diarrhoea is controversial.Therefore, the present study was undertaken to observe thetherapeutic efficacy of parenteral administration of gentamicinand enrofloxacin along with parenteral fluid therapy in thetreatment of neonatal diarrhoeic calves suffering from severedegree of dehydration.

Materials and MethodsSource of neonatal calves: The study was carried out in

neonatal calves in the calf section of livestock farm of CSKHPKV, Palampur (H.P.). During the study period, 149 calveswere born. The diarrhoeic symptoms were observed in newborn calves and 16 calves aged 4-6 days suffering from

diarrhoea with severe degree of dehydration were consideredin this study.

Studies on diseased and healthy calves: The study wasundertaken on 16 diarrhoeic calves. The diarrhoeic calves wereagain divided into 2 groups having 8 calves in each group.Besides, 8 neonatal healthy calves were taken for healthy group.

Cultural examination of faecal samples: Isolation ofbacteria was done on repeated bacteriological culture bystandard bacteriological procedures (Cruickshank et al., 1975).Then, the gram negative bacilli were further identified usingcharacter differentiation Tables by Edwards and Ewing (1972)and Bergey’s manual of determinative bacteriology (1974).

Recording of clinical observations and parameters: Thefaecal consistency and extremity temperature were observed.Besides recording of rectal temperature, respiration rate andpulse rate were done as per the standard method describedby Rosenberger (1979), Michell et al. (1989), Kelly (1984) andRadostits et al. (1994).

Assessment of degree of dehydration: The degree ofdehydration was assessed on the basis of some clinicalparameters as described by Michell et al. (1989).

Collection of blood samples: About 5 ml of venous bloodwas collected in heparinized syringe from each neonatal calfon the day of diarrhoea for estimation of biochemicals andelectrolyte changes viz., plasma glucose, total plasma protein,plasma creatinine, plasma urea nitrogen plasma sodium,plasma potassium and plasma chloride. Apart from this, oneml of blood was collected in EDTA syringe, from each neonatalcalf for examination of pack- cell volume (PCV).

Haematological studies: Immediately after collection of



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blood, PCV was determined as per the methods described byJain (1986).

Biochemicals estimation: The following biochemicalestimations were carried out viz., plasma glucose, total plasmaprotein, plasma urea nitrogen, plasma creatinine. Biochemicalparameters were estimated using specific diagnostic kits onRA-50 (Chemistry analyser)

Electrolytes analysisSodium and potassium: Sodium and potassium were

estimated by using flame photometer as per the methoddescribed by Oser (1965). The values were expressed in mEq/l.

Chloride: Plasma chloride was estimated by colorimetricmethod as described by Frankel et al. (1970) by using chloridekit. The values were expressed in mEq/l.

Therapeutic schedule: Calves showing severe degree ofdehydration in two different groups viz., group I an

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