Research in Veterinary Science 93 (2012) 655–661

Contents lists available at SciVerse ScienceDirect

Research in Veterinary Science

journal homepage: www.elsevier .com/locate / rvsc

Study of the risk factors associated with Neospora caninum seroprevalencein Algerian cattle populations

Farida Ghalmi a,⇑, Bernard China b, Asma Ghalmi a, Darifa Hammitouche a, Bertrand Losson c

a Ecole Nationale Supérieure Vétérinaire d’Alger, Algiers, Algeriab Institut Scientifique de Santé Publique, Brussels, Belgiumc Université de Liège, Faculté de Médecine Vétérinaire, Liège, Belgium

a r t i c l e i n f o a b s t r a c t

Article history:Received 13 May 2011Accepted 12 December 2011

Keywords:Neospora caninumCattleRisk factorsSeroprevalenceAlgeriaIFAT

0034-5288/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.rvsc.2011.12.015

⇑ Corresponding author. Address: Ecole Nationale SBP 161 Hacène Badi, El Harrach, Algiers, Algeria. Tel.: +(0) 21 824481.

E-mail address: fghalmi@yahoo.fr (F. Ghalmi).

Bovine abortions due to Neospora caninum infection were reported worldwide. The situation in Algeriawas unknown. For the evaluation of the prevalence of N. caninum and its associated risk factors, 799 cattlebelonging to 87 farms of the north and northeast of Algeria were analyzed. The cattle were divided intoimported cattle, local cattle and improved cattle corresponding to breeding between imported and localcattle. Sera were examined for the presence of N. caninum antibodies by indirect fluorescence antibodytest. The overall seroprevalence for the 87 farms was 52.87% (41.28–62.71%). The overall animal sero-prevalence was 19.64% (16.82–22.45%). The seroprevalence of N. caninum in local cattle (34.28%) was sig-nificantly higher (p < 0.05) than in modern (16.04%) and improved (18.64%) cattle. The risk factorsanalysis indicated that cattle population, geographical location, dog presence, season, global farmhygiene or the presence of abortion were significantly associated with seroprevalence.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction life and there are no clear signs of protective immunity (Dubey,

Neospora caninum is an obligate intracellular protozoan parasitewhich has emerged as a cause of infectious abortion in cattleworldwide (Dubey et al., 2007). In many countries N. caninum isthe most frequently diagnosed cause of bovine abortion (Dubeyand Lindsay, 1996).

The biological cycle of N. caninum is heteroxenous. Dogs, coyotesand gray wolf (Canis lupus) are the only species recognized as defin-itive hosts, in which the sexual phase of N. caninum cycle occurs,resulting in the shedding of oocysts in the feces (Mc Allister et al.,1998; Gondim et al., 2004; Dubey et al., 2011). It has been estab-lished that bovines are the major intermediate hosts of the parasite.The routes of infection in cattle for N. caninum consist of vertical ortransplacental transmission, and horizontal or oral transmission(Dubey, 1999). However, vertical transplacental infection from in-fected dams to their offspring appears to be the major natural routeof infection, and congenitally infected calves remain persistentlyinfected and can infect their offspring (Anderson et al., 1997).

The pathogenesis of neosporosis in cows is complex (Dubeyet al., 2006) and it is not well understood why some animals abortand others do not. Infected cattle remain carriers of the parasite for

ll rights reserved.

upérieure Vétérinaire d’Alger,213 (0) 21 525132; fax: +213

2003).No fully effective vaccine or treatment is available to prevent or

cure the infection (Dubey et al., 2006). The study of risk factors ofherd infection by N. caninum is important for the development andimplementation of measures to control bovine neosporosis (Dubeyet al., 2007).

For epidemiological studies, serology is the most used tech-niques. The Indirect Fluorescence Antibody Test (IFAT) is often usedas a reference serological test for the detection of N. caninum (Dubeyand Lindsay, 1996). Many serologic surveys of N. caninum infectionin cattle worldwide and the risk factors for N. caninum in cattle havebeen discussed broadly in the literature (Dubey et al., 2007).

In Algeria, the cattle population is estimated to 1.6 million,including 53.55% dairy cattle and representing an important foodsource (MADR, 2008). Despite the massive imports of dairy cowswith high genetic potential, the milk production remains low. It isestimated at 1.38 million tons in 2000 (Ghozlane et al., 2003) either0.26% of the world production. This production is in total inade-quacy with the continued strong growth of the population becauseit covers only 40% of the needs (MADR, 2008). Although theneosporosis has been diagnosed in the main dairy and beef cattle-producing countries, no previous study has been published in theliterature about the disease in Maghreb. In Algeria, there is onlyone published report available on its occurrence that indicated20.47% seropositivity in 781 dogs in Algiers (Ghalmi et al., 2009b).This demonstrates the wide diffusion of N. caninum in this area. Astudy of significant factors that influence the distribution of

656 F. Ghalmi et al. / Research in Veterinary Science 93 (2012) 655–661

N. caninum in this area, could be useful to better know the epidemi-ology of N. caninum infection in Algerian cattle. Therefore, the aimof this study was mainly to identify the risk factors associated withN. caninum seroprevalence in different cattle populations in Algeria.

2. Material and methods

2.1. Studied area

A cross-sectional study was designed to study the relationshipbetween the N. caninum serological status of dairy cattle and vari-ous potential risk factors. The populations of interest were dairyfarms in two distinct regions of Algeria. The geographical locationof the farms is presented in Fig. 1. These farms were located eitherin the northern regions (Algiers County) or in the eastern regions(Bejaia and Setif). The sampled region represented around25, 000 square kilometers.

Region-I was located in North Algeria (36� 490 N; 3� 00 E) andcontain about 727 cattle farms characterized mainly by importedcattle with high genetic potential and region II was in the westof the country (36� 100 N; 5� 240 E). The choice of region-II was jus-tified by the fact that more than half of the local breed cattle arelocated in the eastern part of the country.

The distance between the two regions is approximately 400 km.

2.2. Sampling procedures

The animals that were studied belonged to 87 dairy farms inabove cited regions. In the region of Algiers 72 farms were sampledrepresenting 10% of the farms present (n = 727).

Fig. 1. Map of the analyzed regions. The regions in gray were those where the sampledRouiba, Baraki, Birtouta. The regions of Bejaia and Setif constitute the eastern regions.

A total of 799 serum samples were assessed. They represented7% of the cattle population (n = 11,500) in the studied region(INMV, 2009).

The minimum number of cattle to be tested on each farm wasestablished as 10 (Cannon and Roe, 1982), corresponding to theprobability of detecting at least one seropositive animal per farm.On farms with less than 10 bovines, all of them were tested.

A stratified sampling was performed each stratum was a cattlepopulation. Three cattle populations were considered: importedcattle (IPC), improved cattle (IMC) and local catte (LC). For eachpopulation, the relative precision was similar: 25.5%, 22.2% and27.2% for IPC, IMC and LC respectively.

2.3. Collection of blood and epidemiological data

Sera from 799 bovines belonging to 87 farms were collected.324 cattle from 30 farms were IPC, 370 cattle from 43 farms wereIMC and 105 cattle from 14 farms were LC.

The study period ranged from September 2006 to January 2009.Blood samples were collected by coccygeal venipuncture, usingidentified dry tubes. The serum was removed after centrifugationat 2700g for 10 min and stored at �20 �C until analysis.

Simultaneously, blood was collected from dogs (n = 107) pres-ent in 67 out of the 87 farms. Data for N. caninum seroprevalencein those dogs were published previously (Ghalmi et al., 2009b).

An epidemiological questionnaire was fulfilled by farmers whowere asked to answer the questionnaire in order to obtain informa-tion on the risk factors such as: farm location, herd size, cattlebreed, cattle age, presence of abortion in the five last years, pres-ence of pregnant cows, stadium of pregnancy, presence of dogs,

farms are situated. The Algiers region has been divided into 4 sub-regions, Zeralda,

F. Ghalmi et al. / Research in Veterinary Science 93 (2012) 655–661 657

presence of aborted cows. Additional information such as the dateof collection and the general hygiene of the farm were also col-lected. A farm was considered as positive if at least one cow waspositive.

2.4. Variables

Several factors that could influence the seropositivity of cattleto N. caninum were analyzed. Considering the age of the cattle,the cattle were divided in age classes (<3 years, from 3 to 7 yearsand >7 years).

For breeds, we considered the imported breeds (Holstein, Prim’Holstein, Friesan, Montbeliarde, Fleckvieh), the improved cattlewhich are crossed breeds and the local cattle that include Algeriansbreeds (mainly Brown of Atlas) (Table 2).

For the areas agro-ecological, four classifications were per-formed. The sampling areas were situated either in the seasideregion, in the Mitidja region, Tellian Atlas or Highlands. The Mitidjais a great wet plain (1300 km2) in the south east of Algiers. In ourstudy the Mitidja is represented by the regions of Birtouta andBlida (Fig. 1). The Tellian Atlas consists of a chain of mountainsalong the coast of Mediterranean Sea (region of Bejaia).

The agricultural subdivisions (corresponding approximately toDaïra) were considered (Table 2): Birtouta, Baraki, Rouiba andZeralda. In Algeria, Daïra includes several municipalities.

The gestation status of 461 cows were analyzed, 269 (58.35%)were gestating and 192 (41.65%) were non-gestating.

The presence of dogs in farms and the seasons (the sampleswere taken during the four seasons) were also considered.

The global hygiene of the visited farms was also taken intoaccount. We observed mainly the maintenance of the premisesand animals and we have classified the farms into three categories(good, average and bad). We were inspired by European regulation(EC 852/2004/BEHL15, European parliament, 2004).

2.5. Serological test

Serum samples were analyzed by IFAT to determine the pres-ence of N. caninum specific antibodies, as previously described(Ghalmi et al., 2009a). In order to determine the IFAT cut off, a val-idation step has been performed on 100 sera by comparing IFAT tothe validated ELISA herdcheck (IDEXX LABORATORIES) (Wu et al.,2002) largely used in cattle for the determination of seropreva-lence against N. caninum (Dubey et al., 2007). The sera weretitrated in a twofold dilution from 1/100 to 1/1600 and the speci-ficity, the sensitivity and the coefficient of Kappa were calculatedusing the ELISA has the reference method. The results indicatedthat the best agreement was found when the 1/200 dilution wasused (sensitivity = 90%; specificity = 100% and Kappa = 0.82).Therefore, a positive sample in IFAT was only considered at thedilution 1/200 or upper.

Reactions with tachyzoites presenting a total peripheral fluores-cence were considered as positive. Positive and negative sera wereincluded on each slide. All the IFAT positive samples were con-firmed by immunoblot as previously described (Ghalmi et al.,2009b).

2.6. Data analysis

Statistical differences in proportions were compared using thex2-test (Yates corrected) or Fisher’s exact test. The strength of theassociation between serological status and the epidemiological fac-tors assessed here was estimated by computing the odds ratio (OR)for infection occurrence when the factor was present. A multipleregression was also performed. Specificity, sensitivity, Kappa indexand the 95% confidence interval (CI) were also calculated. A multiple

correspondence analysis (ACM) was performed. The used software’swere Winepiscope 2.0, MedCalc and XLstat. The level of statisticalsignificance used was 5%. If the p values were lower, it was indicatedin the text.

3. Results

3.1. Seroprevalence

The overall herd prevalence of N. caninum antibodies among the87 farms was 52.87% (CI 95%: 41.28–62.71%). Antibodies toN. caninum were detected in 157 cattle out of 799, with a generalseroprevalence of 19.64%. When the different cattle populationswere considered, the seroprevalence values were 16.04%, 18.64%and 34.28% for IPC, IMC and LC, respectively (Table 1).

The seroprevalence in LC was significantly (p < 0.01) higherthan in the two others populations. All the positive samples wereconfirmed as positive by immunoblot. Four profiles were detected(Fig. 2), among the 157 tested sera, 19 (12.9%) 71 (48.3%), 41(27.9%), 16 (10.9%) showed profile 1, profile 2, profile 3 and profile4 respectively.

3.2. Risk factors

Concerning the risk factors associated with seroprevalence, theresults of the statistics are summarized in Table 2. Considering theage of the cattle, when the seroprevalence results were consideredall together, no significant association (p > 0.05) between age clas-ses and seroprevalence was shown. Nevertheless, the seropreva-lence is significantly (p < 0.05, OR = 0.64) less important in youngcattle (<36 months). When the different populations were consid-ered, a significant association (p < 0.01) was found only in im-ported cattle (IPC) with a significant lower prevalence (2.7%) inyoungest cattle (<36 months) than in other age classes (19.81% incattle from 36 to 84 and 21.21% in cattle >84 months).

For breeds, the seroprevalence was evaluated into the differentbreeds present in the studied farms. When the results were takenall together, there was no significant association between breedsand seroprevalence (OR = 1; p > 0.05). Nevertheless, The therewas a strong association between local breeds and seroprevalence(OR = 2.47, p < 0.05).

There was a significant association between agro-ecologicalareas and the seroprevalence (p < 0.05). The belonging to seasideand Tellian Atlas areas were considered as a risk factor. Whenthe high titers were considered, there was a significantly higher(p = 0.02) number of positive samples in Mitidja area for titer1600 (data not shown). At the population level, the IPC and theIMC were considered since the LC were mainly present in the eastpart of Algeria. For IPC there was a significant difference (p < 0.01)between the seroprevalence and the seaside and Mitidja regionswhich is not the case for IMC (p = 0.52).

When the regions were considered (Table 2), there was no sig-nificant association between the farm location and the seropreva-lence (p > 0.05). The belonging to Zeralda (west of Algiers, Fig. 1),Rouiba (east of Algiers) and Bejaia (east of Algeria) region was con-sidered as a risk factor (OR > 1, p < 0.05) although the belonging toBirtouta region (south of Algiers) was shown to be protective(OR < 1, p < 0.05).

In our study, the seroprevalence in pregnant and non-pregnantcows was not significantly different (p > 0.05). The conclusion wasthe same for all cattle populations. Among the 192 pregnant cows,the pregnancy stage was known for 179 (93.23%) of them. Theseroprevalence was significantly (p < 0.01) lower for early pregnantcows (1–3 months) and significantly (p < 0.05) higher for mid-pregnancy cows (4–6 months). If the cows with an abortion history

Table 1Seroprevalence in function in the populations studied.

Populationsof cattle

n (%) Negative Positive Seroprevalence(%) (CI 95%)

IPC 324 (4.55) 272 52 16.04 (11.92–20.07)LC*

IMC 370 (46.30) 301 69 18.64 (14.55–22.64)LC*

LC 105 (13.14) 69 36 34.28 (25.01–43.54)IPC,IMC*

Total 799 642 157 19.64 (16.82–22.45)

* Seroprevalence significantly different (p < 0.01) for the indicated populations.

658 F. Ghalmi et al. / Research in Veterinary Science 93 (2012) 655–661

(n = 54) were considered, there was a significant association withseroprevalence. Interestingly, when the populations were consid-ered, the OR was 9.77 (3.76–25.41), 9.43 (3.26–27.27) and 1.4(0.43–4.54) for IPC, IMC and LC, respectively. Therefore, the associ-ation between the seroprevalence and the fact that cows alreadyaborted in the past was only significant (p < 0.01) for IPC andIMC but not for LC.

When the seasons were considered, the seroprevalence was sig-nificantly higher in the spring time and significantly lower in

Table 2Number of cattle, seroprevalence (%) for N. caninum antibodies by general characteristics; ochi-squared test corrected for design effect.

Variable n Seroprevalence (CI) (%)

Age (months)<36 208 14.90 (9.96–19.83)36–84 456 20.61 (16.81–24.38)P84 135 23.88 (16.46–31.13)

BreedFleckvieh 26 19.23 (15,98–22,01)Holstein 210 21.90 (20,06–21,38)Montbéliarde 88 14.77 (13,21–14,78)Crossed breeds 370 18.64 (14.55–22.64)Local breeds 105 34.28 (25.01–43.54)

Agro-ecological areasMitidja 510 14.90 (13.86–14.90)Seaside 210 24.28 (23.59–24.40)Tellian atlas 53 37.73 (35.17–38.82)Highlands 26 19.23 (15.98–22.01)

Region (Daïra)Zeralda 209 24.28 (23.59–24.40)Baraki 52 13.46 (11.70–14.29)Rouiba 61 36.06 (34.42–37.57)Birtouta 398 11.05 (10.84–11.15)Bejaia 53 37.73 (35.17–38.82)Setif 26 19.23 (15.98–22.01)

GestationYes 269 18.21 (13.49–22.90)No 192 17.70 (12.19–23.20)

Stage of gestation (month)1–3 53 7.5 (0–14)4–6 73 26.02 (15.73–36.26)7–9 53 20.75 (9–31.83)

AbortionYes 54 48.14 (34.50–61.69)No 669 17.04 (14.09–19.90)

Presence of dogsYes 645 22.01 (18.73–25.26)No 154 9.74 (4–13.61)

SeasonsAutumn 348 11.2 (7–14.58)Winter 150 17.33 (11.12–23.47)Spring 192 36.45 (29.45–43.34)Summer 109 20.18 (12.42–27.77)

HygieneGood 507 14.7 (11.55–17.84)Average 225 24 (18.30–29.69)Bad 67 43.7 (31.58–55.81)

S = significant (p < 0.05); NS = not significant (p P 0.05).

autumn. This observation was done for each studied populations(p < 0.01). The distribution of high titers was also significantly dif-ferent with the season since the highest titers were also observedin the spring time (p < 0.01).

The good hygiene status of the farm was a protective factor andthe bad hygiene status of the farm was a risk factor. This observa-tion is independent of the studied population.

There was a significantly (p < 0.01) higher seroprevalence incattle in contact with dogs. At the population level, the associationbetween dogs and seroprevalence in cattle was only significant forIPC (Table 3).

When a multiple regression analysis was performed only thepresence of dogs, the fact that the cow has aborted and the seasonwere significant (p < 0.05).

A multiple correspondence analysis (Fig. 3) allowed visualizingthe factors associated with seropositivity or seronegativity. Thisrepresentation indicated that the variables closest to the positiveserology were the presence of abortion, the seaside region, thebad hygiene of the farm, the spring season and the presence ofdogs.

dds ratio (OR) for seropositive results, 95% confidence interval (CI) and p-value for the

OR (CI) Type of effect

1.01 (0.77–1.33) NS No effect0.642 (0.421–0.993)S Protection1.154 (0.809–1.646)NS No effect1.340 (0.881–2.084)NS No effect

0.88 (0.75–1.04)NS No effect0.973 (0.361–2.621)NS No effect1.208 (0.821–1.778)NS No effect0.682 (0.368–1.264)NS No effect0.888 (0.625–1.262)NS No effect2.471 (1.578–3.869)NS Risk

0.76(0.64–0.90)S Protection0.45 (0.316–0.641)S Protection1.462 (1.001–2.135)S Risk2.691 (1.500–4.841)S Risk0.971 (0.361–2.621)NS No effect

0.99 (0.88–1.12)NS No effect1.471 (1.001–2.150)S Risk0.621 (0.270–1.400)NS No effect2.521 (1.450–4.390)S Risk0.320 (0.220–0.460)S Protection2.690 (1.500–4.840)S Risk0.970 (0.360–2.620)NS No effect

0.87 (0.600–1.270)NS No effect0.81 (0.530–1.230)NS No effect

1.09 (0.56–2.13)NS No effect0.260 (0.009–0.780)S Protection2.130 (1.00–4.550)S Risk1.170 (0.520–2.620)NS No effect

4.76 (2.38–9.53)S Protection4.52 (2.56–8.00)S Risk0.42 (0.280–0.630)S Protection

2.62 (1.49–4.6)S Risk0.380 (0.220–0.670)S Protection

0.73 (0.62–0.87)S Protection0.36 (0.24–0.53)S Protection0.83 (0.52–1.32)NS No effect4.81 (3.26–7.09)S Risk0.95 (0.49–1.84)NS No effect

0.72 (0.59–0.87)S Protection0.23 (0.16–0.34)S Protection1.44 (0.99–2.10)NS No effect3.36 (1.99–5.65)S Risk

Fig. 2. Profiles obtained in Immunoblot. The total proteins of Neospora caninumtachyzoites were separated using a 12% SDS–PAGE, transferred to a nylonmembrane and incubated with bovine sera and revealed using an immunoenzy-matic method. Profile 1 revealed proteins of 79, 46 and 37 kDa. Profile 2 showedproteins of 46 kDa, 37 and 17 kDa; profile 3 showed proteins of 79, 46, 37 and30 kDa and profile 4 revealed proteins of 46, 37, 30 and 17 kDa. A molecular weightladder in kDa is indicated.

F. Ghalmi et al. / Research in Veterinary Science 93 (2012) 655–661 659

4. Discussion

N. caninum is a cause of abortion in cattle. Seroprevalence stud-ies are the most useful epidemiological tool to investigate cattleinfection by this particular parasite. The identification of factorsthat can modify the seroprevalence is a major clue. This identifica-tion could lead to preventive actions allowing the reduction of cat-tle infection. The risk factors for N. caninum infections wereinvestigated previously (Hemphill and Gottstein, 2000).

The fact that 52.87% of the studied herds have at least one sero-positive animal to N. caninum suggests that N. caninum infection iswidespread among dairy herds in Algeria.

The study of risk factors allowed identifying some factors play-ing an important role in the development of N. caninum infection incattle. The comparison of our data with data from others countriesis sometimes difficult due to the fact that these countries weresometimes very different from Algeria regarding to the climate,

Table 3Seroprevalence in function the presence of dogs by population.

Population Dogs Cattle (%) Negative Positive

IPC N = 324 Yes 272 (83.95) 221 51No 52 (16.04) 51 1

IMC N = 370 Yes 298 (80.54) 237 61No 72 (19.45) 64 8

LC N = 105 Yes 75 (71.42) 45 30No 30 (28.57) 24 6

the breed or farming methods. But since there is no real data forthe Northern Africa countries, our work could be considered as pio-neer and would constitute a comparison point for further studies.

Considering the age of cattle, the seroconversion risk can in-crease with time or gestations (Jensen et al., 1999; Rinaldi et al.,2005) suggesting that the horizontal transmission is important insome herds (Dubey et al., 2007). But the literature data are some-times contradictory. In Spain the risk of infection increased withage but in Sweden it is the opposite (Bartels et al., 2006). In ourstudy, age was not significantly correlated with seroprevalence.At the population level, an effect was observed for imported cattlebut not for improved cattle or local cattle. In IPC, there is an in-crease in seroprevalence with the age. For IMC and LC the sero-prevalence was higher for <3 years-old cattle. This indicates thatvertical or neonatal transmission is maybe less frequent in thispopulation compared to horizontal transmission, as shown bythe low level of seroprevalence in <3 years old IPC.

The observed differences can also be related to the probabilityto ingest oocysts and to the farm management.

With respect to breed, several studies showed a positive corre-lation between antibody titers and the different breeds (Bartelset al., 2006). For example, the local Spanish breeds were less sero-positive than the Holstein or mixed breeds (Bartels et al., 2006). InCosta Rica, the Jersey breed was associated with high seropreva-lence (Romero et al., 2002). But the data were sometimes difficultto analyze since many other factors can interfere as the farm man-agement. Moreover, in some studies, no evident correlations werefound between the seroprevalence and this variable (Jensen et al.,1999). In Senegal, the seroprevalence in cattle was estimated to17.9% but the local breed showed a higher prevalence as in ourstudy (Kamga-Waladjo et al., 2010).In our study, there was a signif-icant difference between seroprevalence and the different cattlepopulations. When the populations were considered, the seroprev-alence in IPC (16.04%) and in IMC (18.64%) were not significantlydifferent but interestingly, the seroprevalence was significantlyhigher in local breeds (34.29%). Therefore, it seems that localbreeds are particularly exposed to N. caninum. This could be dueto the fact that these cattle to be bred in extensive mode, spendingmost of the day in pasturing areas probably contaminated byoocysts of stray dogs.

In many different epidemiological studies on dairy cattle, thepresence of dogs in the farm or the number of present dogs wasa risk factor to become seropositive (Dubey et al., 2007). Thisobservation was also performed in our study with a significantlyhigher seroprevalence in cattle in contact with dogs than in cattlein farms without dogs.

Most probably, infected dogs can contaminate the food or thedrink water of cattle with oocysts. These relationships are due tothe fact that dogs shed oocysts in pasturing areas. Repeated inges-tions of oocysts by cattle during a long period of time (severalyears) lead to an increase number of infected animals in herds(Schares et al., 2003).

Some recent studies have confirmed the relationship betweenN. caninum seroprevalence and temperature (Dubey et al., 2007).In our study, the seroprevalence was higher in the spring. It is in

Seroprevalence (%) CI 95% OR (CI95%) p

18.75 (13.97–23.42) 11.77 (1.59–87.17) <0.011.92 (0–3)

20.46 (15.73–25.06) 2.06 (0.94–4.52) 0.0711.11 (3–18.50)

40 (28.68–51.31) 2.67(0.97–7.3) 0.0520 (5–34.60)

Fig. 3. The multiple correspondence analysis indicated that the parameters such as presence of dogs (dogs-1), high farm hygiene level (hygiene-1), the seaside region(seaside), presence of abortion in the farm (abortion-1) and spring season merged with seropositivity (serology-1). In opposite, breed IPC, breed IMC, absence of abortion inthe farm (abortion-0), absence of dog in the farm (Dogs-0), autumn season, region mitidja and the non- aborting cows (aborted-0) merged with seronegativity (serology-0).Some other parameters were not associated with the serological results (hygiene-2, breed-LC, regions highlands and Tellian atlas, season winter and summer, aborted cows).

660 F. Ghalmi et al. / Research in Veterinary Science 93 (2012) 655–661

agreement with data from the USA (Barling et al., 2001) where therisk of seropositivity was higher in the spring. This result may beexplained by the fact that the sporulation of N. caninum oocystsis temperature-dependent.

These climatic observations can be related to the differences inseroprevalences observed in the different geographic areas such asthe Mitidja or the seaside regions. It seems that seaside region ismore favorable to cattle infection by N. caninum. Moreover, theeast of Algeria presented the highest levels of seroprevalencewhich can be related either to the climate or to the abundance ofLC in this region since the LC showed the highest seroprevalence.

Since N. caninum is a major abortion factor in cattle, theparameters linked to abortion have been studied. The gestationstatus was not a risk factor in our study since the pregnant andnon- pregnant cows showed the same level of seropositivity.The same observation was made in all populations. This was sur-prising since pregnancy leads to a modification of the immune re-sponse with an increase of humoral immune response and adecrease in cellular response which is in favor of the multiplica-tion of an intracellular pathogen such as N. caninum. Pregnancy fa-vors N. caninum reactivation leading to an increase in specificantibodies production. But no significant difference in end titerswas observed between pregnant and non-pregnant cows. Whenthe gestation stage was investigated, a significant difference wasobserved in seroprevalence between early stage (1–3 months)and latter stages (>4 months). This indicates that the immune sys-tem of pregnant cows in middle or late stage of gestation is moreexposed or more responding to N. caninum infection. These differ-ences were observed in all three studied populations (IPC, IMCand LC). When the aborted cows were analyzed, a clear associa-tion between seroprevalence and abortion has been observed withan odds ratio of 4.52. It is an indirect evidence that N. caninumcould be involved in abortion in cows in Algeria. Interestingly, this

observation was also made for IPC and IMC but not for LC. Thisindicated that LC are probably less susceptible to abort when theyare infected by N. caninum.

Finally, a clear relationship was showed between the generalhygienic status of the farm and the seroprevalence in cattle. It istherefore obvious that a better hygiene in the farm can reducethe risk of infection by N. caninum.

In conclusion, the analysis of risk factors leading to N. caninuminfection in several cattle populations in Algeria indicated thatsome factors such as the presence of dogs, the aborting cows, theseason, the region or the hygiene of the farm were risk factorsfor N. caninum infection. Taking together, all the above analyzedrisk factors for N. caninum seroprevalence indicate that horizontalinfection resulting from the ingestion of oocysts shed by dogs is themost probable route of N. caninum infection in pastured cattle ofareas in Algiers.

At the prevention level, it seems clear that it is better to avoidcontact between cattle or cattle food and dogs in the farm. It willallow blocking the natural biological cycle of the parasite. Theelimination of aborted fetuses and placentas is also important toavoid contamination of the dogs.

Acknowledgments

This work was supported by the Algerian higher education min-istry and the Belgian technical cooperation. The author’s thank DrAnich for his critical reading of the manuscript.

References

Anderson, M.L., Reynolds, J.P., Rowe, J.D., Sverlow, K.W., Packham, A.E., Barr, B.C.,Conrad, P.A., 1997. Evidence of vertical transmission of Neospora sp infection in

F. Ghalmi et al. / Research in Veterinary Science 93 (2012) 655–661 661

dairy cattle. Journal of the American Veterinary Medical Association 210, 1169–1172.

Barling, K.S., Mc Neill, J.W., Paschal, J.C., Mc Collum Third, F.T., Craig, T.M., Adams,L.G., Thompson, J.A., 2001. Ranch-management factors associated with antibodyseropositivity for Neospora caninum in consignments of beef calves in Texas,USA. Preventive Veterinary Medicine 52, 53–61.

Bartels, C.J., Arnaiz-Seco, J.I., Ruiz-Santa-Quitera, A., Björkman, C., Frössling, J., vonBlümroder, D., Conraths, F.J., Schares, G., van Maanen, C., Wouda, W., Ortega-Mora, L.M., 2006. Supranational comparison of Neospora caninumseroprevalences in cattle in Germany, The Netherlands, Spain and Sweden.Veterinary Parasitology 137, 17–27.

Cannon, R.M., Roe, R.T., 1982. Livestock Diseases Surveys; A Field Manual forVeterinarians. Australian Government Publishing Service, Canberra, pp. 35.

Dubey, J.P., Lindsay, D.S., 1996. A review of Neospora caninum and neosporosis.Veterinary Parasitology 67, 1–59.

Dubey, J.P., 1999. Recent advances in Neospora and neosporosis. VeterinaryParasitology 84, 349–367.

Dubey, J.P., 2003. Review of Neospora caninum and neosporosis in animals. KoreanJournal of Parasitology 41, 1–16.

Dubey, J.P., Buxton, D., Wouda, W., 2006. Pathogenesis of bovine neosporosis.Journal of Comparative Pathology 134, 267–289.

Dubey, J.P., Schares, G., Ortega-Mora, L.M., 2007. Epidemiology and control ofneosporosis and Neospora caninum. Clinical Microbiology Reviews 20, 323–367.

Dubey, J.P., Jenkins, M.C., Rajendran, C., Miska, K., Ferreira, L.R., Martins, J., Kwok,O.C.H., Choudhary, S., 2011. Gray wolf (Canis lupus) is a natural definitive hostfor Neospora caninum. Veterinary parasitology 181, 382–387.

European Parliament, 2004. Regulation (EC) No 852/2004 of the EuropeanParliament and of the Council of 29 April 2004 on the hygiene of foodstuffs.Official Journal of the European Union, L139/1-L, 54p.

Ghalmi, F., China, B., Kaidi, R., Losson, B., 2009a. Evaluation of a SRS2 sandwichcommercial enzyme-linked immunosorbent assay for the detection of anti-Neospora caninum antibodies in bovine and canine sera. Journal VeterinaryDiagnosis and Investigation 21, 108–111.

Ghalmi, F., China, B., Kaidi, R., Losson, B., 2009b. First epidemiological study onexposure to Neospora caninum in different canine populations in the AlgiersDistrict (Algeria). Parasitology International 58, 444–450.

Ghozlane, F., Yakhlef, H., Yaici, S., 2003. Performances de reproduction et deproduction laitière des bovins laitiers en Algérie. Annales de l’Institut NationalAgronomique-El-Harrach 24, 1–2.

Gondim, L.F., McAllister, M.M., Pitt, W.C., Zemlicka, D.E., 2004. Coyotes (Canislatrans) are definitive hosts of Neospora caninum. International Journal ofParasitology 34, 159–161.

Hemphill, A., Gottstein, B., 2000. A European perspective on Neospora caninum.International Journal of Parasitology 30, 877–924.

INMV 2009. Institut National de Médecine Vétérinaire, Alger, Algérie.Jensen, A.M., Björkman, C., Kjeldsen, A.M., Wedderkopp, A., Willadsen, C., Uggla, A.,

Lind, P., 1999. Associations of Neospora caninum seropositivity with gestationnumber and pregnancy outcome in Danish dairy herds. Preventive VeterinaryMedicine 43, 139–140.

Kamga-Waladjo, A.R., Gbati, O.B., Kone, P., Lapo, R.A., Chatagnon, G., Bakou, S.N.,Pangui, L.J., Diop Pel, H., Akakpo, J.A., Tainturier, D., 2010. Seroprevalence ofNeospora caninum antibodies and its consequences for reproductiveparameters in dairy cows from Dakar-Senegal, West Africa. Tropical AnimalHealth Production 42, 953–959.

MADR 2008 , Ministère de l’Agriculture et du Développement Rural, Alger, Algérie.Rinaldi, L., Fusco, G., Musella, V., Veneziano, V., Guarino, A., Taddei, R., Cringoli, G.,

2005. Neospora caninum in pastured cattle , determination of climatic,environmental, farm management and individual animal risk factors usingremote sensing and geographical information systems. Veterinary Parasitology128, 219–230.

Mc Allister, M., Dubey, J.P., Lindsay, D.S., Jolley, W.R., Wills, R.A., Mcguire, A.M.,1998. Dogs are definitive hosts of Neospora caninum. International Journal ofParasitology 28, 1473–1478.

Romero, J.J., Perez, E., Dolz, G., Frankena, K., 2002. Factors associated with Neosporacaninum serostatus in cattle of 20 specialised Costa Rican dairy herds.Preventive Veterinary Medicine 53, 263–273.

Schares, G., Bärwald, A., Staubach, C., Ziller, M., Kloss, D., Wurm, R., Rauser, M.,Labohm, R., Drager, K., Fasen, W., Hess, R.G., Conraths, F.J., 2003. Regionaldistribution of bovine Neospora caninum infection in the German state ofRhineland-Palatinate modelled by Logistic regression. International Journal ofParasitology 33, 1631–1640.

Wu, J.T., Dreger, S., Chow, E.Y., Bowlby, E.E., 2002. Validation of 2 commercialNeospora caninum antibody enzyme linked immunosorbent assays. CanadianJournal of Veterinary Research 66, 264–271.