Preventive Veterinary Medicine 93 (2010) 2532

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Preventive Veterinary Medicine

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Flock-level seroprevalence of, and risk factors for, Neospora caninum among sheep and goats in northern Jordan
Mahmoud N. Abo-Shehada a,*, Marwan M. Abu-Halaweh b
a Parasitology Research Laboratory, Department of Basic Veterinary Medical Sciences, Faculty of Veterinary Medicine, Jordan University of Science and Technology, P.O. Box 3030, Irbid 22110, Jordan b Department of Biotechnology and Genetic Engineering, Faculty of Science, Philadelphia University, Jerash, Jordan

A R T I C L E I N F O

A B S T R A C T

Article history: Received 30 April 2008 Received in revised form 31 July 2009 Accepted 16 August 2009 Keywords: Neospora caninum Small ruminants Sheep Goat PCR Seroprevalence Risk factors Jordan

During the period January 2002 to December 2003, serum samples were collected from 104 small ruminant ocks consisting of 18 sheep ocks, 27 goat ocks and 59 mixed ocks containing both sheep and goats in northern Jordan. Only female animals were sampled. At least 5 females aged over 2 years per ock per species were sampled and examined for anti-Neospora caninum antibodies using ELISA. To increase the chances of detecting positive ocks, sick or older ewes were sampled. Also, N. caninum DNA was investigated in 7 sheep brains using PCR technique and 1 was found positive. The ock-level true seroprevalence in small ruminants was 53% (95% CI: 43,63). The true ock-level seroprevalence was higher in sheep (92%) than goats (12%) (OR = 55; 95% CI: 17,197). Similarly, the individual-level seroprevalence in sheep and goat was 63% and 2% respectively (OR = 25; 95% CI: 16,39). Out of 32 production and health management variables, the presence of dogs with the ock (OR = 3.6, 95% CI: 1.2,10) enhanced seropositivity. Cold temperate climate (OR = 0.1, 95% CI: 0.03,0.4), veterinary supervision (OR = 0.2, 95% CI: 0.06,0.6) and buying healthy animals to replace those culled (OR = 0.3, 95% CI: 0.1,0.97) reduced the risk of seropositivity. Both sheep and goats in Jordan are exposed to N. caninum infection with higher seroprevalence in sheep than goats. The contribution of N. caninum to abortion in small ruminant ock needs to be evaluated. Educating the farmers with regard to the role of dogs in transmitting N. caninum infection is expected to enhance small ruminant health in Jordan. 2009 Published by Elsevier Ltd.

1. Introduction Neospora caninum is an obligate intracellular protozoan parasite of a wide range of mammals (Dubey et al., 2007). It is the only species in the genus Neospora recognized at present (Dubey et al., 1988). The parasite was rst discovered in Norway in dogs (Bjerkas et al., 1984). Clinical neosporosis has been reported in dogs, deer, horses, cattle, goats, sheep (Dubey et al., 2007), alpacas and Ilama (Moore, 2005). The infection causes a variety of clinical disorders including encephalitis,

* Corresponding author. Tel.: +962 2 7201000; fax: +962 2 7095123. E-mail address: mshehada@e-parasite.com (M.N. Abo-Shehada). 0167-5877/$ see front matter 2009 Published by Elsevier Ltd. doi:10.1016/j.prevetmed.2009.08.004

myositis, and fatal ascending paralysis in dogs (Cumming et al., 1988). It is the major cause of bovine abortion among dairy cows around the world (Anderson et al., 1991; Barr et al., 1997). In addition to abortion and stillbirths, fetuses may die in the uterus, mummied, autolyzed or born alive with clinical nervous signs (Dubey et al., 1989; Barr et al., 1997). In sheep, neonatal paralysis due to N. caninum was rst recorded in a congenitally infected lamb in England (Dubey et al., 1990) and clinical ovine neosporosis was reported from Japan in a ewe and its twin fetuses (Kobayashi et al., 2001). In addition, an infected ewe was reported to give birth to a weak and partially ataxic lamb that died one week after birth and histo-pathologic evidence of neosporosis was revealed (Hartley and Bridge, 1975). Experimental infection of

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ewes induced acute and chronic abortion-producing infections (Jolley et al., 1999). Also, N. caninum was associated with abortion in naturally infected sheep was reported (Hassig et al., 2003) and natural N. caninum infection was detected in an aborted goat fetus (Elenic et al., 2004). Experimental horizontal and vertical transmissions of N. caninum are reported in cattle, sheep and goats (Dubey, 2003). Thus, neosporosis in livestock, especially cattle, results in the loss of offspring and milk production (Thurmond and Hietala, 1996, 1997; Harnandez et al., 2001) thereby reducing the availability of food and causing economic losses. The seroprevalence of N. caninum in cattle varies depending on the geographical area and the type of serologic test (Dubey, 2003). Using randomized sampling and standardized enzyme linked-immunosorbent assay among laboratories (von Blumroder et al., 2004), resulted in a high seroprevalence (16.2%) among cattle in Spain compared 1.3% in Sweden (Bartels et al., 2006). However, little is known about seroprevalence in sheep (Otter et al., 1997) and goats. Reported individual-level seroprevalence of N. caninum in sheep varied from 3 of 660 aborted ewes in the UK (Helmick et al., 2002), 9% (of 597) in Sao Paulo State, Brazil (Figliuolo et al., 2004) to 36% (of 202) in Egypt (ElGhayash et al., 2003). In the USA 5 (7%) of 77 examined dairy goats, were seropositive to N. caninum (Dubey et al., 1996). In Egypt, of 88 goats 35% were seropositive to N. caninum (El-Ghayash et al., 2003). However, none of the 24 goats had anti-Neospora antibodies in Taiwan (Ooi et al., 2000). Risk factors associated with Neospora in cattle have been reported in the dairy region of Tizauca in Mexico (Sanchez et al., 2003). The presence of dogs and other animal species on the farms, the access of dogs to the water and food sources of cattle, and consumption of placentas and aborted fetuses by dogs were the main variables analyzed. Dairy cattle farms with dogs had higher seroprevalences of N. caninum than those without (Sanchez et al., 2003) but not beef cattle farms (Dubey et al., 2007). Similarly, the presence of dogs in sheep farms was not associated with higher N. caninum seroprevalence (Figliuolo et al., 2004). Thus, neosporosis is expected to have a negative economic impact on livestock farmers in Jordan and the Middle East. However, there is no information regarding neosporosis in Jordan. This study investigates molecular evidence of the presence of the parasite in Jordan, and the ock-level true seroprevalence of, and risk factors for N. caninum seropositivity in small ruminants in northern Jordan. 2. Materials and methods 2.1. Animals and study area The study area included the ve northern governorates of Jordan: Irbid, Mafraq, Jerash, Ajlon, and Zarqa. The main small ruminant farming activities occur within three climatic zones: the warm desert, cool temperate rainy, and the cool steppe (Anon., 1984).

In Jordan, small ruminants are the most abundant domestic animals, with 74% sheep, 23% goats and 2% cattle (Anon., 2001). Sheep and goats are usually kept together mainly under free range roaming transhumance husbandry with a small number of ocks adapting semi-intensive husbandry methods. Awassi sheep and Baladi goats are the main small ruminant breeds in Jordan. Flocks are grazed during spring to early summer and fed on supplements of fodder (Alfalfa) and concentrates (barley and wheat bran) for the rest of the year. The main sources of drinking water for sheep and goats are: spring water and piped water. The lambing season lasts from November until May. Small ruminant ocks in Jordan adopt health programs that include Brucella, anthrax, Clostridia spp., sheep and goat pox and pest de petit ruminant vaccines only. 2.2. Determination of sample size and sampling No information on the prevalence of N. caninum infections in northern Jordan was available. For sample size calculations, a ock-level prevalence of 50% therefore was assumed. The total number of sheep and goat ocks in northern Jordan is estimated at 10,000 (Anon., 2001). According to Thruseld (1995) 62 ocks need to be sampled to estimate the prevalence with an absolute error of 5%, 95% level of condence and 80% power. A sample of that size would be able to detect an odds ratio (OR)  2 for risk factors present in 50% of controls, and an OR  3 for those present in 20% of controls. The main communal grazing areas were sampled including at least one ock per grazing area. Representative ock samples were selected according to the estimated density in each area. The ocks were numbered in each grazing area, and a number was drawn to be interviewed and sampled. On seven occasions farmers declined sampling for different reasons; did not like blood drawn from their animals, the animal owner was not present to give the permission, and absence of adultmale family members, in which case another number was drawn. A number of ocks (39) from an un-shared grazing areas (either private or common grazing area with no access by other farmers) were included. Thus, 104 ocks (18 sheep, 27 goat and 59 mixed) having 12,093 sheep and 4225 goats were randomly selected. The maximum number of sheep per ock is 620 and the minimum is 20 (quartiles are Q1 = 40, Q2 = 90, and Q3 = 200). The maximum number of goat per ock is 300 and the minimum is 8 (quartiles: 15, 25, and 57). N. caninum transplacental transmission has been induced experimentally in cattle, sheep and goats and the ingestion of oocysts is the only demonstrated mode of postnatal (horizontal) transmission in herbivores (Dubey et al., 2007). In infected cattle herds, morbidity is very high and up to 95% of calves are born infected (Dubey et al., 2007). As no similar data are available on the morbidity of N. caninum infection in sheep and goats, the within ock seroprevalence in infected ocks was assumed to be 50%. According to Cannon and Roe (1982), the sample size required to be 95% condent that disease is present in the ock at a prevalence of 50%, is 5 animals. Due to limited funds, the selection of individual sheep/goats for testing

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included the animals most likely to be infected in the ock (older or in poorer condition). Thus, we sampled 510 female animals of more than 2 years of age per ock per species. Twenty mixed ocks had both species sampled; the other 39 mixed ocks had only the predominant species sampled (sheep in 24; goats in 15). During the period from January 2002 to December 2003, each farm/ock was visited once for blood and data collection. Five samples were collected from 56 ocks, 9 samples from 1 ock and 10 samples from 5 sheep ocks. Five, 6, 9 and 10 samples were collected from 59, 1, 1, and 1 goat ocks respectively. Five millilitres of blood samples were obtained from the jugular veins of each animal (n = 641) in a vacuum tube without anticoagulant. Serum samples were separated and stored in aliquots at 20 8C until used. In addition, brain tissues from six local Awassi ewes and one aborted lamb were obtained from northern Jordan and subjected to DNA extraction and examined for N. caninum by PCR. 2.3. Data collection A questionnaire was designed to collect information in a personal interview with the farmer using closed questions. This information covered small ruminant production and health management practices. Farmers were interviewed by a veterinarian who spoke the same dialect of Arabic as the farmers, to reduce the inaccuracies caused by miscommunication. 2.4. PCR-amplication of N. caninum Genomic DNA was extracted by using the Promega, tissue DNA isolation kit (Promega, USA) and Qiagen DNeasy1 purication (Qiagen, Germany) kit according to the manufacturers instructions from 10 to 20 mg of sheep brains. Nested PCR amplication was performed according to Baszler et al. (1999). 2.5. ELISA An indirect ELISA test kit (Biox) was used to detect N. caninum specic antibodies. The absorbance was determined in ELx800 ELISA reader (BIO-TEK Instruments, Inc. Winooski, VT, USA). According to the manufacturer, this tests sensitivity and specicity at the individual animal level are 95% and 96% respectively (Osawa et al., 1998). 2.6. Statistical analysis 2.6.1. Prevalence estimates with 95% CI Seroprevalences of N. caninum infections were calculated and analyzed at individual- and ock-level for sheep and goats. The test sensitivity and specicity were used to calculate the individual-level true prevalence according to Rogan and Gladen (1978). For calculating the ock-level true seroprevalence, the test sensitivity and specicity were adjusted from individual-level to ock-level according to Jordan (1995), using the Herdacc program, for a median ock size of 90 animals, cut-point of one animal positive indicating a positive ock, 510 animals sampled

per ock and sampling without replacement. Then ocklevel sensitivity and specicity were used to calculate the ock-level true seroprevalence according to Rogan and Gladen (1978). The 95% condence intervals were calculated for individual- and ock-level true seroprevalences. Chi-square and Fishers exact tests were employed to test the signicance between seroprevalences. Statistical analyses were performed by using SPSS software version 11 (SPSS Inc., Chicago, IL, USA) and Epi-Info (CDC, Atlanta, GA, USA). p-Value of <0.05 was considered signicant. 2.6.2. Univariable association between herd-level serological status (ock pos/neg) and potential (ock-level) risk factors Data were analyzed according to the casecontrol design, where N. caninum seropositive and seronegative ocks were compared in relation to exposure to potential risk factors (Thruseld, 1995). A cut-point of one as a minimum number of positive animals for a ock to be considered positive was used. Low frequency categories were grouped under a new category e.g. other. To screen for signicant association in order to identify candidate variables (p < 0.05) for the multivariable model, one risk factor at a time was tested for association with N. caninum seropositivity using chi-square test. For ordered categorical variables, the chi-square test for trends was employed. Odds ratios with 95% condence intervals were calculated. 2.6.3. Multivariable (logistic regression) model In the multivariable model, variables were included in the model by backward procedure. A multivariable logistic regression (Hosmer and Lemeshow, 1989) analysis was then conducted starting with all factors that had a p  0.05 or an OR  0.3 or an OR  3.0 in the univariable analysis. The least-signicant variables (based on Walds statistic) were removed and the logistic regression analysis was rerun. This process was continued, the model tted and the results then compared (both parameter estimates and difference in 2 log likelihood of the model) with those of the previous run to check for confounding. Where there was a change in parameter estimates of more than 30%, the removed variable was considered to be a confounder and included in the model again. This resulted in a model containing variables related to N. caninum seropositivity (p < 0.05). Two-way interaction was tested for signicance using SPSS macro for analyzing two-way interactions and simple slopes (version 1.1). 3. Results 3.1. PCR-amplication of N. caninum One of the seven samples examined had the 275 bp fragments in the rst round PCR and the 227 bp fragments were generated by the nested PCR which conrm the results obtained in the rst run and the presence of N. caninum in the sample. 3.2. Seroprevalence The seroprevalence of N. caninum was higher in sheep (92% and 63%) than goats (12% and 2%), on both

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Table 1 Seroprevalence (%) of Neospora caninum in sheep and goats by ELISA during 20022003 in northern Jordan. Species Flock-level No. examined Small ruminant Sheep Goat 104 62 62 True seroprevalence 53 92 12 95% CI 43,63 85,99 4,20 Individual-level No. examined 641 339 302 True seroprevalence 53 63 2 95% CI 49,57 56,66 0.4,4

ock- (p < 0.001) and individual-levels (p < 0.001) respectively (Table 1). 3.3. Multivariable analysis A total of 11 variables were associated (p < 0.05) with N. caninum seropositivity with univariable analysis, eight production (Table 2a) and three health management (Table 2b) variables. After (backward) elimination process, the nal logistic regression model had four variables, namely, cold temperate climatic zone, source of animal, buying, presence of dogs and veterinary supervision (Table 3). 4. Discussion 4.1. PCR and prevalences The current results provided molecular evidence for N. caninum infection among sheep in northern Jordan. In addition, a high true seroprevalence of N. caninum in sheep and goat ocks in northern Jordan (53%) at both individualand ock-levels (Table 2) was found. The true seroprevalence is a more accurate estimate of seroprevalence. The true seroprevalence is derived from the apparent seroprevalence by adjusting for the imperfect test sensitivity and specicity (Rogan and Gladen, 1978). Though, the test sensitivity and specicity can be used for individual-level true seroprevalence calculation, they must be adjusted taking into account the median ock size, the cutoff number of positive animals indicating a positive ock, number of animals sampled per ock and sampling with or without replacement before they can be used in Rogan and Gladen (1978) equation for ock-level seroprevalence calculation (Jordan, 1995). For example, the ELISA test was reported to have excellent sensitivity and specicity for the diagnosis of N. caninum infection in aborted cattle, but the sensitivity drops 10% (from 98% to 88%) when used to detect infection in N. caninum affected herds (Williams et al., 1999). In such circumstances, calculating the true seroprevalence is recommended to calculate a better estimate. The sampling bias towards older animals and those in poorer health may have biased the estimates of individuallevel seroprevalence and contributed to the high ocklevel seroprevalence as older sheep had increased seroprevalence (Figliuolo et al., 2004). This association with age may also indicate the presence of horizontal transmission by ingestion of environmental sporulated oocysts (Figliuolo et al., 2004) so contributing to the high seroprevalence.

Research into seroprevalence of N. caninum infection in small ruminants is scarce (Dubey, 2003). As far as we know, this is the rst reported ock-level seroprevalence of N. caninum in small ruminants. The ock-level true seroprevalence was higher in ewes (92%) than in nanny goats. Similarly, N. caninum seropositivity was found in 26 of 30 (87%) tested sheep farms in Brazil (Figliuolo et al., 2004). N. caninum-associated abortion was reported in goats in the USA (Barr et al., 1992) and Costa Rica (Dubey and Lindsay, 1996) and neosporosis associated granulomatous encephalitis was found in a goat kid in Brazil (Corbellini et al., 2001). The high seroprevalence of N. caninum among small ruminants reported here might be due to the fact that the dog is an integral part of the small ruminant ock in the Middle East including Jordan. In Jordan, the majority of small ruminant ocks (80%) had two dogs or more (AboShehada et al., 2002). Furthermore, dogs are allowed to feed on dead animals, placenta and aborted fetuses (M.N. Abo-Shehada, unpublished observations). Transhumance is adopted in Jordan and the Middle East where small ruminant ocks share communal grazing areas with other animals including shepherd and stray/feral dogs. Also, in Jordan there is no routine anticoccidials use for coccidiosis control that may reduce N. caninum infection and there has been no change in husbandry methods since the start of this study and the production of the data set to the date of reporting. Furthermore, the differences in seroprevalences among sheep and goats may be explained by differences either in the incidence of vertical transmission, grazing behavior and/or resistance to N. caninum infection in the two small ruminant species. The results indicated a broad dissemination of the infection in ve Governorates in northern Jordan involving three climatic zones. These results may also reect the presence of N. caninum infection in dogs in Jordan. Neosporosis is expected to have a negative economic impact on livestock farmers in Jordan and the Middle East. If not the sheep and goat farmers, certainly the cattle farmers by helping to maintain the N. caninum life cycle, increasing the prevalence in dogs and subsequently contaminating cattle feed and water with a heavier load of oocysts (Dubey, 2003). 4.2. Risk factors In the current results, of 32 production and health management variables, the presence of dogs (OR = 3.6) was the enhancing risk factor. Epidemiological evidence for the

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Table 2a Univariable analysis associations between production management risk factors and ock-level Neospora caninum seroprevalence in small ruminant during 20022003 in northern Jordan (n = 104). Variable A. Climatic zone Category (code) Warm steppe (0) Hot desert (1) Cold temperate (2) Sheep (0) Goat (1) Mixed (2) No (0) Yes (1) Same village (0) Local area (1) Other (2) No (0) Yes (1) No (0) Yes (1) Same village (0) Other (2) Spring (0) Pipes (1) Others (2) Same village (0) Local area (1) No (0) Yes (1) No (0) Yes (1) No (0) Yes (1) No (0) Yes (1) <2 months (0) 26 months (1) No (0) Yes (1) No (0) Yes (1) No (0) Yes (1) No. +ve/total (% +ve) 33/46 (72) 23/28 (82) 5/30 (17) 13/18 (72) 8/27(30) 40/59 (68) 31/45 (69) 30/59 (51) 52/82 (63) 2/11 (18) 7/11 (64) 36/64 (56) 25/40 (63) 40/74 (54) 21/30 (70) 50/78 (64) 11/26 (42) 26/44 (59) 5/11 (46) 30/49 (61) 43/79 (54) 18/25 (72) 15/39 (39) 46/65 (71) 21/34 (62) 40/70 (57) 17/40 (43) 44/64 (69) 30/53 (57) 31/51 (61) 15/19 (79) 46/85 (54) 45/70 (64) 16/34 (47) 4/5 (80) 57/99 (58) 40/67 (60) 21/37 (57) p 0.001

B. Small ruminant species

0.002

C1. Source of animals, buying

0.049

C2. Source of buying animals

0.016

D1. Introduce small ruminants onto farm

0.336

D2. Use outsider rams

0.100

D3. Sources of breeding animals

0.085

E. Drinking water source

0.629

F1. Grazing locality

0.186

F2. Communal grazing

0.001

F3. Grazing and concentrate

0.408

G1. Young separated from dam

0.007

G2. Newborn barn with and without ewe

0.408

G3. Weaning age

0.039

H1. Udder cleaning

0.008

H2. Milking manual

0.309

H3. Improper cleaning of milking utensils

0.465

role of the dog in neosporosis, was provided before the discovery of the dog as a denitive host for this parasite. This was the association between seropositivity to N. caninum and the occurrence of N. caninum abortions in dairy cattle, and presence of dogs as the most important risk factor (Pare et al., 1998; Bartels et al., 1999). Then, many studies reported the association between the presence of dogs and seropositivity to N. caninum in dairy cattle (Sanchez et al., 2003), but not in beef cattle (Dubey et al., 2007), sheep (Figliuolo et al., 2004) or goats. Again, the current results associate dogs with N. caninum seroprevalence in small ruminants. The cold temperate climatic zone was associated with low odds (OR = 0.1). N. caninum was rst discovered as a major cause of abortion in cattle in warm California

(Anderson et al., 1991). Following this report, many studies into the prevalence of neosporosis in temperate Europe did not show a high prevalence and did not indicate a similar importance (Otter et al., 1997). On the other hand higher seroprevalence was reported in this study and from Brazil (Figliuolo et al., 2004) in sheep contrary to a seroprevalence of 0.5% reported from the UK (Helmick et al., 2002). Currently, there is little information regarding the frequency of oocyst shedding from the dog and other canids as denitive hosts or the survivability of the cyst in the environment (Dubey, 2003). However, N. caninum oocysts sporulate in the environment (Dubey, 2003) and the present results suggest an adverse effect on sporulation in the temperate climate.

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Table 2b Univariable analysis associations between health risk factors and ock-level Neospora caninum seroprevalence in small ruminant during 20022003 in northern Jordan (n = 104). Variable A1. Neonatal death Categories (code) No (0) Yes (1) No death (0) <5 (1) 510 (2) >10 (3) Absent (0) Present (1) No (0) Yes (1) Absent (0) Present (1) No (0) Yes (1) No (0) Yes (1) No (0) Yes (1) No (0) Yes (1) No (0) Yes (1) No (0) Yes (1) No (0) Yes (1) No (0) Yes (1) Absent (0) Present (1) Absent (0) Present (1) No. +ve/total (% +ve) 30/50 (60) 31/54 (57) 20/39 (51) 26/32 (81) 8/20 (40) 7/13 (54) 17/34 (50) 44/70 (63) 27/50 (54) 34/54 (63) 24/39 (62) 37/65(57) 48/81 (59) 13/23 (57) 41/75 (55) 20/29 (69) 53/71 (75) 8/33 (24) 7/13 (54) 54/91 (59) 25/47 (53) 36/57 (63) 23/47 (49) 38/57 (67) 29/56 (52) 32/48 (67) 42/74 (57) 19/30 (63) 23/41 (56) 38/63 (60) 15/38 (40) 46/66 (70) p 0.473

A2. Neonatal death per year

0.014

B1. Mastitis

0.150

B2. Abortion

0.233

B3. Respiratory signs

0.400

B4. Loss of weight

0.499

B5. Change in milk production

0.134

C1. Veterinary supervision

0.001

C2. Trust vet

0.465

D. Vaccines used

0.204

E1. Treat against helminthes

0.177

E2. When signs appear

0.09

E3. All animals treated

0.348

F1. Wild animals

0.822

F2. Dogs

0.003

Veterinary supervision provides better animal health care and education to farmers regarding dog health and parasite control. In spite of the fact that no anticoccidial is routinely used in small ruminant farms in Jordan and the lack of prophylactic or therapeutic products for N. caninum infection, the veterinary supervision of small ruminant health was associated with low odds (OR = 0.2) of N.
Table 3 Multivariable logistic regression model of seropositivity to Neospora caninum in 104 small ruminants ocks during 20022003 in northern Jordan. Variable Constant Climatic zone Source of animal, buying Dogs Veterinary supervision Category Cold temperate Yes Present Yes p 0.02 0.01 0.05 0.02 0.01 OR 0.1 0.3 3.6 0.2 95% CI 0.03,0.4 0.1,1.0 1.2,10.2 0.06,0.6

Likelihood ratio of chi-square LR2 x : 48.5 on 4 degrees of freedom, p = 0.01.

caninum seropositivity. This may reect the value of professional small ruminant health management. Congenital N. caninum infection of fetuses from chronically infected ewes results in sequential abortion (Jolley et al., 1999) especially in inherited ocks. Culling is a way to prevent transmission from infected cow to heifer (Dubey, 2003) and from ewe to lamb. Buying healthier animals from N. caninum free ocks as a replacement for culled ones is expected to reduce the odds of ock N. caninum seropositivity as found in the current results. Also, the association between buying new stock and reduced seropositivity may conrm the association of the vertical transmission and ock seropositivity. Research into caprine neosporosis is scarcer compared to sheep. However, goat abortions and neonatal deaths caused by or associated with N. caninum were reported from the USA (Barr et al., 1992; Dubey et al., 1996) and experimental infection of pregnant goats caused abortion and infected fetuses (Lindsay et al., 1995). In the current study, more than half of the examined ocks recorded

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neonatal death (52%) and abortion (52%) as health problems, the contribution of N. caninum to which needs to be evaluated. The current results showed high N. caninum seroprevalence in small ruminant ocks and many of those seropositive ocks suffered from abortion with unknown cause. Thus, if N. caninum is associated with naturally occurring small ruminant abortion, it would appear to be a frequent and an important cause. Testing aborted small ruminants for N. caninum along with the other established causes of abortion in the locality may provide useful information needed to evaluate the role of N. caninum in small ruminant abortion. 5. Conclusions In conclusion, this study demonstrated molecular and serologic evidence for N. caninum infection in the Jordanian small ruminant farms. Sheep had a higher seroprevalence than goats. Veterinary supervision, stray/feral dog control and attention to sheepdog health, are expected to reduce the seroprevalence. Acknowledgement This work received nancial support from the Deanship of Research, University of Science and Technology. References
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