Seroprevalence of Neospora caninum in cows with abortion history and in dogs sharing the same area in the Kars Region, Turkey C. KACAR 1 *, H. I. GOKCE 2, A. AKCA 3, O. GUNGOR 4, S. KAYA 1 1 Department of Obstetrics and Gynaecology, Faculty of Veterinary Medicine, University of Kafkas, 36100 Kars, TURKEY. 2 Department of Internal Medicine, Faculty of Veterinary Medicine, University Mehmet Akif Ersoy, 15100 Burdur, TURKEY. 3 Department of Parasitology, Faculty of Veterinary Medicine, University of Kafkas, 36100 Kars, TURKEY. 4 Department of Obstetrics and Gynaecology, Faculty of Veterinary Medicine, University of Mehmet Akif Ersoy, 15100 Burdur, TURKEY. *Corresponding author: cihan3000@hotmail.com SUMMARY The purpose of this study is to determine the seroprevalence of N. caninum in cows with abortion story and in dogs (n = 40) living in the same area using a competitive enzyme-linked immunosorbent assay (celisa). Among the 11 villages regrouping 78 herds (2082 cows) explored in the Kars region, the total abortion rate was 7.1% (148 cows) and among them 7.4% (11 cows) were seropositive for N. caninum and belonged to 7 villages. The neosporosis seroprevalence was similar for local breeds (7.6%) and for Simmental breed (6.3%) and was slightly higher but not significantly in young females (1.5-3 years old). In the great majority of seropositive cases (81.8%), cows have aborted between the 6 th and the 8.5 th gestational months, corresponding to the winter months. In dogs, seropositivity rate was 12.5% and positive dogs, mainly 1.5-3 years old, stemmed from 2 villages in which seropositive cows were detected. In conclusion, the association of N. caninum in cows and in dogs sharing the same environment in the Kars region was observed, but transmission modalities require further investigations. Keywords: Neospora caninum, cow, dog, abortion, seroprevalence, Turkey, celisa. RÉSUMÉ Séroprévalence de Neospora caninum chez les vaches ayant des antécédents d avortement et chez les chiens partageant le même environnement dans la région de Kars, Turquie L objectif de cette étude est de déterminer la séroprévalence de Neospora caninum chez les vaches ayant avorté et chez les chiens (n = 40) vivant dans le même environnement en utilisant un test ELISA par compétition. Parmi les 11 villages regroupant 78 troupeaux (2082 vaches) explorés dans la région de Kars (Turquie), le taux total d avortement a été de 7,1 % (148 vaches) et 7,4 % d entre elles (11 vaches) se sont avérées séropositives envers N. caninum et provenaient de 7 villages. Des séroprévalences de la néosporose similaires ont été obtenues pour les races locales (7,6 %) et pour la race Simmental (6,3 %) mais la fréquence de résultats séropositifs a été plus élevée, bien que de façon non significative, chez les animaux de moins de 3 ans que chez les plus âgés. Dans leur grande majorité, les vaches séropositives ont avorté entre le 6 ème et le 8,5 ème mois de gestation, qui correspondaient aux mois d hiver. Chez les chiens, le taux de séropositivité a été de 12,5 %, et les animaux positifs, principalement âgés entre 1.5 et 3 ans, provenaient de 2 villages dans lesquels des vaches séropositives ont été détectées. En conclusion, il existe une association entre l infection par N. caninum des vaches et celle des chiens évoluant dans le même environnement mais les modalités de transmission restent encore à explorer. Mots clés : Neospora caninum, vache, chien, avortement, séroprévalence, Turquie, celisa. Introduction Neospora caninum was first detected in 1984 in Norway by BJERKAS et al. [6] in a dog displaying neurological symptoms, as an agent similar to Toxoplasma, but not classified. The agent was named Neospora caninum after it was isolated in a dog in 1988 [14, 15]. The action spectrum of N. caninum is reported to include dogs, cows, horses, sheep, goat and wild animals [15]. Dogs are known to be the final host of N. caninum. Oocysts can be seen for several days in the excreta of infected dogs [17, 20, 24]. Oocysts produce spores 2-3 days later. Oocytes in dog excreta pose certain risks as it is an important source of postnatal infection in cows in European countries because they contaminate drinking water and feed [24]. It has been found in studies conducted in different regions of Turkey that the seroprevalence of N. caninum in cows and dogs ranged from 2% to 32.7% [1, 2, 10, 21, 28]. It was determined by Dubey [16] that N. caninum induced transplacental infection. It has been observed that clinical signs of this infection occurred acutely especially in dogs and cows [15]. Cystic coccids of N. caninum have been reported to cause abortions, stillbirths or weak calves unlikely to survive in cows [4, 11, 15, 25, 30]. Clinical symptoms have been observed to manifest in dogs mostly in the neuromuscular system. Symptoms, though occurring in dogs of all ages, are more pronounced in older dogs. Neurological symptoms include paralyses, spastic hyperextensions, muscular atrophy and myalgias in the lower extremities [5]. In addition, symptoms such as pneumonia dermatitis and myocarditis also occur [3, 4]. The purpose of this study is firstly to determine the seropositivity of N. caninum in different breeds of cows which have exhibited abortions and in dogs living in the same area, and secondly, to identify the relationship between cows and dogs.
344 KACAR (C.) AND COLLABORATORS Material and Methods ANIMALS This study was carried out in herds comprising a total of 2082 cows in 11 villages, corresponding to 78 cattle farms in the Kars region and on the dogs living there. Blood samples were taken from 148 cows of local breeds (n = 132) or Simmental (n = 16) that had aborted, and from 40 dogs living in this region. Blood specimens were transported in a refrigerated state at +4 C to the laboratory. After clotting at 4 C for 6 hours, they were centrifuged at 1 200g for 20 minutes at 4 C in the laboratory and sera were carefully harvested and stored at -20 C until analysis. SEROLOGY N. caninum antibodies in the blood sera from investigated cows and dogs were detected using competitive enzyme-linked immunosorbent assay (celisa) kit (VMRD Inc. Veterinary Medical Research and Development, Pulman, WA, VMRD, USA). Sera were analyzed according to the manufacturer s instructions. The test sensitivity and specificity were 96% and 99%, respectively. STATISTICAL ANALYSIS Data were statistically analyzed using the SPSS software (Statistical Analysis System, Version 16.0) statistical program. Chi-square analysis was used to analyze the difference in seroprevalence between villages, breeds and age groups. Differences were considered as significant with a P value less than 0.05. Results This study was conducted in 11 villages and in 78 farms. Among the 2082 cows involved in this study, the overall abortion ratio in the herds was found to be 7.1% (148/2082), ranged in average from 0.19% (4/2082) to 1.87% (39/2082) according to the considered village and from 3.9% (15/384) to 11.2% (26/232) according to the considered herd out of the 78 explored here (Table I). It appeared that overall abortion cases were concentrated in villages in which great proportions of N. caninum positive farms were concerned. N. caninum seropositivity for all the cows aborting was 7.4% (11/148) and N. caninum seropositivity was detected in 7 of the 11 villages (63.6%) where cows had abortions; among them, the herd N. caninum seroprevalence fluctuated from 0.67% (Dikme, Kaynarlı, Oluklu and Esenyazi villages) to 2.0% (Karacoban village). At the farm level, the individual N. caninum seroprevalence fluctuated from 4.3% to 14.3% (Table I). In the present study N. caninum was not detected in four villages (Table I). According to the breed types, serum N. caninum antibodies were more often detected in cows from local breeds (10/132 i.e. 7.6%) than in Simmental cows (1/16, i.e. 6.3%) but the difference in seroprevalence was not statistically significant (P > 0.05) (Table II). Moreover, among the 9 villages in which Simmental cows were present concentrating 139 cows with abortion story, the N. caninum seroprevalence was 6.5% (9/139) whereas it was higher (22.2%, i.e. 2/9), but not significantly, in the 2 villages in which Simmental cows were absent. As shown in Table III, abortion cases were recorded with a great majority (71.6%) in 2-5 years old cows but in parallel, the N. caninum seropositivity in cows with abortion story was similar in 2-5 years old females (7.5%) and in older ones (7.1%). In addition, whereas a greater abortion rate (37.8%) was found in primiparous cows, the frequencies of serum anti- N. caninum antibodies were closely related in primiparous and multiparous females (8.9% and 6.5%, respectively). According to the pregnancy stage, it was observed that the percentage of abortion was maximal between the 0 th and the 3 th month and that nearly all (9/11 i.e. 81.8%) N. caninum seropositive cows have aborted also during the last tiers of pregnancy. When the months in which abortions occurred are taken into consideration (Table IV), it is clear that abortions start in September and continue until March, peaking at the end of autumn and in winter (from November to February) representing 84.5% of abortion cases. The frequency of seropositive cows for N. caninum among cows aborting was also elevated during the same period and 81.8% of seropositive cows have aborted between December and February. In parallel, in 2 of the 7 villages where N. caninum was found in cows, serum anti-n. caninum antibodies were also detected in 5 dogs out of the 40 investigated, i.e. 12.5% (Table V): in one village (Azat village), N. caninum seropositivity was detected in 4 dogs and in one cow and in the other village (Kümbetli village), only one dog but 3 cows were seropositive. In addition, Simmental cows had previously gained access to the villages where seropositive dogs were living. All seropositive dogs were less than 8 years old and 80% of them were younger than 3 years old (Table V). Discussion It is well established that N. caninum oocytes can cause abortions in cows [11, 12, 15, 16, 25, 30]. CONRATHS et al. [9] and BLUMRÖDER et al. [7] have determined the rate of N. caninum antibodies to be 4.1% and 4.0% respectively, in cows with fertility problems or abortion. Studies have been conducted regarding the increase in seroprevalence of N. caninum in cows and dogs in several regions of Turkey [1, 2, 10, 28]. In an earlier study conducted in the Kars region, a seropositivity of 8.2% was found in the Simmental breed of cows whereas cows from local breeds have been seronegative [1]. By contrast, in the present study, the N. caninum seropositivity was slightly higher (7.6%), although not significantly, in local breeds than in Simmental breed (6.3%), suggesting that N. caninum might have spread from Simmental cows imported to Turkey in the Kars region from Germany in 1994 [1] to local breeds. In one study conducted on dogs in Turkey the seropositivity of N. caninum was reported to be 10.0% [10]. BABETTE and ELISABETH [3] found that 13.0% of dogs had seropositive N. caninum antibodies in Germany. KYAW et al. [19] detected N. caninum in 1.2% of dogs in Thailand using the celisa
N. CANINUM SEROPREVALENCE IN ABORTING COWS AND IN DOGS IN KARS REGION, TURKEY 345 Abortion N. caninum seropositivity Overall 7.1% (148/2082) 7.4% (11/148) Repartition Karacoban (18 farms; 528 cows) 1.87 % (39/2082) 2.0% (3/148) By farm: Mean 7.4% (39/528) 7.7% Extreme values 5.3%-9.7% 2.6%-5.2% Derekoy (6 farms; 86 cows) 0.38% (8/2082) 0% (0/148) By farm: Mean 9.3% (8/86) 0% Extreme values 6.7%-11.1% - Azat (3 farms; 59 cows) 0.19% (4/2082) 0% (0/148) By farm: Mean 6.8% (4/59) 0% Extreme values 6.2%-7.7% - Karakale (3 farms; 59 cows) 0.19% (4/2082) 0% (0/148) By farm: Mean 6.8% (4/59) 0% Extreme values 5.0%-7.7% - Akyar (2 farms; 42 cows) 0.19% (4/2082) 0% (0/148) By farm: Mean 9.5% (4/42) 0% Extreme values 7.1%-10.7% - Dikme (6 farms; 131 cows) 0.43% (9/2082) 0.67% (1/148) By farm: Mean 6.9% (9/131) 11.1% Extreme values 6.1%-8.3% - Kaynarli ( 2 farms; 87 cows) 0.33% (7/2082) 0.67% (1/148) By farm: Mean 8.0% (7/87) 14.3% Extreme values 7.1%-9.7% - Oluklu (7 farms; 124 cows) 0.43% (9/2082) 0.67% (1/148) By farm: Mean 7.3%(9/124) 11.1% Extreme values 5.0%-12.5% - Kirkpinar (9 farms; 384 cows) 0.72% (15/2082) 1.35% (2/148) By farm: Mean 3.9% (15/384) 13.3% Extreme values 2.8%-10.0% 13.3% Kayalik (12 farms; 232 cows) 1.24% (26/2082) 1.35% (2/148) By farm: Mean 11.2% (26/232) 7.7% Extreme values 8.3%-14.3% 3.8% (1/26) Esenyazi (10 farms; 359 cows) 1.10% (23/2082) 0.67% (1/148) By farm: Mean 6.4% (23/359) 4.3% Extreme values 5.0%-7.7% - TABLE I: Abortion rates and N. caninum seropositivity rates in the 2082 investigated cows from the Kars region (Turkey) located in 78 farms among 11 villages. N. caninum seroprevalence Simmental cows (n = 16) 6.3% (1/16) Local breeds (n = 132) 7.6% (10/132) Village with Simmental cows (n = 9) 6.5% (9/139) Village without Simmental cows (n = 2) 22.2% (2/9) TABLE II: N. caninum seroprevalence in cows with abortion story (n = 148) from the Kars region according to the breed and to the presence of Simmental breed within a village among the 11 explored. test. In the current study, the seropositivity rate in dogs (12.5%) was similar. With a sensitivity of 96.0% and specificity of 99.0%, the celisa test used in this study is recommended to be employed in dogs acutely infected by N. caninum, but it is not recommended for screening because of its low antibody titration rate in dogs with subacute and chronic infections [8]. However, it seems important to evaluate with accuracy the N. caninum infection rate in dogs because it has been reported that oocytes in their excreta may infect drinking water and feed, leading to serious problems in cows in European countries [25-27]. In addition, in one study, N. caninum antibody titration in dogs feeding on cattle placenta was found to be negative but it was discovered that the dogs had scattered oocytes [13]. In the Kars region, dogs and cows live together in the same herd and in the same pasture and cattle farmers usually give the dead foetus from aborting cows and membranes of the newborn calves to dogs for food, suggesting that disposed agents may be scattered through dogs. The detection of N. caninum in both dogs and cows from the same villages in the present study suggests a possible horizontal transmission between dogs and cows.
346 KACAR (C.) AND COLLABORATORS Abortion (n = 148) N. caninum positivity (n = 11) Age 2-5 years 71.6% (106/148) 7.5% (8/106) > 5 years 28.4% (42/148) 7.1% (7/42) Reproductive status Primiparous 37.8% (56/148) 8.9% (5/56) Multiparous 62.2% (92/148) 6.5% (6/92) 0-3 months of pregnancy 37.8% (56/148) 1.79% (1/56) 3-5 months of pregnancy 33.8% (50/148) 2.00% (1/50) 6-8.5 months of pregnancy 28.4% (42/148) 21.4% (9/42) TABLE III: Abortion rates and N. caninum seropositivity rates in the 148 cows with abortion story from the Kars region (Turkey) according to the age and to the reproductive status. Month of abortion Abortion (n = 148) N. caninum positivity (n = 11) September 1.4% (2/148) 0% (0/2) October 6.1% (9/148) 0% (0/9) November 14.2% (21/148) 4.8% (1/21) December 16.9% (25/148) 12.0% (3/25) January 27.0% (40/148) 7.5% (3/40) February 26.4% (39/148) 7.7% (3/39) March 8.1% (12/148) 8.3% (1/12) TABLE IV: Abortion rates and N. caninum seropositivity rates in the 148 cows with abortion story from the Kars region (Turkey) according to the month of abortion. N. caninum seropositivity in dogs Overall 12.5% (5/40) Origin Azat Village 6.0 % (4/67) Kümbetli Village 0.7 % (1/142) Age 1.5-3 years 16.0% (4/25) 4-8 years 10.0% (1/10) 9-12 years 0% (0/5) TABLE V: N. caninum seropositivity in dogs living in the Kars region in which the survey was conducted. BLUMRÖDER et al. [7] have detected higher N. caninum seropositivity, in 1-2 years old cows than in older ones. SAN- DERSON et al. [23] and JENSEN et al. [18] have also concluded that N. caninum infection may be related to age in cattle, while DAVISON et al. [11] found no relationship between age and N. caninum infection. In the current study, however, N. caninum antibodies have been mainly detected in young cows (less than 5 years old) (71.6%) with abortion story and in primiparous females although differences with older and/or multiparous cows were not statistically significant. BABETTE and ELISABETH [3] failed to demonstrate any relationship between ages in N. caninum seropositive dogs but BARBER et al. [4] and SWADA et al. [29] also reported that N. caninum infection is related to age in dogs. In this study, albeit seropositivity is greater in young adult dogs (1.5-3 years old), age has not been identified as an important factor (P > 0.05), probably because of the low total number of seropositive dogs. N. caninum infection is known to cause abortions in 12.0-42.0% of cows in Belgium, England, USA, New Zealand and Germany [11, 12, 16, 22]. It has been observed that abortions usually occurred during the 5-6 th months of gestation [11] and that most abortions in cows (70.3%) occurred during the winter months (December, January and February). Similarly, 81.8% of the abortions in cows in which N. caninum seropositivity was detected occurred in winter months in the present study. This shows that abortions mostly occur during the last 2/3 of the gestational period because the cows peculiar to this region generally conceive in spring. As a conclusion, while the seropositivity of N. caninum in aborting cows in the Kars region was determined to be 7.4%, the rate was 12.5% in dogs living in the same area. Taken this and the fact that it has been previously reported that local breeds were not concerned by N. caninum infection, this study highly suggests the occurrence of horizontal contaminations between cows and dogs and between cows from various breeds but further investigations will be required for defining
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