Seroprevalence of Neospora Caninum in Stray Dogs

Problem statement: To investigate antiNeospora caninum antibodies in stray dogs living in Tabriz city, 100 blood samples were collected. S erum samples were screened for detection of antiN. caninum IgG antibodies using Indirect Fluorescent Antibody Test (IFAT; ≥50). Antibodies were seen in 31 (31%) of 100 dogs. Approach: The IFAT antibody titers were as follows: 1:50 in 11 dogs, 1:100 in ten dogs, 1:200 in six dogs, 1:400 in one dog, 1 :800 in two dogs and 1:1,600 in one dog. There were no significant differences in seroprevalence of Neo spora infection between different genders (p>0.05). The seropositive results were increased with age an d differences were statistically significant (p<0.0 5). Results: The results confirm the presence and exposure of s tray dogs to N. caninum in Tabriz city and importance of this protozoan as a cause of disease in dogs of the region. Conclusion: Based on the present study, this is the first report on seroprev al nce of N. caninum in stray dogs of the Tabriz northwest of Iran. This finding confirms the presen c of Neospora infection and the important role of dogs in the region.


INTRODUCTION
Neospora caninum is an apicomplexan protozoon which was described by Dubey et al. (1988aDubey et al. ( , 1988b. The agent can infect domestic dogs (Canis familiaris) (McAllister et al., 1998) as well as an ample variety of hosts including ruminants (Anderson et al., 1991). It has been regarded as the major cause of abortion with great economical impact in dairy cattle industry and neurologic signs in puppies and older dogs (Ruehlmann et al., 1995;Dubey and Lindsay 1996;Dubey, 2003). Although the parasite can be transmitted transplacentally in several hosts and the vertical route is the major mode of its transmission in cattle, the role of the definitive host in spreading the infection through shedding of oocyst is important. Carnivores can acquire infection by ingestion of infected tissues (McAllister et al., 1998;Dijkstra et al., 2001). As oocysts are rarely found in dog feces (Schares et al., 2005), serological surveys provide the main clue for estimating the prevalence of canine neosporosis (Bjorkman et al., 1994). The agent has a cosmopolitan distribution, with reports from various countries of Europe, USA, Canada, Australia, South Africa, Japan and Costa Rica (Dubey and Lindsay, 1996). There is also serological evidence of infection in equatorial Africa and South America (Barber et al., 1997a). Seroprevalence (of infection, not disease) varies from 0.5-17% in Europe (Bjorkman et al., 1994;Rasmussen and Jensen, 1996) and has been reported at 2% in the USA. At present, only limited information is available on the occurrence and prevalence of Neospora infections in dogs in Iran. N. caninum infection was first reported by  in household dogs and dogs living in dairy and beef cattle farms. The presence of antibodies against N. caninum in aborted and healthy dairy cattle was detected and neosporosis importance in cases of cattle abortion is being increasingly demonstrated in Iran (Razmi et al., 2006;Sadrebazzaz et al., 2004;Habibi et al., 2005;Bonfitto et al., 2009;Saravanamurthy et al., 2010;Solhjoo et al., 2010). Nevertheless, no information on the prevalence of N. caninum infection in stray dogs is available in Iran. Because of this reason, the recent investigation was done to evaluate the seroprevalence of Neospora infection in stray dogs of northwest Iran.

Animals:
A total of 100 sera from stray dogs of mixed breeds from districts of Tabriz (63 males, 37 females; <1->6 years of age) were randomly sampled. All of the dogs were examined after the drawing of blood samples. Information regarding access to bovines, clinical signs, gender and age of the animals was also obtained and were registered for retrospective correlation with the anti-N. caninum antibody titers in the positive animals.
Preparation of the serum samples: Blood samples were drawn from cephalic vein of each dog using venopuncture. The samples were centrifuged at 700×g for 15 min to obtain the serum, which was aliquoted and stored in microtubes and kept frozen at -20°C until analysis.
Indirect fluorescent antibody test: The sera were screened for IgG antibodies to N. caninum by commercial Indirect Fluorescent Antibody Test (IFAT; Mega Screen Fluoneospora, Horbranz, Austria). All sera were screened at dilutions of 1:50 in phosphate-buffered saline (pH7.2). Serum samples showing fluorescence at the dilution of 1:50 (cutoff value) were further tittered using twofold serial dilutions (Dubey et al., 1988a(Dubey et al., , 1988b. The entire surface fluorescence of tachyzoite was considered positive (Silva et al., 2007). On each slide, a negative and a positive control were included.

Statistical analysis:
For the statistical analysis of the possible effects of the attributes of different gender and age groups range on the prevalence of anti-N. caninum antibodies, chi-squared test was used. Statistical significance was declared at p≤0.05. The analyses were performed by SPSS 12 statistics program for Windows.

DISCUSSION
Assessing seroprevalence and hence the exposure of dog population of N. caninum, is an important part of investigating the possible transmission routes of the parasite as well as identifying populations in which neosporosis may occur. The IFAT is a well-established test for detecting anti-N. caninum antibodies in dogs (Dubey et al., 1988a;1988b). In Iran, the presence of antibodies against N. caninum in dairy cattle (Sadrebazzaz et al., 2004;Razmi et al., 2006), camels (Sadrebazzaz et al., 2006) and farm and household dogs Malmasi et al., 2007) were reported. Based on the present study, this is the first report on the seroprevalence of in stray dogs of the Tabriz northwest of Iran. This finding confirms the presence of Neospora infection and the important role of dogs in the region. The prevalence of 31% was lesser than the prevalence of 33 and 28% reported by Malmasi et al. (2007) and Haddadzadeh et al. (2007), respectively. This prevalence is similar to the reported prevalence of 22% in urban areas in Argentina (Basso et al., 2001). The higher seroprevalence have been reported in earlier studies on dogs conducted in several countries including USA and Canada (Cheadel et al., 1999), Belgium (Barber et al., 1997b), England (Trees et al., 1993), southern Italy, the Netherlands (Wouda et al., 1999) and Turkey (Coskun et al., 2000). Serologic investigations showed that dogs coming from dairy properties have a greater seroprevalence than those from urban areas, suggesting that farm dogs have a higher risk of exposure to the parasite (Sawada et al., 1998;Wouda et al., 1999;Sanchez et al., 2003).   These higher seroprevalences can be due to the risk factors (consumption of placenta, materials of aborted fetuses, uterine discharge, hunting and close contact with potential intermediate hosts of the parasite (Dijkstra et al., 2002;Fernandes et al., 2004). The presence of anti-N. caninum antibodies in city dogs could be associated with subclinical transplacental transmission through several generations as well. To a lesser extent, dogs could become infected by the horizontal route if they consume sporulated oocysts of N. caninum (Yakhchali et al., 2010;McAllister et al., 1998;Dubey, 1999). For this reason, in this region, stray dogs should not be accessed to bovine placentas or uterine discharge around the farms or slaughterhouse. There was no significant difference in seropositivity between males (n = 21, 68%) and females (n = 10, 32%; p>0.05), in agreement with the results of other surveys (Trees et al., 1993;Barber et al., 1997a;Sawada et al., 1998;Cheadel et al., 1999;Haddadzadeh et al., 2007;Malmasi et al., 2007). In this study, there is a tendency for elevated risk of pathogen contact with increasing age, suggesting postnatal exposure to N. caninum. Similar reports from Brazil (Fernandes et al., 2004) and Iran  confirm this finding. There was an association between seroprevalence and age. The youngest seropositive animal was 2-3 years of age and the oldest was 6-10 years. However, dogs of any age (18 months to 6 years old) may be affected by neosporosis (Knowler and Wheeler, 1995;Patitucci et al., 1997). The role of age in seropositivity suggests that most of the dogs acquire the infection in the postnatal period by means of horizontal transmission. The role of age has been observed in similar studies (Barber and Trees, 1996;Sawada et al., 1998;Wouda et al., 1999;Haddadzadeh et al., 2007;Malmasi et al., 2007). The anti-N. caninum titers encountered varied from 50-1600. Thirty (97%) of 31 seropositive dogs had the titers of 1:800 and lower. None of these cases had previous clinical symptoms of the disease, suggesting that a large portion of infection occurs in a subclinical form. In our study, one of the examined dogs showed hind limb paraplegia and had an anti-N. Caninum IFAT titer of 1:1, 600. This may indicate an acute infection (Barber and Trees, 1996). Clinical and subclinical infections with N. caninum in dogs have been extensively reported worldwide with seroprevalence rates ranging from 0.2-54% (Moore, 2005). The presence of infected stray dog could be a risk factor for the occurrence of N. caninum-associated abortions in cattle. Further investigations could be useful to show the correlation between stray dog neosporosis and cattle abortion in this region.

CONCLUSION
Based on the present study, this is the first report on the seroprevalence of in stray dogs of the Tabriz northwest of Iran. This finding confirms the presence of Neospora infection and the important role of dogs in the region.

ACKNOWLEDGMENT
The researchers wish to thanks the Islamic Azad University, Tabriz Branch, Tabriz, Iran for the financial supports and all laboratory technicians for technical aids in this project.