Serology based comprehensive study of Neospora infection in domestic animals in Hamedan province , Iran

This study was conducted to determine seroprevalence of Neospora infection in cattle, sheep, horses, donkeys, and dogs in Hamedan province, Iran. Blood samples (n=2254) from the animals were collected randomly during 2009 to 2012. Sera were prepared from the collected blood samples, which were then examined for the presence of antibodies against Neospora using enzyme-linked immunosorbent assay (ELISA), Neospora modified direct agglutination test (N-MAT), and indirect fluorescent antibody test (IFAT). The seroprevalence rates of Neospora were found as 17.4% (n=245/1406) in cattle, 2.2% (n=8/358) in sheep, 40.8% (n=49/120) in horses, 52% (n=52/100) in donkeys, and 27% (n=73/270) in dogs. In this study, higher levels of Neospora infection were detected in cattle, horses, donkeys, and dogs. This is the first comprehensive study of Neospora infection in domestic animals in Iran. Further researches on molecular and bioassay studies and designing appropriate control strategies against neosporosis in Iran are necessary and strongly recommended.


INTRODUCTION
Neospora (N.) caninum is a coccidian parasite that was first recognized in dogs from Norway in 1984 (Dubey et al., 2007).Domestic and wild canids such as dog and coyote are definitive hosts, and a wide-range of animals such as cattle, sheep, horses and donkeys may play the role as intermediate hosts for this parasite (Dubey and Schares, 2011).Excretion of N. caninum oocysts could act as a risk factor when these are come out through feces, and finally mixed with environment of definitive hosts; these could cause stillbirths and miscarriages to cattle and other intermediate hosts (Sharifdini et al., 2011).
Study of Neospora infection rate in animals such as dogs and cattle is necessary for comprehensive evaluation of neosporosis (Dubey et al., 2007).Several serological tests including enzyme-linked immunosorbent assay (ELISA), indirect fluorescent antibody test (IFAT), and Neospora modified direct agglutination test (N-MAT) have been using for the diagnosis of this infection (Dubey et al., 2007;Dubey and Schares, 2011).Several serological studies on Neospora infection have been reported in different hosts of Iran (Sadrebazzaz et al., 2006;Haddadzadeh et al., 2007;Hajikolaei et al., 2007;Salehi et al., 2010;Hosseini et al., 2011;Asadpour et al., 2012).However, there is no published comprehensive information about neosporosis in the animals in Hamedan province, Iran.Therefore, this study was conducted for a comprehensive study on Neospora infection in domestic animals (cattle, sheep, horses, donkeys and dogs) in Hamedan province, Iran.Serology: Sera were removed from the blood samples after centrifugation at 1500×g for 10 min, and stored at -20°C until use (Haddadzadeh et al., 2007;Nematollahi et al., 2011).The sera of the ruminants (cattle and sheep), equine (horses and donkeys) and dogs were examined for the presence of antibodies against Neospora using ELISA, N-MAT and IFAT, respectively.

MATERIALS AND METHODS
Enzyme-Linked Immunosorbent Assay (ELISA): Anti-Neospora IgG-antibodies in the ruminant samples were detected using a commercially available ELISA kit (HerdCheck ® Anti-Neospora; IDEXX Laboratories; Switzerland).The presence of antibody was determined by calculating the value% according to the instructions of the manufacturer.A value of ≥40% was considered as positive.

Neospora modified direct agglutination test (N-MAT):
Anti-Neospora antibodies in the equine samples were detected using N-MAT (Hosseini et al., 2011).In brief, using phosphate-buffered saline (PBS) containing 0.2M 2-mercaptoethanol, the sera were double-diluted from 1:10 to 1:80.An amount of 50 μL of each dilution was taken in a well of 96-U-bottom microtiter plate.
Then, 50 μL of tachyzoites suspension (3.5×10 7 /mL; NC-1 strain of N. caninum) resuspended in alkaline buffer [7.02g of NaCl, 3.09g of H3BO3, 24 mL of 1N NaOH, 4g of horse serum albumin (fraction V), 50mg of eosin Y, dH2O to 1L, 0.1% sodium azide as preservative; pH: 8.7] was added to each well of serum dilution, and to positive and negative controls.Then, the wells were mixed properly by pipetting.The plate was then incubated overnight at 37°C with 5% CO2.As per Gharedaghi (2012), a cut-off titer of 1:80 was considered as significant for the presence of antibodies.When the tachyzoites were found as spreading condition on bottom of each well, then it was considered as positive reaction.On the other hand, negative reaction showed button formation at the bottom of each well.

Statistical analysis:
Statistical analysis was performed by using the software package SPSS version 16.0 for windows.Odds Ratios (OR), confidence Interval (CI), χ 2 and p-value were calculated separately for each variable.A p-value ≤0.05 was considered as statistically significant.
In our study, the seroprevalence rate was determined between 12.8-20% among different types of cattle.In a study conducted in Spain, Quintanilla-Gozalo et al. (1999) found that prevalence of N. caninum in dairy cattle was higher than beef cattle.This variation in prevalence might be due to difference in production systems of dairy and beef cattle, rather than differences in breed (Dubey and Schares, 2011).
In our study, no significant correlation was found among different age and gender groups of cattle (p=0.961,p=0.069), horses (p=0.131,p=0.344) and donkeys (p=0.353,p=0.096); unlike to age group in sheep (p=0.015,OR=5.9).In contrast, Razmi et al. (2006) and Gharekhani et al. (2012) reported significant correlations among different age groups.Sadrebazzaz et al. (2004) and Wouda et al. (1999) reported equal levels of seroprevalence in all age groups for most herds.On the other hand, Jensen et al. (1999) suggested that seroprevalence increased with age and depended on sample size.
In the present study, 61.2% cattle were found to be seropositive that had abortion history (Table 2), which was in support to several previous studies (Paré et al., 1998;Anderson et al., 2000;López-Gatius et al., 2005;Dubey and Schares, 2011;Gharekhani et al., 2012).Similarly, Razmi et al. (2006) reported a higher abortion rate in seropositive cattle as compared to seronegative cattle (p<0.05,OR=1.78).The risk of abortion in seropositive cattle was reported as 4 (Václavek et al., 2003), 5.3 (Schares et al., 2004)  The overall seroprevalence rate in sheep (2.2%; Table 2) found in our study was similar to the studies that were conducted in Italy and Australia (Ghaffari et al., 2006;King et al., 2010).In another study in Iran, this rate was reported as 1.13% in aborted sheep, and 1.7% in healthy sheep (Ezatpour et al., 2012).On the other hand, Asadpour et al. (2012) reported that 5.7% of sheep and 8.5% of ovine fetus in Northwest Iran were infected to neosporosis.
In this study, Neospora in horses (40.8%) of >2-3 years age group showed higher seroprevalence as compared to 1-≤2 years of age group, which was similar to that of the findings of Locatelli et al. (2006).The infection rate in riding club horses was higher than rural samples, which might be due to intensive management system and their direct contact with dogs of inside club.No significant correlation was seen between the infection rate and genders, similar to the findings of some other previous studies (Mc-Dole and Gay, 2002;Pitel et al., 2003;Jakubek et al., 2006;Moraveji et al., 2011;Gharedaghi, 2012).
Current survey is the first report of Neospora infection in donkeys in Iran.The donkey carcasses are being used as food for carnivores in zoo, and are fed to stray canids in suburb of villages in Iran; which might be responsible for transmission of infection among animals.
In our finding, Neospora infection rate in stray dogs (52.8%) was higher than shepherd dogs (18%) (p<0.001).This result was in support of Nguyen et al. (2010) who conducted researches in South Korea.Haddadzadeh et al. (2007) and Malmasi et al. (2007) reported high infection rate of Neospora infection in farm dogs as compared to urban and household dogs.Similar to some other studies, the seroprevalence rates in dogs increased with age, which suggested postnatal exposure to N. caninum through horizontal transmission (Malmasi et al., 2007;Haddadzadeh et al., 2007;Yakhchali et al., 2010).High infection was attributed to greater chances for exposure to Neospora over time, increasing the susceptibility of older dogs (Hosseininejad and Hosseini, 2011).There was no significant difference in gender, which was in agreement with some other reports (Haddadzadeh et al., 2007;Nguyen et al., 2010;Hosseininejad and Hosseini, 2011;Sharifdini et al., 2011).Farmers in Iran mostly reared male dogs in their farms.Therefore, the male dogs might have been infected with Neospora more than females (Hosseininejad et al., 2010;Khanmohammadi and Fallah, 2011).However, different serological tests and cut-off values, study design, climatic variations and frequency of canids on the farms and around of animals were the main causes of varied results (Dubey et al., 2007).
Most of the animal breeding farms are traditional in Iran, and the animals had a direct contact with the dogs and other canids.Oocyst-contaminated pastures, fodder, and drinking water were considered as potential sources of postnatal infection in animals.Thus, type of feeding practices might pose on increased level of infection risk (Dubey et al., 2007).
Both horizontal and vertical routes of Neospora transmission in animals are present in Hamedan region.After the confirmation of dog as a final host, the presence of dogs in farm was assumed to provide the higher chance of horizontal transmission through ingestion of oocysts that were shed by the infected dogs (Dubey et al., 2007).

CONCLUSIONS
To the best of our knowledge, this is the first comprehensive study on Neospora infection in domestic animals in Iran.In this study, higher levels of infection were detected in cattle, horses, donkeys and dogs; which could be an important reason for economic losses in this region.Molecular and bioassay studies of Neospora and design of appropriate control strategies against neosporosis in Iran are suggested.

Table 1 :
The seroprevalence of Neospora infection in different age and gender groups in animals.

Table 2 :
The seroprevalence of N. caninum in animals having history of abortion.