Prevalence and Risk Factors Associated to Neospora caninum (Apicomplexa: Toxoplasmatidae) in Pet Dogs From Hamadan, West of Iran, 2016

Neospora caninum as a parasite (Apicomplexa: Toxoplasmatidae) with global prevalence was first reported in puppies from Norway (1). Canine and a wide-range of herbivore animals are the definitive and intermediate hosts for N. caninum (2). The infected dogs may represent neuromuscular neurologic disorders such as progressive flaccid paralysis of hind limbs and jaw, gradual atrophy of muscles, dysphagia, and heart failure (3). Although puppies are generally born without clinical symptoms, they ordinarily present progressing toward ascending paralysis of the rear limbs in 3 weeks after birth (3,4). Diagnosis is impossible by using only clinical signs. Accordingly, serological tools, namely, enzyme-linked immunosorbent assay (ELISA) are proposed to distinguish N. caninum in epidemiologic works. So far, molecular techniques have been used for affirming the serological assessment (2,5). N. caninum infection was detected among 2.1% and 54.6% in various types of dogs, samples, and diagnostic methods in different regions of Iran (6-21). Considering the above-mentioned explanations (Table 1), the present study aimed to assess the infection rate and risk factors related to N. caninum in the Iranian household dogs of Hamadan municipality, located in the west of Iran. Furthermore, a historical mini-review on N. caninum infection in the dogs of Iran is presented in the Discussion Section.


Molecular Examination
The genomic DNA extraction of N. caninum in the whole blood samples of seropositive animals was performed by using the Dyna-Bio TM blood kit (Takapouzist Company, Iran). According to the protocol of Müller et al (22), a pair of Np21 plus and Np6 plus primers (Forward: 5'CCCAGTGCGTCCAATCCTGTAAC3' and Reverse: 5'CTCGCCAGTCAACCTACGTCTTCT3') was used to amplify a 330 bp-fragment-length of NC5 gene. Additionally, the nested polymerase chain reaction (PCR) was performed to amplify a 100 bp-fragment of the PCR product by using 1 µL of each PCR product and a pair of primers (Forward: 5'GTGTTGCTCTGCTGACGTGT-3' and Reverse: 5'-TACCAACTCCCTCGGTTCAC-3') (23).

Data Analysis
The chi-square test (χ2) was used to evaluate the relation among the infection rate and different variables (SPSS 16.0, Chicago, IL, USA) and the probability of ≤ 0.05 was considered significant.

Results
The antibodies to N. caninum were detected in 4 (4.9%, CI 95% = 1.8-8%) out of 184 examined dogs ( Table 2). All seropositive animals were confirmed by nested PCR (Figure 1). The infection rate in female animals (7.6%) was 6.4-fold higher compared to male animals (P = 0.047). The highest infection rate was observed in dogs with 1-2 years old (6.7%) but no significant association was found between infection rate and age groups (P > 0.05). In addition, 5.4% and 3.7% infection rate were found in pure and mixed breed dogs, respectively (P = 0.630). The clinical signs of neosporosis were recorded in 2 seropositive dogs (50%, P < 0.0001, OR = 24.71) while the infection had no connection with a food regime (P = 0.914), housing (P = 0.366), and direct contact with another animal (P = 0.610).

Discussion
The interest of keeping pet animals is progressively in spotlights, and therefore, their health status is crucial in the transmission of infection agents to humans. A concern to N. caninum infection in dogs might play a zoonotic role because of its close biologic relationship with Toxoplasma gondii and the occurrence of accidental oocyst ingestion and/or the consumption of raw meat containing tissue cysts. In addition, antibodies to N. caninum were reported in humans with a deficiency in the immune system (24). According to Yakhchali et al (10), assessing seroprevalence and hence the exposure of dogs to N. caninum are considered as the significant parts of investigating the possible transmission routes of the parasite and identifying dogs in which there is the possibility of neosporosis. Additionally, it should be noted that the negative results observed during the examinations of animals do not necessarily confirm the absence of infection (2).
On the global scale, the seroprevalence rate of N. caninum was estimated in 17.14% of the dog' population (25) while 4.9% of our examined animals were seropositive. This finding contradicts that of the other studies (5,26,27)

Neospora infection in pet dogs
conducted in Turkey (10%), Switzerland (7.3%), Brazil (10-26.5%), the United Kingdom (5.8%-16.6%), and the United States (2-7%). These discrepancies might be ascribed to apply various laboratory methods, climatic variations, and dog population types (6). Low prevalence in the examined dogs may be due to their living conditions, limited direct exposure to potential sources of infection, feeding habits, and the owners' attention for eliminating raw meat from the dogs' diet, along with impossible climates such as low humidity and temperature for a pathogen, which is effective in sporulation time in the region (13,14). The infection rate of N. caninum was significantly higher in female than male dogs (Table 2), which is in agreement with the findings of Goździk et al (28) while in contrast with those of other studies in Turkey (27) and Iran (6,10,12,14). Alexander and Stinson (29) noted that female dogs were progressively susceptible to N. caninum. The hormonal contrasts may have a significant role in host susceptibility to N. caninum. Since estrogen excretion enhances antibody production and androgen suppresses T and B cell immune responses, in females, the immunity to infection can be broken down as a result of various factors such as nutrition, age, pregnancy, and environmental changes (7).
In the current study, the highest infection rate was recognized in dogs of 12-24 months old ( Table 2). In a similar investigation, Basso et al (30) revealed the highest antibodies to N. caninum in dogs of more than one year old (47.7%). Interestingly, the seroprevalence rate increased in dogs with postnatal exposure to N. caninum through horizontal transmission in prior reports in Iran (10,14,15). The high infection in older animals is due to their chance of exposure to infection over time (17).
N. caninum infection in the pure breed (5.4%) was higher than mixed breed (3.7%) with no significant difference. Nearly all reports confirmed that there is no correlation between breed susceptibility to N. caninum with seroprevalence (31). Further, Nazir et al (32) reported that there were no significant differences between seroprevalence and breeds belonging to Alsatian (12.6%), Bully (20.6%), Pug (20.4%), Bullterrier (18.9%), German Shepherd (31.4%), Labrador Retriever (19.6%), Crossbreds (28.7%), and Mongrel breeds (35.4%). In contrast, Robbe et al (33) reported higher seropositivity to N. caninum in pure breed compared to the other type. Supplementary universal investigations are suggested subsequently because of less information about the impact of breeds in the epidemiology of canine neosporosis.
The clinical signs of neosporosis in dogs, usually neurological disorders, are not pathognomonic because they are very helpful in the exact diagnosis of diseases parallel to laboratory tools (4). In our study, musculoskeletal and neurological signs were recorded in 2 infected animals. Additionally, the infection rate had no association with food regime, housing, and direct contact with another animal ( Table 2). The infection with Neospora is associated with animal food materials. Dogs with access to prey or raw food materials outside their home derived from intermediate hosts can increase the risk for seropositivity (5).
In conclusion, the outcomes affirmed the presence and exposure of examined dogs to N. caninum in Hamadan. This investigation, to the best of our knowledge, was the primary presentation of risk factors related to N. caninum infection in Iranian pet dogs. Further investigations on household animal populations are necessary for determining the prevalence and possible routes of infection in dogs from other parts of the country. Finally, using diagnostic screening tools and anti-Neospora compounds in pet dog clinics is recommended for controlling the disease.