Prevalence of Neospora spp. in donkeys in China

This study was conducted to detect specific anti-Neospora antibodies using a commercial competitive-inhibition ELISA kit, and to evaluate the risk factors for Neospora spp. infection. Out of a total of 2,228 donkey sera collected in three provinces in China, 211 (9.5%) were found to be positive for anti-Neospora antibodies. Statistical analysis revealed that age (p = 0.019, OR = 1.62, 95%CI: 1.08-2.44), feeding status (p < 0.001, OR = 3.79, 95%CI: 2.65-5.43), miscarriage history (p = 0.006, OR = 2.56, 95%CI: 1.27-4.01), and contact with dogs (p < 0.001, OR = 2.69, 95%CI: 1.86-3.88) were significant risk factors for Neospora spp. infection. This is the first evidence of Neospora infection in donkeys in China.


Introduction
Neospora spp. are globally distributed obligate intracellular parasites [22] and are closely related to Toxoplasma gondii and Sarcocystis spp., belonging to the phylum Apicomplexa of the Sarcocystidae family [7]. Neospora can infect a wide variety of hosts [8]. Dogs, coyotes and dingoes are definitive hosts of Neospora spp. [13,14,19], and certain other mammal species, including cattle and other ruminants, canids and horses can serve as intermediate hosts [10].
Equine neosporosis caused by two species of Neospora (Neospora caninum and Neospora hughesi), is accompanied by neurologic symptoms and reproductive loss [9,21]. Donkeys (Equus asinus) are closely related to horses and can be infected by some equine pathogens such as the protozoa Theileria equi and Babesia caballi [18]. Seroprevalences of Neospora spp. in donkeys have only been reported from Southern Italy [17], Nigeria [5], Brazil [11] and Mexico [1]. However, until now, no information was available about the prevalence of this protozoal disease in donkeys from China. Thus, the aim of this study was to detect antibodies to Neospora ssp. in donkeys from three provinces in China, and to evaluate the risk factors associated with Neospora seroprevalence.

Materials and methods
Serum samples were randomly collected from the jugular vein of 2,228 donkeys from Shandong province (4°23 0 ∼38°24 0 N, 114°48 0 ∼122°42 0 E), Henan province (31°23 0 ∼36°22 0 N, 110°21 0 ∼116°39 0 E), and Hebei province (36°05 0 ∼42°40 0 N, 113°27 0 ∼119°50 0 E) between November 2015 and June 2017 by local veterinary practitioners *Corresponding author: mqfboy@163.com Parasite 25, 16 (2018) ( Figure 1). Donkeys from each farm were selected randomly using a table of random digits. Several large-scale farms (with more than 300 animals) were not included because the owner did not authorize us to collect samples. Approximately 30% donkeys at each farm were sampled. All of the animals sampled were clinically healthy. Serum samples from backyard donkeys were randomly collected when authorization was obtained from the owners of the donkeys. Serum was obtained through centrifugation at 3000 Âg for 5 min and stored at À20°C until tested. Information about breeds, gender, age, contact with dogs, miscarriage history, and feeding status was acquired from the owners.
A commercial competitive-inhibition enzyme-linked immunosorbent assay kit (cELISA) (VMRD, Pullman, WA, USA) was used to detect Neospora antibodies, according to the manufacturer's instructions [17]. The serum was tested in duplicate and considered positive when the percent inhibition values of both runs were more than 30%.
For the statistical analysis, the SPSS 18.0 software package (IBM, Armonk, NY, USA) was used. The Fisher exact test was used to compare the frequencies among groups. Bivariate and multivariate logistic analyses were used to assess the association between the characteristics of the subjects and the Neospora infection. Variables were included in the multivariate logistic analysis if they had a p value of equal to or less than 0.35 in the bivariate logistic analysis [1]. A p value less than 0.05 was considered statistically significant. Odds-ratios (ORs) with 95% confidence intervals based on likelihood ratio statistics were calculated.
This study was approved by the Animal Ethics Committee of Jilin Agricultural University. Serum samples were collected and handled in accordance with the requirements of the Animal Ethics Procedures and Guidelines of the People's Republic of China.

Results and discussion
Commercial competitive-inhibition ELISA kits have been used widely to detect Neospora antibodies in many kinds of animals including donkeys [17] due to the method's high accuracy, sensitivity and accessibility [4]. We therefore first used this method to assess the serologic frequency of antibodies to Neospora in donkeys from China. Out of a total of 2,228 donkeys, 211 (9.5%) were found to be positive for Neospora antibodies. General data for the 2,228 donkeys studied and seroprevalence of Neospora spp. infection are shown in Table 1.
In the present study, age of donkeys was a significant risk factor for this parasitic infection, on the basis of multivariate logistic analysis. The donkeys in age categories 1 year (p = 0.019, OR = 1.62, 95%CI: 1.08-2.44) were found to have a significantly higher seroprevalence than the other age groups, suggesting that donkeys were exposed to the parasite infection at the early stages of their lives. The same phenomenon was found in a previous study [17]. Moreover, maternal antibodies may have been ingested by colostrum, which may give positive results. This is because the variable is < 1 year, which could include a few months of life and thereby influence the result. Interestingly, nine female donkeys and their offspring were found to be positive for Neospora antibodies. This phenomenon leads us to consider possible vertical transmission of Neospora infection in donkeys in further studies.
Neospora infection can induce clinical neosporosis disease, which notably presents as abortion in ruminants. Worldwide, these abortions are the main reason for economic loss to both the dairy and beef industries [6,15]. In the present study, the result of multivariate logistic analysis showed that donkeys with a history of miscarriages have a significantly higher Neospora seroprevalence than those without (p = 0.006, OR = 2.56, 95%CI: 1.27-4.01). Although no direct evidence showed that the miscarriages of donkeys were caused by Neospora infection, prevention and control of Neospora infection should be carried out in the process of raising donkeys. Moreover, further studies should be conducted to explore the association between miscarriage and Neospora infection. Dogs, the definitive hosts of Neospora, play an important role in the transmission of N. caninum, discharging oocysts into the environment [16,20,22], which is a main risk factor for the occurrence of miscarriages and stillbirths associated with N. caninum in ruminants and other intermediate hosts [2,3,12]. Not surprisingly, the donkeys have a significantly higher seroprevalence when they have contact with dogs compared to those without contact (p < 0.001, OR = 2.69, 95%CI: 1.86-3.88). In China, dogs often act as guard animals on farms, so this may increase the possibility of N. caninum infection. Thus, more protective measures should be implemented to reduce N. caninum infection in donkeys, such driving stray dogs from the farms, etc.
In the present study, backyard donkeys had a significantly higher seroprevalence than those feeding on farms (p < 0.001, OR = 3.79, 95%CI: 2.65-5.43). The most likely explanation is that donkeys feeding in backyards have greater contact with dogs because many dogs are raised in the owner's home as guard animals. However, all farms were positive for the presence of Neospora antibodies. It is therefore impossible to evaluate the role of farms as a risk factor. Further studies should be conducted to explore the role played by neosporosis in reproductive and economic losses to donkey breeding in these regions.

Conclusions
This is the first report of Neospora seroprevalence and risk factors associated with Neospora infection in donkeys from China, providing baseline data for designing and evaluating prevention and control measures. More studies are needed to understand and evaluate the occurrence of transplacental transmission throughout pregnancy. Similarly, the occurrence of neurological diseases or fetal loss in the life of congenitally infected donkeys should also be investigated.