First Report of Seroprevalence and Risk Factors of Neospora caninum Infection in Tibetan Sheep in China

Neospora caninum is an intracellular protozoan parasite which can cause abortion and stillbirth in ruminants. However, there is no information on Tibetan sheep N. caninum infection in China. A total of 2187 serum samples were collected from Tibetan sheep in the major production areas of Luqu, Maqu, and Tianzhu in Gansu province, and Nyingchi in southeast Tibet, China. All samples were analyzed for the presence of antibodies to N. caninum using a competitive-inhibition enzyme-linked immunoassay. Of the 2187 serum samples, 184 (8.4%, 95% CI 7.3-9.6) were tested N. caninum seropositive. The N. caninum seroprevalence ranged from 4.4% (95% CI 1.4–7.4) to 11.3% (95% CI 8.2–14.4) among different regions, seasons, ages, and pregnancies, and there was no statistical significance among those groups (P > 0.05). Seroprevalence in male (10.8% 69/638) (95% CI 8.4–13.2) was significantly higher than in female (7.4% 115/1549) (OR =1.51, 95% CI 6.1–8.7) (P < 0.01). To our knowledge, this is the first report of N. caninum seroprevalence in Tibetan sheep in China, which provides baseline data for the prevention and control of N. caninum infection in Tibetan sheep.


Introduction
Neospora caninum is an obligate intracellular protozoan which can infect a variety of mammals and birds [1]. During the whole life cycle of N. caninum, canids are the exclusive definitive hosts and shed environmentally resistant oocysts [2]. Many ruminants like sheep, goats, and dairy cattle commonly act as the primary intermediate hosts and are subjected to abortion, stillbirths, and neonatal mortalities [3,4]. Both vertical transmission from infected mother to foetus and horizontal transmission from food or water contaminated with oocysts can result in the infection of N. caninum [5].
Though no N. caninum has been found from human tissue till now, antibodies to N. caninum have been detected in human serum [6], suggesting a zoonotic potential of the parasite.
The N. caninum seroprevalence has been reported in sheep and goats worldwide [7][8][9]. In China's Tibetan areas, the prevalence and infection of N. caninum have been reported in black yaks [10,11] and white yaks [12]. However, there is no information about the infection of N. caninum in Tibetan sheep in China. Tibetan sheep is one of China's three major varieties of sheep, mainly inhabiting in the Qinghai-Tibet Plateau [13]. Tibetan sheep produce high quality pelage, provide nutritive and delicious meat, and are economically

Statistical Analysis.
Variables associated with N. caninum infection among Tibetan sheep of different seasons, regions, pregnancy, gender, and age groups were analyzed in a multivariable logistic regression model, and probability (P) value < 0.05 was considered as statistically significant between factors and prevalence. Odds-ratios (OR) with 95% confidence intervals based on likelihood ratio statistics are calculated. All statistical analyses were performed using the SAS (Statistical analysis system, Version 8.0).

Results and Discussion
In this study, the total seroprevalence of N. caninum among the examined  [15], 6% in the Czech Republic [16], 6.6% in Argentina, and 6.4% in Brazil [17], the higher prevalence of 25.9% in Turkey [18] and 23.6% in Thailand [19].
Due to the different investigation sites, different detection methods, sheep breeds, and various sample capacities, it is difficult to compare N. caninum prevalence between these studies, but these studies all confirmed that caprines are truly easy to be infected by N. caninum.
To evaluate the seasonal difference of N. caninum seroprevalence in Tibetan sheep, serum samples were collected in the four seasons during a whole year. The results indicated that N. caninum infection varied in a year ranging from 6.5% in autumn to 11.3% in summer, but the difference was not statistically significant (P > 0.05). High altitude in the Qinghai-Tibet Plateau results in significant temperature differences between day and night, which may, to some degree, weaken the influence of seasons on the N. caninum infection in Tibetan sheep.
Statistically, the N. caninum seroprevalence in male Tibetan sheep (10.8%) (69/638, 95% CI 8.4-13.2) was significantly higher than in the female (7.4%) (115/1549, 95% CI 6.1-8.7) (P < 0.01), which is probably related to the different hormone levels between males and females [20]. It also may be due to horizontal transmission of N. caninum and exposure of older Tibetan sheep for a long period. But this result is not consistent with some previous reports that no significant differences were observed between male and female goats [21][22][23], probably reflecting the breed difference.
In addition, the effects of age and pregnancies on the N. caninum infection were also evaluated among the examined Tibetan sheep, and the results indicated age and pregnancies had no significant effect on the N. caninum seroprevalence (both P > 0.05), although these results were not consistent with some previous studies when the older and pregnant animals were at high risk of being exposed to N. caninum [24].
In conclusion, this is the first report of the N. caninum seroprevalence and risk factors associated with the parasite in Tibetan sheep in China. It provides baseline data for establishing control programs. In the future, further molecular investigations and bioassays on N. caninum infection in Tibetan sheep should be carried out.

Data Availability
The Neospora caninum prevalence data used to support the findings of this study are included within the article.

Ethical Approval
All Tibetan sheep were handled in strict accordance with good animal practice according to the Animal Ethics Procedures and Guidelines of the People's Republic of China, and the study was approved by the Animal Administration and Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

Conflicts of Interest
The authors declare that they have no conflicts of interest.