Seroprevalence and risk factors of Neospora caninum infection among domestic sheep in Henan province, central China

This study aimed to determine the frequency of antibodies to Neospora caninum in domestic sheep raised in Henan province, central China. Serum samples from 779 domestic sheep were collected from March 2015 to May 2016, and antibodies to N. caninum were evaluated using an enzyme-linked immunosorbent assay (ELISA). The results showed an overall IgG positive rate of 7.32% (57/779). The risk factors significantly related to seropositivity to N. caninum in sheep were the age, the presence of dogs, and the rearing system. This is the first report of N. caninum infection and associated risk factors in domestic sheep in central China.


Introduction
Neospora caninum is an obligate intracellular protozoan parasite, which is regarded as one of the leading infectious causes of abortion among cattle around the world [10,22]. It has been detected in a variety of domestic and wild or free living animals, including cattle, goats, sheep, horses and deer [5,7,15,21].
Although cattle represent the most relevant and economically important target host for N. caninum [22,29], the presence of N. caninum infection and transplacental transmission in sheep have also been reported [6,26]. N. caninum was first described as a natural infection in sheep in a congenitally infected lamb in England [8]. Soon afterwards, neosporosis of natural occurrence among sheep was discovered around the world [4,14]. Like the parasite Toxoplasma gondii, N. caninum was also associated with reproductive failure. Traditionally, N. caninum is considered the main cause of abortion and neonatal death in cattle, whereas T. gondii has been thought to be one of the principal agents causing abortion in sheep. However, it was also suggested by recent evidence that N. caninum played an essential role in miscarriage among sheep [11,12].
Although the seroprevalence of N. caninum in sheep has been reported worldwide [1,17,19], limited information is available on the seroprevalence of N. caninum in sheep in China. So far, only the seroprevalence of N. caninum in sheep raised in Qinghai province, western China has been investigated. In total, 10.33% (62/600) of the sheep from Qinghai province were seropositive for N caninum. [13]. Data on the seroprevalence of N. caninum among sheep raised in other provinces of China remain unknown.
Therefore, this study was conducted to investigate the seroprevalence and the risk factors related to seropositivity of N. caninum among domestic sheep in Henan province, central China. The results will lay the groundwork for controlling N. caninum infections among domestic sheep in this region.

Ethics statement
The Ethics Committee of Xinxiang Medical University has reviewed and approved this study (reference no. 2015018).

The location of this study
The current investigation was performed in Henan province which is situated in the central section of China ( Fig. 1), with northern latitude of 31°23 0 À36°22 0 and eastern longitude of 110°21 0 À116°39 0 . It occupies an area of 167,000 km 2 and has a population of about 106.01 million with the Yellow River passing through its central section. The continental monsoon climate is characterized by two distinct seasons, with average precipitation of 530-1380 mm and annual temperatures of 12.1-15.7°C. There are seventeen provincial cities distributed in Henan province, with the city of Zhengzhou as its capital. Three cities including Xinxiang (35°18 0 N, 113°54 0 E), Zhoukou (33°03 0 À34°20 0 N, 114°05 0 À115°39 0 E) and Zhumadian (32°18 0 À33°35 0 N, 113°10 0 À115°12 0 E) were chosen for the collection of specimens since these cities are the main suppliers of ovine meat to Henan province and the neighboring regions.

Sample collection
In total, 779 blood samples were collected from domestic sheep in Henan in the above-mentioned cities, throughout the period from March 2015 to May 2016. To identify the risk factors, the information on sources, gender, age, rearing system of each animal, as well as the presence of dogs in the herd was also collected. After centrifuging blood specimens, serum was collected and preserved in 1.5 mL Eppendorf tubes at a temperature of À80°C, before being tested for antibodies against N. caninum.

Detection of N. caninum antibodies
The commercial ELISA kit (Neospora Ab Test, IDEXX Laboratories Inc., Westbrook, ME, USA) was used to analyze serum samples (diluted 1:100 in sample buffer), based on the manufacturer's instructions. The IDEXX Neospora Ab Test is an enzyme immunoassay for the detection of antibodies against N. caninum in serum and plasma samples of ruminants (cattle, goats, and sheep). The rate of specimen absorbance to positive control absorbance (S/P ratio) was represented as the result. S/ P = OD sample À OD negative control /OD positive control À OD negative control . The S/P ratio of 0.50 was considered the threshold value of N. caninum infection and any specimen with a ratio higher than that was considered positive. The specificity and sensitivity of this N. caninum ELISA kit were 98.3% and 98.6%, respectively [2].

Statistical analyses
A Chi square test was used to analyze variations of effects on N. caninum serum positive rates by risk factors including source, age, gender, rearing system of each animal, and the presence of dogs in the herd. Statistical analyses were performed using the SPSS 20 software for Windows (SPSS Inc., Chicago, IL, USA). A p value lower than 0.05 indicated statistical significance.

Results
In this study, ELISA was conducted on 779 blood samples from domestic sheep to test for N. caninum antibodies. As shown in Table 1, the serum positive rate of N. caninum among domestic sheep from Henan province, central China was 7.32% (57/779). Seropositive rates for sheep from the cities of Zhoukou, Zhumadian, and Xinxiang were 4.71% of 255, 8.82% of 272, and 8.33% of 252, respectively.
The serum positive rate for N. caninum among female sheep (8.01%, 39/487) was higher than that among male sheep (6.16%, 18/292) ( Table 1), but the difference was not statistically significant (p>0.05). The serum positive rate of N. caninum among sheep rose remarkably (p < 0.05) with increasing age. The peak value (11.20%) appeared among sheep no younger than 2 years, followed closely by the intermediate value (6.51%) which appeared among 1-2 year-old sheep. The sheep younger than 1 year exhibited the lowest prevalence value, at 4.47% (Table 1).
In addition, significantly higher seroprevalence was found in sheep raised on the farm with the presence of dogs (11.14%), compared to those without dogs (4.05%) (p < 0.01). Additionally, the seroprevalence obtained in extensively raised sheep was higher than that in intensively and semi-intensively raised samples (p < 0.05), but the difference between intensively and semi-intensively raised sheep was not significant (p > 0.05) ( Table 1).

Discussion
The current investigation showed that the total serum positive rate of N. caninum was 7.32% among domestic sheep in Henan, central China, which was lower than that found in Qinghai province, China (10.33%) [13]. Compared with other countries in the world, it was lower than rates observed in Galicia, Northwest Spain (10.1%) [20], the Czech Republic (12%) [3], São Paulo, Brazil (59.23%) [19], Pernambuco State, Brazil (64.2%) [27], but higher than those observed in New South Wales, Australia (2.2%) [4] and Slovakia (3.7%) [25]. The variations in serum positive rates among different regions are probably related to different sheep breeds, sample capacities, times of investigations, testing methods, as well as geographical and ecological factors.
Prevalence of N. caninum in this work was not found to be associated with gender of sheep, which was consistent with previous results [23,24]. Moreover, the N. caninum serum positive rate among sheep in this study was remarkably correlated with increasing age in a positive manner, which was in line with results in previous reports [19,24,27], indicating that N. caninum was probably transmitted horizontally among the investigated herds.
Dogs play an essential role in N. caninum transmission since they are the final hosts and shed oocysts into the environment [14,18,28]. Sheep can be infected with N. caninum mainly by intake of drink and food containing sporulated oocysts of N. caninum, or by transplacental transmission [11,14,16]. In this study, canine presence on sheep farms was confirmed as one of the risk factors for occurrence of N. caninum infection among sheep, which agreed with findings in previous studies [1,14,24].
Moreover, the serum positive rate among extensively raised sheep was higher than that among intensively and semi-intensively raised samples (p < 0.05), which was consistent with results in previous reports [9,19]. These results suggest that the rearing system is certainly a very important risk factor associated with N. caninum infection On the other hand, semi-intensive sheep farms are usually family-run businesses, which probably have lower hygiene standards and consequently suffer from N. caninum oocysts spreading among their animals. Extensive sheep farms may be more exposed to dogs in the environment or to contaminated stagnant pools, even though oocysts may be more dispersed in the environment.

Conclusions
The existence of N. caninum among sheep in Henan province, central China was revealed for the first time in the current study. Control measures, for example, completely cutting off contact between canines and sheep, are needed on sheep farms. Furthermore, further studies will be needed to determine the impact of N. caninum on sheep reproduction disorders in China.