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

Authors: Lan-Bi Nie; Wei Cong; Yang Zou; Dong-Hui Zhou; Qin-Li Liang; Wen-Bin Zheng; Jian-Gang Ma; Rui Du; Xing-Quan Zhu
Journal: BioMed Research International
 (2018)

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## Abstract

Neospora caninum is an intracellular protozoan parasite which can cause abortion and stillbirth in ruminants. However, there is no information on Tibetan sheepN. 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 toN. caninum using a competitive-inhibition enzyme-linked immunoassay. Of the 2187 serum samples, 184 (8.4%, 95% CI 7.3-9.6) were testedN. caninum seropositive. TheN. 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 ofN. caninum seroprevalence in Tibetan sheep in China, which provides baseline data for the prevention and control ofN. caninum infection in Tibetan sheep.


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## Body

## 1. Introduction

Neospora caninumis an obligate intracellular protozoan which can infect a variety of mammals and birds [1]. During the whole life cycle ofN. 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 ofN. caninum[5]. Though noN. caninum has been found from human tissue till now, antibodies toN. caninum have been detected in human serum [6], suggesting a zoonotic potential of the parasite.TheN. caninum seroprevalence has been reported in sheep and goats worldwide [7–9]. In China’s Tibetan areas, the prevalence and infection ofN. caninum have been reported in black yaks [10, 11] and white yaks [12]. However, there is no information about the infection ofN. 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 important for the local Tibetans. Thus, the objective of this study was to determine the seroprevalence and assess rick factors ofN. caninum infection in Tibetan sheep in China.


## 2. Materials and Methods

### 2.1. The Study Sites

In the present study, serum samples were collected from Tibetan sheep in Gansu province (32°31′′~42°57′′ N, 92°13′′~108°46′′ E), northwest China, and Nyingchi prefecture (26°52′′~30°40′′ N, 92°09′′~98°47′′ E) in southeast Tibet, southwest China. The average elevation of the surveyed areas is more than 3000 meters above sea level and has a plateau continental climate.


### 2.2. Serum Samples

Serum samples were collected from Tibetan sheep via the caudal vein by local veterinary practitioners. A total of 2187 serum samples were collected from farmed Tibetan sheep from Luqu, Maqu, and Tianzhu in Gansu province, and Nyingchi in Tibet, China (Table1). Serum samples were taken to the laboratory and kept at room temperature for 2 hours and then centrifuged at 3000 rpm for 10 min. Serum was separated and stored at – 20°C until further tested. Moreover, a standardized questionnaire was used to record region, age, gender, history of pregnancy, and sampling season.Table 1
Seroprevalence ofNeospora caninumin Tibetan sheep from different regions, genders, seasons, ages, and pregnancies in Gansu and Tibet provinces, China, tested by competitive-inhibition enzyme-linked immunosorbent assay.
Variable Category No. examined Positive (%) 95% CI P-value OR (95% CI) Region Luqu 182 8 (4.4%) 1.4-7.4 >0.05 Reference Maqu 588 48 (8.2%) 5.9-10.4  1.9 (0.9-4.2) Tianzhu 962 90 (9.4%) 7.5-11.2  2.3 (1.1-4.7) Nyingchi 455 38 (8.4%) 5.8-10.9  1.9 (0.9-4.3) Sex Male 638 69 (10.8%) 8.4-13.2 <0.01 1.5 (1.1-2.1) Female 1549 115 (7.4%) 6.1-8.7  Reference Season Spring 480 41 (8.5%) 6.2-11.1 >0.05 1.4 (0.8-2.2) Summer 398 45 (11.3%) 8.2-14.4  1.8 (1.1-2.9) Autumn 479 31 (6.5%) 4.3-8.7  Reference Winter 375 29 (7.7%) 5.0-10.4  1.2 (0.7-2.1) No data 455 38 (8.4%) 5.8-10.9  1.3 (0.8-2.1) Age (year∗) 0-1 447 45 (10.1%) 7.3-12.9 >0.05 1.9 (1.1-3.8) ≥1-2 413 27 (6.5%) 4.2-8.9  1.2 (0.6-2.5) ≥2-3 243 13 (5.4%) 2.5-8.2  Reference ≥3 1084 99 (9.1%) 7.4-10.9  1.8 (0.9-3.2) Pregnancy Not pregnant 581 49 (8.4%) 6.2-10.7 >0.05 1.2 (0.8-1.7) Pregnant 796 59 (7.4%) 5.6-9.2  Reference No data 810 76 (9.4%) 7.4-11.4  1.3 (0.9-1.9) Total  2187 184 (8.4%) 7.3-9.6 95% CI, 95% confidence interval; OR, odds ratio. A n i m a l s   ∗ are grouped by age of year.


### 2.3. Serological Examination

All samples were analyzed for the presence of antibodies toN. caninum using a competitive-inhibition enzyme-linked immunosorbent assay (cELISA) kit (VMRD, Pullman, USA) validated for ruminants according to the manufacturer’s instructions. The test results were expressed as percentage of inhibition (%I) according to the following formula: %I = 100 [1- (Sample OD ÷ NC OD)]. The serum was examined in duplicate and considered positive if more than 30% inhibition was detected.


### 2.4. Statistical Analysis

Variables associated withN. 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).


## 2.1. The Study Sites

In the present study, serum samples were collected from Tibetan sheep in Gansu province (32°31′′~42°57′′ N, 92°13′′~108°46′′ E), northwest China, and Nyingchi prefecture (26°52′′~30°40′′ N, 92°09′′~98°47′′ E) in southeast Tibet, southwest China. The average elevation of the surveyed areas is more than 3000 meters above sea level and has a plateau continental climate.


## 2.2. Serum Samples

Serum samples were collected from Tibetan sheep via the caudal vein by local veterinary practitioners. A total of 2187 serum samples were collected from farmed Tibetan sheep from Luqu, Maqu, and Tianzhu in Gansu province, and Nyingchi in Tibet, China (Table1). Serum samples were taken to the laboratory and kept at room temperature for 2 hours and then centrifuged at 3000 rpm for 10 min. Serum was separated and stored at – 20°C until further tested. Moreover, a standardized questionnaire was used to record region, age, gender, history of pregnancy, and sampling season.Table 1
Seroprevalence ofNeospora caninumin Tibetan sheep from different regions, genders, seasons, ages, and pregnancies in Gansu and Tibet provinces, China, tested by competitive-inhibition enzyme-linked immunosorbent assay.
Variable Category No. examined Positive (%) 95% CI P-value OR (95% CI) Region Luqu 182 8 (4.4%) 1.4-7.4 >0.05 Reference Maqu 588 48 (8.2%) 5.9-10.4  1.9 (0.9-4.2) Tianzhu 962 90 (9.4%) 7.5-11.2  2.3 (1.1-4.7) Nyingchi 455 38 (8.4%) 5.8-10.9  1.9 (0.9-4.3) Sex Male 638 69 (10.8%) 8.4-13.2 <0.01 1.5 (1.1-2.1) Female 1549 115 (7.4%) 6.1-8.7  Reference Season Spring 480 41 (8.5%) 6.2-11.1 >0.05 1.4 (0.8-2.2) Summer 398 45 (11.3%) 8.2-14.4  1.8 (1.1-2.9) Autumn 479 31 (6.5%) 4.3-8.7  Reference Winter 375 29 (7.7%) 5.0-10.4  1.2 (0.7-2.1) No data 455 38 (8.4%) 5.8-10.9  1.3 (0.8-2.1) Age (year∗) 0-1 447 45 (10.1%) 7.3-12.9 >0.05 1.9 (1.1-3.8) ≥1-2 413 27 (6.5%) 4.2-8.9  1.2 (0.6-2.5) ≥2-3 243 13 (5.4%) 2.5-8.2  Reference ≥3 1084 99 (9.1%) 7.4-10.9  1.8 (0.9-3.2) Pregnancy Not pregnant 581 49 (8.4%) 6.2-10.7 >0.05 1.2 (0.8-1.7) Pregnant 796 59 (7.4%) 5.6-9.2  Reference No data 810 76 (9.4%) 7.4-11.4  1.3 (0.9-1.9) Total  2187 184 (8.4%) 7.3-9.6 95% CI, 95% confidence interval; OR, odds ratio. A n i m a l s   ∗ are grouped by age of year.


## 2.3. Serological Examination

All samples were analyzed for the presence of antibodies toN. caninum using a competitive-inhibition enzyme-linked immunosorbent assay (cELISA) kit (VMRD, Pullman, USA) validated for ruminants according to the manufacturer’s instructions. The test results were expressed as percentage of inhibition (%I) according to the following formula: %I = 100 [1- (Sample OD ÷ NC OD)]. The serum was examined in duplicate and considered positive if more than 30% inhibition was detected.


## 2.4. Statistical Analysis

Variables associated withN. 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).


## 3. Results and Discussion

In this study, the total seroprevalence ofN. caninum among the examined Tibetan sheep was 8.4% (184/2187, 95% CI 7.3-9.6). TheN. caninum prevalence in Tibetan sheep varied between regions, ranging from 4.4% in Luqu (8/182, 95% CI 1.4-7.4) to 9.4% in Tianzhu (90/962, 95% CI 7.5-11.2) (Table 1), but the difference was not statistically significant (P> 0.05). Caprine infection ofN. caninum has been reported worldwide, for example, the lower prevalence of 5.6% in Iraq [14], 6.1% in Costa Rica [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 compareN. caninum prevalence between these studies, but these studies all confirmed that caprines are truly easy to be infected byN. caninum.To evaluate the seasonal difference ofN. caninumseroprevalence in Tibetan sheep, serum samples were collected in the four seasons during a whole year. The results indicated thatN. caninuminfection 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 theN. caninuminfection in Tibetan sheep.Statistically, theN. 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 ofN. 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 [21–23], probably reflecting the breed difference.In addition, the effects of age and pregnancies on theN. caninum infection were also evaluated among the examined Tibetan sheep, and the results indicated age and pregnancies had no significant effect on theN. caninum seroprevalence (bothP> 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 toN. caninum[24].In conclusion, this is the first report of theN. 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 onN. caninum infection in Tibetan sheep should be carried out.



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*Source: 2098908-2018-05-29.xml*