Seroprevalence and Risk Factors of Chlamydia abortus Infection in Tibetan Sheep in Gansu Province, Northwest China

Chlamydia abortus, an important pathogen in a variety of animals, is associated with abortion in sheep. In the present study, 1732 blood samples, collected from Tibetan sheep between June 2013 and April 2014, were examined by the indirect hemagglutination (IHA) test, aiming to evaluate the seroprevalence and risk factors of C. abortus infection in Tibetan sheep. 323 of 1732 (18.65%) samples were seropositive for C. abortus antibodies at the cut-off of 1 : 16. A multivariate logistic regression analysis was used to evaluate the risk factors associated with seroprevalence, which could provide foundation to prevent and control C. abortus infection in Tibetan sheep. Gender of Tibetan sheep was left out of the final model because it is not significant in the logistic regression analysis (P > 0.05). Region, season, and age were considered as major risk factors associated with C. abortus infection in Tibetan sheep. Our study revealed a widespread and high prevalence of C. abortus infection in Tibetan sheep in Gansu province, northwest China, with higher exposure risk in different seasons and ages and distinct geographical distribution.


Introduction
Chlamydia, an obligate intracellular gram-negative bacterium, is known to cause a variety of diseases in animals and humans [1,2]. Chlamydiaceae have a single genus Chlamydia that includes nine species; among them C. abortus and C. pecorum can cause diseases in sheep [1][2][3]. In particular, C. abortus is recognized as a major cause of abortion and lamb loss throughout the world, especially in the intensively managed farms [4][5][6]. C. abortus usually causes ulceration of endometrial epithelium resulting in placental infection if infection was acquired during the early stages of that pregnancy. More typically, infection acquired during late gestation will result in abortion in the following gestation and the symptoms caused by C. abortus also include epididymitis, pneumonia, arthritis, and conjunctivitis [7][8][9]. Recent reports described the presence of C. abortus DNA in the eyes of ewes [10,11]. C. abortus not only causes economic loss in the sheep industry, but also induces abortions in humans due to contact with aborting sheep or goats [12][13][14].
Chlamydial infection of sheep has been reported throughout the world [10,[15][16][17][18], including China. However, on account of Tibetan sheep breeding only in northwest China, no more reports were available on epidemiology of C. abortus infection in Tibetan sheep in China except that several reports about C. abortus infection in Tibetan sheep in Qinghai were published in local journals [19,20] and only one article concerning C. abortus infection in Tibetan sheep in Tibet was documented in the international literature [21].
Tibetan sheep is one of the three main original sheep breeds living in the Qinghai-Tibetan Plateau passing through parts of regions of Gansu province, northwest China, where the solar radiation is strong and the temperature and oxygen content are low [19]. Tibetan sheep are recognized as a major  Serum samples were separated from blood samples in local veterinary stations and then were taken to the laboratory and stored at −20 ∘ C until further tested. The detailed information of animals was obtained from local herdsmen by questioning.

Serological Examination.
A commercially available Indirect Hemagglutination Assay (IHA) kit (Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China) was purchased to test antibodies to C. abortus and it was carried out according to the manufacturer's instructions as described previously [20][21][22]. The IHA kit was employed following the standard method described by China CADC, and the sensitivity and specificity of the experiment are 100% and 95%, respectively. The Ministry of Agriculture of China (NY/T 562-2002) has validated the sensitivity and specificity values for the testing kit used in this study. In brief, serum samples were added to 96well V-bottomed polystyrene plates, which were diluted 4fold serially beginning with 1 : 4 to 1 : 1,024. Then, the C. abortus antigen was added, and the plates were shaken gently for 2 min and incubated at 37 ∘ C for 2 h. Each test was performed with positive, negative, and blank controls, and serum samples which had positive reaction at dilutions of 1 : 16 or higher dilutions were considered positive for C. abortus antibodies. Positive results between 1 : 4 and 1 : 16 were considered "suspect" and were retested.

Statistical Analysis.
Differences in the seroprevalence of C. abortus among Tibetan sheep of different geographical origins, genders, seasons, and age groups were analyzed with Chi-square tests using the SPSS software (SPSS Inc., IBM Corporation, Version 19, USA). The differences were considered statistically significant if < 0.05 and the 95% confidence intervals (CI) are also calculated.

Results
In the present study, 323 (18.65%) out of 1732 serum samples from Tibetan sheep in Gansu province were seropositive for C. abortus infection by IHA at a 1 : 16 cut-off ( Table 1). As shown in Table 1, the seroprevalence of C. abortus infection in Tianzhu County ( = 962), Maqu County ( = 588), and Luqu County ( = 182) was 16.32%, 22.62%, and 18.13%, respectively. The numbers of seropositive animals and seroprevalence of individual herd were described in Table 2 Table 1), respectively. The ages of the examined Tibetan sheep varied from 0 years to 3 years or greater and seroprevalence in different age groups ranged from 7.46% to 24.41% ( Table 1). The highest seroprevalence was found in Tibetan sheep older than 3 years (24.41%) and the lowest in 0 years to 3 years (7.46%). Table 3  According to forward stepwise logistic regression, gender of Tibetan sheep was not significant in the logistic regression analysis ( > 0.05) and left out of the final model. Multivariable analysis of such 4 factors showed that C. abortus seropositivity was associated with regions, seasons, and ages. Tibetan sheep in Luqu County was more than 1.4fold increase (OR = 1.40, 95% CI = 1.07-1.84, = 0.014) at risk of C. abortus infection compared to Tibetan sheep in Tianzhu County, and no regional differences were found in Luqu County compared to Tianzhu County ( > 0.05). Ages of Tibetan sheep which were 1 < years ≤ 2 years old (15.82%) had a 2.5-fold increase higher risk of being seropositive compared to that of Tibetan sheep which were 0 < years ≤ 1 years old (7.46%) (OR = 2.48, 95% CI = 1.51-4.07, < 0.001), while Tibetan sheep of 2 < years ≤ 3 and >3 years old had a 3-fold increase (OR = 1.40, 95% CI = 1.72-5.19, < 0.001) and 4.0 times (OR = 4.02, 95% CI = 2.58-6.26, < 0.001) higher risk compared to Tibetan sheep of 0 < years ≤ 1 age group separately (Table 4). In terms of seasons, the risk of C. abortus infection in summer was more than 2.0-fold increase (OR = 1.97, 95% CI = 1.33-2.91, = 0.001) compared to C. abortus infection in winter (Table 4).
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Discussions
Members of the family Chlamydiaceae can result in a broad range of diseases in both humans and animals, including sheep, which could cause health problems and heavy economic losses [23]. In particular, lamb loss in sheep was caused by C. abortus in North America, Africa, and parts of Europe, including UK, in which costs to the farming industry due to C. abortus in sheep were estimated to be £ 20,000,000 per annum [1]. Tibetan sheep surveyed in the present study were free-ranging on the grassland in Tianzhu Tibetan Autonomous County and Gannan Tibetan Autonomous Prefecture, and the meat production of which was not only transported to other Cities of Gansu province including Lanzhou as the provincial capital, but also transported to adjacent provinces including Qinghai, Sichuan, Shanxi, and Ningxia. However, there was little information available about the prevalence of C. abortus in Tibetan sheep in Gansu province, northwest China. In the present study, the overall C. abortus seroprevalence in Tibetan sheep in Gansu province was 18.65%, which is higher than the 12.69% seroprevalence among Tibetan sheep in Tianjun County [19] and 8.01% in Yushu County of Qinghai province, China, by IHA [24]. It is lower than the values of 20.9% in Tibetan sheep in Tibet [21] and 26.3% in sheep in Gansu, China [25]. The different prevalence observed was probably due to differences in animal-welfare, sanitation, climates, and husbandry practices. In view of herds surveyed, although seropositive samples were found in all examined herds, seroprevalence in each herd varied obviously. Many seropositive animals in one tested herd could have an effect on high level of seroprevalence in evaluated region and the high seroprevalence may not be connected with individual region but with high level of infection at the herd level, which may be the main season for high seroprevalence in Gannan.
Chlamydia infection in humans and animals was caused by direct contacts with infected animals. The transmission of Chlamydia occurs mainly through inhalation or ingestion of these infectious dust contaminated materials [26]. Most of the time Tibetan sheep are free-ranging on the grassland and sheds of sheep are simple and crude, which increase the opportunity to be infected with C. abortus. The farms of Tibetan sheep herds were located in mountainous areas belonging to Qinghai-Tibet Plateau in which a lot of yaks lived around with Tibetan sheep. Yaks infected with C. abortus have been reported in Qinghai [22] and Gansu provinces (unpublished), which indicated a high risk as a source of C. abortus infection for Tibetan sheep. This may be one of the possible reasons for the high C. abortus seroprevalence (18.65%) in Tibetan sheep in Gansu, northwestern China.
In the present study, the C. abortus seroprevalence in male and female sheep was 21.51% and 17.39%, respectively. However, there was no significant difference in C. abortus seroprevalence between genders ( > 0.05), which is consistent with the studies of Huang et al. [21] in which they reported negative association between sex and C. abortus prevalence in Tibetan sheep in Tibet, implying that gender may not be a crucial factor for C. abortus infection in Tibetan sheep.
This study revealed that the geographical origin of Tibetan sheep is one of the risk factors associated with C. abortus seroprevalence. Tibetan sheep in Gannan Tibetan Autonomous Prefecture (21.56%) had a higher risk of being C. abortus seropositive compared to Tibetan sheep in Tianzhu Tibetan Autonomous County (16.32%). The geographic differences in prevalence may be related with differences in living environment and husbandry practices. According to local herders' introduction, wild animals including wild yaks, sika deer, and Tibetan antelope were found in field and farms of Gannan Tibetan Autonomous Prefecture, and all of these species are susceptible to C. abortus. In Gannan, people have low awareness of disease prevention and control, and it is difficult for veterinarians to reach the places to implement immunization programs. Therefore, living environment and husbandry practices were two main risk factors associated with C. abortus.
Season is a significant risk factor for C. abortus prevalence due to different climates in different seasons, including diverse temperature, precipitation, and humidity. In addition, the seasonal seroprevalence data may be related to persistence of antibody following abortion. Abortion occurred in spring The Scientific World Journal 5 resulting in generating many antibodies for resistance to C. abortus and the rubbish of abortion may be contacted by other healthy sheep, which led to high seroprevalence in summer. As time went on, the antibody level reached the lowest in winter.
Age of Tibetan sheep (years) as a continuous variable was analyzed in the logistic regression model, and the results showed that the prevalences were different significantly with ages, demonstrating that age is a predisposing factor for C. abortus prevalence. As the growth of the age, the seroprevalence of C. abortus infection went up all the time, indicating that there may be a cumulative likelihood for exposure to C. abortus infection with age in these surveyed regions.
IHA is regarded as a simple, safe, and useful method for examination of C. abortus antibodies, which has been employed in previous serological surveys [21,22]. The sensitivity and specificity values of the testing IHA kit have been validated by the Ministry of Agriculture of China, which demonstrates that the IHA is not only more efficient than the CFT but also more inexpensive than the ELISA [27]. Due to these advantages, it may be the most appropriate commercially available kit for detecting C. abortus infection in Tibetan sheep.

Conclusions
Results of the present study revealed a high C. abortus seroprevalence in Tibetan sheep in Gansu province, which can cause significant economic losses to the local ovine industry and pose a potential threat to Tibetans in these areas. This study also showed that region, season, and age are main risk factors for C. abortus seroprevalence. Therefore, it is necessary to implement integrated control and efficient management measures to prevent and control C. abortus infection in Tibetan sheep in Gansu province.

Ethical Approval
This study was approved by the Animal Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Approval No. LVRIAEC2013-010). The Tibetan sheep from which the serum samples were collected were handled in accordance with good animal practices required by the Animal Ethics Procedures and Guidelines of the People's Republic of China.