# Seroprevalence and Risk Factors ofChlamydia abortus Infection in Tibetan Sheep in Gansu Province, Northwest China

Authors: Si-Yuan Qin; Ming-Yang Yin; Wei Cong; Dong-Hui Zhou; Xiao-Xuan Zhang; Quan Zhao; Xing-Quan Zhu; Ji-Zhang Zhou; Ai-Dong Qian
Journal: The Scientific World Journal
 (2014)

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## Abstract

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.


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## Body

## 1. 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 genusChlamydia that includes nine species; among themC. abortus andC. pecorum can cause diseases in sheep [1–3]. In particular,C. abortus is recognized as a major cause of abortion and lamb loss throughout the world, especially in the intensively managed farms [4–6].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 byC. abortus also include epididymitis, pneumonia, arthritis, and conjunctivitis [7–9]. Recent reports described the presence ofC. 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 [12–14].Chlamydial infection of sheep has been reported throughout the world [10, 15–18], including China. However, on account of Tibetan sheep breeding only in northwest China, no more reports were available on epidemiology ofC. abortus infection in Tibetan sheep in China except that several reports aboutC. abortus infection in Tibetan sheep in Qinghai were published in local journals [19, 20] and only one article concerningC. 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 source of income for local Tibetans, due to its high quality pelage and nutritive and delicious meat. However, it is yet to know whether Tibetan sheep are infected withC. abortusin Gansu Province, northwestern China. The objective of the present study was to evaluate the seroprevalence and risk factors ofC. abortus infection in Tibetan sheep in Tianzhu Tibetan Autonomous County and Gannan Tibetan Autonomous Prefecture, Gansu province, northwest China.


## 2. Materials and Methods

### 2.1. The Study Site

The serum samples used in the present study were collected from Tibetan sheep in Tianzhu Tibetan Autonomous County and Gannan Tibetan Autonomous Prefecture including Maqu County and Luqu County in Gansu Province, northwest China. The Tibetan sheep in Gansu were mainly bred in Tianzhu and Gannan, in which most of the Tibetans also live. The sampling site is between the eastern longitudes of 102°07′-103°46′ and northern latitudes of 36°31′–37°55′ in Tianzhu Tibetan Autonomous County, and the average annual temperatures are from −8 to 4°C. Gannan Tibetan Autonomous Prefecture is located between longitudes 100°45′ to 104°45′ east and latitudes 33°06′ to 35°34′ north, in which the average annual temperatures are from 1 to 13°C.


### 2.2. Serum Samples

Blood samples were collected from 1732 Tibetan sheep in 3 counties in Gansu province from June 2013 to April 2014. The samples were collected randomly from 8 Tibetan sheep herds including 4 random herds in Tianzhu Tibetan Autonomous County and 4 random herds of the farms in Gannan Tibetan Autonomous Prefecture. 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.


### 2.3. 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 toC. abortus and it was carried out according to the manufacturer’s instructions as described previously [20–22]. 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 96-well V-bottomed polystyrene plates, which were diluted 4-fold serially beginning with 1 : 4 to 1 : 1,024. Then, theC. 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 forC. abortus antibodies. Positive results between 1 : 4 and 1 : 16 were considered “suspect” and were retested.


### 2.4. Statistical Analysis

Differences in the seroprevalence ofC. 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 P
<
0.05 and the 95% confidence intervals (CI) are also calculated.


## 2.1. The Study Site

The serum samples used in the present study were collected from Tibetan sheep in Tianzhu Tibetan Autonomous County and Gannan Tibetan Autonomous Prefecture including Maqu County and Luqu County in Gansu Province, northwest China. The Tibetan sheep in Gansu were mainly bred in Tianzhu and Gannan, in which most of the Tibetans also live. The sampling site is between the eastern longitudes of 102°07′-103°46′ and northern latitudes of 36°31′–37°55′ in Tianzhu Tibetan Autonomous County, and the average annual temperatures are from −8 to 4°C. Gannan Tibetan Autonomous Prefecture is located between longitudes 100°45′ to 104°45′ east and latitudes 33°06′ to 35°34′ north, in which the average annual temperatures are from 1 to 13°C.


## 2.2. Serum Samples

Blood samples were collected from 1732 Tibetan sheep in 3 counties in Gansu province from June 2013 to April 2014. The samples were collected randomly from 8 Tibetan sheep herds including 4 random herds in Tianzhu Tibetan Autonomous County and 4 random herds of the farms in Gannan Tibetan Autonomous Prefecture. 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.


## 2.3. 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 toC. abortus and it was carried out according to the manufacturer’s instructions as described previously [20–22]. 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 96-well V-bottomed polystyrene plates, which were diluted 4-fold serially beginning with 1 : 4 to 1 : 1,024. Then, theC. 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 forC. abortus antibodies. Positive results between 1 : 4 and 1 : 16 were considered “suspect” and were retested.


## 2.4. Statistical Analysis

Differences in the seroprevalence ofC. 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 P
<
0.05 and the 95% confidence intervals (CI) are also calculated.


## 3. Results

In the present study, 323 (18.65%) out of 1732 serum samples from Tibetan sheep in Gansu province were seropositive forC. abortus infection by IHA at a 1 : 16 cut-off (Table 1). As shown in Table 1, the seroprevalence ofC. abortus infection in Tianzhu County (n
=
962), Maqu County (n
=
588), and Luqu County (n
=
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. All of the sampled 8 Tibetan sheep herds were serologically positive. Male sheep had a higher prevalence (21.51%) compared to females (17.39%). The positive samples were distributed among four seasons (Spring: n
=
480; Summer: n
=
398; Autumn: n
=
479; Winter: n
=
375) in which the prevalences ofC. abortus infection were 18.54%, 22.11%, 20.46%, and 12.80% (P
<
0.05, 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 summarizes the antibodies titers in different ages, regions, genders, and seasons with IHA titers of 1 : 16 in 188 samples (58.20%), 1 : 32 in 72 samples (22.29%), 1 : 64 in 34 samples (10.53%), 1 : 128 in 14 samples (4.33%), 1 : 256 in 12 samples (3.72%), 1 : 512 in 2 samples (0.62%), and 1 : 1024 in 1 sample (0.31%).Table 1
Seroprevalence ofChlamydia abortus infection in Tibetan sheep associated with different factors in Gansu province, northwest China, by indirect hemagglutination assay (IHA).
Factor
Category
Tested numbers
Positive numbers
Prevalence (%)
Region
Tianzhu County
962
157
16.32
Maqu County
588
133
22.62
Luqu County
182
33
18.13
Sex
Male
530
114
21.51
Female
1202
209
17.39
Season
Spring
480
89
18.54
Summer
398
88
22.11
Autumn
479
98
20.46
Winter
375
48
12.80
Age (yr)
0 < yr ≤ 1
335
25
7.46
1 < yr ≤ 2
392
62
15.82
2 < yr ≤ 3
194
38
19.59
yr > 3
811
198
24.41
Total
1732
323
18.65Table 2
Seroprevalence ofChlamydia  abortus infection in Tibetan sheep in eight herds in two regions in Gansu, China.
Region
Herd numbers
Examined numbers
Positive numbers
Prevalence (%)
Tianzhu
Herd (1)
242
43
17.77
Herd (2)
216
38
17.60
Herd (3)
255
31
12.16
Herd (4)
249
45
18.07
Total
962
157
16.32
Gannan
Herd (5)
238
46
19.33
Herd (6)
159
10
6.29
Herd (7)
224
67
29.91
Herd (8)
149
43
28.86
Total
770
166
21.56Table 3
Seroprevalence ofChlamydia  abortus infection in Tibetan sheep in Gansu, China, determined by indirect haemagglutination (IHA) test.
Biometric data
Category
Antibody  titers
Positive numbers
Tested numbers
Prevalence  (%)
1 : 16
1 : 32
1 : 64
1 : 128
1 : 256
1 : 512
1 : 1024
Age (years)
≤1
16
4
0
3
2
0
0
25
335
7.46
1 < yr ≤ 2
39
12
7
2
2
0
0
62
392
15.82
2 < yr ≤ 3
19
10
4
2
3
0
0
38
194
19.59
yr > 3
114
46
23
7
5
2
1
198
811
24.41
Region
Tianzhu County
83
30
21
12
10
0
1
157
962
16.32
Maqu County
85
35
8
2
1
2
0
133
588
22.62
Luqu County
20
7
5
0
1
0
0
33
182
18.13
Sex
Male
76
24
9
2
2
1
0
114
530
21.51
Female
112
48
25
12
10
1
1
209
1202
17.39
Season
Winter
28
10
4
3
2
0
1
48
375
12.80
Spring
48
20
11
4
6
0
0
89
480
18.54
Autumn
62
22
8
5
1
0
0
98
479
20.46
Summer
50
20
11
2
3
2
0
88
398
22.11
Total
188
72
34
14
12
2
1
323
1732
18.65According to forward stepwise logistic regression, gender of Tibetan sheep was not significant in the logistic regression analysis (P
>
0.05) and left out of the final model. Multivariable analysis of such 4 factors showed thatC. abortus seropositivity was associated with regions, seasons, and ages. Tibetan sheep in Luqu County was more than 1.4-fold increase (OR = 1.40, 95% CI = 1.07–1.84, P
=
0.0
14) at risk ofC. abortus infection compared to Tibetan sheep in Tianzhu County, and no regional differences were found in Luqu County compared to Tianzhu County (P
>
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, P
<
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, P
<
0.001) and 4.0 times (OR = 4.02, 95% CI = 2.58–6.26, P
<
0.001) higher risk compared to Tibetan sheep of 0 < years ≤ 1 age group separately (Table 4). In terms of seasons, the risk ofC. abortus infection in summer was more than 2.0-fold increase (OR = 1.97, 95% CI = 1.33–2.91, P
=
0.0
01) compared toC. abortus infection in winter (Table 4).Table 4
Odds ratios for ages, seasons, and geographical origin of Tibetan sheep are taken as risk factors forChlamydia abortus seroprevalence in Tibetan sheep.
Factor
Category
Tested numbers
Positive numbers
Prevalence (%)
OR (95% CI)
P value
Region
Tianzhu County
962
157
16.32
Reference
Maqu County
588
133
22.62
0.91 (0.56–1.48)
0.694
Luqu County
182
33
18.13
1.40 (1.07–1.84)
0.014
Season
Winter
375
48
12.80
Reference
Spring
480
89
18.54
1.67 (1.08–2.57)
0.022
Autumn
479
98
20.46
1.78 (1.21–2.62)
0.003
Summer
398
88
22.11
1.97 (1.33–2.91)
0.001
Age (yr)
0 < yr ≤ 1
335
25
7.46
Reference
1 < yr ≤ 2
392
62
15.82
2.48 (1.51–4.07)
<0.001
2 < yr ≤ 3
194
38
19.59
2.99 (1.72–5.19)
<0.001
yr > 3
811
198
24.41
4.02 (2.58–6.26)
<0.001


## 4. 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 byC. abortus in North America, Africa, and parts of Europe, including UK, in which costs to the farming industry due toC. 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 ofC. abortus in Tibetan sheep in Gansu province, northwest China.In the present study, the overallC. 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 ofChlamydia 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 withC. 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 withC. abortushave been reported in Qinghai [22] and Gansu provinces (unpublished), which indicated a high risk as a source ofC. abortus infection for Tibetan sheep. This may be one of the possible reasons for the highC. abortus seroprevalence (18.65%) in Tibetan sheep in Gansu, northwestern China.In the present study, theC. abortus seroprevalence in male and female sheep was 21.51% and 17.39%, respectively. However, there was no significant difference inC. abortus seroprevalence between genders (P
>
0.05), which is consistent with the studies of Huang et al. [21] in which they reported negative association between sex andC. abortus prevalence in Tibetan sheep in Tibet, implying that gender may not be a crucial factor forC. abortus infection in Tibetan sheep.This study revealed that the geographical origin of Tibetan sheep is one of the risk factors associated withC. abortus seroprevalence. Tibetan sheep in Gannan Tibetan Autonomous Prefecture (21.56%) had a higher risk of beingC. 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 toC. 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 withC. abortus.Season is a significant risk factor forC. 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 resulting in generating many antibodies for resistance toC. 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 forC. abortus prevalence. As the growth of the age, the seroprevalence ofC. abortus infection went up all the time, indicating that there may be a cumulative likelihood for exposure toC. abortus infection with age in these surveyed regions.IHA is regarded as a simple, safe, and useful method for examination ofC. 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 detectingC. abortus infection in Tibetan sheep.


## 5. Conclusions

Results of the present study revealed a highC. 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 forC. abortus seroprevalence. Therefore, it is necessary to implement integrated control and efficient management measures to prevent and controlC. abortus infection in Tibetan sheep in Gansu province.



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*Source: 193464-2014-10-23.xml*