# Seroprevalence and Risk Factors ofChlamydia Infection in Domestic Rabbits (Oryctolagus cuniculus) in China

Authors: Xiaoting Ni; Siyuan Qin; Zhilong Lou; Hongrui Ning; Xiaolin Sun
Journal: BioMed Research International
 (2015)

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## Abstract

Chlamydia spp. are obligate intracellular bacteria distributed all over the world, known to cause various forms of diseases in animals and humans. In the present study, a serological survey was conducted to detect the seroprevalence and risk factors associated with rabbit chlamydiosis in northeast China, including Liaoning province, Jilin province, Heilongjiang province, and Inner Mongolia Autonomous Region. Antibodies to Chlamydia were determined by indirect hemagglutination assay (IHA). The overall seroprevalence was estimated at 17.88% in total of 800 blood samples. The Chlamydia seroprevalence varied in domestic rabbits from different factors, and genders of domestic rabbits were considered as major risk factors associated with Chlamydia infection. Our study revealed a widespread and high prevalence of Chlamydia infection in domestic rabbits in northeast China, with higher exposure risk in female domestic rabbits. These findings suggested the potential importance of domestic rabbits in the transmission of zoonotic Chlamydia infection, and thus Chlamydia should be taken into consideration in diagnosing rabbit diseases. To our knowledge, there is no report of Chlamydia infection in domestic rabbits in China and the results extend the host range for Chlamydia, which has important implications for public health and the local economy.


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## Body

## 1. Introduction

Chlamydia is a genus comprising important zoonotic obligate intracellular pathogens that affect humans and a wide range of animals [1, 2].Chlamydia infection can cause various diseases in nonhuman mammals and birds, including conjunctivitis, atypical pneumonia, enteritidis, endocarditis, and even abortion, resulting in serious economic losses [3–6]. SeveralChlamydia species are known to infect humans and are of serious public health significance because they may result in atherosclerosis, coronary heart disease, pneumonia, and other severe diseases [7].Chlamydia abortus andChlamydia psittaci are of particular importance because they can cause abortion and psittacosis, respectively, in animals, birds and humans. In Egypt, a study revealed that in symptomatic gynecologically diseased women the seroprevalence ofChlamydia psittaci was 50.0%,Chlamydia abortus was 35.7%, andChlamydia trachomatis was 15.2% [8], and in the genital tract of cattle the main Chlamydiaceae wereChlamydia psittaci andChlamydia abortus [9]. The animal diseases caused by these microorganisms should be given more attention due to their zoonotic potential [1, 7, 10].The breeding of domestic rabbits (Oryctolagus cuniculus) for human consumption has a long radition in China, and rabbit industry is one of green economy industries, which is emerging and promising, with broad space for development and has gone through a long glorious history and its products have brought great wealth to the rural people in China. At present, China plays an important role of breeding rabbits, and domestic rabbits yield high quality meat and hair which are transported to many parts of home and abroad. However, disease is the main challenge for rabbit industry [11, 12]. To date, there is little information about the seroprevalence ofChlamydia and the risk factors associated withChlamydia infection in domestic rabbits in the world. Thus, the present study was aimed at determining the seroprevalence ofChlamydia infection in domestic rabbits in northeast China and identifying factors associated with the presence ofChlamydia antibodies in domestic rabbits in the study area.


## 2. Materials and Methods

### 2.1. Ethics Statement

This study was approved by the Animal Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Approval no. LVRIAEC2013-010). The domestic rabbits, 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.


### 2.2. Collection and Preparation of Serum Samples

A total of eight hundred domestic rabbits were randomly sampled from Liaoning province, Jilin province, Heilongjiang province, and Inner Mongolia Autonomous Region. From each rabbit, approximately 2 mL of blood was collected from the marginal ear vein or postmortem. After centrifugation at 3,000 rpm for 10 min, sera were collected and stored at −20°C until use. Handling of rabbits was in compliance with the Animal Ethics Procedures and Guidelines of the People’s Republic of China. Information about species, ages, geographic origin, and gender was acquired from breeders.


### 2.3. Serological Examination

Antibodies toChlamydia were tested by indirect hemagglutination antibody (IHA) using a commercially marketed kit (Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu Province, China). The procedures are according to the manufacturer’s instructions and previous descriptions [13, 14]. The IHA kit was performed following the standard protocol recommended by Chinese Center for Animal Diseases Prevention and Control, and the sensitivity and specificity of the test are 100% and 95%, respectively [13]. The sensitivity and specificity values for the testing kit used in this study have been validated by The Ministry of Agriculture of the People’s Republic of China (NY/T 562–2002). In brief, 75 μL of the IHA dilution solution was transferred into 96 well V-bottomed polystyrene plates with 25 μL of serum added and diluted in a fourfold series from 1 : 4 to 1 : 64. The plates were shaken for 2 min and then incubated at 37°C for 2 h without shaking. The test was considered positive when a layer of agglutinated erythrocytes was formed in wells at dilutions of 1 : 16 or higher, and positive and negative controls were included in each test. Those sera which showed dubious results were picked out for retest.


### 2.4. Statistical Analyses

Regions, genders, age groups, and breeds of rabbits were analyzed in a multivariable logistic regression model, and probability (P) value < 0.05 was considered as statistically significant. Odds ratios (OR) with 95% confidence intervals are also calculated. All statistical analyses were performed using the PASW Statistics 18.0 (SPSS Inc., IBM Corporation, Somers, NY).


## 2.1. Ethics Statement

This study was approved by the Animal Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Approval no. LVRIAEC2013-010). The domestic rabbits, 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.


## 2.2. Collection and Preparation of Serum Samples

A total of eight hundred domestic rabbits were randomly sampled from Liaoning province, Jilin province, Heilongjiang province, and Inner Mongolia Autonomous Region. From each rabbit, approximately 2 mL of blood was collected from the marginal ear vein or postmortem. After centrifugation at 3,000 rpm for 10 min, sera were collected and stored at −20°C until use. Handling of rabbits was in compliance with the Animal Ethics Procedures and Guidelines of the People’s Republic of China. Information about species, ages, geographic origin, and gender was acquired from breeders.


## 2.3. Serological Examination

Antibodies toChlamydia were tested by indirect hemagglutination antibody (IHA) using a commercially marketed kit (Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu Province, China). The procedures are according to the manufacturer’s instructions and previous descriptions [13, 14]. The IHA kit was performed following the standard protocol recommended by Chinese Center for Animal Diseases Prevention and Control, and the sensitivity and specificity of the test are 100% and 95%, respectively [13]. The sensitivity and specificity values for the testing kit used in this study have been validated by The Ministry of Agriculture of the People’s Republic of China (NY/T 562–2002). In brief, 75 μL of the IHA dilution solution was transferred into 96 well V-bottomed polystyrene plates with 25 μL of serum added and diluted in a fourfold series from 1 : 4 to 1 : 64. The plates were shaken for 2 min and then incubated at 37°C for 2 h without shaking. The test was considered positive when a layer of agglutinated erythrocytes was formed in wells at dilutions of 1 : 16 or higher, and positive and negative controls were included in each test. Those sera which showed dubious results were picked out for retest.


## 2.4. Statistical Analyses

Regions, genders, age groups, and breeds of rabbits were analyzed in a multivariable logistic regression model, and probability (P) value < 0.05 was considered as statistically significant. Odds ratios (OR) with 95% confidence intervals are also calculated. All statistical analyses were performed using the PASW Statistics 18.0 (SPSS Inc., IBM Corporation, Somers, NY).


## 3. Results

Antibodies toChlamydia were detected in 143 of 800 (17.88%, 95% CI 15.22–20.53) domestic rabbits. Of these, antibody titers were 1 : 16 in 22, 1 : 32 in 18, 1 : 64 in 33, 1 : 128 in 30, 1 : 256 in 27, 1 : 512 in 9, and 1 : 1024 in 4, respectively (Table 3). TheChlamydia seroprevalence varied in domestic rabbits from different regions, ranging from 15.00% (95% CI 10.05–19.95) to 20.50% (95% CI 14.91–26.10) (Table 1), but the differences were not significantly different. The juvenile rabbits had higher seroprevalence (19.83%) than that in adults (15.18%). The seroprevalence ofChlamydia was diverse in different breeds of domestic rabbits; the highest level was 20.88% in Chinese White Rabbits, followed by 17.61% in New Zealand Rabbits and 15.20% in California Rabbits (Table 1). However, the differences were not statistically significant (P>0.05).Table 1
Seroprevalence ofChlamydia infection in domestic rabbits by age, gender, and breed in China, as detected by indirect haemagglutination assay (IHA).
Factor
Category
Number examined
Number positive
% (95% CI)
Age
Juvenile
464
92
19.83 (16.20–23.46)
Adult
336
51
15.18 (11.34–19.02)
Gender
Male
319
45
14.11 (10.29–17.93)
Female
481
98
20.34 (16.78–23.97)
Breed
Chinese White Rabbit
249
52
20.88 (15.84–25.93)
California Rabbit
250
38
15.20 (10.75–19.65)
New Zealand Rabbit
301
53
17.61 (13.31–21.91)
Region
Liaoning province
200
39
19.50 (14.01–24.99)
Jilin province
200
41
20.50 (14.91–26.10)
Heilongjiang province
200
33
16.50 (11.36–21.64)
Inner Mongolia Autonomous Region
200
30
15.00 (10.05–19.95)
Total
800
143
17.88 (15.22–20.53)In terms of gender, seroprevalence ofChlamydia in female (20.34%) was higher than that in male (14.11%), and gender was considered as major risk factors associated withChlamydia infection in domestic rabbits (P<0.05, Table 2). And the risk ofChlamydia infection in female was more than 1.56-fold increase (OR = 1.56, 95% CI = 1.06–2.29, P<0.024) compared toChlamydia infection in male (Table 2).Table 2
Odds ratios for genders of domestic rabbits are taken as risk factors forChlamydia seroprevalence in domestic rabbits.
Gender
Number tested
Number positive
Prevalence (%)
OR (95% CI)
P value
Male
319
45
14.11 (10.29–17.93)
Reference
Female
481
98
20.34 (16.78–23.97)
1.56 (1.06–2.29)
0.024Table 3
Seroprevalence and antibody titers ofChlamydia infection in domestic rabbits, as detected by indirect haemagglutination (IHA) test.
Biometric data
Category
Antibody  titers
Number  positive
Number  tested
Prevalence (%)
1 : 16
1 : 32
1 : 64
1 : 128
1 : 256
1 : 512
1 : 1024
Gender
Female
17
16
20
18
16
7
4
98
319
20.34
Male
5
2
13
12
11
2
0
45
481
14.11
Age
Infancy
6
6
8
13
6
2
1
42
175
24.00
Juvenile
8
6
12
9
10
4
1
50
289
17.30
Adult
8
6
13
8
11
3
2
51
336
15.18
Region
Liaoning province
5
8
8
10
7
1
0
39
200
19.50
Jilin province
6
4
8
9
9
3
2
41
200
20.50
Heilongjiang province
6
4
11
4
6
2
0
33
200
16.50
Inner Mongolia Autonomous Region
5
2
6
7
5
3
2
30
200
15.00
Breed
Chinese White Rabbit
9
6
14
8
10
4
1
52
249
20.88
California Rabbit
6
5
8
8
9
1
1
38
250
15.20
New Zealand Rabbit
7
7
11
14
8
4
2
53
301
17.61
Total
22
18
33
30
27
9
4
143
800
17.88


## 4. Discussion

Chlamydia spp. are gram-negative bacteria that cause various significant diseases in humans and animals all over the world, leading to health problems and serious economic losses. In the present study, the overall seroprevalence ofChlamydia infection in domestic rabbits was 17.88%, which was higher than that observed previously in cats (5.9%) [7], dogs (12.1%) [15], pet birds (10.80%) [16], pigeon (6.80%) [17], red deer (9.60%) [18], cattle (4.75%) [19] and dairy cattle (7.25%) [20], but lower than in sows (62.70%) [21], wild boar (63.60%) [22], and feral Canada geese (93.80%) [23] using the same serological test (IHA) in China. Differences inChlamydia seroprevalence are likely due to differences in animal welfares, animal categories, climates, and husbandry practices. Today, there is an increasing demand for rabbit meat in China, and the safety and sanitary quality of meat pose a potential threat to local residents and other people in exported regions. Therefore, it is important to understand the extent ofChlamydia infection in domestic rabbits in China.In the present study,Chlamydia seroprevalence in juvenile rabbits (19.83%) was higher than that in adult rabbits (15.18%), but the differences were not statistically significant (P>0.05), which was consistent with the previous study [14]. And the reason why juveniles were more susceptible than adults and subadults may be that the juveniles had lower immunity compared with adults. Moreover, theChlamydia seroprevalence between male and female rabbits was significantly different (P<0.05), implying that gender is a crucial factor forChlamydia infection in domestic rabbits.This study revealed that gender is a significant risk factor forChlamydia prevalence in domestic rabbits, with females having a higher seroprevalence than males. TheChlamydia seroprevalence in male and female domestic rabbit was 14.11% and 20.34%, respectively. This tendency is consistent with the studies of Tibetan pigs (females, 17.61%; males, 12.72%) [24] and also concurs with that observed in pet parrots (females, 41.22%; males, 30.06%) [14], in wild boars in Germany (females, 83.3%; males, 42.9%) [25] and Italy (females, 45.95%; males, 38.8%) [22]. Females and males were exposed toChlamydia in the same environment, and in our study females are more sensitive to the pathogen than males for each of the breeds in each region group, which may be due to variation in immune response or antibody persistence rates between females and males.In the present study, there was no significant difference inChlamydia seroprevalence in rabbits in different provinces (P>0.05). The statistically similar seropositivity suggests that the pathogen could be mostly transmitted by the direct contact route in domestic rabbits, which is little affected by environmental change. Thus, further studies are needed to illuminate the potential effect ofChlamydia on the geographical origin of domestic rabbits.Chlamydia has been reported to be associated with abortion in horses, rabbits, guinea pigs, mice, and pigs [26]. More importantly,Chlamydia can cause devastating consequences of infertility, ectopic pregnancy, chronic pelvic pain, or even abortion in humans [8, 27], especially for people exposed toChlamydia infection by working with rabbits, which increased the possibility to be infected [14, 28]. Thus, further studies are needed to illuminate the potential effect ofChlamydia on reproduction of domestic rabbits. Moreover, serological investigation ofChlamydia infection in human (particularly pregnant women) working in rabbit farms in northeast China is also warranted.Among different breeds of domestic rabbits,Chlamydia seroprevalence in Chinese White Rabbit (20.88%) was higher than that in New Zealand Rabbit (17.61%) and California Rabbit (15.20%), but the differences were not statistically significant, which was consistent with the previous study, and the animal species factor was calculated and was found to be of no significance [29]. Further investigations should be studied about the correlation ofChlamydia infection with the animal species.In conclusion, the results of the present study revealed thatChlamydia infection in domestic rabbits is highly prevalent in northeast China, but this severe situation has been neglected in the past. Therefore, it is important to carry out integrated control strategies and measures to prevent and controlChlamydia infection in domestic rabbits in China. This is the first report ofChlamydia seroprevalence in domestic rabbits in China.



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