Abstract
Coxiella burnetii is the pathogenic agent of Q fever, a zoonosis that occurs worldwide. In China, the organism infects people and a wide range of wild and farmed animals. However, little is known about C. burnetii infection in farmed ruminants in northeastern China, and there appears to have been no previous study on sika deer. The present study was conducted to determine the seroprevalence of C. burnetii in farmed ruminants in this region of China. A total of 3147 domestic ruminants (784 sheep, 1016 cattle, and 1347 sika deer) were randomly collected for this study. An indirect enzyme-linked immunosorbent assay kit was used to detect the antibodies to C. burnetii. The results showed an overall seroprevalence of 16.7% in domestic ruminants (24.9%, 13.5%, and 12.3% in cattle, sheep, and sika deer, respectively). This is the first report of C. burnetii seroprevalence in sika deer in China. Protective and hygiene measures should be applied in farm management to reduce the risk of exposure of C. burnetii to humans.
Introduction
Q
In China, infection has been detected in humans as well as in a wide range of wild, domestic, and farmed animals, such as cattle, goats, dogs, pigs, mice, sheep, and horses (El-Mahallawy et al. 2014). Cattle and sheep are the main reservoir species (El-Mahallawy et al. 2014). Q fever is not a reportable disease in China, and little is known about the incidence of C. burnetii infection in farmed ruminants, and especially sika deer, in the three northeastern-most provinces and the adjacent Inner Mongolia Autonomous Region (Ni et al. 2011). Thus, the objective of the present study was to determine the seroprevalence of C. burnetii infection in important farmed ruminants in northeastern China, with an emphasis on sika deer.
Materials and Methods
Ethics statement
This study was approved by the Animal Ethics Committee of Jilin Agricultural University. The ruminants from which blood was collected were handled in accordance with good animal practices required by the Animal Ethics Procedures and Guidelines of the People's Republic of China.
Serum samples and serological examination
A total of 3147 farmed ruminants (784 sheep, 1016 cattle, and 1347 sika deer) from four administrative regions of northeastern China (Liaoning, Jilin, and Heilongjiang Provinces, and Inner Mongolia Autonomous Region) were randomly selected for this study in 2013 and 2014 (Table 1). Sheep and cattle blood samples were collected from 11 administrative districts across the four regions. Sheep blood samples were randomly collected from 22 flocks (each ≤100 sheep). Cattle samples were randomly collected from 19 cattle herds (seven farms with ≤50 cattle, eight farms with 50–150 cattle, and four farms with >50 cattle). Sika deer blood samples were collected from 16 farms each with at least 100 head registered. Sika deer from each farm were selected randomly using a table of random digits. Small-scale farms (with fewer than 100 animals) were not included because the owner was often not present to give permission. In the case of sika deer, approximately 5% on each farm were sampled.
All of the animals sampled were clinically healthy. The blood samples were taken to the laboratory, kept at room temperature for 2 h, and centrifuged at 3000×g for 10 min. Clear serum was removed and stored at −20°C until testing. Information about species, geographic origin, and gender was acquired from farm administrators. A commercial enzyme-linked immunosorbent assay (ELISA) (LSIVET Ruminant Milk/Serum Q Fever ELISA COXLS LSI, Lissieu, France) was used to detect the presence of C. burnetii immunoglobulin G (IgG) phases 1 and 2 specific antibodies, according to the manufacturer's instructions. The antigen is a sheep strain (phase 1–2). Sensitivity and specificity of this ELISA test were 87% and 100%, respectively (manufacturer's data) (Cardinale et al. 2014). Positive samples were retested.
Statistical analyses
Exploratory analysis was performed to examine variables potentially associated with exposure to C. burnetii infection. All factors were studied in a bivariate logistic regression model. A probability (p) value<0.05 was considered as statistically significant between levels within factors and interactions. Odds ratios (ORs), with 95% confidence intervals (CI) based on likelihood ratio statistics, were reported. All statistical analyses were performed using SPSS (Release 18.0 standard version, SPSS Inc., Chicago, IL).
Results and Discussion
A total of 525 (16.7%, 95% CI 15.38–17.99) out of 3147 serum samples from farmed ruminants in northeastern China tested positive by ELISA for C. burnetii antibodies (Table 1). Overall values for cattle, sheep, and sika deer were 24.9%, 13.5%, and 12.3%, respectively. To our knowledge, this is the first study of seroprevalence of C. burnetii infection in farmed sika deer in China.
The seroprevalence of 12.3% (166/1347, 95% CI 10.57–14.08) in domestic sika deer in this study was lower than that reported among cervids in some other countries. Seroprevalences of 20.96% and 14.41% were reported in Pennsylvania and New York, respectively (Kirchgessner et al. 2012), 36% in Spain using the same method (González-Barrio et al. 2013), and 23% in The Netherlands, as determined using duplex quantitative PCR (Rijks et al. 2011). However, the prevalence we detected in China was higher than that in wild Korean water deer (9.18%) using the same method (Shin et al. 2014) and in red deer (3.64%) in the southwest Iberian Peninsula (Castillo et al. 2010). Several factors may contribute to the differences in seroprevalence of C. burnetii in cervids, such as their living conditions, geographic and climatic conditions, species, and serologic tests used.
The sika deer is of particular economic interest in China. Velvet antlers produced by these deer are considered one of the three treasures in northeastern China (Qin et al. 2014). Deer farming has become an important source of income locally, but has been hindered by the occurrence of some infectious diseases. C. burnetii was considered to play an important role in reproductive failure among farmed red deer (González-Barrio et al. 2013). This pathogen may, directly or indirectly, also be a cause of significant economic losses for farmers in northeastern China.
In the present study, cattle (24.9%, 253/1016) appeared to have a significantly higher risk of being seropositive than did sheep (13.5%, 106/784) or sika deer (12.3%, 166/1347) (p<0.05). The highest seroprevalence was found in Inner Mongolia Autonomous Region (17.8%, 117/656), followed by Jilin Province (17.5%, 189/1079), Heilongjiang Province (16.6%, 127/767), and Liaoning Province (14.3%, 92/645). However, rates of seroprevalence of C. burnetii were not statistically significantly different among these four regions.
Diagnosis of C. burnetii infections is commonly based on herd history and clinical symptoms. It is difficult to isolate the bacterium from clinical samples due to its fragility and fastidious growth requirements. Moreover, subclinical infection in carrier animals is typical (Saegerman et al. 2015). Serological methods, such as immunofluorescence assay (IFA) and ELISA, have been widely used for Q fever diagnosis (Ni et al. 2011, González-Barrio et al. 2013, Klaasen et al. 2014, Shin et al. 2014). We used the ELISA method because of its convenience and high sensitivity. This commercially produced kit has been widely used for Q fever diagnosis in bovines, sheep, goats, and other sensitive species (manufacturer's data).
This survey of C. burnetii seroprevalence in domestic ruminants in northeastern China suggests a considerable prevalence of current or past infection in the farmed ruminant population. However, our study has some weaknesses. Seropositivity does not necessarily indicate current infection, but we did not try to detect the current presence of the pathogen in sampled animals. Further studies should be conducted with this aim. Moreover, the situation of human infection in the investigated areas was ignored. C. burnetii is highly contagious and very resistant in the environment. People living in a Q fever endemic area are at risk of getting infected. Thus, more research should be carried out to detect antibodies to C. burnetii in both humans and animals, aiming to investigate the possible linkage between humans and animals.
Conclusion
The present survey indicated a widespread exposure to C. burnetii among farmed ruminants in northeastern China. This information is useful for formulating and implementing locally applicable control measures for Q fever, and these measures can be carried out by local animal and human healthcare workers. To our knowledge, the present study is the first to document the seroprevalence of C. burnetii in sika deer in China.
Footnotes
Acknowledgments
Project support was provided by the national support program (no. 2010BAD04B01) and Quality Inspection Special Public Welfare Industry Research (no. 201410061). Prof. David Blair at School of Marine and Tropical Biology, James Cook University, Townsville, Queensland, Australia, is thanked for copy-editing the manuscript and improving the English language of the manuscript.
Author Disclosure Statement
No competing financial interests exist. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the funding agencies.