VECTOR-BORNE AND ZOONOTIC DISEASES Volume 15, Number 7, 2015 ª Mary Ann Liebert, Inc. DOI: 10.1089/vbz.2014.1749
First Report of Coxiella burnetii Seroprevalence in Tibetan Sheep in China Ming-Yang Yin,1,2,* Si-Yuan Qin,1,3,* Qi-Dong Tan,1,4 Sheng-Yong Feng,1 Guang-Xue Liu,1 Dong-Hui Zhou,1 and Xing-Quan Zhu1,2
Abstract
Query (Q) fever is a vector-borne zoonosis caused by the obligate intracellular pathogen Coxiella burnetii. Animals, including dogs, cats, cattle, and sheep, can be infected by C. burnetii. However, little information is available about C. burnetii infection in Tibetan sheep in China. Antibodies against C. burnetii in Tibetan sheep serums, which were collected from Maqu County, Tianzhu County and Nyingchi Prefecture, China, between 2011 and 2013, were examined by enzyme-linked immunosorbent assay (ELISA). Overall, 14.39% (304/2112, 95% confidence interval [CI] 12.90–15.89) of the examined Tibetan sheep were positive for C. burnetii infection. The C. burnetii seroprevalence in male and female Tibetan sheep was 12.94% (95% CI 10.26–15.61) and 14.98% (95% CI 13.18–16.78), respectively. The seroprevalence varied from 13.19% (95% CI 10.08– 16.30) to 14.96% (95% CI 12.31–17.62) in Tibetan sheep in different areas. In addition, the seroprevalence ranged from 13.16% (95% CI 9.83–16.50) to 15.10% (95% CI 13.15–17.04) in different age groups. The results of the present survey indicate that C. burnetii infection is highly prevalent in Tibetan sheep in China. This is the first report of C. burnetii infection in Tibetan sheep in China, extending the host range for C. burnetii. Further studies are warranted to understand better the epidemiological situation of C. burnetii transmission in these areas and elsewhere. Key Words:
Coxiella burnetii—Tibetan sheep—Seroprevalence—China.
Introduction
C
oxiella burnetii, a small Gram-negative bacterium, is the main cause for a vector-borne zoonosis Query (Q) fever (Parker et al. 2006, Tutusaus et al. 2014). Since 1935 in Queensland, Australia, C. burnetii infection in humans has been reported increasingly around the world (Kaplan and Bertagna 1955). Q fever infection is usually symptomless and can be misdiagnosed as other flu-like illnesses (Cutler et al. 2007). Acute Q fever can induce acute or atypical pneumonia and hepatitis, whereas the chronic form is usually accompanied by endocarditis or vascular infection (Brouqui et al. 1993, Parker et al. 2006). C. burnetii can also infect many animal hosts, such as wild mammals, pets, chickens, birds, cattle, pigs, horse, yaks, sheep, goats, and even ticks (Rodolakis 2009, Cooper et al.
2011, Kirchgessner et al. 2012, Sprong et al. 2012, Bjork et al. 2014, Meredith et al. 2014, Muramatsu et al. 2014). C. burnetii infection can lead to abortion, stillbirth, and reproductive failure in livestock, such as cattle, sheep, and goats (Snedeker and Sikora 2014). The abortion rate was estimated to range from 3% to 80% (Arricau-Bouvery and Rodolakis 2005). The most common route of C. burnetii infection is through inhalation of contaminated aerosols. Therefore, the environment plays an important role in C. burnetii transmission (Schimmer et al. 2011). C. burnetii excreted via urine, feces, milk, and birth products of infected animals is highly infectious and highly prevalent in almost every flock, causing significant economic losses worldwide (Berri et al. 2001, Arricau-Bouvery and Rodolakis 2005, Guatteo et al. 2006, van Asseldonk et al. 2013). The Tibetan sheep is unique for
1 State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu Province, P.R. China. 2 College of Veterinary Medicine, Hunan Agricultural University, Changsha, Hunan Province, P.R. China. 3 College of Animal Science and Technology, Jilin Agricultural University, Changchun, Jilin Province, P.R. China. 4 College of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui Province, P.R. China. *These authors contributed equally to this work
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its high-quality pelage and famous for its nutritive and delicious meat. Raising Tibetan sheep is a major source of income for Tibetans in China. Gansu and Tibet are the major Tibetan sheep producers in China. However, there has been no report about C. burnetii infection in Tibetan sheep in China. The objective of the present investigation was to determine the seroprevalence of C. burnetti infection in Tibetan sheep in China. These data should provide baseline information for the execution of control strategies against C. burnetti infection in Tibetan sheep in these provinces and elsewhere in China. Materials and Methods
tracted from blood samples and then kept at -20C until analysis (Wu et al. 2011). Information about location, age, gender, and pregnancy of female Tibetan sheep was obtained during the process of sampling. Detection by enzyme-linked immunosorbent assay
Antibodies against C. burnetii in serum samples were examined by using the Q fever LSI enzyme-linked immunosorbent assay (ELISA) kit (LSI, Lissieu, France) according to the manufacturer’s protocol and previous descriptions (Klaasen et al. 2014). Each test included positive and the negative controls. Those samples with suspicious results were retested.
Ethics statement
Statistical analysis
This study was approved by the Animal Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (approval no. LVRIAEC2011-008). The Tibetan sheep 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.
Differences in C. burnetii seroprevalence in Tibetan sheep among different variables, including region, age, gender, and pregnancy of female Tibetan sheep, were analyzed using a chi-squared test in Analytics Software (PASW) Statistics 18. Results were considered statistically significant when p < 0.05. These variables, with the exception of pregnancy of Tibetan sheep, were also evaluated by multivariate logistic regression analysis to test C. burnetii seroprevalence. The risk factors were included in the model when p < 0.05.
Sampling sites
The Tibetan sheep examined came from Maqu County and Tianzhu County, Gansu Province, and Nyingchi Prefecture, Tibet, western China. Maqu county (3306¢–3430¢N, 10045¢–10229¢E) is located in the south of Gansu province with an average altitude of 2960 meters and an average annual temperature of 1.7C. Tianzhu County (3631¢–3755¢N, 10207¢–10346¢E) is situated in the middle of Gansu Province with an average annual temperature ranging from -8 to 4C. Nyingchi Prefecture is situated in southeastern Tibet (2921¢–3015¢N, 9327¢–9517¢E). The average altitude of Nyingchi Prefecture is 3000 meters with an average annual temperature of 8.5C. Collection and preparation of serum samples
A total of 2112 Tibetan sheep from Maqu County (695), Tianzhu County (962), and Nyingchi Prefecture (455) were sampled from 2011 to 2013. The blood samples were collected from the jugular vein and were stored at 37C for 2 h and then centrifuged for 5 min at 3000 rpm. Serum was ex-
Results
A total of 304 (14.39%, 95% confidence interval [CI] 12.90–15.89) out of the 2112 Tibetan sheep serum samples were positive for C. burnetii antibodies by ELISA (Table 1). Of these, positivity for antibodies to C. burnetii varied from 13.19% (95% CI 10.08–16.30) to 14.96% (95% CI 12.31– 17.62) in different regions, but the difference was not significant ( p = 0.69). No statistically significant difference was found in C. burnetii seroprevalence between male Tibetan sheep (12.94%, 95% CI 10.26–15.61) and female Tibetan sheep (14.98%, 95% CI 13.18–16.78, p = 0.23). The C. burnetii seroprevalence in Tibetan sheep of different age groups ranged from 13.16% (95% CI 9.83–16.50) to 15.10% (95% CI 13.15–17.04) (Table 1), but the difference was not statistically significant ( p = 0.50). The C. burnetii seroprevalence in female Tibetan sheep of different pregnancy groups ranged from 13.49% (95% CI 10.51–16.48) to 16.95% (95% CI 10.18–23.72) (Table 2), but the difference was not statistically significant ( p = 0.67). In the multivariable
Table 1. Prevalence of Antibodies to Coxiella burnetii in Tibetan Sheep by Region, Gender, and Age of Tibetan Sheep in China Using Enzyme-Linked Immunosorbent Assay Variable Region Gender Age
Category
No. tested
No. positive
Maqu county Tianzhu county Nyingchi Prefecture Male Female £1 year 2 year ‡3 year
695 962 455 603 1509 412 395 1305 2112
104 140 60 78 226 55 52 197 304
Total CI, confidence interval.
% (95% CI) 14.96 14.55 13.19 12.94 14.98 13.35 13.16 15.10 14.39
(12.31–17.62) (12.33–16.78) (10.08–16.30) (10.26–15.61) (13.18–16.78) (10.07–16.63) (9.83–16.50) (13.15–17.04) (12.90–15.89)
p value 0.69 0.23 0.50
C. burnetii INFECTION IN TIBETAN SHEEP IN CHINA
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Table 2. Prevalence of Antibodies to Coxiella burnetii in Tibetan Sheep by Pregnancy of Female Tibetan Sheep in China Using Enzyme-Linked Immunosorbent Assay Variable
Category
No. tested
No. positive
0 1 2 3 4
504 280 381 226 118 1509
68 39 63 36 20 226
Pregnancy
Total
% (95% CI) 13.49 13.93 16.54 15.93 16.95 14.98
(10.51–16.48) (9.87–17.98) (12.81–20.27) (11.16–20.70) (10.18–23.72) (13.18–16.78)
p value 0.67
CI, confidence interval.
regression analysis, no risk factors could be included in the final model because all of the p values were above 0.05. Discussion
The overall C. burnetii seroprevalence in Tibetan sheep obtained in the present study was 14.39% (95% CI 12.90– 15.89), which was lower than that in sheep (37.5%) and goats (28.8%) tested by ELISA in Portugal (Anasta´cio et al. 2011), in sheep (33.6%) in Iran (Esmaeili et al. 2014), and in goats (24.2%) and in sheep (18.5%) in Gambia (Klaasen et al. 2014), but higher than that in goats in Gansu Province (5.6%, 15/270) (Yang et al. 1993) and Shandong Province (13.14%, 51/388) (Sun 1999) of China and in the United States (8%) (Sondgeroth et al. 2013). C. burnetii seroprevalence in Tibetan sheep is also higher than that in yaks in Maqu County, Gansu Province (13.59%, 75/552) (Yin et al, in press). This may be attributed to different investigation regions and methods used. In the present study, the C. burnetii seroprevalence in Tibetan sheep in Maqu County was the highest (14.96%) compared with that in two other regions. But the difference was not statistically significant ( p = 0.69), which was different from the result of a previous study (Esmaeili et al. 2014). Ticks are the important vectors transmitting C. burnetii, and the number of species of ticks in Gansu Province and the Tibetan Autonomous Region of Xinjiang are 20 and eight, respectively (Shi et al. 2004, Wang et al. 2011). The role of ticks in the transmission of C. burnetii to Tibetan sheep needs to be elucidated. There was no significant difference in the C. burnetii seroprevalence between male Tibetan sheep (12.94%, 95% CI 10.26–15.61) and females (14.98%, 95% CI 13.18–16.78), which is similar to previous reports (Esmaeili et al. 2014, Javad et al. 2014). This is likely because male and female Tibetan sheep were feeding in the same pastures and thus had relatively similar opportunities to contact grass or water contaminated with C. burnetii. Tibetan sheep that were 3 years old had the highest C. burnetii seroprevalence of 15.10%, which is consistent with that of a previous report indicating that C. burnetii seroprevalence also increased with age (Schimmer et al. 2011). Older Tibetan sheep seem to be more likely to be infected by C. burnetii (Ruiz-Fons et al. 2010), but age was not a risk factor for C. burnetii infection in Tibetan sheep ( p > 0.05); the result was in accordance with a previous study ( Javad et al. 2014). The reason why older Tibetan sheep seem to be more likely to be infected by C. burnetii may be that contact with C. burnetii may increase with growth.
The C. burnetii seroprevalence in female Tibetan sheep ranged from 13.49% (95% CI 10.51–16.48) in the group with no pregnancies to 16.95% (95% CI 10.18–23.72) in the group with four pregnancies; however, the difference is not statistically significant ( p > 0.05). Van den Brom (2013) showed that pregnancy in sheep and goats is the risk factor for C. burnetii infection. C. burnetii infection may cause abortion in female Tibetan sheep, and pregnant Tibetan sheep may have increased the risk of C. burnetii infection. Conclusion
The present survey revealed, for the first time, that C. burnetii infection was highly prevalent in Tibetan sheep in China. Therefore, immediate measures need to be taken to control it. Further studies are warranted to better understand the epidemiological situation of C. burnetii transmission in these areas and elsewhere. Acknowledgments
Project support was provided by the ‘‘Special Fund for Agro-scientific Research in the Public Interest’’ (grant no. 201303037) and the Science Fund for Creative Research Groups of Gansu Province (grant no. 1210RJIA006). Author Disclosure Statement
No completing 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. References
Anasta´cio, S, Tavares, N, Carolino, N, Sidi-Boumedine, K, et al. Serological evidence of exposure to Coxiella burnetii in sheep and goats in central Portugal. Vet Microbiol 2011; 167:500–505. Arricau-Bouvery, N, Rodolakis, A. Is Q fever an emerging or re-emerging zoonosis? Vet Res 2005; 3:327–349. Berri, M, Souriau, A, Crosby M, Crochet, D, et al. Relationships between the shedding of Coxiella burnetii, clinical signs and serological responses of 34 sheep. Vet Rec 2001; 16:502– 505. Bjork, A, Marsden-Haug, N, Nett, RJ. First reported multistate human Q fever outbreak in the United States, 2011. Vector Borne Zoonotic Dis 2014; 2:111–117. Brouqui, P, Dupont, HT, Drancourt, M, Berland, Y, et al. Chronic Q fever. Ninety-two cases from France, including 27 cases without endocarditis. Arch Intern Med 1993; 5:642– 648.
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Cooper, A, Hedlefs, R, Ketheesan, N, Govan, B. Serological evidence of Coxiella burnetii infection in dogs in a regional centre. Aust Vet J 2011; 89:385–387. Cutler, SJ, Bouzid, M, Cutler, RR. Q fever. J Infection 2007; 4:313–318. Esmaeili, S, Bagheri Amiri, F, Mostafavi, E. Seroprevalence survey of Q fever among sheep in northwestern Iran. Vector Borne Zoonotic Dis 2014; 3:189–192. Guatteo, R, Beaudeau, F, Berri, M, Rodolakis, A, et al. Shedding routes of Coxiella burnetii in dairy cows: Implications for detection and control. Vet Res 2006; 6:827–833. Javad, A, Mohammad, K, Mojtaba, K, Maryam, AL, et al. Risk factors of Q fever in sheep and goat flocks with history of abortion. Comp Clin Path 2014; 3:625–630. Kaplan, MM, Bertagna, P. The geographical distribution of Q fever. Bull World Health Organ 1955; 5:829–860. Kirchgessner, MS, Dubovi, EJ, Whipps, CM. Seroepidemiology of Coxiella burnetii in wild white-tailed deer (Odocoileus virginianus) in New York, United States. Vector Borne Zoonotic Dis 2012; 12:942–947. Klaasen, M, Roest, HJ, van der Hoek, W, Goossens, B, et al. Coxiella burnetii seroprevalence in small ruminants in the Gambia. PLoS One 2014; 9:e85424. Meredith, AL, Cleaveland, SC, Denwood, MJ, Brown, JK, et al. Coxiella burnetii (Q-fever) seroprevalence in prey and predators in the United Kingdom: Evaluation of infection in wild rodents, foxes and domestic cats using a modified ELISA. Transbound Emerg Dis. 2014; doi: 10.1111/ tbed.12211. [Epub ahead of print] Muramatsu, Y, Usaki, N, Thongchai, C, Kramomtong, I, et al. Seroepidemiologic survey in Thailand of Coxiella burnetii infection in cattle and chickens and presence in ticks attached to dairy cattle. Southeast Asian J Trop Med Public Health 2014; 45:1167–1172. Parker, NR, Barralet, JH, Bell, AM. Q fever. Lancet 2006; 9511:679–688. Rodolakis, A.Q fever in dairy animals. Ann NY Acad Sci 2009; 1:90–93. Ruiz-Fons, F, Astobiza, I, Barandika, JF, Hurtado, A, et al. Seroepidemiological study of Q fever in domestic ruminants in semi-extensive grazing systems. BMC Vet Res 2010; 6:3. Schimmer, B, Luttikholt, S, Hautvast, JL, Graat, EA, et al. Seroprevalence and risk factors of Q fever in goats on commercial dairy goat farms in the Netherlands, 2009–2010. BMC Vet Res 2011; 7:81.
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Shi, ZY, Yang, YS, Li Q, Zhao, HB. Study on species and geographic distribution of ticks in Gansu Province. Chin J Vet Sci Tech 2004; 8:48–49. (In Chinese). Snedeker, KG, Sikora, C. Q fever in Alberta, Canada: 1998– 2011. Zoonoses Public Health 2014; 2:124–130. Sondgeroth, KS, Davis, MA, Schlee, SL. Seroprevalence of Coxiella burnetii in Washington State domestic goat herds. Vector Borne Zoonotic Dis 2013; 11:779–783. Sprong, H, Tijsse-Klasen, E, Langelaar, M. Prevalence of Coxiella burnetii in ticks after a large outbreak of Q fever. Zoonoses Public Health 2012; 1:69–75. Sun, T. Investigations on rickettsiosis in Shandong Province. China Public Health 1999; 10:24–25. (In Chinese) Tutusaus, J, Lo´pez-Gatius, F, Serrano, B, Monleo´n, E, et al. Serological and shedding patterns after Coxiella burnetii vaccination in the third gestation trimester in dairy cows. Acta Vet Hung 2014; 2:145–154. van Asseldonk, MA, Prins J, Bergevoet, RH. Economic assessment of Q fever in the Netherlands. Prev Vet Med 2013; 1:27–34. Van den Brom, R, Moll, L, van Schaik, G, Vellema, P. Demography of Q fever seroprevalence in sheep and goats in the Netherlands in 2008. Prev Vet Med 2013; 109:76–82. Wang, Z, Cao, WL, Liu, QS, Yan, S, et al. Survey of ticks on yaks of mountain grasslands in Xinjiang. Xingjiang Agri Sci 2011; 48:1514–1519. (In Chinese). Wu, SM, Huang, SY, Fu, BQ, Liu, GY, et al. Seroprevalence of Toxoplasma gondii infection in pet dogs in Lanzhou, Northwest China. Parasit Vectors 2011; 4:64. Yang, YS, Wang, DH, Shi, SZ, Zhang, J, et al. Seroprevalence of Q fever about cattle and sheep. Chin J Vet Sci Tech1993; 12:19–20. (In Chinese) Yin, MY, Tan, QD, Qin, SY, Hu, LY, et al. First serologic survey of Q fever in free-range yaks in China. Iran J Vet Res (in press).
Address correspondence to: Xing-Quan Zhu State Key Laboratory of Veterinary Etiological Biology Lanzhou Veterinary Research Institute Chinese Academy of Agricultural Sciences Lanzhou, Gansu Province 730046 The People’s Republic of China E-mail: [email protected]
First Report of Coxiella burnetii Seroprevalence in Tibetan Sheep in China Ming-Yang Yin,1,2,* Si-Yuan Qin,1,3,* Qi-Dong Tan,1,4 Sheng-Yong Feng,1 Guang-Xue Liu,1 Dong-Hui Zhou,1 and Xing-Quan Zhu1,2
Abstract
Query (Q) fever is a vector-borne zoonosis caused by the obligate intracellular pathogen Coxiella burnetii. Animals, including dogs, cats, cattle, and sheep, can be infected by C. burnetii. However, little information is available about C. burnetii infection in Tibetan sheep in China. Antibodies against C. burnetii in Tibetan sheep serums, which were collected from Maqu County, Tianzhu County and Nyingchi Prefecture, China, between 2011 and 2013, were examined by enzyme-linked immunosorbent assay (ELISA). Overall, 14.39% (304/2112, 95% confidence interval [CI] 12.90–15.89) of the examined Tibetan sheep were positive for C. burnetii infection. The C. burnetii seroprevalence in male and female Tibetan sheep was 12.94% (95% CI 10.26–15.61) and 14.98% (95% CI 13.18–16.78), respectively. The seroprevalence varied from 13.19% (95% CI 10.08– 16.30) to 14.96% (95% CI 12.31–17.62) in Tibetan sheep in different areas. In addition, the seroprevalence ranged from 13.16% (95% CI 9.83–16.50) to 15.10% (95% CI 13.15–17.04) in different age groups. The results of the present survey indicate that C. burnetii infection is highly prevalent in Tibetan sheep in China. This is the first report of C. burnetii infection in Tibetan sheep in China, extending the host range for C. burnetii. Further studies are warranted to understand better the epidemiological situation of C. burnetii transmission in these areas and elsewhere. Key Words:
Coxiella burnetii—Tibetan sheep—Seroprevalence—China.
Introduction
C
oxiella burnetii, a small Gram-negative bacterium, is the main cause for a vector-borne zoonosis Query (Q) fever (Parker et al. 2006, Tutusaus et al. 2014). Since 1935 in Queensland, Australia, C. burnetii infection in humans has been reported increasingly around the world (Kaplan and Bertagna 1955). Q fever infection is usually symptomless and can be misdiagnosed as other flu-like illnesses (Cutler et al. 2007). Acute Q fever can induce acute or atypical pneumonia and hepatitis, whereas the chronic form is usually accompanied by endocarditis or vascular infection (Brouqui et al. 1993, Parker et al. 2006). C. burnetii can also infect many animal hosts, such as wild mammals, pets, chickens, birds, cattle, pigs, horse, yaks, sheep, goats, and even ticks (Rodolakis 2009, Cooper et al.
2011, Kirchgessner et al. 2012, Sprong et al. 2012, Bjork et al. 2014, Meredith et al. 2014, Muramatsu et al. 2014). C. burnetii infection can lead to abortion, stillbirth, and reproductive failure in livestock, such as cattle, sheep, and goats (Snedeker and Sikora 2014). The abortion rate was estimated to range from 3% to 80% (Arricau-Bouvery and Rodolakis 2005). The most common route of C. burnetii infection is through inhalation of contaminated aerosols. Therefore, the environment plays an important role in C. burnetii transmission (Schimmer et al. 2011). C. burnetii excreted via urine, feces, milk, and birth products of infected animals is highly infectious and highly prevalent in almost every flock, causing significant economic losses worldwide (Berri et al. 2001, Arricau-Bouvery and Rodolakis 2005, Guatteo et al. 2006, van Asseldonk et al. 2013). The Tibetan sheep is unique for
1 State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu Province, P.R. China. 2 College of Veterinary Medicine, Hunan Agricultural University, Changsha, Hunan Province, P.R. China. 3 College of Animal Science and Technology, Jilin Agricultural University, Changchun, Jilin Province, P.R. China. 4 College of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui Province, P.R. China. *These authors contributed equally to this work
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its high-quality pelage and famous for its nutritive and delicious meat. Raising Tibetan sheep is a major source of income for Tibetans in China. Gansu and Tibet are the major Tibetan sheep producers in China. However, there has been no report about C. burnetii infection in Tibetan sheep in China. The objective of the present investigation was to determine the seroprevalence of C. burnetti infection in Tibetan sheep in China. These data should provide baseline information for the execution of control strategies against C. burnetti infection in Tibetan sheep in these provinces and elsewhere in China. Materials and Methods
tracted from blood samples and then kept at -20C until analysis (Wu et al. 2011). Information about location, age, gender, and pregnancy of female Tibetan sheep was obtained during the process of sampling. Detection by enzyme-linked immunosorbent assay
Antibodies against C. burnetii in serum samples were examined by using the Q fever LSI enzyme-linked immunosorbent assay (ELISA) kit (LSI, Lissieu, France) according to the manufacturer’s protocol and previous descriptions (Klaasen et al. 2014). Each test included positive and the negative controls. Those samples with suspicious results were retested.
Ethics statement
Statistical analysis
This study was approved by the Animal Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (approval no. LVRIAEC2011-008). The Tibetan sheep 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.
Differences in C. burnetii seroprevalence in Tibetan sheep among different variables, including region, age, gender, and pregnancy of female Tibetan sheep, were analyzed using a chi-squared test in Analytics Software (PASW) Statistics 18. Results were considered statistically significant when p < 0.05. These variables, with the exception of pregnancy of Tibetan sheep, were also evaluated by multivariate logistic regression analysis to test C. burnetii seroprevalence. The risk factors were included in the model when p < 0.05.
Sampling sites
The Tibetan sheep examined came from Maqu County and Tianzhu County, Gansu Province, and Nyingchi Prefecture, Tibet, western China. Maqu county (3306¢–3430¢N, 10045¢–10229¢E) is located in the south of Gansu province with an average altitude of 2960 meters and an average annual temperature of 1.7C. Tianzhu County (3631¢–3755¢N, 10207¢–10346¢E) is situated in the middle of Gansu Province with an average annual temperature ranging from -8 to 4C. Nyingchi Prefecture is situated in southeastern Tibet (2921¢–3015¢N, 9327¢–9517¢E). The average altitude of Nyingchi Prefecture is 3000 meters with an average annual temperature of 8.5C. Collection and preparation of serum samples
A total of 2112 Tibetan sheep from Maqu County (695), Tianzhu County (962), and Nyingchi Prefecture (455) were sampled from 2011 to 2013. The blood samples were collected from the jugular vein and were stored at 37C for 2 h and then centrifuged for 5 min at 3000 rpm. Serum was ex-
Results
A total of 304 (14.39%, 95% confidence interval [CI] 12.90–15.89) out of the 2112 Tibetan sheep serum samples were positive for C. burnetii antibodies by ELISA (Table 1). Of these, positivity for antibodies to C. burnetii varied from 13.19% (95% CI 10.08–16.30) to 14.96% (95% CI 12.31– 17.62) in different regions, but the difference was not significant ( p = 0.69). No statistically significant difference was found in C. burnetii seroprevalence between male Tibetan sheep (12.94%, 95% CI 10.26–15.61) and female Tibetan sheep (14.98%, 95% CI 13.18–16.78, p = 0.23). The C. burnetii seroprevalence in Tibetan sheep of different age groups ranged from 13.16% (95% CI 9.83–16.50) to 15.10% (95% CI 13.15–17.04) (Table 1), but the difference was not statistically significant ( p = 0.50). The C. burnetii seroprevalence in female Tibetan sheep of different pregnancy groups ranged from 13.49% (95% CI 10.51–16.48) to 16.95% (95% CI 10.18–23.72) (Table 2), but the difference was not statistically significant ( p = 0.67). In the multivariable
Table 1. Prevalence of Antibodies to Coxiella burnetii in Tibetan Sheep by Region, Gender, and Age of Tibetan Sheep in China Using Enzyme-Linked Immunosorbent Assay Variable Region Gender Age
Category
No. tested
No. positive
Maqu county Tianzhu county Nyingchi Prefecture Male Female £1 year 2 year ‡3 year
695 962 455 603 1509 412 395 1305 2112
104 140 60 78 226 55 52 197 304
Total CI, confidence interval.
% (95% CI) 14.96 14.55 13.19 12.94 14.98 13.35 13.16 15.10 14.39
(12.31–17.62) (12.33–16.78) (10.08–16.30) (10.26–15.61) (13.18–16.78) (10.07–16.63) (9.83–16.50) (13.15–17.04) (12.90–15.89)
p value 0.69 0.23 0.50
C. burnetii INFECTION IN TIBETAN SHEEP IN CHINA
421
Table 2. Prevalence of Antibodies to Coxiella burnetii in Tibetan Sheep by Pregnancy of Female Tibetan Sheep in China Using Enzyme-Linked Immunosorbent Assay Variable
Category
No. tested
No. positive
0 1 2 3 4
504 280 381 226 118 1509
68 39 63 36 20 226
Pregnancy
Total
% (95% CI) 13.49 13.93 16.54 15.93 16.95 14.98
(10.51–16.48) (9.87–17.98) (12.81–20.27) (11.16–20.70) (10.18–23.72) (13.18–16.78)
p value 0.67
CI, confidence interval.
regression analysis, no risk factors could be included in the final model because all of the p values were above 0.05. Discussion
The overall C. burnetii seroprevalence in Tibetan sheep obtained in the present study was 14.39% (95% CI 12.90– 15.89), which was lower than that in sheep (37.5%) and goats (28.8%) tested by ELISA in Portugal (Anasta´cio et al. 2011), in sheep (33.6%) in Iran (Esmaeili et al. 2014), and in goats (24.2%) and in sheep (18.5%) in Gambia (Klaasen et al. 2014), but higher than that in goats in Gansu Province (5.6%, 15/270) (Yang et al. 1993) and Shandong Province (13.14%, 51/388) (Sun 1999) of China and in the United States (8%) (Sondgeroth et al. 2013). C. burnetii seroprevalence in Tibetan sheep is also higher than that in yaks in Maqu County, Gansu Province (13.59%, 75/552) (Yin et al, in press). This may be attributed to different investigation regions and methods used. In the present study, the C. burnetii seroprevalence in Tibetan sheep in Maqu County was the highest (14.96%) compared with that in two other regions. But the difference was not statistically significant ( p = 0.69), which was different from the result of a previous study (Esmaeili et al. 2014). Ticks are the important vectors transmitting C. burnetii, and the number of species of ticks in Gansu Province and the Tibetan Autonomous Region of Xinjiang are 20 and eight, respectively (Shi et al. 2004, Wang et al. 2011). The role of ticks in the transmission of C. burnetii to Tibetan sheep needs to be elucidated. There was no significant difference in the C. burnetii seroprevalence between male Tibetan sheep (12.94%, 95% CI 10.26–15.61) and females (14.98%, 95% CI 13.18–16.78), which is similar to previous reports (Esmaeili et al. 2014, Javad et al. 2014). This is likely because male and female Tibetan sheep were feeding in the same pastures and thus had relatively similar opportunities to contact grass or water contaminated with C. burnetii. Tibetan sheep that were 3 years old had the highest C. burnetii seroprevalence of 15.10%, which is consistent with that of a previous report indicating that C. burnetii seroprevalence also increased with age (Schimmer et al. 2011). Older Tibetan sheep seem to be more likely to be infected by C. burnetii (Ruiz-Fons et al. 2010), but age was not a risk factor for C. burnetii infection in Tibetan sheep ( p > 0.05); the result was in accordance with a previous study ( Javad et al. 2014). The reason why older Tibetan sheep seem to be more likely to be infected by C. burnetii may be that contact with C. burnetii may increase with growth.
The C. burnetii seroprevalence in female Tibetan sheep ranged from 13.49% (95% CI 10.51–16.48) in the group with no pregnancies to 16.95% (95% CI 10.18–23.72) in the group with four pregnancies; however, the difference is not statistically significant ( p > 0.05). Van den Brom (2013) showed that pregnancy in sheep and goats is the risk factor for C. burnetii infection. C. burnetii infection may cause abortion in female Tibetan sheep, and pregnant Tibetan sheep may have increased the risk of C. burnetii infection. Conclusion
The present survey revealed, for the first time, that C. burnetii infection was highly prevalent in Tibetan sheep in China. Therefore, immediate measures need to be taken to control it. Further studies are warranted to better understand the epidemiological situation of C. burnetii transmission in these areas and elsewhere. Acknowledgments
Project support was provided by the ‘‘Special Fund for Agro-scientific Research in the Public Interest’’ (grant no. 201303037) and the Science Fund for Creative Research Groups of Gansu Province (grant no. 1210RJIA006). Author Disclosure Statement
No completing 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. References
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Address correspondence to: Xing-Quan Zhu State Key Laboratory of Veterinary Etiological Biology Lanzhou Veterinary Research Institute Chinese Academy of Agricultural Sciences Lanzhou, Gansu Province 730046 The People’s Republic of China E-mail: [email protected]
