Seroprevalence of Chlamydia abortus infection in yak (Bos grunniens) in Tibet, China

Chlamydia spp. are prevalent zoonotic pathogens that infect a wide variety of host species. Chlamydia abortus (C. abortus) infection in yaks has been reported in Gansu and Qinghai province, China. However, no data about C. abortus infection are available in yaks in Tibet, China. A total of 938 serum samples was collected from yaks in Tibet, China and specific antibodies against C. abortus were detected by the enzyme-linked immunosorbent assay (ELISA). The results showed that the overall seroprevalence of C. abortus in yaks was 104/938 (11.1 %, 95 % confidence interval [CI] 9.1–13.1). The prevalence in female and male yaks was 59/556 (10.6 %, 95 % CI 8.0-13.2) and 45/382 (11.8 %, 95 % CI 8.5–15.0), respectively with no significant difference (p > 0.05). The seroprevalence of antibodies to C. abortus in yaks ranged from 8.0 to 18.2 % among the six different areas, and the difference was also without statistical significance (p > 0.05). The prevalence among different age groups ranged from 7.0 to 15.9 %, with a higher prevalence among 1 to 2 years age category. The results demonstrate the presence of C. abortus infection in yaks in Tibet and may pose a risk for the general yak populations in addition to its potential impact on public health and the local Tibetan economy. To our knowledge, this is the first seroprevalence survey of C. abortus in yaks in Tibet, China.


Introduction
Chlamydia spp. are obligate intracellular bacteria with a unique biphasic developmental cycle, which can infect a wide range of animals and humans [1]. Eleven species of the family Chlamydiaceae have been identified along with six newly characterized candidate species [2][3][4][5][6]. Among them, infections in cattle with Chlamydia (C). abortus, C. pecorum, C. psittaci, C. suis, C. gallinacea and C. pneumoniae have been reported in many countries around the world, such as Australia, Ireland, Zimbabwe, Poland and China [7][8][9][10][11][12]. C. abortus is an important pathogen in cattle, capable of causing, premature birth, stillborn or weak offspring [8,13]. It can also cause zoonotic infection, especially in pregnant women, where infection can lead to pelvic inflammatory disease, placental dysfunction and lateterm fetal death [14].
There are approximately 14 million yaks in the Qinghai-Tibetan Plateau area of China, representing about 90 % of the yak population in the world. The yak is very important to native Tibetans, because of its high-quality fur, wool, meat and milk. C. abortus is an important pathogen of yak, capable of causing abortion in pregnant yak [15]. While data on the seroprevalence of C. abortus in yaks is available for Qinghai and Gansu provinces of China [16,17], there is no information on the prevalence of C. abortus is in yaks in Tibet, China to the best of our knowledge. In this study, we investigated the seroprevalence of C. abortus and risk factors including location, gender and age of yaks in Tibet.

Materials and methods
The study was carried out in Nierong county and Seni district of Naqu city, north Tibet in June 2019. Naqu City has an average altitude of 4,500 m. It is the highest place in China and called "the roof of the world".
A total of 938 blood samples were collected randomly from apparently healthy yaks. Four hundred were collected in Nierong County area (Xiaqu, Seqing), including 247 females and 153 males while 538 samples were collected in the Seni district (Luoma, Namaqie, Dasha, Mufa), including 309 females and 229 males. The blood samples were placed at room temperature for 4 h and quickly transferred to the laboratory, and centrifuged at 3000 g for 10 min to obtain sera that were stored at -80 ℃ until use.
Anti-C. abortus IgG antibodies were detected with ELISA kits according to the manufacturer's instructions (ID Screen® Chlamydophila abortus Indirect Multispecies, IDVET Innovative Diagnostics, Montpellier, France). The optical density (OD) was measured at 450 nm by an ELISA microplate reader (Multiskan™ FC, Thermo Fisher Scientific™, USA). Signal to noise ratio was calculated according to the following formula: S/P (%) = 100 × (OD sample / OD positive control).
Where S: tested sample, P: positive control. Sera samples with S/P ratios higher than 60 % were considered positive.
The differences in infection rate based on location, genders and age groups of yaks were analyzed by Chisquare test in SPSS (Statistical Analysis System, Version 22.0). Values of p < 0.05 were considered statistically significant.
According to our results, the seroprevalence of yak C. abortus in Tibet was lower than that in Qinghai (17.66 %) and Gansu provinces (16.22 %) of China [16,17], and in India (35 %) [18], but higher than the prevalence reported in Ireland and Poland [8,11]. This difference may be related to the sample collection, feeding method, species and age of animals, as well as different detection methods, such as indirect hemagglutination assay (IHA) and ELISA which can cause a significant difference in seroprevalence rates because of differences in test sensitivity.
There was no significant difference in the prevalence in female and male yaks (p > 0.05), which was consistent with the results of white yaks in Gansu [17] and yaks in Qinghai Province [16]. The same results have been observed in studies on other animal species such as the Tibetan pigs in Tibet, China [19]. These findings suggested that gender might not be a key factor in C. abortus infection in Tibet animal populations.
Tibetan yaks basically live at an altitude of 5,000 m above sea level. High altitude is the most important ecological challenge for yaks. In this study, antibodies against C. abortus in yak varied from 8.0 to 18.2 % between regions. The highest seroprevalence for C. abortus was in Xiaqu township. The more intensive husbandry  found in townships such as Xiaqu suggests that close contact at feeding may be a risk factor for the increased prevalence of C. abortus.
Among the different age groups, a higher prevalence (15.9 %) was detected in the 1 to 2 year age category but was not significant (p > 0.05), This pattern of age-related seroprevalence has also been observed in previous surveys of yaks elsewhere in China [16,17] and was also not different from reports in cattle in Guangzhou, China [20]. High seroprevalence among members of the 1-2 years age category could be due to lower resistance or immunity, chronic infection, or sustained infection in yaks of this age.
Tibet is one of four major pastoral areas and an important grassland animal husbandry production base in China. There are about 4 million yaks in Tibet which makes it an economically important livestock species. Yaks abortion caused by C. abortus infection in Qinghai province has been reported previously [15]. However, the prevalence of C. abortus in yak population in the vast Tibet area is not known. The current study showed an overall serological positive ratio of 11.1 % by a C. abortus antibody-specific ELISA kits. According to local herdsmen, yak flocks suffered abortions in Tibet in recent years. We believe that some of the yak abortion cases might be the outcome of C. abortus infection in the yak population, although C. abortus-related abortion has not been investigated. C. abortus is a known zoonotic bacterial pathogen. Several reports have demonstrated that humans can acquire infection through contact with infected animals or aborted feteus and placenta, resulting in severe health problems including atypical pneumonia in men and abortion or pelvic inflammatory diseases in women [21,22]. It might be a potential risk for public health.
In conclusion, the results showed a C. abortus seroprevalence of 11.1 % in yaks in Tibet, China. To our knowledge, the present report is the first to document the seroprevalence of C. abortus infection in yaks in Tibet, China. Further work is required to establish the significance of C. abortus infection in Tibetan yaks.