Prevalence, Diagnosis, and Vaccination Situation of Animal Chlamydiosis in China

Since the first case of Chlamydia infection in duck had been reported in 1956 and the first case from domestic animal had been reported in 1979 in China, the chlamydia prevalence in China was heavily according to the published data. The Chlamydia in avian prevalence has been reported at least 11 provinces, Chlamydia in sheep and goats at least 11 provinces, in swine at least 15 provinces, in cows at least 13 provinces and in yaks at least 5 provinces with result of IHA detection. Different diagnostic method such as CFT, ELISA and ABC-ELISA (avidin-biotin-complex ELISA) had been established besides IHA. The inactivated vaccines have been developed with isolated strains from sheep, goats, swine and cows. These inactivated vaccines have been used since 1980s and Chlamydia prevalence in China has been successfully controlled in domestic animal. However, the inactivated vaccines of Chlamydia isolated from avian species have not been successful, although a series of experimental vaccine have been done. Due to the unsustainable eradication plan of Chlamydia in China, sporadic outbreak in animal would happen if the vaccinations were suspended and economy lose in some farmers. Although Chlamydia prevalence in China has a long history, however, almost all published studies are in Chinese, which, in some degree, blocked scientists in other countries to understand the prevalence situation and control measures of Chlamydia in China.

of Chlamydia in China. However, almost all these achievements were reported only in Chinese, rather than in English, which presents a challenge for scientists all over the world to understand the situation of Chlamydia prevalence and control measures in China.

ChlAmydiA PRevAlenCe
Following the first report of Chlamydiosis in China from a duck in Beijing in 1959 (15), prevalence of avian Chlamydia has been reported in 10 provinces, including Beijing, Tianjin, Jiangsu, and Guangdong due to lack of effective vaccines (16).
The first instance of livestock Chlamydiosis was found in ruminants in the Qinghai province by scientists from the Lanzhou Veterinary Research institute (LVRI), Chinese Academy of Agricultural Sciences (CAAS), in 1979 (17). Chlamydia was detected in sheep and goats in at least 11 provinces, in swine in 15 provinces, in bovines in 13 provinces, and in yaks in 5 provinces, using the indirect hemagglutination assay (IHA) test; this prevalence if the disease caused a huge economic loss in China (Figure 1). Although the seropositive rate was high in different animals in China, similar seropositive rates existed in other countries. For example, a German study reported Chlamydia antigen prevalence in sheep to range between 5 and 71% (18) and a Polish study reported the prevalence of Chlamydial infection in birds in Europe to range between 6.3 and 19.2% (19). There was uncertainty about the accuracy of the results of Chlamydia detected in animals using the IHA technique in China. However, compared with the results from other countries, which used McCoy cell culture isolation, the IHA technique may reflect the real prevalence of Chlamydia in China.

DiAGnoStiC MethoD
Before 1984, antibodies to Chlamydia were detected by using the complement fixation (CF) method in China. However, this technique was cumbersome and time consuming (88). Thus, a new technique, IHA, was developed. The IHA technique had a higher sensitivity and specificity than CF, and has been used to determine the prevalence of Chlamydia in domestic animals in China since the 1980s (88). It has also been used as a high throughput method of seroprevalence detection. Positive results are detectable within 2 h, but false positive and negative readings are possible, since scoring is subjective to the researcher's observation (88). Besides  For pathogen diagnosis, chicken egg isolation and Giemsa staining were combined to detect Chlamydia. The yolk sac membranes from dead chicken embryos were spread on slides and fixed with methanol or through heating, and Giemsa stain was used to stain them for half an hour or overnight. Chlamydia elementary body (EB) and inclusion were detected by light microscopy (91).
The PCR and real-time PCR tests, although highly sensitive and used to detect Chlamydia in different animals in other countries (92)(93)(94)(95)(96) have seldom been used on a large scale due to high cost. The omp1 gene, which is very conservative, was used as a target gene to detect Chlamydia in different animals when IHA results were ambiguous (16,97). In China, the PCR-RFLP method was developed and used only to identify Chlamydia species isolated from animals (11), while recombinase polymerase amplification (RPA) was used to identify C. abortus (98). However, IHA is considered as a simple, safe, and reliable means of testing C. abortus antibodies, and has been employed in previous serological investigations (79,99).

PRevention AnD ContRol oF ChlAMYDioSiS vaccination
Vaccination is one of the most important methods of disease prevention in animals. The inactivated vaccine of C. abortus plays a huge role to control the spreading of the disease in China. Simply, the process for developing inactivated vaccine is as follows: Chlamydia seeds were isolated from the yolk sac membranes of 7-day-old chicken embryos between the 4 and 8th days after inoculation. The titer of Chlamydia used for vaccines was at least 10 12 ELD 50 /0.4 ml. The yolk sac membrane was disrupted with a homogenizer and filtered through a mesh strainer. Formalin (0.3%) was used to inactivate Chlamydia with an equal volume of 206 adjuvant (SEPPIC, France) and mixed under 40 Mpa of pressure. After 7 days of inactivation with formalin, the safety and efficacy of the vaccine were tested using specific pathogen free (SPF) embryos and mice (100)(101)(102).
Chlamydia inactivated vaccines for sheep and goat have been used since 1981-1986 (100). During that time, 120,000 sheep and goats in Qinghai, Gansu province, and Xinjiang Uygur Autonomous region were vaccinated and the abortion rate declined sharply. No abortion happened due to vaccination in pregnant sheep and goats. A total of 2,000,000 ml (about 700,000 doses) of inactivated vaccine was produced and used in 1988 (100). The duration of immunity was at least 2 years, but 75% of the sheep and goats were protected from infection in the 4th year after vaccination (100,103). Besides, the inactivated vaccine could be used after a 2 year storage period at 4°C (100). A similar inactivated vaccine of Chlamydia isolated from goat was studied by Zhang in the Inner Mongolia Autonomous Region (33). According to this report, 5,099 goats belonging to 51 groups were vaccinated with inactivated vaccine and at least 90% of the goats were protected. Other regions, such as Huachi County, Gansu province, showed similar results of very high prevalence of Chlamydia in goats during 1981-1986, with abortion rate in goats being 20-40%. The disease was controlled when inactivated vaccine was used, reducing the abortion rate of the vaccinated groups to 3.3-6.5%, compared with the control group abortion rate of 14.03-14.3%, during 1986-1988 (102).
Chlamydia of swine was also detected and isolated in Qinghai province and the GuangxiZhuang Autonomous Region (55,56,88). According to these reports, abortion happened among sows and the highest positive rate of abortion was 56.1% in one of the groups in which Chlamydia was detected using the complement fixation test (CFT). Two strains were isolated using 7-day-old SPF eggs, and the inactivated vaccine was produced and tested (101). A total of 1,080 sows were vaccinated in two farms and each sow was immunized subcutaneously with 3 ml of inactivated vaccine (101). After 3 months, 482 sows that had been vaccinated and 439 sows that were not vaccinated were studied; only 1.45% abortion rate was observed in the vaccinated group, while 29.53% abortion rate was observed in the non-vaccinated group (101). These results showed that the inactivated vaccine provided good infection protection (101). Subsequently, 10,594 sows were vaccinated in Qinghai, Shanxi province, and Guangxi Zhuang Autonomous regions. The duration of immunity was at least 1 year when 2 ml of vaccine was injected (104). The vaccine remained active after storage at 4-8°C for 1 year (101).
Chlamydia in bovines was reported firstly in China in 1986 (70). In 1990, 2 strains (CCS10 and CCS15) were isolated from cows in a farm in Shaanxi province (105). However, the strain used for the inactivated vaccine was isolated in 2006 (102). This isolated strain (SX5) from a farm in Shaanxi province was tested and the LD 50 /0.4 ml remained at 10 −12 after at least 5 times propagation in SPF eggs. The minimal effective dosages for the vaccine was 3 ml for adult dairy cow and 1.5 ml for calf. The average protection was about 94.4%, while the duration of immunity was 10 months (102).
Although formalin-inactivated C. abortus vaccines have been used in China since 1985, their production was discontinued because of lack of good manufacturing practices (GMP), causing  (11) sporadic outbreaks in sheep (46), yaks (106), and other animals.
However, there is no information about Chlamydia prevalence in recent years from Dulan county (100), Delingha (56), Qinghai province, and Huachi county (101), Gansu province, where Chlamydia was first isolated and animals were vaccinated with inactivated vaccine. On the contrary, farms near the original places, such as in Wulan county, Guinan county, Haiyan county, Gonghe county, Huzhu county, Huangyuan county, and Tianjun County, Qinghai province, reported that Chlamydia caused huge abortion among sheep, goats, and yaks in recent years (11,35,38,68,75,84,87,(107)(108)(109). Interestingly, during the investigation, shepherds reported that dead Tibetan antelopes were found and their eyes were obviously blind (personal communication). They also reported that Chlamydia spread among different groups of sheep that had never been in contact with each other, suggesting that wild animals may play a very important role to spread Chlamydia to domestic animal. Therefore, based on this information, we can conclude that the original source of Chlamydia infections in China is wild animals in Qinghai province. Besides inactivated vaccine, the subunit vaccine for Chlamydia in ewes, which has three different doses of major outer-membrane protein from genetically engineered Chlamydia psittaci, was developed by the State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, China. The study analyzed the antibody responses in ewes vaccinated with the subunit vaccine of rCps-MOMP. The sera of ewes were detected before vaccination and at different times post-vaccination. Experimental results indicated that multilocus intramuscular injection in the neck region with a dose of 0.5 mg per ewe could stimulate good immune response (110). Because of the good security and immunity protection, a new veterinary drug certificate was awarded by the Ministry of Agriculture of the People's Republic of China (111). However, there are few reports of the promotion and application of the submit vaccine in China (112).
Although some successes were obtained for controlling Chlamydia in livestock in China since the inactivated vaccines were introduced, each year huge economic losses are caused by avian Chlamydia in chicken production (28). A subunit vaccine and a recombinant adenovirus live vector vaccine have been developed (16,113). However, the recombinant adenovirus vector vaccine has not been approved by the government due to potential biological concerns, and no further data about the subunit vaccine has been published. Avian Chlamydia is still serious in China due to a lack of effective and safe vaccine. Moreover, the disease is a potential risk for human health. Therefore, further investigation into the development of vaccines is necessary. tReAtMent Antibiotics such as tetracycline, oxytetracycline, and penicillin sodium are used to treat Chlamydiosis in animals in China. However, since 2014, to regulate the use of veterinary antibiotics in China, the government established a veterinary prescription drug management system, including measures for administration of veterinary prescription and over-the-counter drugs. The quality of antibacterial drug products has been improving every year. The quality of sampling inspection is maintained at more than 95%, whereas the rate of residues of veterinary drugs in livestock and poultry products remains stable at more than 97%. Thus, the use of antibiotics has been greatly reduced.

FutuRe PeRSPeCtive
Although IHA plays a very important role in detecting Chlamydia in animals in China, it does not reflect the real situation of Chlamydiosis prevalence in animals. However, Hagemann JB (114) reported that aborting sheep exhibited a strong antibody response to surface (MOMP, MIP, and Pmp13G) and virulenceassociated (CPAF, TARP, and SINC) antigens. While the latter disappeared within 18 weeks following abortion in majority of the animals, antibodies to surface proteins persisted beyond the duration of the study. In contrast, experimental non-abortion infected sheep developed antibodies mainly to surface antigens (MOMP, MIP, and Pmp13G), all of which did not persist. This indicates that new diagnostic methods need to be established to improve the accuracy of disease diagnosis and provides scientific basis for controlling animal Chlamydiosis.

ConCluSion
Since the first case of avian Chlamydia was reported in China, Chlamydia infection has been observed in different animals in most areas of China, which causes serious economic losses each year. Several diagnostic techniques, including CF, IHA, ELISA, ABC-ELISA, egg isolation, and PCR, have been studied and used in China. Formalin inactivated vaccines of Chlamydia from sheep, goat, swine, and cow were developed and have been used since 1981 in those areas where animals are threatened by Chlamydia. Because Chlamydiosis was considered an unimportant disease in animals by the Chinese government and no eradiation plan has implemented, there are sporadic outbreaks of Chlamydia in domestic animals in some areas, especially where vaccination has been suspended. However, the abortion rate was down sharply when inactivated vaccines for Chlamydia were used in domestic animals. This may have contributed to the lack of large-scale outbreak of Chlamydiosis in domestic animals in the last 30 years. The most important problem now is avian Chlamydiosis, which has a seropositive rate of 10-50% with IHA and easily spreads from birds to humans. Due to lack of effective vaccines, avian Chlamydiosis may become a public health problem in China.(Reorder)

AuthoR ContRiButionS
Substantial contribution to the references of the work: ZLi, ZLou, YF. Draft and/or critical revision of the manuscript: JZ. Final approval of the version to be published: JZ, ZLi, ZLou, YF.