# Identification and Characterization ofChlamydia abortus Isolates from Yaks in Qinghai, China

Authors: Zhaocai Li; Xiaoan Cao; Baoquan Fu; Yilin Chao; Jinshan Cai; Jizhang Zhou
Journal: BioMed Research International
 (2015)

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## Abstract

Recently, the yak population has exhibited reproductive disorders, which are considered to be associated withChlamydia abortus (C. abortus) in Qinghai, China. In this study, a total of 9 aborted fetuses (each from a different herd) and 126 vaginal swab samples from the 9 herds were collected and analyzed.C. abortusDNA was detected from all of the 9 aborted fetuses and 30 of the 126 vaginal swab samples (23.81%) from yak cows in the selected herds. FourC. abortus strains were isolated from embryonated egg yolk sacs inoculated with foetal organ suspensions. The isolatedC. abortus strains were further identified, which showed identical restriction profiles with theC. abortus reference strain usingAluI restriction enzyme in the RFLP test. Moreover, the isolatedC. abortus strains andC. abortus-positive vaginal swab samples were genotyped by multiple loci variable number tandem repeat analysis and all belonged to the genotype 2 group. These findings suggested thatC. abortus played a substantial role in yak abortion in Qinghai, China.


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## Body

## 1. Introduction

Chlamydia abortus (C. abortus) is endemic in ruminants throughout the world.C. abortus can efficiently colonize the placental trophoblasts and is one of the causative agents of abortion and foetal loss in sheep, goats, and cattle in many countries [1–3]. The pathogen has also been associated with cases of abortion in pigs, horses, rabbits, guinea pigs, and mice [4] and represents a zoonotic risk to humans [5, 6]. Although this bacterium is distributed worldwide and can infect a range of hosts,C. abortus is considered a very homogeneous species with low genetic heterogeneity [7, 8]. A previous study has developed a multiple loci variable number tandem repeat (VNTR) analysis (MLVA) system to explore the diversity ofC. abortus. Five selected loci with polymorphism have been shown to be suitable for genotyping ofC. abortus strains, which results in the classification of all strains studied into six genotypes. Notably, the classification of theC. abortus strains into different genotypes has shown to be, to a large extent, related to their geographical origin [8].The yak (Bos grunniens) is one of the most remarkable domestic animals found in the cold mountainous areas of the Himalayan region, at 3 000 meters above sea level. There are about 15 million yaks existing in the world. More than 90% of the yak population lives in the Qinghai-Tibetan Plateau, China. They provide meat, milk, wool, transportation, and home heating for the local people. Thus, the yak is the basis for much of the agricultural economy of the vast rural regions where few other domestic animals will survive. In general, female yaks can only be pregnant once every 2 years or twice in 3 years, and each yak cow produces a single offspring. In addition to the low reproductive rate, yaks are suspected to be infected with a large variety of pathogens includingChlamydia [9, 10], which significantly affect the production of yak resulting in great economic losses. Several studies have shown the seroprevalence ofChlamydia infection in yak herds with abortions in different regions in China [11, 12]. Therefore, the aim of the present study is to identify and characterize theC. abortus strains prevalent in yaks in Qinghai, China.


## 2. Materials and Methods

### 2.1. Samples

During the birth season of 2012, yak flocks in several regions of Qinghai province, China, have demonstrated the problem of abortion. A serological analysis suggested thatChlamydia infection was incriminated. For further diagnosis, a total of 9 samples from aborted fetuses in Guinan County (six herds) and Haiyan County (three herds), Qinghai province, each of which was from different herds, were sent to Center for Animal Disease Control and Prevention in Qinghai province and Lanzhou Veterinary Institute for further diagnosis. Another 126 vaginal swabs were taken from yak cows in these 9 herds, some of which were taken from yak cows that suffered from abortion, and the others were taken from pregnant yak cows that had not delivered or aborted yet.


### 2.2. PCR Analysis

DNA was isolated from fetal tissues and vaginal swab samples using the QIAamp DNA Kit (Qiagen) according to the kit’s instructions. The DNA samples were then subjected to PCR detection forChlamydia. The genus-specific primers were targeted to thepmp gene ofChlamydia (FP: 5′-ATGAAACATCCAGTCTACTGG-3′; RF: 5′-TTGTGTAGTAATATTATCAAA-3′) and PCR reaction was performed as described previously [13].


### 2.3. Isolation ofChlamydia Strain

For isolation, a 10% (v/v) suspension of foetal organ samples (liver, spleen, and lung) was prepared in SPG buffer (0.25 M sucrose, 10 mM sodium phosphate, and 5 mM L-glutamic acid) containing 1 mg/mL of streptomycin and 1 mg/mL of kanamycin. 0.4 mL of the suspension was inoculated into the yolk sac of 7-day-old specific pathogen free embryonated chicken eggs. The eggs were incubated at 37°C and observed daily and were harvested while they died during 4–10 days after inoculation for further passage. Blind passages were made when no embryonic death had been observed. The presence of chlamydial progeny in yolk sacs was detected byC. abortus-specific PCR. To identify the chlamydial inclusion body, positive cultures were further inoculated in McCoy cells monolayer, followed by Giemsa staining.


### 2.4. PCR-RFLP Analysis

For identification of the isolatedChlamydia strains, PCR-RFLP analysis of the helicase genes clone 8 andomp2 gene was performed as described previously [14, 15]. The reference strains ofC. abortus SX5 (isolated from cattle),C. psittaci CpL (isolated from chicken), andC. pecorum E58 were kept in our laboratory [16, 17]. Amplified DNA fragments of helicase genes clone 8 andomp2 were digested with 3 U ofAluI in 30 μL final volume overnight at 37°C, respectively. The resulting fragments were subjected to electrophoresis in 2% agarose gel at 60 V for 2 h and examined under UV illumination.


### 2.5. MLVA Typing of the Isolates and Clinical Samples

A set of VNTR loci, namely, ChlAb_300, ChlAb_457, ChlAb_581, ChlAb_620, and ChlAb_914, were used as the genotyping markers. PCR was performed to amplify the tandem repeat sequences from theC. abortus isolates and theChlamydia DNA positive vaginal swab samples, respectively. The primers and PCR procedure were described previously [8]. The obtained amplicons were then sequenced at Shanghai Sangon Biotech (Shanghai, China). The numbers of repeats in the set of VNTR loci for eachChlamydia abortus isolate or sample were recorded. Therefore, an allelic profile for eachC. abortus isolate or vaginal swab sample was obtained as an ordered string of allele numbers corresponding to the number of repeat units at each MLVA locus. To determine the MLVA genotype, the assessed allelic profile was compared with the reference genotyping data [8].


## 2.1. Samples

During the birth season of 2012, yak flocks in several regions of Qinghai province, China, have demonstrated the problem of abortion. A serological analysis suggested thatChlamydia infection was incriminated. For further diagnosis, a total of 9 samples from aborted fetuses in Guinan County (six herds) and Haiyan County (three herds), Qinghai province, each of which was from different herds, were sent to Center for Animal Disease Control and Prevention in Qinghai province and Lanzhou Veterinary Institute for further diagnosis. Another 126 vaginal swabs were taken from yak cows in these 9 herds, some of which were taken from yak cows that suffered from abortion, and the others were taken from pregnant yak cows that had not delivered or aborted yet.


## 2.2. PCR Analysis

DNA was isolated from fetal tissues and vaginal swab samples using the QIAamp DNA Kit (Qiagen) according to the kit’s instructions. The DNA samples were then subjected to PCR detection forChlamydia. The genus-specific primers were targeted to thepmp gene ofChlamydia (FP: 5′-ATGAAACATCCAGTCTACTGG-3′; RF: 5′-TTGTGTAGTAATATTATCAAA-3′) and PCR reaction was performed as described previously [13].


## 2.3. Isolation ofChlamydia Strain

For isolation, a 10% (v/v) suspension of foetal organ samples (liver, spleen, and lung) was prepared in SPG buffer (0.25 M sucrose, 10 mM sodium phosphate, and 5 mM L-glutamic acid) containing 1 mg/mL of streptomycin and 1 mg/mL of kanamycin. 0.4 mL of the suspension was inoculated into the yolk sac of 7-day-old specific pathogen free embryonated chicken eggs. The eggs were incubated at 37°C and observed daily and were harvested while they died during 4–10 days after inoculation for further passage. Blind passages were made when no embryonic death had been observed. The presence of chlamydial progeny in yolk sacs was detected byC. abortus-specific PCR. To identify the chlamydial inclusion body, positive cultures were further inoculated in McCoy cells monolayer, followed by Giemsa staining.


## 2.4. PCR-RFLP Analysis

For identification of the isolatedChlamydia strains, PCR-RFLP analysis of the helicase genes clone 8 andomp2 gene was performed as described previously [14, 15]. The reference strains ofC. abortus SX5 (isolated from cattle),C. psittaci CpL (isolated from chicken), andC. pecorum E58 were kept in our laboratory [16, 17]. Amplified DNA fragments of helicase genes clone 8 andomp2 were digested with 3 U ofAluI in 30 μL final volume overnight at 37°C, respectively. The resulting fragments were subjected to electrophoresis in 2% agarose gel at 60 V for 2 h and examined under UV illumination.


## 2.5. MLVA Typing of the Isolates and Clinical Samples

A set of VNTR loci, namely, ChlAb_300, ChlAb_457, ChlAb_581, ChlAb_620, and ChlAb_914, were used as the genotyping markers. PCR was performed to amplify the tandem repeat sequences from theC. abortus isolates and theChlamydia DNA positive vaginal swab samples, respectively. The primers and PCR procedure were described previously [8]. The obtained amplicons were then sequenced at Shanghai Sangon Biotech (Shanghai, China). The numbers of repeats in the set of VNTR loci for eachChlamydia abortus isolate or sample were recorded. Therefore, an allelic profile for eachC. abortus isolate or vaginal swab sample was obtained as an ordered string of allele numbers corresponding to the number of repeat units at each MLVA locus. To determine the MLVA genotype, the assessed allelic profile was compared with the reference genotyping data [8].


## 3. Results

### 3.1.Chlamydia Related Abortion

During April and May of 2012, yak flocks have demonstrated the problem of abortion in several regions of Qinghai province, China. The abortion occurred at the end of gestation (1~2 months before delivery) without any premonitory signs. A serological analysis of several abortion diseases performed on the female’s serum sample suggested thatChlamydia was incriminated (data not shown). In the present study, we investigated the 9 aborted fetuses and 126 vaginal swab samples from corresponding herds byChlamydia genus-specific PCR analysis. TheChlamydia DNA was found in all of the aborted fetuses (Figure 1).Chlamydia DNA was also present in 30 of the 126 vaginal swab samples (23.81%) from yak cows in herds with abortion (Table 1). The results suggested that the yak abortion might be associated withChlamydia infection.Table 1
Detection ofC. abortus by PCR from vaginal swab samples.
Herds
Herd size
Number of vaginal swabs
Positive PCR: number (and %)
MLVA genotype of the positive samples
GN-1
340
21
4 (19.05)
2
GN-2
203
12
2 (16.67)
2
GN-3
280
18
3 (16.67)
2
GN-4
267
15
5 (33.33)
2
GN-5
271
10
4 (40.00)
2
GN-6
189
7
4 (57.14)
2
HY-1
334
19
6 (31.58)
2
HY-2
265
11
1 (9.09)
2
HY-3
310
13
1 (7.69)
2
Total
126
30 (23.81)Figure 1
Detection ofChlamydia DNA from the 9 aborted yak fetuses from different herds. Marker: 100 bp DNA ladder.


### 3.2. Isolation and Identification of YakC. abortus Isolates

After several sets of passages, four yakChlamydia strains were isolated from the aborted yak foetal organ samples using embryonated chicken egg yolk sac culture, as evidenced by the presence ofC. abortus DNA when examined by PCR assay. To identify the chlamydial inclusion body, the positive cultures were inoculated in McCoy cells monolayer, followed by Giemsa staining. Figure 2 showed the chlamydial inclusions in McCoy cells after 48 h of inoculation. The isolated strains were named as GN-1, GN-4, GN-6, and HY-1 according to their origin region and herd identifier. NoChlamydia strain could be isolated from the other aborted yak fetuses since noC. abortus DNA could be detected from the yolk sacs of embryonated eggs after a series of 5 passages.Figure 2
Perinuclear chlamydial inclusions (arrows) in McCoy cells when infected with the isolated strain GN-6. ×400, Giemsa.For further species identification of the four isolates, amplicons of helicase genes clone 8 andomp2 gene were subjected to PCR-RFLP analysis. As shown in Figure 3, PCR using helicase genes clone 8 primers produced a band of 479 bp from the four isolates and reference strain ofC. abortus SX5. A positive band was also observed from theC. psittaci CpL; however, no band was observed from theC. pecorum E58 (Figure 3(a)). When the amplified DNA fragments of helicase genes clone 8 were digested with theAluI enzyme, all isolates exhibited a profile with two bands of 271 bp and 208 bp, the same as that of theC. abortus SX5 reference strain. However, the intact amplified fragment was observed without any cleavage fromC. psittaci CpL strain afterAluI enzyme digestion, which differentiated it from the four yak isolates (Figure 3(b)). PCR usingomp2 primers produced a band of 587 bp from the four isolates and reference strains ofC. abortus SX5 andC. pecorum E58 (Figure 3(c)). Following digestion with theAluI enzyme, all isolates exhibited the same profile with two bands of 352 bp and 235 bp as theC. abortus SX5 strain, whileC. pecorum E58 exhibited a different profile with two bands of 397 bp and 193 bp (Figure 3(d)). Therefore, the yak isolates were identified asC. abortus strains.Figure 3
Identification ofC. abortus isolates by PCR-RFLP analysis. (a) Helicase gene clone 8 PCR products. (b) RFLP analysis byAluI restriction enzyme of clone 8 PCR products. (c)omp2 PCR products. (d) RFLP analysis byAluI ofomp2 PCR products. Marker: 100 bp DNA ladder.
(a)
(b)
(c)
(d)


### 3.3. MLVA Genotyping ofC. abortus Isolates and Clinical Samples

In this study, we determined the genotype of yakC. abortus isolates and the 34 clinical samples by using the MLVA typing method. The five polymorphic loci, namely, ChlAb_457, ChlAb_581, ChlAb_620, ChlAb_914, and ChlAb_300, were amplified and sequenced, and the repeated units in each locus were recorded. The results showed that the same allelic profile (1-1-2-1-3) was shared by all the isolates and clinical samples, which matched the MLVA genotype 2 ofC. abortus (see Figure S1 in Supplementary Material available online at http://dx.doi.org/10.1155/2015/658519 and Table 1) [8]. Therefore, we classified the yak isolates into MLVA genotype 2. The results may also suggest that the MLVA genotype 2 ofC. abortus is the predominant strains prevalent in yaks in Qinghai, China.


## 3.1.Chlamydia Related Abortion

During April and May of 2012, yak flocks have demonstrated the problem of abortion in several regions of Qinghai province, China. The abortion occurred at the end of gestation (1~2 months before delivery) without any premonitory signs. A serological analysis of several abortion diseases performed on the female’s serum sample suggested thatChlamydia was incriminated (data not shown). In the present study, we investigated the 9 aborted fetuses and 126 vaginal swab samples from corresponding herds byChlamydia genus-specific PCR analysis. TheChlamydia DNA was found in all of the aborted fetuses (Figure 1).Chlamydia DNA was also present in 30 of the 126 vaginal swab samples (23.81%) from yak cows in herds with abortion (Table 1). The results suggested that the yak abortion might be associated withChlamydia infection.Table 1
Detection ofC. abortus by PCR from vaginal swab samples.
Herds
Herd size
Number of vaginal swabs
Positive PCR: number (and %)
MLVA genotype of the positive samples
GN-1
340
21
4 (19.05)
2
GN-2
203
12
2 (16.67)
2
GN-3
280
18
3 (16.67)
2
GN-4
267
15
5 (33.33)
2
GN-5
271
10
4 (40.00)
2
GN-6
189
7
4 (57.14)
2
HY-1
334
19
6 (31.58)
2
HY-2
265
11
1 (9.09)
2
HY-3
310
13
1 (7.69)
2
Total
126
30 (23.81)Figure 1
Detection ofChlamydia DNA from the 9 aborted yak fetuses from different herds. Marker: 100 bp DNA ladder.


## 3.2. Isolation and Identification of YakC. abortus Isolates

After several sets of passages, four yakChlamydia strains were isolated from the aborted yak foetal organ samples using embryonated chicken egg yolk sac culture, as evidenced by the presence ofC. abortus DNA when examined by PCR assay. To identify the chlamydial inclusion body, the positive cultures were inoculated in McCoy cells monolayer, followed by Giemsa staining. Figure 2 showed the chlamydial inclusions in McCoy cells after 48 h of inoculation. The isolated strains were named as GN-1, GN-4, GN-6, and HY-1 according to their origin region and herd identifier. NoChlamydia strain could be isolated from the other aborted yak fetuses since noC. abortus DNA could be detected from the yolk sacs of embryonated eggs after a series of 5 passages.Figure 2
Perinuclear chlamydial inclusions (arrows) in McCoy cells when infected with the isolated strain GN-6. ×400, Giemsa.For further species identification of the four isolates, amplicons of helicase genes clone 8 andomp2 gene were subjected to PCR-RFLP analysis. As shown in Figure 3, PCR using helicase genes clone 8 primers produced a band of 479 bp from the four isolates and reference strain ofC. abortus SX5. A positive band was also observed from theC. psittaci CpL; however, no band was observed from theC. pecorum E58 (Figure 3(a)). When the amplified DNA fragments of helicase genes clone 8 were digested with theAluI enzyme, all isolates exhibited a profile with two bands of 271 bp and 208 bp, the same as that of theC. abortus SX5 reference strain. However, the intact amplified fragment was observed without any cleavage fromC. psittaci CpL strain afterAluI enzyme digestion, which differentiated it from the four yak isolates (Figure 3(b)). PCR usingomp2 primers produced a band of 587 bp from the four isolates and reference strains ofC. abortus SX5 andC. pecorum E58 (Figure 3(c)). Following digestion with theAluI enzyme, all isolates exhibited the same profile with two bands of 352 bp and 235 bp as theC. abortus SX5 strain, whileC. pecorum E58 exhibited a different profile with two bands of 397 bp and 193 bp (Figure 3(d)). Therefore, the yak isolates were identified asC. abortus strains.Figure 3
Identification ofC. abortus isolates by PCR-RFLP analysis. (a) Helicase gene clone 8 PCR products. (b) RFLP analysis byAluI restriction enzyme of clone 8 PCR products. (c)omp2 PCR products. (d) RFLP analysis byAluI ofomp2 PCR products. Marker: 100 bp DNA ladder.
(a)
(b)
(c)
(d)


## 3.3. MLVA Genotyping ofC. abortus Isolates and Clinical Samples

In this study, we determined the genotype of yakC. abortus isolates and the 34 clinical samples by using the MLVA typing method. The five polymorphic loci, namely, ChlAb_457, ChlAb_581, ChlAb_620, ChlAb_914, and ChlAb_300, were amplified and sequenced, and the repeated units in each locus were recorded. The results showed that the same allelic profile (1-1-2-1-3) was shared by all the isolates and clinical samples, which matched the MLVA genotype 2 ofC. abortus (see Figure S1 in Supplementary Material available online at http://dx.doi.org/10.1155/2015/658519 and Table 1) [8]. Therefore, we classified the yak isolates into MLVA genotype 2. The results may also suggest that the MLVA genotype 2 ofC. abortus is the predominant strains prevalent in yaks in Qinghai, China.


## 4. Discussion

The yak populations seem to be susceptible to most pathogenic agents that affect cattle. Serological studies showed thatBrucella,Toxoplasma gondii, and bovine viral diarrhea virus infection in yaks commonly occurred in Qinghai province, China [18–20]. In recent years,Chlamydia infection has attracted attention. Several Chinese literatures showed a seroprevalence rate (9.37%~57.14%) in yak populations in different areas of Qinghai-Tibet Plateau [21, 22]. As early as 1988, Shuai et al. reported that abortions in yaks in a part of Qinghai province were diagnosed as being caused byChlamydia abortus infection [23]. Ma surveyed the prevalence ofBrucella andChlamydia in Qinghai province in 2002, and the results showed the incidence of abortion in yak due toChlamydia may be of significance [9]. However, there has been no direct detection or isolation of Chlamydiae from yaks. In this study, we succeeded to isolate and characterize chlamydial strains responsible for yak abortion. The 9 aborted fetus samples were analyzed by species-specific PCR forC. abortus. DNA ofC. abortus was detected from all of the samples, suggesting thatC. abortus infection was responsible for the yak abortion cases. The inoculation of infected foetal organ extract into chicken embryos led to the isolation of fourC. abortus strains. Some reports also showed that, in addition toC. abortus,C. psittaci andC. pecorum were also associated with cases of abortion in sheep and goat [24, 25]. Due to the high degree of phenotypic similarity and for further identification of the isolated strains, it is necessary to differentiate the species from each other. It has been suggested thatC. abortus can be differentiated fromC. psittaci andC. pecorum by RFLP analysis of PCR products obtained with primers specific to clone 8 of theC. abortus helicase gene andomp2 gene and that these primers allow species-specific identification [14, 26]. The present study showed that PCR-RFLP with clone 8 andomp2 primers is adequate for the species identification ofC. abortus and the result further confirmed that the yak abortion cases were associated withC. abortus infection.C. abortus is an obligate intracellular bacterium which can colonize very different types of placenta (ruminant, porcine, murine, and human), causing abortion during the last third of the gestation period. This disease is especially important in yaks, one of the natural hosts in Qinghai-Tibet Plateau, because of the economic losses it causes. However, chlamydial diseases in yaks are not well understood, partially because they have received relatively little attention. In addition, the bacteria also present a potential zoonotic risk to pregnant women, since several cases of human abortion have been reported as being associated with exposure to aborted sheep or goat caused byC. abortus [5, 6]. Therefore, there is a clear need for improved control strategies for this issue including more effective diagnosis and control measures. For diagnosis, isolation of the pathogens from aborted samples (fetuses and placentae) represents the gold standard for definitive diagnosis. However, isolation of bacteria requires obtaining samples in optimal conditions (they must be fresh, with little or no contamination, and free of toxic factors) that contain a threshold number of live and viable microorganisms. This process is labor intensive and time consuming and therefore not suitable for routine diagnostics or large-scale epidemiological investigations. Indirect hemagglutination (IHA) test based on theC. abortus elementary bodies is the most common and available reagent for routine diagnosis of theC. abortus antibodies in China. However, cross-reactions withC. psittaci andC. felis can be observed [27, 28]. Detection ofChlamydia specific DNA by PCR is more sensitive and specific than serological tests and therefore recommended. Conventional and real-time PCR have been widely used to identifyC. abortus in clinical samples. Further study is now necessary to get a better understanding of the epidemiology of the bacterium in the yak populations by integration of these techniques in routine diagnostics.For prevention, vaccination may be the most effective way of prevention ofC. abortus related abortion. A live, avirulentC. abortus 1B vaccine has been developed and found to suppress abortions and prevent the persistence of subclinical infection in sheep and goat [28]. In China, inactivated vaccines againstC. abortus for sheep and cattle were developed and used in several regions such as Gansu, Xinjiang, Yunnan, Inner Mongolia, Ningxia, and Shaanxi [16, 29]. However, up to now, there is no effective vaccine available for yak chlamydiosis. It is worth noting that the same vaccination measure may present different efficacies against differentChlamydia abortus strains due to genetic heterogeneity between the vaccine and target strains. For instance,C. abortus strains such as LLG and POS isolated in Greece seem to display genetic heterogeneity and exhibit phenotypic differences in antibody cross-reactivity [30]. The live 1B vaccine was more effective against the AB16 strain than against the LLG and POS strains [31]. In addition, the strains 1B and AB16 belonged to MLVA genotype 2, while LLG and POS strains belonged to MLVA genotype 6 [8]. Therefore, the genetic characters of the local epidemic strains should be clearly understood when developing a new vaccine or vaccination. In the present study, we analyzed the genotype of theC.abortus isolates and field samples by MLVA as described previously [8]. The result showed that both the isolates and the field epidemic strains all belonged to the same MLVA genotype, genotype 2. This may imply that field epidemic strains share a similar genetic background and that vaccination measures targeting the most prevalent serotype might be effective for most of the yak herds in Qinghai, China.


## 5. Conclusions

The results from PCR and bacteria isolation showed thatC. abortus is responsible for yak abortion in some areas of Qinghai province, China. It is necessary to carry out further research on prevalence and distribution ofC. abortus to better understand its impact in yak health and production in Qinghai-Tibet Plateau. Pertinent measures to control and preventC. abortus transmission among animals also need to be implemented.



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*Source: 658519-2015-04-28.xml*