# First Report ofChlamydia abortus in Farmed Fur Animals

Authors: Zhaocai Li; Ping Liu; Xiaoan Cao; Zhongzi Lou; Kinga Zaręba-Marchewka; Monika Szymańska-Czerwińska; Krzysztof Niemczuk; Bo Hu; Xue Bai; Jizhang Zhou
Journal: BioMed Research International
 (2018)

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## Abstract

Chlamydia (C.) abortus, a globally distributed obligate intracellular bacterium, has attracted increasing interest according to its veterinary importance and zoonotic nature.C. abortus can infect a variety of animals and cause foetal loss in livestock resulting in economic loss. In this study, the samples collected from two farms of foxes (n=20), raccoon dogs (n=15) and minks (n=20), were investigated byChlamydiaceae- andChlamydia species-specific real-time PCR. The results showed that all the tested foxes (20/20) and raccoon dogs (15/15) harboredChlamydia spp., while 5% of minks (1/20) were positive forChlamydia spp.C. abortus was identified in all positive samples as the dominantChlamydia species, withC. pecorum DNA coexistence in some of the rectal samples (7/20) taken from foxes. Phylogenetic analysis based on specific gene fragments of 16S rRNA, IGS-23S rRNA, andompA revealed that all sequences obtained in this study were assigned to theChlamydiaceae family with high similarity toC. abortus S26/3 and B577 previously identified in ruminants. This is the first report confirming that farmed foxes, raccoon dogs, and minks carryC. abortus. Further studies are needed to fully elucidate the epidemiology and pathogenicity of this pathogen in farmed fur animals as well as the potential risks to public health.


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## Body

## 1. Introduction

Chlamydia spp. are obligate intracellular bacteria widely distributed throughout the world and responsible for a variety of diseases in humans and animals. To date, eleven species have been identified along with five newly characterized candidate species in the single genus of the familyChlamydiaceae [1–5].Chlamydia abortus(C. abortus) has attracted increasing interest due to its veterinary importance and zoonotic potential.C. abortus can infect a variety of mammalian hosts and is associated with abortion cases in ruminants, pigs, horses, rabbits, guinea pigs, and mice [6]. In addition, infection withC. abortus in nonmammalian hosts has also been reported recently [7, 8]. Infections ofC. abortus are common in ruminants such as sheep, goats, and cattle in many countries around the world, causing tremendous economic loss for the livestock industry [9]. Infected animals may be a source of infection in humans and lead to severe outcomes including respiratory disorder and miscarriage [10, 11]. The wide distribution, broad-host infection, and zoonotic potential ofC. abortus may exhibit an increasing but neglected risk to public health. Therefore, there is an urgent need to investigate the presence of this zoonotic pathogen in animals closely related to humans and evaluate the potential health risk.Foxes, raccoon dogs, and minks are captive-bred wild animals raised as fur animals with substantial populations in northern Europe, northern America, and China [12]. These animals are suspected to be infected with a wide range of pathogens including several important zoonotic ones, such as rabies virus, hepatitis E virus, and influenza virus [13–15]. However, there are as yet no reports on the prevalence of chlamydiosis in these animals. Indeed, reproductive failure cases of abortion and stillbirth in foxes, raccoon dogs, and minks were commonly observed in some farms with unknown reasons in Northeast China. Thus, a survey was conducted in China to acquire information concerning farmed fur animal species as possible reservoirs ofChlamydia spp.


## 2. Materials and Methods

Samples from fur animals came from two farms located in the suburbs of Changchun city, Jilin province, China, and sampling was performed in November 2017 in a local slaughterhouse. All samples were collected before the slaughtering of the animals and immediately transported on ice to the laboratory. A formal ethical approval is not required for this kind of study and sampling was performed by authorized veterinarians during routine medical and veterinary activities. A total of 55 sampled animals included 9 male and 11 female foxes, 12 male and 3 female raccoon dogs, and 16 male and 4 female minks (Table1). In total, rectal swabs (n=55), whole blood (n=55) samples, conjunctive swabs (n=35), and vaginal swabs (n=18) were collected from apparently healthy minks, while the foxes and raccoon dogs came from farm with an abortion history. Samples were stored at −80°C until use. DNA extraction from blood (200 μl), vaginal swabs, and conjunctive swabs was performed using the commercial TIANamp Genomic DNA Kit (Tiangen Biotech Co., LTD) following the manufacturer’s protocol. For rectal swabs, the QIAamp DNA Stool Mini Kit (Qiagen) was used for DNA extraction. All DNA samples were eluted in 100 μl of elution buffer and screened using aChlamydiaceae-specific real-time PCR targeting the 23S rRNA gene fragment, as previously described by Ehrichtet al. [16]. AllChlamydiaceae-positive samples were retested with specific real-time PCR assays to identifyChlamydia species includingC. abortus,C. pecorum,C. psittaci,C. felis, andC. suis [17]. An analytical cut-off value of 39.0 was selected corresponding to the defined lower limit of detection of the test. Any Ct value above this defined limit would, thereafter, be considered unreliable. For further quantification DNA concentration from the positive samples, the target fragments for each detecting primer pairs were chemically synthesized and cloned into pMD18T vector (Takara). Quantification of the recombinant plasmids was done on a Nanodrop ND-2000 (Thermo, US), and 10-fold dilutions (6×107 copies to 6×101 copy/μl) were used as positive controls to establish a standard curve for quantification. TheChlamydia DNA copies in the samples were tested and calculated according to the formulation obtained from the standard curves.Chlamydiaceae positive DNA samples with a high copies based on real-time PCR were used for DNA sequencing to confirm the identity ofChlamydiaspp. Specific fragments ofompA, 16S rRNA, and 16S rRNA-23S rRNA intergenic spacer together with 23S rRNA domain I (IGS-23S rRNA) were amplified with previously published primer sets [8]. PCR amplicons were sent to an external company (Shanghai Sangon Biotech, China) for sequencing. The obtained sequences were deposited in the GenBank database with the following accession numbers: MH532474-MH532478 (16S rRNA), MH537631-MH537633 (IGS-23S rRNA), and MH542157-MH542161 (ompA).Table 1
Results summary of farmed fur animals testing.
species of animals Gender no. of tested /Chlamydiaceae positive samples no. ofC. abortus positive/Chlamydiaceae positive samples no. ofC. pecorum positive/Chlamydiaceae positive samples type of sample rectal swabs blood vaginal swabs conjunctive swabs rectal swab blood vaginal swabs conjunctive swabs rectal swabs blood vaginal swabs conjunctive swabs fox Male 9/9 9/2 — 9/7 9/9 2/2 — 7/7 1/9 0/2 — 0/7 Female 11/11 11/5 11/9 11/9 11/11 5/5 9/9 9/9 6/11 0/5 0/9 0/9 Total 20/20 20/7 11/9 20/16 20/20 7/7 9/9 16/16 7/20 0/7 0/9 0/16  raccoon dog Male 12/12 12/2 — 12/6 12/12 2/2 — 6/6 0/12 0/2 — 0/6 Female 3/3 3/1 3/1 3/2 3/3 1/1 1/1 2/2 0/3 0/1 0/1 0/2 Total 15/15 15/3 3/1 15/8 15/15 3/3 1/1 8/8 0/15 0/3 0/1 0/8  mink Male 16/1 16/0 — — 1/1 N/A — — 0/1 N/A — — Female 4/0 4/0 4/0 — N/A N/A N/A — N/A N/A N/A — Total 20/1 20/0 4/0 — 1/1 N/A N/A — 0/1 N/A N/A — Note: N/A: not investigated; —: no sample collected.The data were analysed using MEGA 5.05 software [18]. Amplicons were subjected to BLAST analysis against the GenBank database (NCBI) to identify related entries and aligned with a panel ofChlamydiareference strains. To assess the phylogenetic relationship betweenChlamydia spp. and the tested samples, phylogenetic trees for 16S rRNA (1358bp) and IGS-23S rRNA (990 bp) as well asompA (940 bp) were constructed by the neighbor-joining method with 1,000 replicates’ bootstrap using the Maximum Composite Likelihood model with MEGA 5.05.


## 3. Results and Discussion

The results of real-time PCR detection are presented in Table1 and Table S1. Among 55 tested animals, 36 were shedders ofChlamydiaceae giving a prevalence rate of 65.45%. Foxes and raccoon dogs demonstrated the highest level ofChlamydiaceae prevalence at 100%. Positive results forChlamydiaceae were obtained for all rectal swabs from foxes and raccoon dogs irrespective of gender. DNA ofChlamydiaceae was also present in several blood (n=10) and conjunctive swab (n=24) samples from foxes and raccoon dogs. For the females, vaginal swab samples revealed a high rate of positive results forChlamydiaceae in foxes (9/11) and raccoon dogs (1/3). Further testing with species-specific real-time PCR identified the detected agent in allChlamydiaceae positive samples asC. abortus. Moreover in 7 rectal swabs from foxes the coexistence ofC. pecorum was confirmed. No otherChlamydia species was identified. Only one rectal swab from a male mink was tested positive forChlamydiaceae and determined asC. abortus with a relatively low level of DNA copies.To further confirm the presence ofChlamydiaspp., amplification and sequencing of 16S rRNA, IGS-23S rRNA, andompA gene fragments were carried out on high positive samples. The 16S rRNA gene amplicons were successfully obtained from 2 samples originating from foxes (MH532477, MH532478) and 3 samples originating from raccoon dogs (MH532474-MH532476), IGS-23S rRNA from 1 fox (MH537633) and 2 samples from raccoon dogs (MH537631, MH537632), andompA from 1 fox (MH542161) and 4 samples from raccoon dogs (MH542157-MH542160), while the process of obtaining amplicons from mink samples failed. Dendrograms were constructed on the basis ofompA, 16S rRNA, and IGS-23S rRNA gene fragments aligning withChlamydiaspecies reference sequences available in GenBank and altogether showed a similar topology. All sequences obtained in this study were assigned toC. abortus within theChlamydiaceae family (Figure 1). In addition, phylogenetic analysis showedompA sequences were clustered with the strainsC. abortus S26/3 and B577 hosted by ruminants as the closest relative.Figure 1
Phylogenetic tree based on 16S rRNA gene fragment (1358 bp) (a), 16S-23S intergenic spacer and full length of 23S rRNA domain I fragment (990 bp) (b), andompA gene fragment (940 bp) (c). Representative sequences of establishedChlamydiaceae species as well as the sequences obtained from this study (Genbank accession number was shown in bold) were included.S.negevensisstrain Z was used as an outgroup. Based on these alignments, phylogenetic trees were constructed by the neighbor-joining method using the Maximum Composite Likelihood model with MEGA 5.05.
(a) (b) (c)Chlamydiosis is common in livestock, poultry, companion, and wild animals [19, 20]. In the available literature, there is a lack of reports about chlamydiosis cases in fur animals. In this study, evidence for the existence ofC. abortus in farmed fur animals was shown for the first time. It seemed that the gastrointestinal tract provided a major endemic habitat forC. abortus in farmed foxes and raccoon dogs, although the genital tract was generally considered as the colonized site for this pathogen in female animals. AnotherChlamydia species found in this study as a coexisting pathogen in a few rectal swabs in foxes wasC. pecorum, pathogen of the koala, and commonly found in the gastrointestinal tract of cattle [21, 22]. These results suggested thatC. abortuswas the dominantChlamydia species carried by tested animals. However, due to the limited number of samples investigated in this study, more evidence is required to reveal the relationship betweenC. abortusinfection and reproductive disorder in foxes and raccoon dogs. In addition, it is not clear whether fur animals are the natural hosts ofChlamydia spp. such asC. abortus or if infection occurs via other vectors. It is worth mentioning that the feed for fur animals in China always contains fish, chicken, and occasionally pig products. As has been reported, the prevalence ofChlamydia spp. in chickens and pigs is quite common in China [23, 24]. Therefore, feeding should be considered as a possible way for farmed fur animals to become infected withChlamydia spp.C. abortus is well known for its zoonotic nature. Frequent human contact with domestic livestock at work increases the risk of infection. According to data published in 2002, the average number of notified cases of human chlamydiosis originating fromC. abortus infected animals was approximately 100 per year in Germany [25]. Although the possible transmission ofC. abortus from farmed fur animals to humans has not been investigated yet, environmental contamination by the faecal shedding of this zoonotic agent may become a potential source of infection for workers exposed to infected animals during their daily activities.


## 4. Conclusions

This is the first report confirming that farmed fur animals, foxes, raccoon dogs, and minks, mainly harboredC. abortus, whileC. pecorum occasionally coexists in foxes. Further studies are needed to fully elucidate the epidemiology and pathogenicity ofC. abortus in farmed fur animals. Moreover, an evaluation of the potential health risk to fur farm personnel and the creation of a strategy to preserve human safety are required.



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*Source: 4289648-2018-11-26.xml*