Background

Crocodile farms in Thailand are very famous sites for the national tourism industry and well-known leather providers for international fashion products. There are over 1.2 million Siamese crocodiles (Crocodylus siamensis) across 1415 registered farms, based on the database of the Fisheries Department from 2021 [1]. However, an outbreak of Chlamydia sp. infection caused economic devastation and the loss of thousands of young crocodiles in 2012–2013, after which the infection became an endemic in Thailand [2, 3].

Chlamydia sp. is a Gram-negative obligate intracellular bacterium characterized by a unique biphasic developmental cycle that can infect a wide range of hosts, including Siamese crocodiles [4,5,6]. A survey on its molecular diversity revealed very high diversity, wide distribution, and high abundance of Chlamydia sp. in the hosts and the environment settings [7]. Infected crocodiles can either be asymptomatic or show nonspecific clinical signs, e.g., conjunctivitis, pharyngitis, ascites, depression, anorexia, death [5, 8, 9], kyphoscoliosis, and stunted growth. Diagnoses are based on gross and histopathological examination and molecular testing for 16S/23S rRNA and major outer membrane protein (ompA) genes. A new species, Chlamydia crocodili, was reported in 2021 [10]. Although chlamydiosis causes high mortality in juvenile crocodiles, our knowledge regarding its pathogenesis and risk factors is very limited. Crocodile farming management and hygiene in Thailand is diverse, and certain animal husbandry activities can pose risks for Chlamydia sp. infections. This study aims to investigate the epidemiology and potential risk factors associated with Chlamydia sp. infections on Siamese crocodile farms in Thailand.

Methods

Study design and sample collection

This cross-sectional epidemiological study was conducted between July and December 2019. Crocodile farms were selected from the list from the Department of Fisheries, Thailand according to the consent of the farm owners and the availability of the farm during the sample collection period. The number of farms from each region (North, Central, East, Northeast, West, and South) and the sample size from each farm were calculated with a 90% level of confidence, 10% precision, and an assumed 25% prevalence for Chlamydia sp. infection. As a result, samples were collected from 486 live crocodiles from 40 farms across six regions in Thailand. Crocodiles were randomly chosen within the farm for sample collection. Swab samples were collected using sterile rayon swabs (Puritan Medical Products Company, ME, USA) from three sites: the conjunctiva, pharynx, and cloaca. All samples were transported in transport media (sucrose/phosphate/glutamate buffer containing 500 μg/mL streptomycin, 500 μg/mL vancomycin, 50 μg/mL gentamycin, and 2.5 μg/mL fungizone) at 4 ℃ and stored at − 80 ℃ until DNA extraction.

Risk factor analyses

Information about the type of pen, previous crocodile health status, and farm management practices was collected by interviewing farm practitioners. The 26 risk factors collected were as follows: the primary farm objectives, crocodile sources, crocodile species, presence of aquatic and avian livestock on the farm, presence of nearby livestock, feed source, food storage, feed additives, management of leftover feed, pen floor type, presence of shade, ratio of dry and wet area, pond preparation, pond cleaning practice, water source, water reservoir, reuse of treated wastewater from the treatment pond, routine water quality checking practice, frequency of water replacement for juveniles, water replacement after discovering a dead crocodile, dead crocodile management, routine health monitoring, previous health problems, signs of depression, and vertebral deformities. The data from each factor were categorized and underwent further statistical analysis of infection risks.

Univariate logistic regression was performed to calculate the odds ratios and 95% confidence intervals for the risk factor analyses between different factors and infection. The Chi-square test was used to determine P-values. Statistical significance was set at P < 0.05. All tests were conducted in IBM SPSS Statistics for Windows Ver. 25 (Statistical Package for the Social Sciences, IBM Corp., Armonk, NY, USA).

Molecular detection of Chlamydiaceae

Genomic DNA was extracted from conjunctival, pharyngeal, and cloacal swabs using the Genomic DNA Mini Kit (Geneaid, New Taipei City, Taiwan). The extracted DNA was suspended in 30 µL of Tris–EDTA buffer and stored at −  20 ℃ until analysis. Semi-nested PCR using primers specific to the ompA gene was performed using a previously published method [11]. Briefly, primers A and B were used in the first round of PCR, and primers B, and C were used in the second round of PCR. In the first round, 25 μL of the PCR mixture contained 2 µL of template DNA, 2.5 µL of 10 × Mg2+-free buffer, 1.5 mM of Mg2+ solution, 1 mM of dNTPs, 2.5 units of i-Taq DNA polymerase (iNtRON Biotechnology, Inc., South Korea), and 0.5 µM of each primer. The cycling parameters were as follows: 2 min at 94 °C for initial denaturing, followed by 35 cycles of 30 s at 94 °C, 30 s at 58 °C, and 30 s at 72 °C, and termination at 72 °C for 7 min. After the first round of PCR, the second round of PCR was performed using primers B and C and the product from the first round as a template. The PCR mixture and PCR parameters were the same as those of the first round of PCR, except the use of Mg2+ at a final concentration of 3 mM. The second round of PCR generated a product 165 bp in size. A positive control (recombinant plasmid DNAs harboring the ompA gene fragment), negative control (nuclease-free water), and extraction of negative control (phosphate-buffered saline) were included in each PCR run.

Phylogenetic marker amplification, DNA sequencing, and DNA analysis

Two Chlamydia-positive samples from each region (in total 12 samples from 6 regions) were randomly selected for ompA gene sequencing. A 1058 bp fragment of the ompA gene was amplified with primers CTU (5′ ATGAAA AAA CTC TTG AAA TCG G 3′) and CTL (5′ CAA GAT TTT CTA GAY TTC ATYTTG TT 3′) [12]. Briefly, 25 μL of PCR reaction mixture contained 2 μL of template DNA, 2.5 μL of 10 × PCR buffer containing 1.5 mM Mg2+, 1 mM dNTPs mix, 0.5 μL of i-Taq DNA polymerase (iNtRON Biotechnology, Inc., South Korea), and 0.5 μM each of forward and reverse primer. PCR was performed under the conditions of 2 min at 94 °C for initial denaturing, followed by 35 cycles of 30 s at 94 °C, 30 s at 58 °C, and 30 s at 72 °C, and was terminated at 72 °C for 7 min. The PCR product was purified and then directly sequenced using the Sanger sequencing method by U2Bio sequencing service (U2Bio Co., Ltd, South Korea). The phylogenetic tree based on a 992 bp nucleotide sequence of the ompA gene fragment was generated by MEGA11 version 11.0.13 using the Neighbor-Joining method with a bootstrap value based on 1000 replicates [13]. The sequences were compared with the corresponding nucleotide sequences from other Chlamydia species. All the sequences used in this study were retrieved from the GenBank database. The nucleotide identity of ompA sequence alignment was calculated using the Sequence Identity and Similarity program (http://imed.med.ucm.es/Tools/sias.html).

Results

Chlamydia sp. was detected in 189 samples from 26 farms, representing a prevalence of 65% of all farms. Positive samples were found in all regions of Thailand (Table 1 and Fig. 1), with the highest prevalence detected in the Western region (75%). Chlamydia sp. was detected in different swab sites (Table 2), with the highest rate in the cloaca (98.9%), followed by the pharynx (57.1%) and conjunctiva (51.5%).

Table 1 Detection rate and prevalence of Chlamydia sp. on crocodile farms in Thailand in 2019
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Fig. 1
figure 1

Provinces in Thailand with PCR results positive for Chlamydia sp. See Additional Table S1 for the numbers of farms and crocodiles tested for each province. 1: Lamphun, 2: Lampang, 3: Kalasin, 4: Mahasarakham, 5: Roi Et, 6: Nakhon Sawan, 7: Uthaithani, 8: Kanchanaburi, 9: Chainat, 10: Lopburi, 11: Nakhon Ratchasima, 12: Suphanburi, 13: Singburi, 14: Phra Nakhon Si Ayutthaya, 15: Saraburi, 16: Nakhon Nayok, 17: Prachinburi, 18: Chachoengsao, 19: Chonburi, 20: Petchaburi, 21: Trang, 22: Satun, and 23: Songkhla

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Table 2 Detection rate of Chlamydia sp. from conjunctival, pharyngeal, and cloacal swabs
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Although 12 Chlamydia-positive samples were sequenced for nearly the full-length of the ompA gene, only 3 Chlamydia-positive samples were successfully sequenced because of the low DNA quantities of the other samples. The sequenced samples originated from the Eastern (31–02), Northeastern (34–09), and Western region (39–03) and were submitted to GenBank (NCBI) and registered under accession number OP913412.1-OP913414.1. The ompA phylogeny demonstrated that sample 34–09 was grouped into the same group as C. crocodili (Fig. 2), and this sample exhibited 100% sequence similarity with C. crocodili strain No. 12 (accession no. NZ CP060791.1). Samples 31–02, and 39–03 were clustered in a different phylogenetic group. The nucleotide sequences of these samples were 86.99% and 85.28% identical to Chlamydia caviae GPIC (accession no. NC 003361.3) and C. crocodili strain No. 12 (accession no. NZ CP060791.1), respectively.

Fig. 2
figure 2

The ompA phylogenetic tree. A total of 992 bp nucleotide sequence was used for phylogenetic construction using MEGA11. Numbers show the percentage of times each branch was found in 1000 bootstrap replicates. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The black circle indicates the samples obtained in this study

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Risk factor analyses and odd ratios of various factors for Chlamydia sp. infection in crocodiles are presented in Table 3 and Additional file 1. The following factors significantly correlated with Chlamydia sp. detection: water source (P = 0.003), reuse of treated wastewater from the treatment pond (P = 0.04), disposal of leftover feed (P = 0.022), water replacement frequency for juvenile crocodiles (P = 0.001), and water replacement following the presence of a deceased crocodile (P = 0.036). Chlamydia sp. detection also exhibited a tendency to correlate with the presence of nearby livestock areas (P = 0.056).

Table 3 Notable risk factors of Chlamydia sp. infection on Siamese crocodile farms (P-value < 0.05)
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Discussion

Chlamydia sp. causes diseases in a vast variety of vertebrates [6, 14, 15]. In reptiles, this pathogen is commonly reported in crocodilian species, accounting for approximately 57% of all reported cases [14]. Chlamydia sp. infection in crocodiles has been reported in several countries in Africa, Thailand, North America, and Australia [2, 6, 8, 15]. However, epidemiological studies seem to be limited, and the information available is mostly from sporadic reports of diagnosed cases.

One study of the prevalence of Chlamydia sp. infection, in farmed Siamese crocodiles in the central region of Thailand during 2012–2013, [2] demonstrated different results than our present study (74% and 44%, respectively). The higher detection rate observed in the preceding study could potentially be attributed to the sampling methodology in which specimens were obtained from dead crocodiles that previously had shown clinical manifestations depression and anorexia. This differs from our current investigation, in which samples were collected from live crocodiles. Another study also investigated the prevalence of Chlamydia sp. on farms in the Northeastern region of Thailand [3] and reported a prevalence of 48.9%, whereas we found a prevalence of 27.5% (Additional file 2). The observed disparity could potentially arise from variations in the timing of sample collection. Specifically, the previous study collected samples from January to June, whereas we collected samples from July to December. It is noteworthy that the potential impact of the data collection period within a year on pathogen detection has not been investigated yet and should be elucidated in a future study.

The cloacal swab provided the highest detection rate compared with the conjunctiva and pharynx (Table 2), which is in agreement with the results of a previous study [3]. However, this finding contradicts the study of Paungpin et al. [9] in which pharyngeal swabs revealed a 100% Chlamydia sp. infection rate. This may be explained by the fact that the samples were collected from severely moribund or dead crocodiles. In our current study, samples were randomly collected from animals with various degrees of clinical signs, which may have led to a lower detection rate of pharyngeal and conjunctival swabs.

Based on the results of our study, feces, and cloacal secretion can play an important role in horizontal disease transmission among crocodiles in affected ponds. Furthermore, cloacal swabs can provide the highest sensitivity and are recommended as the preferred sample collection technique in animals with mild to moderate clinical signs and on farms with low morbidity rates. Additionally, this technique is low-invasive, and does not damage the skin that may lead to poor leather quality. Cloacal swabs also have the following advantages: ease of animal handling, good access to collection sites, time-efficient, and a low risk of traumatic injury to animals and researchers compared with other swab sites, including conjunctiva, and pharynx. Performing pharyngeal swabs is more difficult and presents the highest risk of occupational hazard for researchers and curators while swabbing restrained animals, especially adult crocodiles. Hence, we suggest the cloaca as the sample collection site for Chlamydia sp. detection in live crocodiles.

Sequencing and phylogenetic analysis of ompA revealed that sample 34–09 was 100% identical to C. crocodili strain No. 12, whereas samples 31–02, and 39–03 were grouped together in another cluster. A previous report demonstrated that based on ompA gene characterization, Chlamydia crocodili detected in Siamese crocodiles in the Central region of Thailand may be divided into three different genotypes [2]. Thus, Chlamydia crocodili detected in our study may contain at least two genotypes. However, these genotypes should be further investigated by whole genome sequencing.

The previous study demonstrated variations in Chlamydia sp. infection risks among crocodiles emanating from different companies. Many crocodile farms do not breed the crocodile within their farms but receive growing crocodile from the breeder farms. These may pose the risk for disease outbreak if the breeder farm has no measure for disease prevention before sending the crocodile to the growing farm or if the growing farm has no quarantine measure. However, our study analyzed the risks of both farms that had breeding stock within their farm and farms that received growing crocodile from the breeder and found no correlation to the detection of the Chlaymydia sp. (Additional file 1). All risk factors we identified in the present study to be associated with the finding of Chlamydiae sp. were environmental and management-related factors. Thus, further studies of the detection of the pathogen in the environment of the farm are required for confirmation of the source of the Chlamydia sp.

Many farmers feed crocodiles with carcasses of other dead livestock, which poses the risk of Chlamydia sp. outbreaks among crocodiles due to infected livestock carcasses. In our study, the presence of livestock in the vicinity of crocodile farms did not pose a significant risk factor but only a trend toward significance (P = 0.056). In addition, crocodiles in Thailand are usually farmed in open areas, which enables potential disease transmission between free-ranging animals, particularly Chlamydia psittaci from wild birds, to captive crocodiles. This spillover phenomenon have occurred and caused major losses in equine production and posed a risk to human health [4, 16,17,18]. Wild pigeons are distributed across all regions of Thailand and are often carriers of Chlamydia sp., which can be found in both respiratory and gastrointestinal organs of even asymptomatic pigeons [19]. In birds, C. psittaci occurs as an endemic infection affecting 1–5% of the bird population globally, with recovered birds acting as lifelong asymptomatic shedding carriers [4, 15]. Because of the abovementioned reasons, interactions between wild birds, and crocodiles are considered a potential risk factor for Chlamydia sp. infection.

Other characteristics of the pond environment, including water depth and the presence of shade, were not identified as risk factors for Chlamydia sp. infection. This finding is in accordance with the study by Inchuai et al. [3]. Although water usage and drainage on farms were not found to be risk factors in a previous report [3], using a natural water source was a significant risk factor in our study. The data on the water treatment protocols used on the farm before using in the farm were collected. However, these protocols, including the types of chemicals or natural substances used and the duration of treatment, varied highly among farms and could not be grouped for further analysis. The frequency of water replacement was a risk factor in a previous study by Inchuai et al. [3]. This frequency affects crocodile health because water replacement reduces waste and ammonia levels [20, 21]. In this study, it was observed that extending the duration between water replacements to more than two weeks significantly elevated the risk of Chlamydia sp. infections in juvenile crocodiles (Table 3).

During the survey, when sick, and dead crocodiles were found on the farms, farmers treated the animals with drugs (of unknown type and amount) and increased the frequency of water replacements according to the advice of the companies that provide crocodiles and the Fisheries District Officers. This may result in drug contamination in the environment, and the antimicrobial resistance risk. Our study revealed that the reuse of treated wastewater from the water treatment pond is a risk factor for Chlamydia sp. detection. However, data regarding the water treatment protocol were not collected. Nevertheless, reusing wastewater may result in reinfections on the farm or environmental contamination.

A notable characteristic of Chlamydia sp. is its persistence across different temperatures and climates [22]. In Thailand, C. psittaci can thrive with effective infectivity at the high temperature of 56 ℃ for 72 h. Moreover, Chlamydia sp. could be retrieved from nature without prior, isolation and cultivation of the natural host cells [21, 23]. Such resilience of Chlamydia sp. is in accordance with the environmental risk factors identified in the present study: using natural water sources, not disposing of leftover feed, and not replacing water after the death of a crocodile. Mitigating Chlamydia sp. infections on crocodile farms can potentially be achieved by implementing biosecurity measures and environmental sample testing.

Conclusions

The present study demonstrated that Chlamydia sp. infection in Siamese crocodile farming in Thailand during July to December 2019 was 65%. For Chlamydia sp. detection, cloacal swabs were superior to conjunctival and pharyngeal swabs due to their higher sensitivity in detecting Chlamydia sp., as well as their lower invasiveness. Phylogenetic analysis revealed two potential Chlamydia sp. genotypes. The following risk factors associated with Chlamydia sp. detection were determined: the use of natural water sources, reuse of treated wastewater from the water treatment pond, no disposal of leftover feed, low frequency of water replacement in juvenile crocodiles, and no water replacement after the death of a crocodile. These data provide useful information for establishing proper guidelines and concepts of disease management and control to prevent disease transmission on crocodile farms. Nevertheless, more detailed studies regarding environmental Chlamydia sp. contamination and biosecurity are needed. Moreover, studies regarding antibiotic susceptibility and pathogen resistance are required to establish efficient treatment regimens.