Prevalence and Molecular Characterization of Cryptosporidium serpentis in Captive Snakes in China

Cryptosporidium spp. are obligate intracellular protozoan parasites known to affect animals including humans, mammals, birds, fish, amphibians, and reptiles, and have been found in over 90 countries (Xiao et al., 2004a; Chen and Huang, 2012; Liu et al., 2015). Cryptosporidium species can be transmitted by direct contact with infected individuals or animals, as well as orally via contaminated food and water (Liu et al., 2014). Clinical symptoms vary depending on the age, species, and immune status of the host, and environmental conditions (Liu et al., 2014). Immunocompromised individuals aren't necessarily at higher risk of contracting an infection, but they are at higher risk of having a more severe disease progression and negative outcomes (Izadi et al., 2012; Zhang et al., 2018; Sannella et al., 2019).

Cryptosporidiosis is an important disease in reptiles, as it is highly contagious and associated with a high mortality rate. The first study on Cryptosporidium in snakes was conducted in 1977, wherein Cryptosporidium was detected in captive snakes with severe hypertrophic gastritis (Brownstein et al., 1977). Subsequently, infections have been reported in at least 57 reptilian species (Xiao et al., 2004b). Cryptosporidium serpentis, Cryptosporidium parvum, Cryptosporidium parvum mouse genotype, Cryptosporidium muris, Cryptosporidium sp., Cryptosporidium tyzzeri, Cryptosporidium varanii, and Cryptosporidium andersoni have been reported in various snakes worldwide (Plutzer and Karanis, 2007; Kuroki et al., 2008; Pedraza et al., 2009; Rinaldi et al., 2012; Yimming et al., 2016). However, 2 species are recognized as specific for reptiles: the gastric parasite C. serpentis and intestinal parasite Cryptosporidium varanii (syn. Cryptosporidium saurophilum; Fayer, 2010). Cryptosporidium serpentis has not been reported in humans; nevertheless, this species affects the health of snakes (Fayer, 2010). Furthermore, all C. serpentis infections occur in the stomach–abomasum of their respective hosts and cause a chronic, progressive disease that leads to death, thus eliminating valuable and biologically important ophidians (Graczyk et al., 1996). Cryptosporidium varanii is an intestinal parasite found primarily in lizards (Pavlasek and Ryan, 2008; Xiao et al., 2004b) and can cause anorexia, progressive weight loss, abdominal swelling, and high mortality, particularly in juvenile lizards (Koudela and Modrý, 1998; Dellarupe et al., 2016). As snake cryptosporidiosis results in the natural death of a snake or requires obligatory euthanasia to prevent spread of the pathogen, the economic loss can be immense.

There are approximately 2,800 different snake species reported around the world, out of which 200 species have been discovered in China. As global markets for live plants and animals have grown, the exotic pet trade is gaining attention over the past decade (Lockwood et al., 2019) and snakes have become increasingly popular pets (Pedraza-Díaz et al., 2009). Moreover, in China, snakes have long been considered a delicacy and are also utilized in traditional medicine (Zhou and Jiang, 2005). In recent years, the consumption of snake meat has become increasingly popular because of its taste and nutritive properties (Liu et al., 2020). Furthermore, swallowing raw gall bladder of snakes is a common practice in traditional Chinese medicine for its therapeutic effects (Sun and Li, 2004). Snakes with asymptomatic Cryptosporidium infections are difficult to diagnose (Xiao et al., 2019) and the aim of this study was to assess the current status of Cryptosporidium species prevalence in captive snakes in China using a molecular approach and to comprehend zoonotic implications of cryptosporidiosis.

This study was performed following the recommendations of the Guide for the Care and Use of Laboratory Animals of the Ministry of Health, China. Before the initiation of the experiments, the protocol of the current study was reviewed and approved by the Institutional Animal Care and Use Committee of the Sichuan Agricultural University under permit. No animals were harmed during the sampling process. Permission was obtained from snake farm owners or shop managers before the collection of fecal specimens.

A total of 609 samples were collected from the following 4 regions in the Sichuan province and Beijing City between March 2019 and June 2019 (Fig. 1): Chengdu (n = 12), Duzhou (n = 359), Ziyang (n = 6), and Beijing fangshan district (n = 227). The snakes in Dazhou and Ziyang are commercial snakes used for food, whereas those in Chengdu and Beijing are pet snakes. The fecal samples of the following snakes were sampled: black king snake, corn snake, Boulenger, Asian king snake, garter snake, king rat snake, sand boa, king snake, black rat snake, ball python, green snake, striped racer, Indo-Chinese rat snake, and cobra (Table I). Each snake was kept in a separate cage. Approximately 200 mg of fresh feces was collected from the bottom of each cage after defecation using sterile disposal latex gloves and immediately placed into individual disposable plastic bags. No obvious clinical signs were observed at the time of sampling, and the species and source were recorded at the same time. All fecal specimens were stored in 2.5% potassium dichromate solution at 4 C until further processing.

The fecal specimens were washed 3 times in distilled water and centrifuged at 3,000 g for 10 min to remove the potassium dichromate. Genomic DNA was extracted from approximately 200 mg of each processed fecal specimen using an E.Z.N.A. Stool DNA kit (Omega Biotek Inc., Norcross, Georgia) according to the manufacturer's recommended instructions. The extracted DNA was stored at −20 C until molecular analysis was performed.

A nested polymerase chain reaction (PCR) targeting a ∼830–base-pair (bp) fragment of the SSU rRNA sequence was performed to determine the Cryptosporidium species/genotype. The primers were F1 (5′-CCCATTTCCTTCGAAACAGGA-3′) and R1 (5′-TTCTAGAGCTAATACATGCG-3′) for the primary PCR, and F2 (5′-AAGGAGTAAGGAACAACCTCCA-3′) and R2 (5′-GGAAGGGTTGTATTATTAGATAAAG-3′) for the secondary PCR (Xiao et al., 1999). The annealing temperature was 55 and 58 C for the primary and secondary PCR, respectively. TaKaRa Taq DNA Polymerase (TaKaRa Bio Inc., Tokyo, Japan) was used for PCR amplification. Positive and negative controls were included in each amplification. Secondary PCR products were subjected to electrophoresis in a 1.5% agarose gel and visualized by staining with ethidium bromide.

All positive PCR products were directly sequenced on an ABI PRISM 3730 DNA Analyzer (Applied Biosystems, Foster, California), using a BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems). Nucleotide sequences obtained in the present study were subjected to BLAST (http://www.ncbi.nlm.nih.gov/blast/), for analyzing the sequence similarity with reference sequences downloaded from the GenBank (http://www.ncbi.nlm.nih.gov). The sequences were aligned using Clustal X 2.0 (http://www.clustal.org/) to determine the Cryptosporidium species/genotypes. The nucleotide sequences generated in the present study have been deposited in GenBank under accession numbers MN474006, MN474009, MN474001, MN474011, MN545606, MN545609, MN545610, MN545612, MN545613, MN545618, MN545619, and MN545622 (Fig. 2).

A neighbor-joining tree was constructed to assess the genetic relationship among the Cryptosporidium subtypes obtained in the present study and those identified in previous studies using the MEGA 7 software (http://www.megasoftware.net/). The sequences of the barcode region of Cryptosporidium were trimmed using trimAl (Capella-Gutiérrez et al., 2009). Evolutionary distances were calculated using the Kimura 2-parameter model. The reliability of the trees was assessed by bootstrap analysis with 1,000 replicates.

Statistical analyses were performed using SPSS version 22.0 (SPSS Inc., Chicago, Illinois). A chi-square test was used to compare the prevalence of Cryptosporidium in different species. Results with P < 0.05 were considered statistically significant.

The overall prevalence of Cryptosporidium in captive snakes was 1.97% (12/609; 95% confidence interval [CI]: 1.1–3.4%). There were no significant difference in the prevalence of Cryptosporidium between the 4 locations tested, which ranged from 0 to 16.67% (χ² = 2.473, df = 3, P > 0.05) (Table I). The prevalence in different snake species ranged from 2.78% to 16.95% (Table I) and differed significantly between the 7 species (χ² = 9.699, df = 4, P < 0.05).

Twelve samples were successfully sequenced, and all SSU rRNA PCR-positive samples were phylogenetically analyzed. According to the sequence data and genetic tree, the infection was caused by C. serpentis (Fig. 2; Table I). In the SSU rRNA gene, there were 1–5 nucleotide differences between the captive snake isolates and other known C. serpentis isolates. Nucleotide sequence comparison revealed genetic diversity between the 2 sequences from the 12 isolates. MN474011 contained 3 single-nucleotide polymorphisms (SNPs) within the 43-, 44-, and 83-bp regions of the SSU rRNA gene sequence of Cryptosporidium serpentis (addition: A, C, A), whereas isolate MN474006 contained 4 SNPs within the 41-bp (transversion: C/G), 61-bp (transversion: C/A), 108-bp (transition: C/T), and 132-bp (transversion: C/A) regions compared to the snake Cryptosporidium serpentis AF151376. The nucleotide sequences of MN474010, MN474009, MN545618, MN545612, MN545606, MN545622, MN545609, MN545610, MN545613, and MN545619, are the same as AF151376.

Cryptosporidium species have been reported in snakes from 9 different countries, including developed (USA, Australia, Italy, Japan, and Spain) and developing countries (India, Brazil, Thailand, and China; Table II; Brownstein et al., 1977; Xiao et al., 2004a; Chen and Qiu, 2012; Díaz et al., 2013; Yimming et al., 2016; Bercier et al., 2017; Akhila et al., 2018). In this study, the prevalence of Cryptosporidium was 1.97%, which is consistent with previous reports on the captive oriental rat snakes in Guangxi province in China (3/141, 2.1%; Graczyk et al., 1996; Zhou and Jiang, 2005; Karim et al., 2014; Bercier et al., 2017; Akhila et al., 2018; Xiao et al., 2019). However, the prevalence of Cryptosporidium-positive snakes in our study was lower than that of snakes in central China (Xiao et al., 2019). The 239 samples we collected in Beijing and Chengdu were sourced from pet snake shops. In our study, Cryptosporidium prevalence (5%) in pet snakes was similar to Cryptosporidium prevalence in Italian pet snakes (4.8%; Rinaldi et al., 2012). More importantly, Cryptosporidium was not detected in the commercial snake samples collected in Dazhou and Ziyang. Commercial snakes include cobras, Asian king snakes, and striped racers, which are used as a food source for consumers. The results demonstrate the occurrence of Cryptosporidium infection in pet snakes. However, the prevalence of Cryptosporidium infection in snakes from farms that provide snake meat was 0%. These results reveal that snakes bred under commercial conditions are relatively safe for consumption.

The prevalence in this study was lower than that of snakes in other countries where Cryptosporidium is found in snakes (Table II). This difference may be explained by several factors, such as the number of samples, geography of the source region, host health status, and raising and management systems. In our study, the relatively lower prevalence of Cryptosporidium may be because snakes were kept in separate cages and had a lower feeding density, which reduces the possibility of transmission between infected and healthy snakes. In addition, pet snake owners take precautionary measures to remove parasites, avoid food sourced from the wildlife by breeding their mice, and also breed the pet snakes themselves. Snake eggs are directly brought into the farms and raised. During the feeding process, farm owners break newborn chicken and pork meat into pieces for feeding. Great attention is paid to achieving isolation from the external environment and the prevention of diseases.

In our study, only C. serpentis was detected in snakes, and all C. serpentis isolates showed the C. serpentis genotype A 18S rDNA sequence described by Xiao et al. (2004b). Cryptosporidium serpentis is an important parasite in snakes and is usually found in the gastric epithelium (Yimming et al., 2016). Moreover, C. serpentis was the only species identified in reptiles (Xiao et al., 2004b) until recently. Later studies have found that C. serpentis also occurs in cows and Alaskan caribou (Chen and Qiu, 2012; Paiva et al., 2013). Snakes infected with C. serpentis have increased mortality (Rinaldi et al., 2012; Paiva et al., 2013).

In conclusion, this is the first report of the prevalence of Cryptosporidium in snakes in southwest and north China. Moreover, this is the first time C. serpentis was detected in black king snakes, corn snakes, king snakes, ball python, striped racer, and Indo-Chinese rat snakes. Significantly, all of the above are common pet snakes in China and their proximity to humans can affect the health of handlers. Therefore, our findings contribute to the knowledge of Cryptosporidium distribution among snakes in China and provide baseline data for the implementation of effective measures and strategies to control and prevent snake and human infection with Cryptosporidium. However, there is a need to assess further the distribution of this group of parasites within the snake population as well as the potential risk that some of the Cryptosporidium species may pose to humans. Therefore, larger-scale surveys of snake cryptosporidiosis in various areas of China are needed to evaluate those in China and to assess the public health significance of the parasite better.

The study was financially supported by the National Science and Technology Department “13th five-year” Special Subproject of China (No.2016YFD0501009) and Chengdu Research Base of Giant Panda Breeding (CPF2017-12, CPF2015-09). The funders contributed to the study design and data collection.