Identification of Cryptosporidium viatorum XVa subtype family in two wild rat species in China

Background Cryptosporidium viatorum is a minor Cryptosporidium pathogen in humans. Currently, there is limited information regarding the prevalence and genotypes of C. viatorum in animals in China. Methods In this study, 228 faecal samples were collected from two wild rat species (Leopoldamys edwardsi and Berylmys bowersi) in Chongqing Municipality and Guangdong Province, China. These specimens were analyzed for C. viatorum and then subtyped it using PCR and sequence analysis of the small subunit ribosomal RNA (SSU rRNA) and 60-kilodalton glycoprotein (gp60) genes, respectively. Results A total of 25 (11.0%) faecal samples were tested positive for C. viatorum by SSU rRNA assay. Of these samples, 4 (3.6%) came from L. edwardsi and 21 (18.0%) from B. bowersi. Of the 25 C. viatorum-positive samples, 17 were successfully amplified at the gp60 gene locus, which represented four subtypes belonging to two subtype families, including XVa (XVaA6, XVaA3g, XVaA3h) and XVc (XVcA2G1). Phylogenetic analysis based on the gp60 amino acid sequences indicated that all of the C. viatorum isolates grouped together, supporting the conclusion that C. viatorum from the wild rats represent two subtype families. Conclusions These results indicate an occurrence of C. viatorum XVa subtype family from rats which is genetically identical to those found in humans. Our findings suggest that wild rats may be a potential source of human cryptosporidiosis.


Background
Cryptosporidiosis is caused by Cryptosporidium spp. and is a serious enteric disease of humans and animals worldwide [1,2]. Humans can become infected by ingesting food and water contaminated with Cryptosporidium oocysts [3]. To date, more than 78 species/genotypes of Cryptosporidium are known to infect humans and animals [4]. Cryptosporidium viatorum is a novel parasite that has emerged in the last decade, and is a minor Cryptosporidium species to infect humans [5].
Cryptosporidium viatorum was first found in the faecal samples of travellers returning to Britain from the Indian subcontinent in 2012 [6]. Later, C. viatorum was found in two Swedish patients in 2013 [7]. Since then, C. viatorum infection has been recorded in patients with underdeveloped or impaired immunity (including children and AIDS patients) living in developing countries such as Nigeria, Ethiopia, Colombia and India [8][9][10][11][12][13]. Recently, there was also one case of C. viatorum found in western Australian human populations (2015-2018) [14]. The clinical symptoms of C. viatorum infection in humans include diarrhoea, fever, headache, abdominal pain, nausea, vomiting and marked weight loss [1].
There have been reports of C. viatorum infection (XVa subtype family) in humans, but very limited information is available regarding C. viatorum infection in animal hosts [15,16]. Recently, Koehler et al. [16] reported C. viatorum infection (XVb subtype family) in Australian swamp rats, suggesting that it is endemic to native rats in Australia. In addition, the identification of C. viatorum in urban wastewater and combined sewer overflows indicated that this human pathogen may be common in China as well [17]. Very recently, Zhao et al. [15] found that C. viatorum (XVc and XVd subtype families) was prevalent (7.3%) in wild rats in Hainan Province of China. However, to date, it is unknown whether C. viatorum XVa subtype family which is in humans is present in animal hosts, including in wild rats. Wild rodents are one of the largest of the mammalian groups [18]. They have a wide distribution and can transmit numerous pathogens to other animals and to humans [19].
In China, Leopoldamys edwardsi and Berylmys bowersi are two common rat species with a wide distribution.
To determine whether C. viatorum XVa subtype family which is in humans is present in L. edwardsi and B. bowersi, we performed an investigation of the prevalence and the subtypes of C. viatorum in these two wild rat species in China.
Specific identification of the two wild rat species was determined by PCR-based sequencing of the mitochondrial cox1 gene based on a previous method [20]. The obtained sequences were compared with sequences in GenBank using the BLASTn algorithm. The cox1 sequences of the two wild rat species were found to have a sequence identity of more than 99% to previously published sequences for L. edwardsi and B. bowersi from China and Vietnam (GenBank: KM434322 and JN105105, respectively).

DNA extraction and genotyping of C. viatorum
Fresh faecal specimens were subjected to direct DNA extraction using the E.Z.N.A. ® Stool DNA Kit (Omega Biotek Inc., Norcross, GA, USA) according to the manufacturer's protocols. The extracted DNA was stored at − 20 °C for subsequent molecular analysis. Nested PCR was used to amplify and sequence regions of the small subunit ribosomal RNA (SSU rRNA) gene from Cryptosporidium spp. [21]. The 60-kilodalton glycoprotein (gp60) gene was used to classify the subtype levels of C. viatorum [22]. Positive and negative controls were included in each amplification. Amplification products were examined using 1.5% agarose gel containing GoldView TM (Solarbio, China) and observed under UV light.

Sequence and phylogenetic analysis
Positive PCR products were sent to Sangon Biotech (Shanghai, China) for sequencing in both directions. The sequences were aligned with known reference sequences available on GenBank using the Basic Local Alignment Search Tool (BLAST). The genotypes of C. viatorum were identified using Clustal X 1.83 [23]. Available gp60 amino acid sequences of C. viatorum were aligned using MAFFT 7.122 [24], and ambiguous sites and regions were excluded using Gblocks 0.91b with default parameters [25]. Phylogenetic analysis was performed using MEGA 5.0 [26]. The p-distance model was selected as the most suitable one. Neighbor-joining (NJ) tree was calculated based on 1000 bootstrap replicates.

Statistical analysis
Statistical analysis was performed using SPSS V20.0 (IBM, Chicago, IL, USA). The following variables were analysed using the Chi-square test: variation in the prevalence of C. viatorum (y), wild rat species (x1), gender (x2) and different geographic region (x3). Each variable was included in a Binary Logit Model and as an independent variable in a multivariate regression analysis. The best model was judged by Fisher's scoring algorithm. All tests were two-sided. P < 0.05 was considered statistically significant. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were estimated to explore the strength of the association between C. viatorum-positivity and the test conditions.

Results
Of the 228 wild rats collected, 25 (11.0%) were found to be C. viatorum-positive based on PCR amplification of the partial, small subunit (SSU) rRNA gene ( Table 1). The SSU rDNA sequences from the two wild rat species exhibited more than 99.4% identity to previously published sequence from a human in Kenya (GenBank: JX978271). The overall prevalence of C. viatorum in L. edwardsi was 3.6% (4/111) and in B. bowersi it was 18.0% (21/117). The prevalence of C. viatorum in female rats (8.9%) was somewhat lower than in male rats (13.3%), although this difference was not statistically significant (P > 0.05).
Phylogenetic analysis based on SSU rDNA sequences demonstrated that all of the C. viatorum isolates (including human isolates) grouped together with high statistical support (bootstrapping frequencies = 100) (not shown), indicating that all of the isolates from the present study represent C. viatorum. In addition, the gp60 gene provided clear evidence that the present four subtypes (XVaA6, XVaA3g, XVaA3h and XVcA2G1) of C. viatorum from the wild rats represented two subtype families (XVa and XVc) with strong bootstrap support (Fig. 1).

Discussion
Cryptosporidium viatorum has been documented in many countries [6][7][8][9][10][11][12][13], and infection can cause persistent gastrointestinal diseases in humans. The prevalence of C. viatorum in patients may be caused by their lack of natural or acquired resistance, as well as by behavioural and habitual activities related to environmental and socio-economic determinants [10]. Recently, C. viatorum was found in rats in Australia [16] and Hainan Province of China [15], indicating that these rats can act as a natural host. In the present study, C. viatorum was found in wild rats in China, which warrants further consideration into whether wild rats will become a potentially important avenue for C. viatorum transmission to humans and other animals.
The gp60 subtyping tool has been used to divide Cryptosporidium into 15 subtype families (numbered I-XV) according to their gp60 sequence [21,27]. Stensvold et al. in 2015 [22] has classified a number of C. viatorum sequences isolated from humans into six subtypes (XVaA3a-XVaA3f ). Recently, a new subtype (XVaA3g) of C. viatorum has been found in an Australian patient [14]. To our knowledge, to date, only the XVa subtype family has found in C. viatorum-positive patients [22] and wastewater in China [17]. Interestingly, the results of the present study provide clear evidence that the three subtypes (XVaA6, XVaA3g and XVaA3h) from the wild rats belong to the XVa subtype family which is genetically identical to those found in humans, suggesting that wild rats may have a potential for zoonotic transmission, and must be considered as a potential threat to human health.
Our results indicate an occurrence of C. viatorum XVa subtype family in wild rats, indicating that C. viatorum XVa subtype family may be an extensive host range. In fact, in previous studies, XVa subtype family of C. viatorum were not found in rats (Edwardʼs long-tailed rats and Australian swamp rats) [15,16]. However, we should be cautious whether the finding of XVa subtype family in rats represents a natural infection needs to be further confirmed with additional datasets. Unfortunately, we are unable to determine the source of infection and transmission dynamics of C. viatorum XVa subtype family in investigated rats due to the lack of C. viatorum data from humans in the investigated areas. In addition, three novel subtypes (XVcA2G1a, XVcA2G1b and XVdA3) of C. viatorum were identified in Edwardʼs long-tailed rats in Hainan Province, China. However, these subtypes of C. viatorum were not found in the present investigation. The difference may be related to the geographical origin [22], the health status of the animals at the time of sampling, and the overall sample sizes. A previous study [15] and the present study provide strong evidence that C. viatorum infection is prevalent in wild rats in China, suggesting that wild rats infected with C. viatorum may pose a threat to human health. Importantly, the C. viatorum XVa subtype family identified in the present study has significant public health implications, suggesting that these animals represent a potential zoonotic risk for the transfer of the pathogen in China. More in-depth studies are needed to understand the transmission of this C. viatorum XVa subtype family in rats. In addition, we believe that an increase in the number of wild rats from broader geographical locations and diversity of rat species surveyed for C. viatorum may help gain a much-improved understanding of the role of wild rats in the zoonotic transmission of C. viatorum.

Conclusions
The present results indicate an occurrence of C. viatorum XVa subtype family in rats which are genetically identical to those found in humans. Our findings suggest that L. edwardsi and B. bowersi may play a potential role in the transmission of C. viatorum to other animals and humans. Future studies investigating the molecular epidemiology of C. viatorum in wild rats are needed to better clarify the risks and modes of transmission.