Occurrence and molecular characterization of Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi, and Blastocystis sp. in captive wild animals in zoos in Henan, China

Background Captive wild animals in zoos infected with Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi, and Blastocystis sp. can be sources of zoonotic infections and diseases. Therefore, to investigate the distribution of these pathogens in captive wild animals of zoos in Henan, China, a total of 429 fresh fecal samples were collected from six zoos in Henan, China. The infection rates of Cryptosporidium spp., G. duodenalis, E. bieneusi, and Blastocystis sp. were determined by PCR analysis of corresponding loci. Positive results for Cryptosporidium (C. parvum and C. hominis) were subtyped based on the (gp60) gene. Results The overall prevalence was 43.1% (185/429), and the prevalence of Cryptosporidium, Giardia duodenalis, Enterocytozoon bieneusi, and Blastocystis sp. were 2.8% (12/429), 0.5% (2/429), 20.8% (89/429), and 19.1% (82/429), respectively. Five Cryptosporidium species, namely, C. hominis, C. parvum, C. muris, C. andersoni, and C. macropodum, were identified in this study. Cryptosporidium parvum was further subtyped as IIdA19G1. Two Giardia duodenalis assemblages (A and E) were also identified. A total of 20 Enterocytozoon bieneusi genotypes were detected, including 18 known (BEB6, D, HND-1, CD7, SDD1, Henan-IV, KIN-1, CHK1, Peru8, Henan-V, CHG11, CHG-1, CHS9, CHG21, Type-IV, CHC9, CM5, and CHB1) and 2 novel genotypes (CHWD1 and CHPM1). A total of nine subtypes of Blastocystis sp. (ST1, ST2, ST3, ST5, ST6, ST7, ST10, ST13, and ST14) were identified in captive wild animals in zoos in the present study. Cryptosporidium andersoni, nine Enterocytozoon bieneusi genotypes, and five Blastocystis subtypes were here first identified in new hosts. Conclusions Our study has expanded the host ranges of these four pathogens. The data indicate that animals in zoos can commonly be infected with these four zoonotic pathogens, and animals in zoos are potential sources of zoonotic infections in humans. Supplementary Information The online version contains supplementary material available at 10.1186/s12917-021-03035-0.


Background
Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi, and Blastocystis sp. are four common opportunistic pathogens with wide host ranges that include livestock, wildlife, and humans [1][2][3][4]. Infections with these pathogens can cause diarrhea and several other gastrointestinal illnesses in humans and animals [1][2][3][4]. The fecal-oral route is the main transmission pathway of the four pathogens, and infection can also result from contaminated food or water [2,4].
Currently, at least 44 valid species and about 70 genotypes of Cryptosporidium have been described, and at least 20 species and 5 genotypes have been detected in humans [4,5]. Giardia duodenalis is considered a species complex with at least eight distinct assemblages (A-H), and assemblages A and B are infectious to humans and other mammals as well as a wide range of hosts [6]. Over 474 Enterocytozoon bieneusi genotypes were distributed in several genetically isolated populations comprising 11 major groups in a phylogenetic analysis, including zoonotic group 1 and several host-adapted groups [3,7]. Among 17 approved subtypes (ST1-ST17) of Blastocystis sp., ST1-ST9 and ST12 have been observed in humans. Two subtypes (ST9 and ST12) are specific to humans, and the remaining subtypes have been detected in non-human hosts [8,9].
For many animal species, life in a zoo is very different from natural free-range conditions. Due to the limitations of living space, the prevalence of parasites in captive animals is often higher than that of wild animals [10]. Animal keepers can be in close contact with animals through feeding, washing, and cleaning, and visitors can indirectly contact animals by petting or by giving food. Previous studies have found Cryptosporidium and Blastocystis in zoo animals and their keepers [11][12][13]. There is a potential for zoonotic transmission between animals and humans in zoos [14]. The objectives of this study are to examine the prevalence and determine the genetic distributions of Cryptosporidium, G. duodenalis, E. bieneusi, and Blastocystis and to identify genotypes/assemblages of human health importance..

Cryptosporidium species and subtypes
Five Cryptosporidium species, namely C. hominis, C. parvum, C. andersoni, C. muris, and C. macropodum were identified in this study ( Table 2). The Cryptosporidium hominis and C. parvum samples were further subtyped based on gp60 gene sequence analysis, with all C. parvum identified as subtype IIdA19G1. Cryptosporidium hominis was not successfully identified. The three gp60 sequences showed 99.7% nucleotide sequence identity to the isolates from dairy cattle (MF074761) and Homo sapiens (JQ796092) from China.

Giardia duodenalis assemblages
Two Giardia duodenalis assemblages, A and E were detected based on SSU rRNA and gdh loci ( Table 2). Assemblage A shared 100% similarity with the sequence from Brazilian Panthera (HM134217), and Assemblage E was identical to the isolate derived from dairy cattle in China (KF843926).

Discussion
In the present study, the Cryptosporidium prevalence was 2.8%, which is lower than the rates (70.0%) reported in Xining Zoo in China [15] and zoo mammals (35.8%) in Barcelona, Spain [16]. However, the infection rate was higher than those in zoos (2.0%) in Alberta and Manitoba of Canada [17] and Zhengzhou Zoo (1.6%) in China [18]. For Giardia duodenalis, the prevalence was 0.5%, which is lower than the rate found in most studies conducted in zoos worldwide, for example the 2.5% in Zhengzhou Zoo in China [18], 3.3% in the zoo in Aprilia, Italy [19], and 24.0% in a zoological garden in Poland [20]. The overall infection rate of E. bieneusi was 20.8%, lower than in a previous study conducted in the zoo (32.5%) of Howard County in America [21] and higher than in Zhengzhou Zoo (15.8%) in China [18]. Concerning Blastocystis, the infection rate was 19.1%, which was higher than the 6.6% found in four zoos in Italy [21] and 12.3% in a zoological garden in Poland [20]. In contrast, the prevalence was lower than that (40.2%) in Qinling Zoo in China [8]. Infection rates may be influenced by many factors, including the zoo management model, living conditions, the immune status of the animals, climate, and geography. This study demonstrates a high sample prevalence and diversity of intestinal parasites in captive wild animals in zoos in Henan, China. The present study indicated that Giardia duodenalis was only found in Kaifeng Zoo, and Cryptosporidium spp. was found in Kaifeng Zoo and Swan Lake Zoo. Enterocytozoon bieneusi and Blastocystis sp. were found in 6 zoos. It can be concluded that Enterocytozoon bieneusi and Blastocystis sp. are common in some zoos in Henan Province, and among the 6 zoos, the infection rate of Enterocytozoon bieneusi is different. The highest infection rate is Zhengzhou Zoo and the lowest is Kaifeng Zoo. The result of Blastocystis sp. is consistent with E. bieneusi. So the prevalence and diversity varied with geographic region.
Zoonotic Cryptosporidium species (C. hominis, C. parvum, C. muris, and C. andersoni) and C. macropodum were identified in the present study. Cryptosporidium hominis and C. parvum are responsible for most cases of cryptosporidiosis in humans [22,23]. Cryptosporidium parvum subtyping revealed IIdA19G1 in our study that has previously been found in humans, dairy cattle, and yaks in China [24]. Since the first report of C. muris in human samples in 2000, evidence of human infection with C. muris has been accumulating [25]. Including diarrhea patients cattle, sheep, and the cactus mouse [24,26], C. andersoni was found in a south China tiger in the present study, thereby expanding the host range of C. andersoni. Cryptosporidium macropodum (only detected in Australia previously) [27] was detected for the first time in a white kangaroo in China.
Similar to previous reports [28], we identified two G. duodenalis assemblages (A and E) in the Bactrian camel. Assemblage A was one of the two species of G. duodenalis most commonly detected in human samples, and this assemblage has also been detected in livestock, companion animals, and non-human primates (NHPs) [2]. Assemblage E has been reported as an assemblage with host specificity, mainly infecting cattle, sheep, goats, and pigs. However, there are still some studies reporting the presence in human [29]. Therefore, the data indicate that animals in these zoos may serve as reservoirs of G. duodenalis assemblages with the potential for zoonotic infection in humans. However, this speculation needs further research and data to confirm.
A total of 20 Enterocytozoon bieneusi genotypes were observed in 89 positive specimens. BEB6 was the predominant E. bieneusi genotype in the current study; it has been found in NHPs, sheep and goats, companion animals (cats and dogs), chinchillas, rabbits, meerkats, and bats. The genotypes D, type IV, and Peru8 were previously detected in humans, goats, pigs, and NHPs, and they have been frequently found in different water sources, suggesting the likelihood of zoonotic or waterborne transmission [30][31][32][33]. Indeed, previous reports have found genotype BEB6, D, type IV, and Peru8 in humans and wildlife in various countries [22,34,35]. Therefore, these findings indicate that zoonotic transmission to humans and between wildlife species may occur in the zoos investigated in the present research. We also found the newly identified genotypes CHPM1and CHWD1 in Table 3 Enterocytozoon bieneusi ITS genotypes identified in this study white-lipped deer and patas monkeys, respectively. Interestingly, the isolation of novel genotypes from whitelipped deer, as well as the three novel genotypes identified by the study of Li et al., suggest that genetic variability in deer-derived E. bieneusi may be common [3]. Phylogenetic analysis showed that genotypes D, Peru8, SDD1, HND-I, Type-IV, KIN-1, Henan-IV, Henan-V, CHPM1, and CHWD1 were clustered in Group 1, which was composed of zoonotic genotypes. Genotypes CHG11, CHG-1, BEB6, CM5, CHC9, and CHS9 were clustered in Group 2, and other data indicated that the genotypes in Group 2 may have zoonotic potential. Accumulating evidence indicates that some Group 2 genotypes (I, J, BEB4, and BEB6) can also be detected in humans. A total of nine subtypes were detected in Blastocystis, including ST1, ST2, ST3, ST5, ST6, ST7, ST10, ST13, and ST14. Studies indicated that ST1 to ST9 and ST12 have the ability to infect humans [8]. For example, ST1, ST3, and ST5 have been found in NHPs and their keepers, in southern hairy-nosed wombats and their keepers, and in pigs and their workers, respectively [12,13]. Although the fecal samples of animal keepers were not considered in the present study, the possibility of transmission between animals and animal keepers is undeniable. Therefore, subtypes ST1, ST2, ST3, and ST5 may have potential risk for zoonotic transmission between animals and humans. Present study indicated that the dominant species of Blastocystis subtypes in NHPs is ST1-ST3, which is consistent with other results [36][37][38][39]. In addition, we found that ostriches can be infected by ST5, a subtype that has been isolated sporadically from many other animals, including NHPs, camels, the black rhinoceros, and rodents [13,40]. ST6 was found in blue-eared pheasant and peafowl, and ST7 was found in turkeys and the whooper swan, similar to previous findings [36]. ST10 was previously identified in fallow deer and camels [41]. Similarly, ST10 was detected in sika deer and yellow deer in the present study. Only ST14 was found in Bactrian camels here, which is consistent with previous findings [36]. The above results demonstrate that these subtypes can infect animal species in zoos as well as humans, and thus more attention should be paid to these parasites. Some species, genotypes and subtypes of these parasites were detected in some new hosts in present study. However, what can not be determined is that whether these animals are natural hosts or carriers of theses pathogens and whether these parasites can cause infections in new hosts. Animals could get infected via eating food or drinking water which contain viable pathogens. Considering the specificity of zoo environment, where different species of animals are kept in seperate areas, the risk of cryptosporidiosis transmission through contaminated food or water seems relatively low. Due to the rarity of wild captive animals and the limitation of the amount of pathogen infection, it is difficult for us to demonstrate cross-transmission in different species of wild captive animals. We only successfully infected BALB/c mice with the oocysts of C. muris [42]. Efforts should be made in the following study to conduct more investigative research on these problems.

Conclusion
Our results indicate that animals in zoos can be infected with human pathogenic Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi, and Blastocystis sp. These animals can serve as reservoirs of human cryptosporidiosis, giardiasis, microsporidiosis, and blastocystosis. Effort should be made to conduct more experimental work to reveal the genetic characteristics and assess the zoonotic risks of these parasites.

Study area and sample collection
Between October 2018and June 2020, a total of 429 fresh fecal samples (77 animal species) were collected from captive animals in 6 zoos in Henan, China; these were Xinxiang Zoo (n = 23), Kaifeng Zoo (n = 36), Luoyang Zoo (n = 27), Shangqiu Zoo (n = 24), Jiaozuo Swan Lake Zoo (n = 120) (private zoo), and Zhengzhou Zoo (n = 199) (Fig. 2). Some of the specimens tested in this study were animals imported from abroad and were ill (slight or severe diarrhea, and some adult worms in fecal samples) (Table S1). One specimen per animal was used in this study. Only the central portion of the fecal material was collected during sampling to ensure no environmental contamination. Each fresh sample was collected into a sterile glove, labeled, and placed into a container with ice packs and immediately sent to the lab for DNA extraction.

DNA extraction
200 mg of fecal samples were used to extract DNA with an E.Z.N.A. ® Stool DNA Kit (Onmega Biotek Inc., Norcross, GA, USA), according to the manufacturer's instructions, and the extracted DNA was stored at − 20 °C before PCR analysis. The quality of the DNA extracted was determined by using the NanoDrop absorbance ratio.

PCR amplification
All of the samples were amplified by nested PCR to identify Cryptosporidium spp. and Giardia duodenalis based on the small subunit (SSU) rRNA gene and the glutamate dehydrogenase (gdh) gene [43,44], respectively. Positives for Cryptosporidium (C. parvum and C. hominis) were subtyped based on the 60-kDa glycoprotein (gp60) gene [45]. Enterocytozoon bieneusi and Blastocystis sp. were identified based on the ITS region [46] and the SSU rRNA gene [47], respectively (Table S3). The amplification was performed in 25 μL reaction mixtures. The first reaction mixture contained 1 μL of extracted DNA. The second reaction mixture contained 1 μL of the first PCR amplification product. The KOD Plus DNA polymerase (Toyobo Co., Ltd., Osaka, Japan) was used for all PCR amplification. Positive and negative control samples (distilled water) were included in each PCR assay, and two replicates of each PCR were run for all of the samples. The final PCR products were subjected to 1.0% agarose gel electrophoresis and visualized by staining with DNAGREEN (Tiandz, Inc., Beijing, China).

Sequencing and phylogenetic analysis
All of the final positive PCR products were sequenced using the ABI PRISM ™ 3730 XL DNA Analyzer with the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA), and two-directional Fig. 2 Neighbor-joining tree of Enterocytozoon bieneusi ITS genotypes. Phylogenetic relationships of ITS nucleotide sequences of the Enterocytozoon bieneusi genotypes identified in this study and other reported genotypes. The phylogeny was inferred by a neighbor-joining analysis. Bootstrap values were obtained using 1000 replicates; those with values > 50% are shown on the nodes. The genotypes in this study are marked by empty triangles, and the novel genotypes are marked by filled triangles sequencing was used to ensure accuracy. To identify different species or genotypes, sequences obtained were aligned with the reference sequences in GenBank (http:// blast. ncbi. nlm. nih. gov) using the software Clustal X 2.1 (http:// www. clust al. org/). The phylogenetic relationships of E. bieneusi genotypes were analyzed by the neighbor-joining algorithm in MEGA 7.0 (http:// www. megas oftwa re. net/). Bootstrap values were calculated by analyzing 1000 replicates. The established nomenclature system was used in the naming of E. bieneusi ITS genotypes [48].

Statistical analysis
The infection rates with 95% confidence intervals (CI) were calculated by Wald's method in SPSS 22.0 version (SPSS Inc., Chicago, IL, United States). Differences in corresponding infection rates among locations were examined by the Chi-square test, and differences were considered significant at P < 0.05.