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..

Results

Occurrence of Cryptosporidium, Giardia duodenalis, Enterocytozoon bieneusi, and Blastocystis

The overall infection rate was 43.1% (185/429, 95% CI: 39.33–48.77%, χ2 = 25.048, df = 5, P < 0.001) among six zoos. The prevalence of Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi, and Blastocystis sp. were 2.8% (12/429, 95% CI: 1.23–4.36%, χ2 = 23.613, df = 5, P < 0.001), 0.5% (2/429, 95% CI: 0–1.11%, χ2 = 21.936, df = 5, P < 0.001), 20.8% (89/429, 95% CI: 16.89–24.59%, χ2 = 25.877, df = 5, p < 0.001), and 19.1% (82/429, 95% CI: 16.24–23.85%, χ2 = 7.696, df = 5, p > 0.05), respectively (Table 1). Co-infection results showed that 29 samples were infected by two kinds of parasites; the infected species were sika deer (n = 6), white kangaroos (n = 4), macaques (n = 4), black-and-white colobus monkeys (n = 3), two giraffes (n = 2), a Bactrian camel (n = 1), a patas monkey (n = 1), a peafowl (n = 1), a pony (n = 1), a leopard (n = 1), a golden sub-nosed monkey (n = 1), a white-browed monkey (n = 1), a green monkey (n = 1), a squirrel monkey (n = 1), and a northern pigtail macaque (n = 1).

Table 1 Occurrence of Cryptosporidium spp., G. duodenalis, E. bieneusi, and Blastocystis sp. in this study
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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.

Table 2 Distribution of Cryptosporidium and Giardia duodenalis in this study
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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).

Enterocytozoon bieneusi genotypes

A total of 20 genotypes of Enterocytozoon bieneusi were identified in the present study, including 18 known genotypes: 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. However, a novel genotype CHPM1 was found in a patas monkey, and CHWD1 was found in a white-lipped deer. Additionally, SDD1, BEB6, CD7, HDN-1, CHG-1, CHC9, D, Peru8, and Type-IV were identified for the first time in animal hosts. The most prevalent E. bieneusi genotype was BEB6 (32/89, 36.0%) followed by D (16/89, 18.0%) (Table 3). Compared with genotype D (KX383624), novel genotypes CHPM1 and CHWD1 had one and three SNPs based on the ITS region, respectively (Table S2). Phylogenetic analysis of E. bieneusi showed that genotypes D, Peru8, SDD1, HND-I, Type-IV, KIN-1, Henan-IV, Henan-V, CHPM1, and CHWD1 were clustered in Group 1, whereas CHG11, CHG-1, BEB6, CM5, CHC9, and CHS9 were clustered into Group 2. CHG21, CD7 and CHB1, CHK1were clustered into Group 9, Group 11 and Group10, respectively (Fig. 1).

Table 3 Enterocytozoon bieneusi ITS genotypes identified in this study
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Fig. 1
figure 1

Locations where specimens were collected in this study. The figure was originally designed by the authors using the software ArcGIS 10.2. No copyright permission was required. The original vector diagram imported in ArcGIS was adapted from Natural Earth (http://www.naturalearthdata.com)

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Blastocystis subtypes

A total of nine Blastocystis subtypes were found, including ST1, ST2, ST3, ST5, ST6, ST7, ST10, ST13, and ST14. However, the other six subtypes were identified in new hosts for the first time: ST2, ST3, ST5, ST6, ST7, and ST10 were detected in ponies, an orangutan, a gorilla, sika deer, white kangaroos, a blue eared-pheasant, a whooper swan, and giraffes. The most prevalent Blastocystis subtype was ST5 (19/86, 22.1%) followed by ST10 (18/86, 20.1%) (Table 4).

Table 4 Distribution of Blastocystis sp. in the wildlife in this study
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Discussion

In the present study, the Cryptosporidium prevalence was 2.8%, which is lower than the rates (70.0%) reported in ** 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) [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., Bei**g, 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 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.clustal.org/). The phylogenetic relationships of E. bieneusi genotypes were analyzed by the neighbor-joining algorithm in MEGA 7.0 (http://www.megasoftware.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.