This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Non-human primates (NHPs) are confirmed as reservoirs of Cryptosporidium spp., Giardia intestinalis, and Enterocytozoon bieneusi. In this study, 197 fresh fecal samples from 8 NHP species in Qinling Mountains, northwestern China, were collected and examined using multilocus sequence typing (MLST) method. The results showed that 35 (17.8%) samples were positive for tested parasites, including Cryptosporidium spp. (3.0%), G. intestinalis (2.0%), and E. bieneusi (12.7%). Cryptosporidium spp. were detected in 6 fecal samples of Macaca mulatta, and were identified as C. parvum (n=1) and C. andersoni (n=5). Subtyping analysis showed Cryptosporidium spp. belonged to the C. andersoni MLST subtype (A4, A4, A4, and A1) and C. parvum 60 kDa glycoprotein (gp60) subtype IId A15G2R1. G. intestinalis assemblage E was detected in 3 M. mulatta and 1 Saimiri sciureus. Intra-variations were observed at the triose phosphate isomerase (tpi), beta giardin (bg), and glutamate dehydrogenase (gdh) loci, with 3, 1, and 2 new subtypes found in respective locus. E. bieneusi was found in Cercopithecus neglectus (25.0%), Papio hamadrayas (16.7%), M. mulatta (16.3%), S. sciureus (10%), and Rhinopithecus roxellana (9.5%), with 5 ribosomal internal transcribed spacer (ITS) genotypes: 2 known genotypes (D and BEB6) and 3 novel genotypes (MH, XH, and BSH). These findings indicated the presence of zoonotic potential of Cryptosporidium spp. and E. bieneusi in NHPs in Qinling Mountains. This is the first report of C. andersoni in NHPs. The present study provided basic information for control of cryptosporidiosis, giardiasis, and microsporidiosis in human and animals in this area.
Cryptosporidiosis, giardiasis, and microsporidiosis, 3 emerging infectious diseases caused by Cryptosporidium, Giardia, and microsporidia in humans and animals, have been reported both in developed countries and in those under development in last decades [1-3]. Clinical signs of these diseases are normally chronic diarrhea in immune-competent hosts, but these infections may lead to significant morbidity and mortality in immune-compromised or -suppressed hosts, such as AIDS patients [1,4].
Accurate identification of species/genotype/subtype is essential for characterization of transmission pattern and control of parasitic diseases. Until now, there are 27 valid species of Cryptosporidium and over 70 genotypes have been described in the literature, and at least 14 species and 5 genotypes were detected in humans [3,5]. More than 50 species of Giardia have been proposed during 1920-1930, but only 6 species are accepted by most researchers on the basis of the morphology of the trophozoites/cysts [4]. Giardia intestinalis (syn. G. lamblia, G. duodenalis), the only species that can infect humans, contains at least 7 valid assemblages (A-G) within species. The assemblages A and B are responsible for most of human infections [4]. Likewise, some Enterocytozoon bieneusi genotypes are mostly host-adapted in nature, and some are zoonotic [2]. Epidemiological surveys indicated that humans acquire infections of Cryptosporidium spp., G. intestinalis, and E. bieneusi mainly through the fecal-oral route via direct person-to-person or animal-to-person contact, or ingestion of contaminated water or food by animal manure [1,4]. Zoonotic Cryptosporidium spp., G. intestinalis, and E. bieneusi have been detected in domestic livestock, companion and wild animals. Their hosts have been believed to play significant roles in this context, contributing to parasite oocysts/cysts in large proportion [1,2,4].
Data from several studies suggested that non-human primates (NHPs) might be sources of cryptosporidiosis, giardiasis, and microsporidiosis for human infections [6-11], since they have close relationships with human and can harbor zoonotic species/genotypes/subtypes. The infections of human-pathogenic Cryptosporidium spp., G. intestinalis, and E. bieneusi in NHPs have been documented in Asia, America, and Africa [6-11]. In Uganda, potential cross-transmissions between human and mountain gorillas for G. intestinalis assemblage A and C. parvum were reported [6]. In China, the respective prevalences of human-pathogenic Cryptosporidium spp. and G. intestinalis infections were 10.9% and 8.3%, respectively, in Macaca mulatta from Guizhou [7]. The zoonotic C. hominis and G. intestinalis were also detected in M. fascicularis from a commercial animal facility in Guangxi of China [8]. E. bieneusi was reported in 5 NHP species from Guangdong, Yunnan, Guangxi, Henan, and Sichuan provinces, including M. fascicularis (67.7%), M. mulatta (8.8%), M. fuscata (33.3%), Trachypithecus leucocephalus (13.6%), and Rhinopithecus roxellana (9.5%) [9].
Qinling Mountains, the natural boundary between the North and South of China, are mainly situated in Shaanxi province. The mountains support huge varieties of endangered wild and captive animals, such as R. roxellana, Budorcas taxicolor, Nipponia nippon, and Ailuropoda melanoleuca. Here, 2 largest captive animal sites in Shaanxi province at the foot of Qinling Mountains were selected to determine the prevalence and species/genotype/subtypes distribution of Cryptosporidium spp., G. intestinalis and E. bieneusi in captive NHPs, and to unravel their public health significance.
MATERIALS AND METHODS
From December 2013 to July 2014, 197 fresh fecal specimens from 8 species of NHPs were collected from 2 largest captive wild animal farms in Qinling Mountains (Table 1) according to the legal requirements of guide for the Care and Use of Laboratory Animals of the Ministry of Health, China, and approval of the Research Ethics Committee of Northwest A&F University (No. NWAFREC-2013-09-0002). One farm covers over 173 ha and houses 10,000 wild animals belonging to almost 300 species comprising mammals, birds, and reptiles. The other (5.3 ha) harbors 600 animals which were rescued from Qinling Mountains. Each site is near to human habitats and is visited by more than 100 million or 50 million individuals each year. Visitors are allowed to watch them from a short distance. All NHP species of 2 origins are kept in mono-specific groups sharing each respective pens/cages. All NHPs are housed in cages/pens littered with natural materials such as the ground bark. Fresh stool samples from the rectum of each of rare NHPs were collected, placed immediately in disposable plastic bags, and stored at 4˚C for further study. All animals in this study showed no clinical symptoms.
Genomic DNA was extracted from each fecal specimen using the E.Z.N.A® Stool DNA Kit (OMEGA Bio-tek, Inc, Georgia, USA) according to manufacture-recommended procedures, and stored at -20˚C for molecular analysis. The gene loci of triose phosphate isomerase (tpi), small subunit ribosomal RNA (SSU rRNA), beta giardin (bg), and glutamate dehydrogenase (gdh) loci were used to determine G. intestinalis assemblages according to protocols described previously [10]. The 18S rRNA and Cryptosporidium oocyst wall protein (COWP) genes were used to indicate Cryptosporidium species/genotypes [10,12]. The 60 kDa glycoprotein (gp60) gene was employed specifically for detection and assignment of Cryptosporidium species/genotypes and subtypes that are infective to humans [13]. To further analyze C. andersoni subtypes, 4 minisatellite loci (CM-MS1, CM-MS2, CM-MS3, and CM-MS16) were used [12]. A 392 bp fragment of the internal transcribed spacer (ITS) gene was amplified by nested PCR as previously to genotype E. bieneusi [9].
The TaKaRa Ex Taq (TaKaRa Shuzo Co., Ltd., Otsu, Japan) was used to amplify all target loci. The PCR reactions were conducted in 25 μl reaction mixtures containing 1×Ex Taq buffer (Mg2+ free), 2 mM MgCl2, 0.2 mM each deoxy-ribonucleoside triphosphate (dNTP), 0.4 μM each primer, 0.625 U TaKaRa ExTaq DNA polymerase, and 2 μl of DNA template (or 1 μl of the first PCR amplicon). For the amplication of SSU rRNA for G. intestinalis, 1 μl dimethyl sulfoxide (DMSO) was added additionally. Replicate analysis was done at each locus with both positive and negative controls. The secondary PCR products were visualized on a UV transilluminator after electrophoresis in 1.5% agarose gel and stained with ethidium bromide to validate amplification efficiency.
All positive PCR products were sequenced directly by Shanghai Sangon Biological Engineering Biotechnology Company using ABI 3730xl automated DNA sequencer (Big Dye Terminator Chemistry, Applied Biosystems, Foster City, California, USA). The species/assemblages/genotypes/subtypes of enteric parasites detected were determined by the alignment with reference sequences from the GenBankTM database using BLAST program (http://www.ncbi.nlm.nih.gov). Phylogenetic trees were implemented in the program Mega 5.0 (http://www.megasoftware.net/) using a neighbor-joining method. Bootstrap analysis was used to assess the robustness of clusters with 1,000 replicates.
RESULTS
Of 197 samples examined, 35 (17.8%) were positive for tested parasites. The differences in prevalence among infections of different parasites in NHPs were compared using the χ2 test in SPSS 17.0 (http://www-01.ibm.com/software/analytics/spss/), with statistically significant at P<0.05. The differences of infection rates for 3 parasites were not statistically significant (P>0.05). Cryptosporidium spp. were detected in 6 fecal samples from M. mulatta (7.0%), with the overall infection rate of 3.0% in NHPs (Table 1), which was higher than that reported in laboratory M. fascicularis (0.5%) and NHPs (0.7%) in China, newly captive Papio anubis (2.6%) in Kenya, but lower than that in M. mulatta (10.9%) in China, P. anubis (11.9%) and Cercopithecus aethiops (29.3%) of Ethiopia [7,10,14,15].
G. intestinalis was identified in 4 specimens of M. mulatta (3.5%) and S. sciureus (5.0%), with the overall prevalence in NHPs in this study was 2.0% (Table 1). Different infection rates of G. intestinalis have been reported in different NHPs in China and other countries: 47% in Lemur catta in Italy, 18% in NHPs in Brazil, 9% in mountain gorillas in Rwanda, 8.5% in M. mulatta, and 2.2% in NHPs in China [10,16-18].
E. bieneusi was detected in 25 (12.7%) specimens from 5 captive NHP species, including C. neglectus (25.0%), P. hamadrayas (16.7%), M. mulatta (16.3%), S. sciureus (10.0%), and R. roxellana (9.5%) (Table 1). Recently, E. bieneusi frequently presented in P. troglodytesin (33.3%) in the Slovak Republic, captive P. anubis (12.3%) and P. troglodytesin (2.6%) in Kenya, M. fascicularis (18.5%) and NHPs (11.4%) in China, and P. troglodytesin (4.5%) in Cameroon [8,15,19]. The differences of infection status of these parasites may be related to the species of NHP, sample sizes, examination methods, different management systems, the timing of specimen collection, and geo-ecological conditions.
DISCUSSION
In the present study, of all pathogens, single infection of C. andersoni, C. parvum, G. intestinalis, and E. bieneusi was presented in fecal samples of M. mulatta, S. sciureus, C. neglectus, P. hamadrayas, and R. roxellana. Double infections of C. andersoni and E. bieneusi were co-occurred only in 4 samples of M. mulatta, but no other mixed infections were observed. These results suggested that single pathogen infection was more common in NHPs in this area.
Previous studies indicated that 5 Cryptosporidium species, namely C. hominis, C. parvum, C. felis, C. muris, and C. ubiquitum, have been detected in NHPs [1,7,10]. In the present study, sequence analysis revealed the presence of 2 Cryptosporidium species, namely C. andersoni (n=5, GeneBank accession no. KJ917574 to KJ917578, KJ917580 to KJ917584) and C. parvum (n=1, KJ917579, KJ917585) in M. mulatta (Table 1). The successful amplification of gp60 gene was only observed for the fecal sample with the infection of C. parvum. BLSAT search and alignment of the obtained gp60 gene sequence (KJ917586) from M. mulatta had 100% identity with the isolates Cp12 (GU214367) and D536 (FJ897784) from diarrheal patients in England and India [20,21], which indicated C. parvum isolate in this study belonged to the subtype IId A15G2R1 (KJ917586) based on the “genotypic” nomenclature described previously [20]. Based on gp60, subtype families Ia, Ib, Id, Ie, If, and Ii of C. hominis and IIc of C. parvum have been reported in NHPs [7,10,20]. In China, the subtype IId of C. parvum was detected in pre-weaned dairy cattle, hamsters, urban wastewater, and HIV-positive patients [22]. In the present study, 1 C. parvum gp60 subtype IId A15G2R1 was identified in M. mulatta and identical to the isolate from human patients in England and India [20,21]. This is the first report of subtype IId in NHPs, and it suggested that NHPs in Qinling Mountains may be potentially responsible for some zoonotic transmission of C. parvum.
C. andersoni, the common species in ruminants, was detected in this study. The zoonotic potential of C. andersoni was controversial in previous studies. The presence of partial 18S rRNA that very likely represented C. andersoni was confirmed in a male HIV-positive patient in France [23], and C. andersoni was detected by PCR/RFLP and DNA sequencing of 18S rRNA and COWP genes in 3 diarrheal patients in England between 1985 and 2000 [24]. However, no shedding oocysts were observed in M. fascicularis under either both normal or immunosuppressive conditions by orally inoculated with oocysts of 2 different C. andersoni Kawatabi types and C. muris RN-66 [25]. Herein, Cryptosporidium oocysts from M. mulatta with average size of 5.62×7.36 μm were observed under optical microscope using modified acid-fast technique, and it was identified as C. andersoni by sequencing of 18S rRNA (KJ917574 to KJ917578) and COWP (KJ917580 to KJ917584) genes. To subtype C. andersoni isolates in this study, each isolate of C. andersoni was successfully amplified and sequenced at 4 loci. Sequence analysis (KJ917587 to KJ917606) indicated haplotypes A4, A4, A4, and A1 at loci MS1, MS2, MS3, and MS16, respectively, forming 1 MLST subtype A4, A4, A4, and A1, which was identical to C. andersoni isolates from cattle in Henan, Jilin, Guangxi, Heilongjiang, Sichuan, and Shaanxi of China [12]. These findings may indicate C. andersoni infecting certain species of NHPs, such as M. mulatta, or distinct biological characteristics of different C. andersoni isolates. Therefore, further researches on animal adaptation and infectivity of C. andersoni obtained will be studied to illuminate zoonotic potential of C. andersoni in the future.
Of 8 G. intestinalis assemblages, assemblages A and B were commonly detected in NHPs, with the assemblage B dominating [4,10,26]. The assemblage E, restricted to ruminants, was also found in Ptephrosceles tephrosceles in western Uganda by real-time PCR and sequence analysis of ef1-a, SSU rRNA, tpi, and gdh genes, which suggested that livestock-to-primate transmission was most likely in assemblage E [27]. In the present study, 4 G. intestinalis-positive samples in M. mulatta (n=3) and S. sciureus (n=1) were successfully sequenced at 4 gene loci (KJ917607 to KJ917622) (Table 1). BLAST search against NCBI nucleotide sequence database showed that G. intestinalis isolates in this study were all belonged to G. intestinalis assemblage E. The reasons may be due to the fact of assemblage E being predominant in NHPs here, or small numbers of NHPs investigated. Considering a study based on PCR and DNA sequence analysis of tpi gene showed the assemblage E as the second dominating assemblage in human in Egypt [26], the NHPs in this study may have zoonotic potential for transmission of G. intestinalis.
Sequence analysis indicated no sequence differences of SSU rRNA gene (KJ917607 to KJ917610) among 4 G. intestinalis-positive samples, with 99% identity to G. intestinalis assemblage E isolates from cattle (KF843921) and goats (AY826210). The intra-genotypic diversity of 4 G. intestinalis isolates was observed at loci of tpi (KJ917619 to KJ917622), bg (KJ917611 to KJ917614), and gdh (KJ917615 to KJ917618) genes (Table 2). Four single nucleotide polymorphisms (SNPs) were detected at the tpi locus, forming 3 new assemblage E subtypes (KJ917619 to KJ917622). There were 2 SNPs among bg gene sequences, forming 1 new subtype (KJ917611, KJ917613) and 1 known subtype reported in Bison bonasus (FJ472822). Three subtypes were detected at the gdh gene locus, with 2 new subtypes (KJ917615, KJ917616) and 1 same to isolates Ly20 from cattle (KF843923). These results indicated complicate variability of G. intestinalis assemblage E isolates from different hosts and origins. One subtype in each of bg and gdh loci in this study had 100% identity with isolates from bovine in China (KF843923, FJ472822), which suggested animal-to-animal transmission may exist for G. intestinalis.
In the present study, 25 specimens were amplified successfully at the ITS region of E. bieneusi. Sequence analysis indicated that these samples belonged to 5 distinct genotypes, including genotypes D (n=10, KM591953 to KM591962), BEB6 (n=4, KM591947 to KM591950) and 3 novel genotypes, namely MH (n=7, KM591963 to KM591969), XH (n=2, KM591951, KM591952) and BSH (n=2, KM591945, KM591946). The genotype D is the dominating genotype, and has been detected in M. mulatta (n=6), R. roxellana (n=2), and P. hamadrayas (n=2). The genotype BEB6 previously described in cat and sheep [2,11] was found in 4 fecal samples from R. roxellana. The novel genotype MH was found only in M. mulatta (n=7), with 2 SNPs to genotype D (GQ406055). Phylogenetic analysis (Fig. 1) showed that the novel genotype MH was closely related to genotype D and clustered in Group 1a that was regarded as zoonotic previously [2]. Considering that the genotypes D has been reported in humans, NHPs and urban wastewater [9,28,29], NHPs in Qinling Mountains would have risk for zoonotic transmission potential. The new genotypes XH and BSH were observed in respective fecal samples from S. sciureu (n=2) and C. neglectus (n=2). The genotype BSH contained 6 SNPs compared with BEB6 (KJ668737) grouped into genotypes of Group 2 reported only in animals [2]. Sequence comparisons revealed that the sequences of genotypes XH had 4 SNPs compared to genotypes CD5 (KJ668732) and CM4 (KF543866). In the phylogenetic tree, the novel genotype XH clustered with genotype CD5 from dog and genotype CM4 from NHPs in China [10,28], and this cluster was located in a solitary clade and was proposed as Group 9 (Fig. 1).
In conclusion, the infections of Cryptosporidium spp., G. intestinalis, and E. bieneusi were detected in captive NHPs in Qinling Mountains. C. andersoni and C. parvum subtype IId A15G2R1 were firstly reported in NHPs. The assemblage E was the only G. intestinalis assemblage detected in NHPs in this study, and new subtypes at 4 loci were also found. Zoonotic E. bieneusi genotypes D and host-adapted genotype BEB6 were both presented in NHPs, and 3 novel genotypes were found. These results suggested that captive NHPs in Qinling Mountains would have significant zoonotic potential. Therefore, integrity measure should be implemented to control infections of Cryptosporidium spp., G. intestinalis and E. bieneusi in NHPs, and special care should also be taken by the animal attendants, animal care specialists, veterinarians, scientists and visitors to zoonotic transmission of cryptosporidiosis, giardiasis and microsporidiosis.
Notes
The authors declare that they have no competing interests.
This study was supported by grants from the Program for New Century Excellent Talents in University (grant no. NCET-13-0489), the State Key Program of National Natural Science Foundation of China (grant no. 31330079), the Fund for Basic Scientific Research (grant no. ZD2012010), and the Open Funds of the State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (grant no. SKLVEB2013KFKT007).
Fig. 1.
Phylogenetic relationships of E. bieneusi genotypes identified in this study and other genotypes previously deposited in GenBank as inferred by the neighbor-joining analysis of ITS sequences based on genetic distances calculated by the Kimura 2-parameter model. Bootstrap values greater than 50% from 1,000 replicates are shown on nodes. Each sequence from GenBank is identified by the accession number, host origin, and the genotype designation. Genotypes identified in this study are indicated by the symbol (◆).
Table 1.
Distribution of Cryptosporidium spp., G. intestinalis and E. bieneusi in non-human primates (NHPs) in Qinling Mountains, northwestern China
1. Xiao L, Fayer R. Molecular characterisation of species and genotypes of Cryptosporidium and Giardia and assessment of zoonotic transmission. Int J Parasitol 2008;38:1239-1255.
2. Thellier M, Breton J. Enterocytozoon bieneusi in human and animals, focus on laboratory identification and molecular epidemiology. Parasite 2008;15:349-358.
6. Nizeyi JB, Cranfield MR, Graczyk TK. Cattle near the Bwindi Impenetrable National Park, Uganda, as a reservoir of Cryptosporidium parvum and Giardia duodenalis for local community and freeranging gorillas. Parasitol Res 2002;88:380-385.
8. Ye J, Xiao L, Li J, Huang W, Amer SE, Guo Y, Roellig D, Feng Y. Occurrence of human-pathogenic Enterocytozoon bieneusi, Giardia duodenalis and Cryptosporidium genotypes in laboratory macaques in Guangxi, China. Parasitol Int 2014;63:132-137.
9. Karim MR, Wang R, Dong H, Zhang L, Li J, Zhang S, Rume FI, Qi M, Jian F, Sun M, Yang G, Zou F, Ning C, Xiao L. Genetic polymorphism and zoonotic potential of Enterocytozoon bieneusi from nonhuman primates in China. Appl Environ Microbiol 2014;80:1893-1898.
10. Karim MR, Zhang S, Jian F, Li J, Zhou C, Zhang L, Sun M, Yang G, Zou F, Dong H, Li J, Rume FI, Qi M, Wang R, Ning C, Xiao L. Multilocus typing of Cryptosporidium spp, and Giardia duodenalis from non-human primates in China. Int J Parasitol 2014;18:1039-1047.
11. Karim MR, Dong H, Yu F, Jian F, Zhang L, Wang R, Zhang S, Rume FI, Ning C, Xiao L. Genetic diversity in Enterocytozoon bieneusi isolates from dogs and cats in China: host specificity and public health implications. J Clin Microbiol 2014;52:3297-302.
12. Zhao GH, Ren WX, Gao M, Bian QQ, Hu B, Cong MM, Lin Q, Wang RJ, Qi M, Qi MZ, Zhu XQ, Zhang LX. Genotyping Cryptosporidium andersoni in cattle in Shaanxi Province, Northwestern China. PLoS One 2013;8:e60112.
13. Abeywardena H, Jex AR, Koehler AV, Rajapakse RP, Udayawarna K, Haydon SR, Stevens MA, Gasser RB. First molecular characterization of Cryptosporidium and Giardia from bovines (Bos taurus and Bubalus bubalis) in Sri Lanka: unexpected absence of C. parvum from pre-weaned calves. Parasit Vectors 2014;7:75.
14. Legesse M, Erko B. Zoonotic intestinal parasites in Papio anubis (baboon) and Cercopithecus aethiops (vervet) from four localities in Ethiopia. Exp Parasitol 2004;90:231-236.
15. Li N, Xiao L, Wang L, Zhao S, Zhao X, Duan L, Guo M, Liu L, Feng Y. Molecular surveillance of Cryptosporidium spp, Giardia duodenalis and Enterocytozoon bieneusi by genotyping and subtyping parasites in wastewater. PLoS Negl Trop Dis 2012;6:e1809.
16. Berrilli F, Prisco C, Friedrich KG, Di Cerbo P, Di Cave D, De Liberato C. Giardia duodenalis assemblages and Entamoeba species infecting non-human primates in an Italian zoological garden: zoonotic potential and management traits. Parasit Vectors 2011;4:199.
17. David ÉB, Patti M, Coradi ST, Oliveira-Sequeira TC, Ribolla PE, Guimarães S. Molecular typing of Giardia duodenalis isolates from nonhuman primates housed in a Brazilian zoo. Rev Inst Med Tro SP 2014;56:49-54.
19. Sak B, Kvác M, Petrželková K, Květonová D, Pomajbíková K, Mulama M, Kiyang J, Modrý D. Diversity of microsporidia (Fungi: Microsporidia) among captive great apes in European zoos and African sanctuaries: evidence for zoonotic transmission? Folia Parasitol 2011;58:81-86.
20. Pangasa A, Jex AR, Nolan MJ, Campbell BE, Haydon SR, Stevens MA, Gasser RB. Highly sensitive non-isotopic restriction endonuclease fingerprinting of nucleotide variability in the gp60 gene within Cryptosporidium species genotypes and subgenotypes infective to humans and its implications. Electrophoresis 2010;31:1637-1647.
21. Ajjampur SS, Liakath FB, Kannan A, Rajendran P, Sarkar R, Moses PD, Simon A, Agarwal I, Mathew A, O'Connor R, Ward H, Kang G. Multisite study of cryptosporidiosis in children with diarrhea in India. J Clin Microbiol 2010;48:2075-2081.
22. Wang R, Zhang L, Axen C, Bjorkman C, Jian F, Amer S, Liu A, Feng Y, Li G, Lv C, Zhao Z, Qi M, Dong H, Wang H, Sun Y, Ning C, Xiao L. Cryptosporidium parvum IId family: clonal population and dispersal from Western Asia to other geographical regions. Sci Rep 2014;4:4208.
23. Guyot K, Follet-dumoulin A, Lelievre E, Sarfati C, Rabodonirina M, Nevez G, Cailliez JC, Camus D, Dei-Cas E. Molecular characterization of Cryptosporidium isolates obtained from humans in France. J Clin Microbiol 2001;39:3472-3480.
24. Leoni F, Amar C, Nichols G, Pedraza-Diaz S, Mclauchlin J. Genetic analysis of Cryptosporidium from 2414 humans with diarrhoea in England between 1985 and 2000. J Med Microbiol 2006;55:703-707.
25. Masuno K, Fukuda Y, Kubo M, Ikarashi R, Kuraishi T, Hattori S, Kimura J, Kai C, Yanai T, Nakai Y. Infectivity of Cryptosporidium andersoni and Cryptosporidium muris to normal and immunosuppressive cynomolgus monkeys. J Vet Med Sci 2014;76:169-172.
26. Foronda P, Bargues MD, Abreu-acosta N, Periago MV, Valero MA, Valladares B, Mas-Coma S. Identification of genotypes of Giardia intestinalis of human isolates in Egypt. Parasitol Res 2008;103:1177-1181.
28. Zhang X, Wang Z, Su Y, Liang X, Sun X, Peng S, Lu H, Jiang N, Yin J, Xiang M, Chen Q. Identification and genotyping of Enterocytozoon bieneusi in China. J Clin Microbiol 2011;49:2006-2008.
29. Li W, Kiulia NM, Mwenda JM, Nyachieo A, Taylor MB, Zhang X, Xiao L. Cyclospora papionis, Cryptosporidium hominis and humanpathogenic Enterocytozoon bieneusi in captive baboons in Kenya. J Clin Microbiol 2011;49:4326-4329.
30. Wang H, Zhao G, Chen G, Jian F, Zhang S, Feng C, Wang R, Zhu J, Dong H, Hu A, Wang M, Zhang L. Multilocus genotyping of Giardia duodenalis in dairy cattle in Henan, China. PLoS One 2014;9:e100453.
31. Geurden T, Goossens E, Levecke B, Vercammen F, Vercruysse J, Claerebout E. Occurrence and molecular characterization of Cryptosporidium and Giardia in captive wild ruminants in Belgium. J Zoo Wildl Med 2009;40:126-130.
First report of Blastocystis sp. and Enterocytozoon bieneusi in raptors Zhen-Qiu Gao, Si-Yuan Qin, Lin-Hong Xie, Guang-Rong Bao, Xingzhou Wang, Ya Qin, Xuetao Han, Xiaoming Yu, Cong-Cong Lei, Xiao-Tian Zhang, Ming-Yuan Yu, He-Ting Sun, Shuo Liu Frontiers in Veterinary Science.2025;[Epub] CrossRef
Distribution of human-pathogenic Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi in crab-eating macaques in China Huilin Zhang, Huiyang Chen, Chaoyue He, Wenchao Li, Falei Li Frontiers in Microbiology.2025;[Epub] CrossRef
Emergence of Cryptosporidium parvum IIc Subtype and Giardia duodenalis Assemblage E in AIDS Patients in Central China: Evidence for Neglected Transmission Dynamics Zhuolin Tao, Ke Hong, Peixi Qin, Hui Liu, Chunqun Wang, Jigang Yin, Xin Li, Guan Zhu, Min Hu Microorganisms.2025; 13(8): 1731. CrossRef
Subtyping of Nonhuman Primate-Adapted
Cryptosporidium hominis
in
Macaca Fascicularis
and
Macaca mulatta
in Yunnan Province, Southwestern China
Yue-Yue Zhang, Yang Zou, Ya-Qi Li, Ping-Ping Ma, Zhong-Li Liu, Shuai Wang, Xiao-Lin Sun Vector-Borne and Zoonotic Diseases.2023; 23(7): 364. CrossRef
Presence and genetic diversity of enteric protists in captive and semi-captive non-human primates in côte d’Ivoire, Sierra Leone, and Peru Pamela C. Köster, Juan Lapuente, Andrea Pizarro, Laura Prieto-Pérez, Ramón Pérez-Tanoira, Alejandro Dashti, Begoña Bailo, Aly S. Muadica, David González-Barrio, Rafael Calero-Bernal, Francisco Ponce-Gordo, David Carmena International Journal for Parasitology: Parasites and Wildlife.2022; 17: 26. CrossRef
Intestinal Protists in Captive Non-human Primates and Their Handlers in Six European Zoological Gardens. Molecular Evidence of Zoonotic Transmission Pamela C. Köster, Eva Martínez-Nevado, Andrea González, María T. Abelló-Poveda, Hugo Fernández-Bellon, Manuel de la Riva-Fraga, Bertille Marquet, Jean-Pascal Guéry, Tobias Knauf-Witzens, Annika Weigold, Alejandro Dashti, Begoña Bailo, Elena Imaña, Aly S. Frontiers in Veterinary Science.2022;[Epub] CrossRef
Prevalence and genetic diversity of Enterocytozoon bieneusi in nonhuman primates in Northern and Central China Peiyang Zhang, Shengyong Feng, Ting Jia, Shuyi Han, Chenglin Zhang, Hongxuan He International Journal for Parasitology: Parasites and Wildlife.2022; 17: 273. CrossRef
High zoonotic potential of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi in wild nonhuman primates from Yunnan Province, China Fanfan Shu, Shujiao Song, Yanting Wei, Falei Li, Yaqiong Guo, Yaoyu Feng, Lihua Xiao, Na Li Parasites & Vectors.2022;[Epub] CrossRef
Decline in Cryptosporidium Infection in Free-Ranging Rhesus Monkeys in a Park After Public Health Interventions Ruilian Jia, Xi Wen, Yaqiong Guo, Lihua Xiao, Yaoyu Feng, Na Li Frontiers in Cellular and Infection Microbiology.2022;[Epub] CrossRef
Ecological and public health significance of Enterocytozoon bieneusi Wei Li, Lihua Xiao One Health.2021; 12: 100209. CrossRef
Occurrence and potentially zoonotic genotypes of Enterocytozoon bieneusi in wild rhesus macaques (Macaca mulatta) living in Nanwan Monkey Island, Hainan, China: a public health concern Wei Zhao, Huan-Huan Zhou, Guang-Xu Ren, Yu Qiang, Hui-Cong Huang, Gang Lu, Feng Tan BMC Veterinary Research.2021;[Epub] CrossRef
Occurrence and molecular characterization of Cryptosporidium spp. and Giardia duodenalis among captive mammals in the Bangladesh National Zoo Md Robiul Karim, Junqiang Li, Farzana Islam Rume, Sm. Mostafizur Rahaman Sumon, Abu Sadeque Md Selim, Nazmul Hoda, Longxian Zhang Parasitology International.2021; 84: 102414. CrossRef
Investigation of giardiasis in captive animals in zoological gardens with strain typing of assemblages in China Hua Liu, Bin Wang, Jianhai Yin, Zhongying Yuan, Yanyan Jiang, Jing Zhang, Jianping Cao, Yujuan Shen, Hui Liu Parasitology.2021; 148(11): 1360. CrossRef
Risk Evaluation of Pathogenic Intestinal Protozoa Infection Among Laboratory Macaques, Animal Facility Workers, and Nearby Villagers From One Health Perspective Jian Li, Yijing Ren, Haiying Chen, Weiyi Huang, Xinyu Feng, Wei Hu Frontiers in Veterinary Science.2021;[Epub] CrossRef
Genotyping of Enterocytozoon bieneusi among captive long-tailed macaques (Macaca fascicularis) in Hainan Province: High genetic diversity and zoonotic potential Wei Zhao, Huanhuan Zhou, Hairong Jin, Lijie Sun, Pingyung Li, Meicen Liu, Mingyan Qiu, Liulian Xu, Feiyang Li, Tianming Ma, Shanshan Wang, Feifei Yin, Lihua Li, Xiuji Cui, Jasper Fuk-Woo Chan, Gang Lu Acta Tropica.2020; 201: 105211. CrossRef
Occurrence and multilocus genotyping of Giardia duodenalis in captive non-human primates from 12 zoos in China Xueping Zhang, Liqin Wang, Xinting Lan, Jiaming Dan, Zhihua Ren, Suizhong Cao, Liuhong Shen, Junliang Deng, Zhicai Zuo, Shumin Yu, Ya Wang, Xiaoping Ma, Haifeng Liu, Ziyao Zhou, Yanchun Hu, Hualin Fu, Changliang He, Yi Geng, Xiaobin Gu, Guangneng Peng, Yu PLOS ONE.2020; 15(2): e0228673. CrossRef
Prevalence and genotypic identification of Cryptosporidium spp. and Enterocytozoon bieneusi in captive Asiatic black bears (Ursus thibetanus) in Heilongjiang and Fujian provinces of China Sheng-Nan Wang, Yun Sun, Huan-Huan Zhou, Gang Lu, Meng Qi, Wei-Shi Liu, Wei Zhao BMC Veterinary Research.2020;[Epub] CrossRef
Multilocus sequence typing of Enterocytozoon bieneusi in crab-eating macaques (Macaca fascicularis) in Hainan, China Li Chen, Na Li, Yaqiong Guo, Jianguo Zhao, Yaoyu Feng, Lihua Xiao Parasites & Vectors.2020;[Epub] CrossRef
A retrospective epidemiological analysis of human Cryptosporidium infection in China during the past three decades (1987-2018) Aiqin Liu, Baiyan Gong, Xiaohua Liu, Yujuan Shen, Yanchen Wu, Weizhe Zhang, Jianping Cao, Luther A. Bartelt PLOS Neglected Tropical Diseases.2020; 14(3): e0008146. CrossRef
Prevalence and molecular characterization of Giardia duodenalis in dogs in Israel Harold Salant, Sharon Kuzi, Danielle Navarro, Gad Baneth Comparative Immunology, Microbiology and Infectious Diseases.2020; 73: 101548. CrossRef
Population genetic analysis suggests genetic recombination is responsible for increased zoonotic potential of Enterocytozoon bieneusi from ruminants in China Zhenjie Zhang, Jingbo Ma, Xitong Huang, Xi Wen, Wen Jiang, Li Chen, Na Li, Yaqiong Guo, Longxian Zhang, Lihua Xiao, Yaoyu Feng One Health.2020; 11: 100184. CrossRef
Host Specificity of Enterocytozoon bieneusi and Public Health Implications Wei Li, Yaoyu Feng, Monica Santin Trends in Parasitology.2019; 35(6): 436. CrossRef
Intestinal Parasites and the Occurrence of Zoonotic Giardia duodenalis Genotype in Captive Gibbons at Krabokkoo Wildlife Breeding Center, Thailand Sahatchai Tangtrongsup, Duanghatai Sripakdee, Suchinda Malaivijitnond, Rungroj Angkuratipakorn, Michael Lappin Frontiers in Veterinary Science.2019;[Epub] CrossRef
Molecular prevalence and subtyping of Cryptosporidium hominis among captive long-tailed macaques (Macaca fascicularis) and rhesus macaques (Macaca mulatta) from Hainan Island, southern China Wei Zhao, Huanhuan Zhou, Hairong Jin, Meicen Liu, Mingyan Qiu, Lihua Li, Feifei Yin, Jasper Fuk-Woo Chan, Gang Lu Parasites & Vectors.2019;[Epub] CrossRef
Genotypes and public health potential of Enterocytozoon bieneusi and Giardia duodenalis in crab-eating macaques Li Chen, Jianguo Zhao, Na Li, Yaqiong Guo, Yuanyuan Feng, Yaoyu Feng, Lihua Xiao Parasites & Vectors.2019;[Epub] CrossRef
Prevalence and diversity of Cryptosporidium spp. in bamboo rats (Rhizomys sinensis) in South Central China Zilin Wei, Qiang Liu, Wei Zhao, Xiumei Jiang, Ying Zhang, Aiyun Zhao, Bo Jing, Gang Lu, Meng Qi International Journal for Parasitology: Parasites and Wildlife.2019; 9: 312. CrossRef
Cryptosporidium parvum and Cryptosporidium hominis subtypes in crab-eating macaques Li Chen, Suhui Hu, Wen Jiang, Jianguo Zhao, Na Li, Yaqiong Guo, Chenghong Liao, Qian Han, Yaoyu Feng, Lihua Xiao Parasites & Vectors.2019;[Epub] CrossRef
Molecular characterization and distribution of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi from yaks in Tibet, China Yayun Wu, Yankai Chang, Xiangqian Zhang, Yuancai Chen, Dongfang Li, Lu Wang, Shuangjian Zheng, Rongjun Wang, Sumei Zhang, Fuchun Jian, Changshen Ning, Jiakui Li, Longxian Zhang BMC Veterinary Research.2019;[Epub] CrossRef
Zoonotic potential of Giardia lamblia and control of giardiasis Fantinatti* Maria Insights in Veterinary Science.2019; 3(1): 001. CrossRef
Cryptosporidium parvum in captive primates of Parque Municipal Danilo Galafassi, Paraná, Brazil Alessandra Snak, Luis Eduardo da Silveira Delgado, Silvia Cristina Osaki Semina: Ciências Agrárias.2019; 40(2): 987. CrossRef
Prevalence and molecular characterization of Cryptosporidium spp. and Giardia duodenalis in deer in Henan and Jilin, China Jianying Huang, Zhenjie Zhang, Yiqi Zhang, Yong Yang, Jinfeng Zhao, Rongjun Wang, Fuchun Jian, Changshen Ning, Wanyu Zhang, Longxian Zhang Parasites & Vectors.2018;[Epub] CrossRef
Prevalence and genotypes of Enterocytozoon bieneusi in China Sha-Sha Wang, Rong-Jun Wang, Xian-Cheng Fan, Ting-Li Liu, Long-Xian Zhang, Guang-Hui Zhao Acta Tropica.2018; 183: 142. CrossRef
Host specificity of Enterocytozoon bieneusi genotypes in Bactrian camels (Camelus bactrianus) in China Meng Qi, Junqiang Li, Aiyun Zhao, Zhaohui Cui, Zilin Wei, Bo Jing, Longxian Zhang Parasites & Vectors.2018;[Epub] CrossRef
Molecular characterization and new genotypes of Enterocytozoon bieneusi in pet chipmunks (Eutamias asiaticus) in Sichuan province, China Lei Deng, Wei Li, Zhijun Zhong, Yijun Chai, Leli Yang, Hang Zheng, Wuyou Wang, Hualin Fu, Min He, Xiangming Huang, Zhicai Zuo, Ya Wang, Suizhong Cao, Haifeng Liu, Xiaoping Ma, Kongju Wu, Guangneng Peng BMC Microbiology.2018;[Epub] CrossRef
Giardia duodenalisin primates: Classification and host specificity based on phylogenetic analysis of sequence data O. Brynildsrud, K. R. Tysnes, L. J. Robertson, J. J. Debenham Zoonoses and Public Health.2018; 65(6): 637. CrossRef
Enterocytozoon bieneusi in Minks (Neovison vison) in Northern China: A Public Health Concern Xiao-Xuan Zhang, Ruo-Lan Jiang, Jian-Gang Ma, Chao Xu, Quan Zhao, Guangyu Hou, Guo-Hua Liu Frontiers in Microbiology.2018;[Epub] CrossRef
Molecular characterization and new genotypes of Enterocytozoon bieneusi in minks (Neovison vison) in China Wei Cong, Si-Yuan Qin, Qing-Feng Meng Parasite.2018; 25: 34. CrossRef
Prevalence and multi-locus genotypes of Enterocytozoon bieneusi in black-boned sheep and goats in Yunnan Province, southwestern China Dan Chen, Sha-Sha Wang, Yang Zou, Zhao Li, Shi-Chen Xie, Lian-Qin Shi, Feng-Cai Zou, Xing-Quan Zhu, Jian-Fa Yang, Guang-Hui Zhao Infection, Genetics and Evolution.2018; 65: 385. CrossRef
High prevalence of Enterocytozoon bieneusi zoonotic genotype D in captive golden snub-nosed monkey (Rhinopithecus roxellanae) in zoos in China Fuchang Yu, Yayun Wu, Tongyi Li, Jianke Cao, Jiantang Wang, Suhui Hu, Huili Zhu, Sumei Zhang, Rongjun Wang, Changshen Ning, Longxian Zhang BMC Veterinary Research.2017;[Epub] CrossRef
Molecular characterisation of protist parasites in human-habituated mountain gorillas (Gorilla beringei beringei), humans and livestock, from Bwindi Impenetrable National Park, Uganda Matthew J. Nolan, Melisa Unger, Yuen-Ting Yeap, Emma Rogers, Ilary Millet, Kimberley Harman, Mark Fox, Gladys Kalema-Zikusoka, Damer P. Blake Parasites & Vectors.2017;[Epub] CrossRef
Molecular characterization of Enterocytozoon bieneusi isolates in laboratory macaques in north China: zoonotic concerns Hang Yang, Yongchao Lin, Yijing Li, Mingxin Song, Yixin Lu, Wei Li Parasitology Research.2017; 116(10): 2877. CrossRef
Molecular Epidemiology of Cryptosporidiosis in China Yaoyu Feng, Lihua Xiao Frontiers in Microbiology.2017;[Epub] CrossRef
Multilocus genotyping of Giardia duodenalis in captive non-human primates in Sichuan and Guizhou provinces, Southwestern China Zhijun Zhong, Yinan Tian, Wei Li, Xiangming Huang, Lei Deng, Suizhong Cao, Yi Geng, Hualin Fu, Liuhong Shen, Haifeng Liu, Guangneng Peng, Bi-Song Yue PLOS ONE.2017; 12(9): e0184913. CrossRef
Giardia duodenalis Infections in Humans and Other Animals in China Junqiang Li, Haiyan Wang, Rongjun Wang, Longxian Zhang Frontiers in Microbiology.2017;[Epub] CrossRef
Dominance of the Enterocytozoon bieneusi genotype BEB6 in red deer (Cervus elaphus) and Siberian roe deer (Capreolus pygargus) in China and a brief literature review Wei Zhao, Jianguang Wang, Ziyin Yang, Aiqin Liu Parasite.2017; 24: 54. CrossRef
Public health significance of zoonotic Cryptosporidium species in wildlife: Critical insights into better drinking water management Alireza Zahedi, Andrea Paparini, Fuchun Jian, Ian Robertson, Una Ryan International Journal for Parasitology: Parasites and Wildlife.2016; 5(1): 88. CrossRef
Molecular survey of Enterocytozoon bieneusi in sheep and goats in China Ke Shi, Mengjie Li, Xiaoxing Wang, Junqiang Li, Md Robiul Karim, Rongjun Wang, Longxian Zhang, Fuchun Jian, Changshen Ning Parasites & Vectors.2016;[Epub] CrossRef
Prevalence, risk factors and multilocus genotyping of Enterocytozoon bieneusi in farmed foxes (Vulpes lagopus), Northern China Xiao-Xuan Zhang, Wei Cong, Zhi-Long Lou, Jian-Gang Ma, Wen-Bin Zheng, Qiu-Xia Yao, Quan Zhao, Xing-Quan Zhu Parasites & Vectors.2016;[Epub] CrossRef
The zoonotic potential of Giardia intestinalis assemblage E in rural settings Khaled A. Abdel-Moein, Hossam Saeed Parasitology Research.2016; 115(8): 3197. CrossRef
Identification ofGiardia lambliaAssemblage E in Humans Points to a New Anthropozoonotic Cycle Maria Fantinatti, Alexandre R. Bello, Octavio Fernandes, Alda M. Da-Cruz Journal of Infectious Diseases.2016; 214(8): 1256. CrossRef
First Report of the Human-Pathogenic Enterocytozoon bieneusi from Red-Bellied Tree Squirrels (Callosciurus erythraeus) in Sichuan, China Lei Deng, Wei Li, Xingming Yu, Chao Gong, Xuehan Liu, Zhijun Zhong, Na Xie, Shuangshuang Lei, Jianqiu Yu, Hualin Fu, Hongwei Chen, Huailiang Xu, Yanchun Hu, Guangneng Peng, Tara C Smith PLOS ONE.2016; 11(9): e0163605. CrossRef
Prevalence of Cryptosporidium spp., Enterocytozoon bieneusi, Encephalitozoon spp. and Giardia intestinalis in Wild, Semi-Wild and Captive Orangutans (Pongo abelii and Pongo pygmaeus) on Sumatra and Borneo, Indonesia Anna Mynářová, Ivona Foitová, Martin Kváč, Dana Květoňová, Michael Rost, Helen Morrogh-Bernard, Wisnu Nurcahyo, Cathleen Nguyen, Supriyadi Supriyadi, Bohumil Sak, Govindhaswamy Umapathy PLOS ONE.2016; 11(3): e0152771. CrossRef
Cryptosporidium spp., Giardia intestinalis, and Enterocytozoon bieneusi in Captive Non-Human Primates in Qinling Mountains
Fig. 1. Phylogenetic relationships of E. bieneusi genotypes identified in this study and other genotypes previously deposited in GenBank as inferred by the neighbor-joining analysis of ITS sequences based on genetic distances calculated by the Kimura 2-parameter model. Bootstrap values greater than 50% from 1,000 replicates are shown on nodes. Each sequence from GenBank is identified by the accession number, host origin, and the genotype designation. Genotypes identified in this study are indicated by the symbol (◆).
Fig. 1.
Cryptosporidium spp., Giardia intestinalis, and Enterocytozoon bieneusi in Captive Non-Human Primates in Qinling Mountains
