Multilocus typing of Cryptosporidium spp. and Giardia duodenalis from non-human primates in China☆
Graphical abstract
Introduction
Cryptosporidium spp. and Giardia duodenalis are ubiquitous parasites in humans, domesticated animals and other mammalian, avian and reptilian vertebrates (Appelbee et al., 2005). Similar to other animals and humans, non-human primates (NHPs) are commonly infected with these parasitic pathogens (Ekanayake et al., 2006, Berrilli et al., 2011, Li et al., 2011, Ye et al., 2012). Both parasites are transmitted by the faecal-oral route, via either direct contact or ingestion of contaminated food or water (Xiao et al., 2004, Smith et al., 2006). In captive NHPs, these protozoa can disperse rapidly due to their monoxenous life cycle, low infective dose and short prepatent period.
Due to the paucity of molecular characterisations of these protozoa in NHPs, the importance of this group of hosts in the ecology of disease transmission remains ambiguous. Recently human pathogenic genotypes and subtypes of Cryptosporidium and Giardia have been reported in NHPs from China, Kenya, Italy, Belgium and the Netherlands (Levecke et al., 2009, Berrilli et al., 2011, Li et al., 2011, Ye et al., 2012). Thus, infected NHPs in zoological gardens, breeding farms and research facilities constitute a risk of transmission of diseases to animal caretakers and visitors. On the other hand, human carriers of these pathogens can infect captive NHPs by close contact or via food and water (Miller et al., 1990, Hamlen and Lawrence, 1994, Berrilli et al., 2011, Ye et al., 2012). Consequently, the infected NHPs can serve as reservoirs of human cryptosporidiosis and giardiasis (Ye et al., 2012).
Using molecular tools, it has been revealed that there is an extensive genetic variation within the genus Cryptosporidium, with 26 Cryptosporidium spp. and more than 70 genotypes being recognised (Zhang et al., 2013). To date, five Cryptosporidium spp. including Cryptosporidium hominis, Cryptosporidium parvum, Cryptosporidium felis, Cryptosporidium muris and Cryptosporidium ubiquitum have been detected in NHPs (Xiao and Fayer, 2008, Feng, 2010, Li et al., 2011, Ye et al., 2012). In addition, various subtype families and subtypes have been reported in C. hominis and C. parvum based on DNA sequence analysis of the 60 kDa glycoprotein gene (gp60). Among them, subtype families Ia, Ib, Id, Ie, If and Ii of C. hominis and IIc of C. parvum have been reported in NHPs (Feng et al., 2011, Li et al., 2011, Ye et al., 2012). Similarly, G. duodenalis has at least eight genotypes or assemblages (A to H) based on characterisation of the triosephosphate isomerase (tpi), ssrRNA, β-giardin (bg), glutamate dehydrogenase (gdh) and other genes (Ey et al., 1997, Thompson et al., 2000, Sulaiman et al., 2003, Read et al., 2004, Feng and Xiao, 2011). Assemblages A and B infect humans and a broad range of other hosts including NHPs (Vitazkova and Wade, 2006, Levecke et al., 2009, Feng and Xiao, 2011). Furthermore, assemblage A is divided into three subassemblages such as I, II and III, whereas assemblage B includes many subtypes (Feng et al., 2008, Geurden et al., 2009, Mahdy et al., 2009). Other assemblages (C to H) are mostly host-adapted (Monis et al., 2003, Sulaiman et al., 2003, Feng and Xiao, 2011). To date assemblages A, B and E have been detected in NHPs, with subtypes of assemblage B dominating (Karanis and Ey, 1998, Johnston et al., 2010, Feng and Xiao, 2011, Ryan and Cacciò, 2013).
Despite these advances in molecular epidemiology of Cryptosporidium spp. and G. duodenalis, there are still very few studies on these parasites in NHPs not only in China but also in other regions of the world. The present study was carried out with the aim of the molecularly characterizing Cryptosporidium spp. and G. duodenalis from NHPs in China to better understand their zoonotic potential.
Section snippets
Ethics statement
This study was conducted in accordance with the Chinese Laboratory Animal Administration Act 1988. Before performing the study, the protocol was reviewed and approved by the Research Ethics Committee of the Henan Agricultural University, Zhengzhou PR China. Samples were obtained from faeces of the animals after the permission of owners or authorities responsible for animals.
Specimen collection
A total of 2,660 fresh faecal specimens were collected between 2006 and 2013 in China from 26 species of NHPs residing in
Prevalence of Cryptosporidium
On microscopic examination using Sheather’s sugar flotation technique, 13 (0.5%, 13/2,660) specimens were positive for Cryptosporidium (Table 1). Based on PCR analysis of the 18S rRNA locus, Cryptosporidium was detected in 19 (0.7%) of the 2,660 faecal specimens, belonging to the species C. hominis (73.7%, 14/19) and C. muris (26.3%, 5/19). Cryptosporidium was found in four of 26 NHP species studied including rhesus macaques (0.7%, 9/1,316), cynomolgus monkeys (1.0%, 8/778), slow lorises
Discussion
In this study, Cryptosporidium was identified at a very low rate by both microscopic examination and PCR assay in NHP faecal specimens. On 18S rRNA locus-based PCR analysis, Cryptosporidium was detected at a rate of 0.7% in NHPs, including 0.7% in rhesus macaques, 1.0% in cynomolgus monkeys, 10.0% in slow lorises and 6.7% in Francois’ leaf monkeys. Low prevalence of cryptosporidiosis was reported recently in laboratory cynomolgus monkeys (0.5%) from Guangxi in China (Ye et al., 2014), and newly
Acknowledgements
This study was supported in part by the International Cooperation and Exchange Projects of the National Natural Science Foundation of China (No. 31110103901), the State Key Program of National Natural Science Foundation of China (31330079), the Key National Science and Technology Specific Projects, China (No. 2012ZX10004220-001), the Program for Science and Technology Innovative Research Team in University of Henan Province, China (012IRTSTHN005).
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Note: Nucleotide sequence data reported in this paper are available in the GenBank database under accession numbers GU319778–GU319786 and KF679722–KF679746.