Occurrence and multilocus genotyping of Giardia duodenalis in captive non-human primates from 12 zoos in China

Giardia duodenalis is a common enteric protozoan that infects a range of hosts including humans and other mammals. Multilocus genotyping of G. duodenalis in captive non-human primates (NHPs) from zoos in China is limited. In this study, we evaluated 302 NHP fecal samples collected from 32 different NHP species. The primates were from 12 zoos distributed across eight provinces and two municipalities (Chongqing and Beijing) of China. The overall infection rate was 8.3% (25/302). The six G. duodenalis-positive zoos and their infection rates were: Suzhou Zoo (40.0%, 4/10), Yangzhou Zoo (22.2%, 2/9), Dalian Zoo (16.7%, 4/24), Chengdu Zoo (12.8%, 6/47), Guiyang Forest Wildlife Zoo (12.1%, 7/58), and Changsha Zoo (4.7%, 2/43). Molecular analysis of three loci, beta-giardin (bg), triose phosphate isomerase (tpi), and glutamate dehydrogenase (gdh), showed high genetic heterogeneity, and seven novel subtypes (BIII-1, MB10-1, WB8-1, B14-1, MB9-1, DN7-1, and BIV-1) were detected within assemblage B. Additional analysis revealed 12 different assemblage B multilocus genotypes (MLGs), one known MLG and 11 novel MLGs. Based on phylogenetic analysis, 12 assemblage B MLGs formed two main clades, MLG-SW (10–12, 18) and MLG-SW (13, 14, 16, 17), the other four MLG-SW (15, 19, 20, 21) were scattered throughout the phylogenetic tree in this study. Using multilocus genotyping, this study expands our understanding of the occurrence of Giardia infection and genetic variation in Giardia in captive non-human primates from zoos in China.

To date, most Chinese studies evaluating G. duodenalis infection in NHPs have focused on a single zoo or localized area. Only three studies have extended their investigation to include a larger geographical region [8,15,16]. Ongoing epidemiological surveys on intestinal zoonotic parasites of G. duodenalis, expanded previous studies to large-scale investigation of zoos and NHP species in China. We used multilocus genotyping to evaluate 302 NHP fecal samples (including 32 primate species) from 12 zoos distributed across eight Chinese provinces and two municipalities (Chongqing and Beijing), to better understand G. duodenalis infection in captive NHPs throughout China.

Ethics statement
This study was reviewed and approved by the Institutional Animal Care and Use Committee of Sichuan Agricultural University under permit number ZXP-2018303052. Prior to the collection of fecal specimens from NHPs, permission was obtained from the owners.

Sample collection
Fecal samples from 302 NHPs (including 32 primate species) were collected from March 2018 to January 2019 (S1 Table). The samples from 12 zoos are distributed throughout China (Fig 1), including Beijing Zoo (n = 12), Chengdu Zoo (n = 47), Changsha Zoo (n = 43), Chongqing Zoo (n = 33), Dalian Zoo (n = 24), Guiyang Forest Wildlife Zoo (n = 58), Guangzhou Zoo (n = 8), Kunming Zoo (n = 16), Nanjing Zoo (n = 16), Shaanxi Rare and Wildlife Zoo (n = 26), Suzhou Zoo (n = 10), and Yangzhou Zoo (n = 9). The 12 zoos have adequate facilities to accommodate the different species of primates in indoor enclosure, different species live on separated places, and the feed managements are according to the Standard Rule of Chinese Association of Zoological Gardens. All animals' samples were collected by visiting once. At the time of faecal collections, there were no reported case of diarrhoea in the NHPs. Fresh feces were collected and packed in clear, self-sealing, disposable plastic bags marked with ID numbers, and transported in ice-filled foam boxes. Samples were stored in 2.5% potassium dichromate at 4˚C until DNA was extracted.
The PCR primers and protocol used in this study were previously described [15]. The PCR reactions for the bg, tpi and gdh loci were conducted in 25 μL reaction mixtures containing of 12.5 μL 2× Taq PCR Master Mix (KT201-02, Tiangen, Beijing, China), 8.5 μL deionized water (Tiangen, Beijing, China), 2 μL DNA, and 1 μL each of set primers, respectively. The primers and annealing temperatures for the three genes were listed in Table 1. Secondary PCR products were visualized by 1% agarose gel electrophoresis and staining with Golden View.

Sequence analysis
All positive secondary PCR products were sequenced by BGI Tech Solutions (Liuhe Beijing) Co., Limited and were sequenced in both directions. Sequences were aligned with reference sequences from the GenBank database using BLAST (http://blast.ncbi.nlm.nih.gov) and Clustal X (http://www.clustal.org/). To evaluate the MLGs of G. duodenalis, we only included specimens that were successfully subtyped at all three loci and sequences with ambiguous positions (double peaks) were not included for phylogenetic analyses. Sequences were concatenated for each positive isolate to form a multilocus sequence (bg + tpi + gdh). All concatenated MLGs were used in a neighbour-joining analysis using the Kimura-2 parameter model calculated with MEGA 7 (http://www.megasoftware.net/). Representative nucleotide sequences obtained in this study were deposited in GenBank under the accession numbers: MK909127, MK909131, MK909135, MK909136, MK952610, MK952598, and MK952606.
A phylogenetic, evolutionary tree based on concatenated sequences was constructed to better understand the diversity and relationship between MLGs in NHPs and humans (Fig 2). Of the 12 MLGs we identified, 11 clustered with the NHPs isolates and 1 MLG (MLG21) clustered with human isolates from Sweden. The MLGs formed two main clades, MLG-SW (10)(11)(12)18) and MLG-SW (13,14,16,17). MLG-SW (15,19,20,21) was scattered throughout the phylogenetic tree. The role of NHPs in the transmission of G. duodenalis to humans is not clear; however, the occurrence of assemblage B detected in captive NHPs suggests transmission from humans or an adaptation to primate host [4,8].