Intestinal Parasites and the Occurrence of Zoonotic Giardia duodenalis Genotype in Captive Gibbons at Krabokkoo Wildlife Breeding Center, Thailand

Intestinal parasitic infections can have an impact on health and growth of wildlife. The current study aims were to determine the prevalence of intestinal parasites and to molecular characterize Giardia duodenalis and Cryptosporidium spp. in captive gibbons at Krabokkoo Wildlife Breeding Center, Thailand. Fifty-five gibbons, 2 agile- (Hylobates agilis), 38 lar- (Hylobates lar) and 15 pileated gibbons (Hylobates pileatus) were included in this study. Fecal samples were collected individually at Krabokkoo Wildlife Breeding Center, Chachoengsao province, eastern Thailand, in November 2013. Intestinal parasitic infections were examined by zinc sulfate centrifugation flotation and by a commercially available immunofluorescent assay (IFA) for detection of G. duodenalis and Cryptosporidium spp.. Polymerase chain reaction targeting the Giardia glutamate dehydrogenase (gdh), beta- giardin (bg), triose phosphate isomerase (tpi) genes, and the Cryptosporidium small subunit-rRNA and heat-shock protein (hsp70) following by DNA sequencing were performed on the IFA positive samples. The overall prevalence of intestinal parasitic infection in gibbons at Krabokkoo Wildlife Breeding Center was 12.7% (95%CI: 5.3–24.5), Strongyloides spp. eggs or larvae were present in all positive samples. Co-infections with G. duodenalis were detected in 1.8% (95%CI: 0.1–9.7) of the samples. Based on the sequencing results of the three genes, the IFA Giardia positive isolate typed as the zoonotic genotype B. Since the data reveals the occurrence of zoonotic Giardia genotype, good hygiene management is suggested to prevent the transmission of this pathogen from gibbon to human, and vice versa.

Intestinal parasitic infections can have an impact on health and growth of wildlife. The current study aims were to determine the prevalence of intestinal parasites and to molecular characterize Giardia duodenalis and Cryptosporidium spp. in captive gibbons at Krabokkoo Wildlife Breeding Center, Thailand. Fifty-five gibbons, 2 agile-(Hylobates agilis), 38 lar-(Hylobates lar) and 15 pileated gibbons (Hylobates pileatus) were included in this study. Fecal samples were collected individually at Krabokkoo Wildlife Breeding Center, Chachoengsao province, eastern Thailand, in November 2013. Intestinal parasitic infections were examined by zinc sulfate centrifugation flotation and by a commercially available immunofluorescent assay (IFA) for detection of G. duodenalis and Cryptosporidium spp.. Polymerase chain reaction targeting the Giardia glutamate dehydrogenase (gdh), beta-giardin (bg), triose phosphate isomerase (tpi) genes, and the Cryptosporidium small subunit-rRNA and heat-shock protein (hsp70) following by DNA sequencing were performed on the IFA positive samples. The overall prevalence of intestinal parasitic infection in gibbons at Krabokkoo Wildlife Breeding Center was 12.7% (95%CI: 5.3-24.5), Strongyloides spp. eggs or larvae were present in all positive samples. Co-infections with G. duodenalis were detected in 1.8% (95%CI: 0.1-9.7) of the samples. Based on the sequencing results of the three genes, the IFA Giardia positive isolate typed as the zoonotic genotype B. Since the data reveals the occurrence of zoonotic Giardia genotype, good hygiene management is suggested to prevent the transmission of this pathogen from gibbon to human, and vice versa.

INTRODUCTION
Intestinal parasitic infections are the most common causes of gastrointestinal diseases in captive wildlife. These infections can cause a wide range of clinical signs, from subclinical infections to malabsorption, abdominal pain, diarrhea, vomiting, anemia, severe dehydration, and death (1)(2)(3). As the living area is limited, stress and other factors such as artificial environment, poor diet or the presence of humans lead to the high risk of infection and weaken the natural resistance of the host, making the clinical illness possible (4). The weakened health condition of these captive animals can have a negative impact on their reproduction which is of major concern in the zoos and wildlife breeding facilities of captive or endangered species (3,5).
Currently, there is no information available regarding intestinal parasitic infection in captive gibbons in breeding facilities in Thailand. Knowing background prevalence of gastrointestinal parasites can be beneficial in the health management program in gibbons for the reproduction at the Krabokkoo Wildlife Breeding Center. The aims of this study were, therefore, to determine the prevalence of intestinal parasites and to molecular characterize Giardia duodenalis and Cryptosporidium spp. isolates to determine the potential of zoonotic transmissions of these pathogens from captive gibbons at Krabokkoo Wildlife Breeding Center, Thailand.

Study Area
Krabokkoo Wildlife Breeding Center is located in Chachoengsao province, eastern Thailand, at the coordinates of 13 • 28 ′ 5.05 ′′ N, 101 • 35 ′ 37.30 ′′ E (Figure 1) and at 47 meters above the sea level. In November 2013, this facility accommodated four species of gibbons, 65 white-handed (Hylobates lar), 15 pileated (Hylobates pileatus), 2 agile (Hylobates agilis), and 2 crown (Nomascus spp.) gibbons. The gibbons were separated among species and were housed individually or in groups. Siblings or a family were housed together. They were fed with vegetable-and fruit-based diet and water was supplied in a bowl. Drinking water was replaced on a daily basis. All gibbons were dewormed every 3 months. The temperature are 23-27 • C in winter (mid-Octobermid-February), 35-40 • C in summer (mid-February-mid-May), and 28-35 • C in rainy season (mid-May-mid-October) (32).

Fecal Sample and Data Collection
Fifty-five fecal samples were collected from white-handed (H. lar) (n = 38), pileated (H. pileatus) (n = 15), and agile (H. agilis) (n = 2) gibbons during the November of 2013. The samples used in the study were part of the study on genetic diversity of macaques and Hylobatidae gibbons in Thailand. Each fecal sample was collected from the ground (care was taken not to have soil contamination), kept in a labeled plastic bag and stored at 4 • C until examination. Sex, age, cage and identification number were recorded at the time of collection. Fecal samples were shipped on ice to the Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai, Thailand within a week and the fecal consistencies were determined upon arrival.

Microscopic Examination of Fecal Samples
After Zinc Sulfate Centrifugal Flotation and IFA for Giardia duodenalis and Cryptosporidium spp. Detection Fecal samples were examined for the presence of intestinal parasitic eggs, larvae, protozoal cysts and oocysts using microscopic examination after zinc sulfate centrifugal flotation (33). Giardia duodenalis and Cryptosporidium spp. infections were determined using a commercially available direct immunofluorescent assay (IFA) (MeriFluor R Cryptosporidium/Giardia Test Kit, Meridian Diagnostic Corporation, Cincinnati, OH). Prior IFA, the fecal samples (3 grams) were concentrated using sucrose gradient centrifugation technique as previously described (34). IFA was carried out according to the manufacturer's instruction.

DNA Isolation and Molecular Detection of Giardia duodenalis Infection
Three hundred microliters of each Giardia or Cryptosporidium IFA positive fecal concentrate were subjected to DNA extraction using the FastDNA R kit (MP Biomedicals, Solon, OH, USA) following an established protocol (35).

DNA Sequencing and Phylogenetic Analysis
The PCR products were purified using QIAquick Gel Extraction Kit (Qiagen, OH, USA) and purified PCR product was evaluated by nucleotide sequencing using a commercially available service (1st Base Laboratory, Selangor, Malaysia). For each target gene, the obtained sequences from both directions were aligned and a consensus sequence was generated and compared with nucleotide sequences from the nucleotide database from the GenBank using BLAST analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Phylogenetic and molecular analyses were conducted using the MEGA 6.06 program (43). Multiple sequence alignments were performed using MUSCLE (44), and the phylogenetic analyses were performed by the Maximum Likelihood method based on the Kimura 2-parameter model. The consensus tree was obtained after bootstrap analysis with 500 replications. Reference strains of the different assemblages were retrieved from the GenBank and included for comparative phylogenetic analyses.

Statistical Analyses
A sample was considered positive for gastrointestinal parasites if parasitic eggs or larvae were detected by light microscopic examination after zinc sulfate centrifugal flotation. A sample was considered positive for Giardia and Cryptosporidium if at least one (oo)cyst was detected by either microscopic examination or IFA. Gibbons were grouped by species, age (<10 years, ≥10 years), and sex. Overall prevalence and 95% confidence interval (95%CI) were calculated.
Associations of age category, sex, fecal consistency, gibbon species, and parasitic infestation results were analyzed using Fisher's Exact test. A P < 0.05 was considered statistically significant. All statistical analyses were performed using STATA statistical software release 10.1 (Stata Corp., College Station, Texas, USA).

Microscopic Examination of Fecal Samples After Zinc Sulfate Centrifugal Flotation and IFA for Giardia duodenalis and Cryptosporidium spp. Infections
Characteristics of gibbons and samples and the descriptive statistics are shown in Table 1. Of 55 fecal samples, Strongyloides spp. eggs or larvae were detected in 7 samples by microscopic examination after zinc sulfate centrifugal flotation. Giardia cysts were detected in one fecal sample by IFA. Cryptosporidium oocysts were not detected by IFA in any fecal samples, therefore, PCR assays were not performed.

Giardia duodenalis Sequences and Phylogenetic Analyses
DNA fragments of the only IFA Giardia positive sample (G29) were successfully amplified and typed as assemblage B by the three genes (gdh, bg, and tpi). The gdh sequence of G29 showed 99% homology to the assemblage B gdh sequences recovered from a water sample and a beaver in Canada, an ostrich in Brazil, a human and a dog in Australia, and a human from India (Figure 2). The G29 gdh sequence has 3 SNPs (single-nucleotide polymorphism) at position 12 (A vs. T), 93 (A vs. G), and 199 (A vs. G), when compared to those sequences mentioned previously; however, neither of these SNPs resulted in amino acid change. The beta-giardin sequence of G29 showed 100% homology to the assemblage B from human from Thailand and India and 99.9% homology to assemblage B human isolates from Kenya, Egypt, Brazil, and Ethiopia (Figure 3). Sequences from tpi gene contained ambiguous nucleotides at position 108 (T or G) and 443 (A or T). When translating the G29 tpi sequence to amino acids, substitution of T with G at position 108 did not cause amino acid change, whilst substitution of A with T at position 443 resulted in an amino acid change from Valine to Aspartic acid. Variants of tpi sequences showed 99-100% homology to the tpi sequences recovered from rhesus macaque, long-tailed macaque, and gibbons from China, Sumatran Orangutan from Indonesia, beaver from Canada, cat from Japan, rabbit from Nigeria, and humans from Canada, Malaysia, and Spain (Figure 4). From the phylogenetic analyses of gdh, bg and tpi genes, the G. duodenalis isolate in this study was placed into BIV, BI, and BIV branch, respectively (Figures 2-4).

Statistical Analyses
Due to the low detection of the parasites and the small sample size, the power to detect associations between any risk factors and infections was insufficient.

DISCUSSION
The current study represents the first report of the intestinal parasites as well as G. duodenalis and Cryptosporidium spp. prevalence rates and Giardia genotypes in captive gibbons in Thailand. The prevalence of these infections were commonly high and ranged from 25 to 100% in either free-ranging (5, 7-10, 22, 28, 31, 45, 46) or captive non-human primates (4, 7, 11, 13-15, 20, 23-26, 46, 47). In the current study, overall prevalence rates of nematodes, G. duodenalis, and Cryptosporidium spp. in gibbons were 12.7, 1.8, and 0%, respectively. These prevalence rates, however, were comparable to the previous report of 0-16.4% in gibbons in zoological parks in China (48). The low prevalence in this study may be from collecting a single fecal sample from each animal. Parasitic eggs, Giardia cysts and Cryptosporidium oocysts are intermittent shed in the feces, therefore, three or more fecal samples from animals can increase the sensitivity of intestinal parasites (33). In addition, the low detection rate of Giardia and the lack of Cryptosporidium spp. were also because of the number of cysts/oocysts were below the detection limit of the diagnostic tests used in the study (49).
The most common helminthic species detected in NHP were Strongyloides spp., Trichuris spp., Oesophagostomum spp., Ascaris spp. and hookworms (5,7,22,45,46). A similar pattern of gastrointestinal parasites was also observed in captive gibbons in a zoo in China (13). In a study in 23 wild white-handed gibbons at Khao Yai National Park, Thailand, Trichuris spp. and Ternidens spp. were the most prevalent helminthic parasites detected (91.3%), followed by Strongyloides fuelleborni (56.5%) (9). However, in the current study, only Strongyloides spp. eggs or larvae were detected in the fecal samples. Strongyloides spp. are soil-transmitted nematodes with an estimated 370 million people infected worldwide (50). In this study, the detection of Strongyloides spp. in feces is less likely to be from contaminated soil as fecal samples were carefully collected not be contaminated with soil before the storage in a plastic bag. These nematodes can cause a chronic and persistent strongyloidiasis in the infected host because of the autoinfective life cycle (51) and cause diarrhea, hyperinfection syndrome, dissemination, and death in immunocompromised hosts. Strongyloides stercoralis is a primary species infecting human; however, the infections of primates' parasites S. fuelleborni fuelleborni and S. fuelleborni kellyi have also been reported (8). The molecular characterization of Strongyloides positive samples is suggested since microscopic identification is insufficient for species identification and determination of its zoonotic potential. In this study, the species identification was not performed but this finding has raised concerns regarding the zoonotic potential. Since the FIGURE 4 | Phylogenetic tree of Giardia isolate based on the sequence of triose phosphate isomerase (tpi) gene from a gibbon in this study by the Maximum Likelihood algorithm using the MEGA 6.06 program. Sequences obtained from GenBank are indicated by their accession numbers. Percentage bootstrap supports (500 replicates) are shown by numbers at the respective nodes. Bold texts represent the Giardia detected in this study.
fatal strongyloidiasis cases of gibbons in Thailand has been reported (1) and Strongyloides spp. is also an important parasitic helminth of humans (8), an effective anthelminthic program is recommended.
Giardia duodenalis and Cryptosporidium spp. are important intestinal protozoans in non-human primates. These pathogens can cause a wide range of clinical signs, from subclinical to malabsorption, abdominal pain, failure to thrive, acute or chronic diarrhea especially in young, old and immunecompromised animals (3,52,53). The organisms are commonly found in both free-ranging and captive non-human primates with the prevalence from 0-70% to 0-48%, for Giardia and Cryptosporidium infections, respectively (19,21,22,24,31,47,54). In Thailand, a low prevalence rate (1/23, 4.35%) of Cryptosporidium spp. infection has been previously reported in wild white-handed gibbons at Khao Yai National Park in Thailand. IFA was used for the detection of Giardia cysts or Cryptosporidium oocysts in repeatedly collected fecal samples that ranged from 3 to 25 samples per gibbon, resulting in a total of 324 samples (9). In that study, there was no Giardia detected. In this study, in contrast, no Cryptosporidium oocysts were detected in all fecal samples and Giardia cysts were detected in only one fecal sample of 55 samples. These findings could be due to that the numbers of cysts or oocysts of these pathogens were low and were below the detection limit of the IFA tests. The Giardia positive sample, in this study, typed as assemblage BIV and BI by gdh and tpi and bg genes, respectively. This finding is in agreement with previous reports that assemblage B was predominant in NHP (18,23,27,28). Although the prevalence of Giardia infection in this study is low, the identification of G. duodenalis assemblage B may suggest the potential of zoonotic or anthroponotic transmissions in this area.
The limitations of this study are the small sample size and the nature of single sample collected from each animal; selection bias may have led to an underestimation of the prevalence rates. A larger sample size or more frequent collection of gibbon's feces are needed for further studies. This study had inadequate power to detect associations between any risk factors and infections. In addition, we analyzed gibbons' species, age, sex, and diarrhea status; however, other important risk factors, e.g., season, diet, or water source could be suggested for future study to help in prevention and control of intestinal parasitic infection in this population.

AUTHOR CONTRIBUTIONS
ST, SM, and ML designed the study. SM and RA performed fecal sample collection. DS performed the microscopic fecal examination, ST performed IFA and molecular analyses. ST analyzed sequences. ML provided laboratory supplies. ST interpreted the results and wrote the manuscript. All authors read and approved the final manuscript.