Prevalence of Gastrointestinal Parasites in Zoo Animals and Phylogenetic Characterization of Toxascaris leonina (Linstow, 1902) and Baylisascaris transfuga (Rudolphi, 1819) in Jiangsu Province, Eastern China

Simple Summary A study in eastern China has found that over 40% of zoo animals are infected with gastrointestinal parasites, which pose a threat to their welfare and the health of visitors and veterinarians. More than 11 parasite species were identified in the study conducted at Zhuyuwan Zoo, including two species of Ascaris, and they detected Paramphistomum spp. eggs in the captive Père David’s deer and Fasciola spp. eggs in sika deer, which had not been previously reported in Chinese zoos. The study highlights the need for prevention and control measures to be implemented to tackle parasitic diseases in zoo animals. Abstract The burden of gastrointestinal parasites in zoo animals has serious implications for their welfare and the health of veterinarians and visitors. Zhuyuwan Zoo is located in the eastern suburb of Yangzhou city in eastern China, in which over 40 species of zoo animals are kept. In order to understand the infection status of GI parasites in Zhuyuwan Zoo, a total of 104 fresh fecal samples collected randomly from birds (n = 19), primates (n = 19), and non-primate mammals (n = 66) were analyzed using the saturated saline flotation technique and nylon sifter elutriation and sieving method for eggs/oocysts, respectively. Two Ascaris species were molecularly characterized. The results showed that the overall prevalence of parasitic infection was 42.3% (44/104). The parasitic infection rate in birds, primates, and non-primate mammals were 26.3% (5/19), 31.6% (6/19), and 50.0% (33/66), respectively. A total of 11 species of parasites were identified, namely, Trichostrongylidae, Capillaria sp., Trichuris spp., Strongyloides spp., Amidostomum sp., Toxascaris leonina, Baylisascaris transfuga, Parascaris equorum, Paramphistomum spp., Fasciola spp., and Eimeria spp. Paramphistomum spp. eggs were first detected from the captive Père David’s deer, and Fasciola spp. eggs were first reported from sika deer in zoo in China. A sequence analysis of ITS-2 and cox1 showed that the eggs isolated from the African lion (Panthera leo Linnaeus, 1758) were T. leonina, and the eggs from the brown bear (Ursus arctos Linnaeus, 1758) were B. transfuga. The public health threat posed by these potential zoonotic parasitic agents requires attention. These results lay a theoretical foundation for prevention and control of wild animal parasitic diseases at zoos in China.


Introduction
Wild animals are not only important assets to the natural world but are also considered companions to humans.Nonetheless, the rapid increase in the global human population over the past six decades has resulted in significant ecological changes and a loss of wildlife habitats [1].Consequently, the survival of wild animals has been jeopardized, with certain species teetering on the brink of extinction [2,3].Additionally, both captive and wild animals contribute to the spread of various diseases.Keeping wild animals in zoos can worsen the problem of parasitic infections, posing a significant threat to endangered species and sometimes causing unexpected declines in local populations [4].
The prevalence of GI parasites in zoo animals poses a significant health concern, with symptoms such as apathy, colic, diarrhea, malaise, and weight loss [5].Despite the global prevalence of GI parasites in wildlife [6], there has been limited research conducted on their prevalence in zoo animals within eastern China.Prevention and control of parasitic diseases in wildlife is the responsibility of zoo veterinarians [7].To effectively evaluate and regulate the impact of intestinal parasites on animal populations, including zoonotic pathogens, it is essential to assess their prevalence within wildlife populations.This becomes even more crucial during the COVID-19 pandemic, as concerns regarding emerging zoonoses have heightened [8].Furthermore, such assessments are vital for ensuring the health and safety of zoo veterinarians and tourists [9].
Traditionally, the detection and identification of Ascarididae eggs have relied on microscopy, which is a method that requires expertise and knowledge but can sometimes lead to identification errors [10,11].For example, there is misidentification between Toxocara canis (Werner, 1782) eggs and Toxocara cati (Schrank, 1788) eggs, and some pollens are morphologically similar to Ascarididae eggs.However, in recent years, molecular techniques have emerged as valuable tools for distinguishing and classifying Ascarididae species [12].Particularly, several genetic markers such as internal transcribed spacer 2 (ITS-2) in the nuclear ribosomal DNA (rDNA) region and cytochrome c oxidase subunit 1 (cox1) in the mitochondrial DNA (mtDNA) region have proven to be useful in investigating genetic diversity within the family Ascarididae [13][14][15].However, Blouin [16] suggests that cox1 may provide more reliable results for genetic comparison compared to ITS in closely related nematode species.
In the present study, we aimed to determine the occurrence and variety of intestinal parasites in fecal samples collected from wild animals residing in the Zhuyuwan Zoo, located in the Yangzhou region of eastern China, and we utilized molecular genetic techniques to identify two Ascarididae species at the ITS-2 locus and subsequently compared their genetic divergence within an evolutionary tree at the cox1 locus.These results lay a foundation for prevention and control of wild animal parasitic diseases at a zoo in Jiangsu Province, eastern China.

Study Site
The study was conducted at Zhuyuwan Zoo in Yangzhou city, Jiangsu Province, located in eastern China (Figure 1).Yangzhou city, situated on the lower reaches of the Yangtze River, has a temperate continental monsoon climate with four distinct seasons.The area has a mild climate with ample sunshine and rainfall, which creates favorable conditions for the occurrence and transmission of parasitic diseases.The zoo relocated from Slender West Lake to Zhuyuwan in 2004.Currently, the zoo accommodates more than 40 species of mammals, birds, and amphibians from Asia, Africa, and the Americas.

Sample Collection
In 2021, a total of 104 fecal samples were randomly collected from different animals living in Zhuyuwan Zoo.Of them, 19 samples were collected from 15 species of birds, 19 from 8 species of primates, and 66 from 17 species of non-primate mammals.Trained animal handlers conducted the collection of fecal samples (~50 g) in the morning.The samples were stored in zipper bags and placed in a ~4 °C sample box for storage, and processed within 6 h of collection.

Microscopy
Nematode eggs and coccidian oocysts were detected using the saturated saline floatation method [17].Briefly, 10 g of feces were diluted with 15 mL of saturated saline, filtered through a filter with a pore size of 250 µm, and the filtrate was centrifuged for 5 min at 800× g.A coverslip was placed over the surface of the supernatant, and it was viewed under a microscope after 3 min.The eggs of trematodes were examined using the nylon sieve washing method [17], i.e., 10 g feces diluted with water passed through 60 mesh (aperture = 250 µm) sieve and 260 mesh (aperture = 57 µm) sieve successively, and then the filter residue in the 260 mesh sieve was washed with water until the final filtrate was clear.Finally, the sediment in the sieve mesh was observed under a microscope.The eggs of nematode and trematode were observed and identified under a 40× objective lens.Unsporulated oocysts were incubated in 2.5% potassium dichromate (K2Cr2O7) for 5-7 days.After centrifugation at 800× g for 8 min, the supernatant was discarded, and the precipitate was resuspended in saturated saline and centrifuged at 700× g for 8 min.The sporulation oocysts from the supernatant were observed under the 40× objective.All identifications were performed as previously described [17][18][19][20].Among nematode eggs, Trichuris and Strongyloides egg are easy to be identified according to their characteristic morphology and structure, in which Trichuris eggs are lemon shaped, yellow, or brown in color, with a thick smooth shell and a conspicuous polar plug at both ends, whereas Strongyloides egg

Sample Collection
In 2021, a total of 104 fecal samples were randomly collected from different animals living in Zhuyuwan Zoo.Of them, 19 samples were collected from 15 species of birds, 19 from 8 species of primates, and 66 from 17 species of non-primate mammals.Trained animal handlers conducted the collection of fecal samples (~50 g) in the morning.The samples were stored in zipper bags and placed in a ~4 • C sample box for storage, and processed within 6 h of collection.

Microscopy
Nematode eggs and coccidian oocysts were detected using the saturated saline floatation method [17].Briefly, 10 g of feces were diluted with 15 mL of saturated saline, filtered through a filter with a pore size of 250 µm, and the filtrate was centrifuged for 5 min at 800× g.A coverslip was placed over the surface of the supernatant, and it was viewed under a microscope after 3 min.The eggs of trematodes were examined using the nylon sieve washing method [17], i.e., 10 g feces diluted with water passed through 60 mesh (aperture = 250 µm) sieve and 260 mesh (aperture = 57 µm) sieve successively, and then the filter residue in the 260 mesh sieve was washed with water until the final filtrate was clear.Finally, the sediment in the sieve mesh was observed under a microscope.The eggs of nematode and trematode were observed and identified under a 40× objective lens.Unsporulated oocysts were incubated in 2.5% potassium dichromate (K 2 Cr 2 O 7 ) for 5-7 days.After centrifugation at 800× g for 8 min, the supernatant was discarded, and the precipitate was resuspended in saturated saline and centrifuged at 700× g for 8 min.The sporulation oocysts from the supernatant were observed under the 40× objective.All identifications were performed as previously described [17][18][19][20].Among nematode eggs, Trichuris and Strongyloides egg are easy to be identified according to their characteristic morphology and structure, in which Trichuris eggs are lemon shaped, yellow, or brown in color, with a thick smooth shell and a conspicuous polar plug at both ends, whereas Strongyloides egg are oval, thin-shelled and with larvae.In addition, the eggs of Haemonchus spp., Ostertagia spp., Trichostronglus spp.(belonging to Trichostrongylidae), and Oesophagostomum spp.(belonging to Cyathostomidae) possess similar sizes (73~95 µm × 34~50 µm), morphologies (ovoid in shape), and structures (containing numbers of embryo cells); it is difficult to distinguish between these species [17,18].In this study, therefore, eggs with the above similar morphological structure were classified as Trichostrongylidae parasites.

DNA Isolation and PCR Amplification
About 100 eggs recovered from the African lions (Panthera leo) and the brown bears (Ursus arctos) were ground five times in liquid nitrogen, each time for 1 min.The subsequent steps for DNA extraction followed the guidelines outlined in the MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0 (TaKaRa, Tokyo, Japan).
A standard PCR-based sequencing technique targeting ITS-2 locus (~300 bp, specific primers) was used to detect the Toxascaris leonina and Baylisascaris transfuga in the feces from an African lion and a brown bear, as previously reported [21,22].Additionally, a standard PCR protocol was used to amplify the cox1 gene sequence, utilizing generic primers reported by Gasser et al. [19].The expected size of the PCR product (cox1) was approximately 450 bp (Table 1).The PCR amplification process followed these cycling conditions: an initial denaturation step at 94 • C for 5 min, followed by 35 cycles of denaturation at 94 • C for 30 s, annealing at 60 • C (for ITS-2) or 50 • C (for cox1) for 30 s, extension at 72 • C for 60 s, and a final extension step at 72 • C for 5 min.Subsequently, we purified the PCR products using the MiniBEST DNA Fragment Purification Kit Ver.4.0 (TaKaRa, Tokyo, Japan) and subjected them to 1.0% agarose gel electrophoresis, followed by staining with ethidium bromide.The gel was then transilluminated and photographed using a gel imaging system (Bio-Rad, Hercules, CA, USA).

Sequence and Phylogenetic Analysis
All PCR products exhibited a single band.The purified PCR products were sent to Beijing Genomics Institute (BGI, Beijing, China) for Sanger sequencing.Subsequently, the sequencing results were analyzed using MegAlign with the clustal/W method and BLAST using highly similar sequences.
Phylogenetic analysis of T. leonina and B. transfuga was conducted based on the cox1 loci, and additional isolates from GenBank were included.The phylogenetic trees were constructed using the MEGA 5 software [23].To determine the most suitable model, Mod-elTest in MEGA 5 was employed, and the Tamura-Nei [24,25] was utilized for Maximum Likelihood (ML), Neighbor-Joining (NJ), and Maximum Parsimony (MP) analyses.The reliability of the results was assessed through bootstrap analyses comprising 1000 replicates.Trichuris suis was selected as an outgroup.

PCR Amplification Analysis
A total of 23.9 ng/µL and 7.8 ng/µL of DNA were extracted from about 100 eggs collected from an African lion and a brown bear, respectively.At the ITS-2 locus, a band of 300 bp in size was amplified only using TleoF-NC2R specific primers from the eggs from an African lion (Figure 3A).Similarly, a band of 301 bp was amplified only using NITSF-NITSR primers from the eggs isolated from a brown bear (Figure 3B).The NC13-NC2 specific Animals 2024, 14, 375 6 of 13 primers were employed as a control to verify the presence of parasite DNA in each sample.At the cox1 locus, a band of about 450 bp in size were successfully amplified using JB3-JB4.5 generic primers from Toxascaris leonina (African lion) and Baylisascaris transfuga (brown bear) and sequenced, respectively (Figure 3C).Afr L refers to samples recovered from African lion feces and Bn B refers to samples recovered from brown bear feces.

PCR Amplification Analysis
A total of 23.9 ng/µL and 7.8 ng/µL of DNA were extracted from about 100 eggs collected from an African lion and a brown bear, respectively.At the ITS-2 locus, a band of 300 bp in size was amplified only using TleoF-NC2R specific primers from the eggs from an African lion (Figure 3A).Similarly, a band of 301 bp was amplified only using NITSF-NITSR primers from the eggs isolated from a brown bear (Figure 3B).The NC13-NC2 specific primers were employed as a control to verify the presence of parasite DNA in each sample.At the cox1 locus, a band of about 450 bp in size were successfully amplified using JB3-JB4.5 generic primers from Toxascaris leonina (African lion) and Baylisascaris transfuga (brown bear) and sequenced, respectively (Figure 3C).Afr L refers to samples recovered from African lion feces and Bn B refers to samples recovered from brown bear feces.

Discussion
The overall prevalence of GI parasites in animals living in Zhuyuwan Zoo was 42.3% (44/104), which is similar to the results observed in zoo animals in Xining of China [20], Slovenia (45%, 337/741) [21], and Poland (48%, 34/71) [22].However, the infection rate of GI parasites in the present study was higher than that in twenty-four zoological gardens of China (26.51%, 317/1196) [23], Nepal (19.54%, 17/87) [24], and France (32.2%, 99/307) [25], and lower than that in Sichuan of China [26], Italy (80%, 24/30) [27], and Brazil (71.1%, 27/38) [28].These differences may be related to the ecological environment and sampling season.The present study found a higher occurrence of helminths (39.4%, 41/104) compared to protozoans (5.8%, 6/104), similar to the observations in a zoological garden in Kenya, which revealed a higher occurrence of helminths (64.4%, 203/315) and a lower occurrence of protozoans (17.1%, 54/315) [29].Interestingly, the prevalence of protozoa is lower than that of helminths in primates and non-primate mammals, whereas in birds the prevalence of protozoa infection is higher than that of helminths.This may be related to the environment in which these birds and primates and non-primate mammals live.These Animals 2024, 14, 375 10 of 13 birds are wading birds whose feeding environment relies heavily on silt, which contains some fecal matter and some infectious Eimeria coccidia, thus making them susceptible to disease transmission [30].Of course, it may also be due to birds being more susceptible to Eimeria, as Eimeria coccidia causes the most severe damage to poultry (belonging to birds).Additionally, our results revealed that the positive rates of GI parasites in birds, primates, and non-primate mammals were 26.3% (5/19), 31.6%(6/19), and 50.0%(33/66), respectively.Interestingly, the positive rate of mixed infection with two or more parasites was only 5% (5/104), which is consistent with previous reports (7%, 55/741) [21].In addition, no tapeworms were detected in this study.
In this study, Trichuris spp.had a 5.8% (6/104) infection rate, second only to Trichostrongylidae (22.1%, 23/104).Strongyloides eggs were detected in fecal samples from the Ring-tailed lemurs (Lemur catta) and red pandas (Ailurus fulgens).Strongyloides parasites can infect their hosts through skin penetration or ingestion [31] and reproduce asexually in the host's intestinal wall, contributing to their high infection rates [32].Rondon, Ortiz, Leon, Galvis, Link and Gonzalez [33] reported that 21.6% (40/185) of fecal samples from neotropical primates were positive for Strongyloides spp.Studies on parasites infecting nonhuman primates are essential for better understanding the potential threat of zoonotic transmission to humans [34], especially given the ongoing processes of pervasive land use change and biodiversity loss [35] and the COVID-19 pandemic [36].In addition, Trichuris spp., as a zoonotic parasite, may increase the risk of mixed infection with intestinal pathogens [37].Therefore, Zhuyuwan Zoo needs to pay more attention to prevention and control of Trichuris spp.and Strongyloides spp.
The Père David's deer (Elaphurus davidianus) and the sika deer (Cervus nippon) are National Protected Animals in China [38].Here, Paramphistomum spp.eggs were first detected in the captive Père David's deer in a zoo in China, and Fasciola spp.eggs were detected from the feces of sika deer.Paramphistomum spp.and Fasciola spp.have different degrees of pathogenicity to animals, and Fasciola parasites are an important zoonotic pathogen.So, Zhuyuwan Zoo needs to pay special attention to prevention and control of Paramphistomum spp.and Fasciola spp.
For decades, there has been considerable debate surrounding the systematics of members of the Ascaridomorpha (including Anisakidae, Ascarididae, Ascaridiidae and Toxocaridae) [39,40].With the increasing utilization of molecular biology methods, there has been a growing ability to examine and identify ascarids [41].This is particularly important because the eggs of many ascarid species share similar morphological characteristics, potentially leading to misidentification [42].In the present study, two Ascaris species were identified using molecular biological methods in this study.It was found that T. leonina could only be amplified using TleoF-NC2R primers and B. transfuga could only be amplified using NITSF-NITSR primers, respectively.The results of a phylogenetic tree showed that T. leonina was closer to T. canis than to T. cati at cox1 locus, which was similar to the result reported by Xie et al. [15].
Toxascaris spp.and Baylisascais spp., belonging to the family Ascarididae, are common GI parasites parasitized in carnivores such as members of Canidae, Felidae, and Ursidae.T. leonina mainly resides in the gastrointestinal tract of dogs, cats, and lions [43,44], which carries the potential risk to infect humans due to their close relationship with zoonotic ascarids like Toxocara canis and Toxocara cati [45,46].Baylisascaris transfuga has been documented in all extant members of the family Ursidae worldwide, except for the spectacled bear [47,48].The possibility of B. transfuga causing illness in humans has been suggested, and it poses a threat to public health [42,49].Previous research has shown that B. transfuga larvae can migrate within the tissues of various animals like chickens [50], rabbits [51], mice [52], and Mongolian jirds [53] from their original locations in the intestine.However, the occurrence of visceral larva migrans (VLM) has only been observed in mammals [49].Therefore, there is a potential risk for humans who come into contact with infectious eggs of B. transfuga in the environment [48].In addition, the other five GI parasites detected in this study also are potentially dangerous for humans.A previous study has shown that anthelmintic drugs could effectively control soil-transmitted helminth infections [54].Therefore, we recommend that zoos appropriately use albendazole or praziquantel for helminths eradication to reduce the risk of infection to tourists and veterinarians.

Conclusions
The prevalence of GI parasites was 42.3% (44/104) in Zhuyuwan Zoo in China.Paramphistomum spp.eggs were first detected from the captive Père David's deer and Fasciola spp.eggs were first reported from sika deer in zoo in China.Ascaridoid parasite eggs from the African lions were identified as T. leonina, and those from the brown bear were identified as B. transfuga.Of 11 GI parasites detected in this study, seven are potentially dangerous for humans.The public health threat posed by these potential zoonotic parasitic agents requires attention.

Figure 1 .
Figure 1.Geographical location of the sampling site, Zhuyuwan Zoo.

Figure 1 .
Figure 1.Geographical location of the sampling site, Zhuyuwan Zoo.

Figure 4 .
Figure 4. Evolutionary relationships of T. leonina and B. transfuga inferred using ML, NJ and MP analyses of cox1 sequences.Toxascaris leonina recovered from the African lion was genetically divided into the general Toxascaris and B. transfuga recovered from the brown bear was genetically divided into the general Baylisascaris which were indicated with blue and red rings, respectively.The general Toxocaris and Ascaris were indicated with yellow and green rings, and the Outgroup was purple, respectively.The numbers along branches indicate bootstrap values derived from different analyses in the order: ML/NJ/MP.Values

Figure 4 .
Figure 4. Evolutionary relationships of T. leonina and B. transfuga inferred using ML, NJ and MP analyses of cox1 sequences.Toxascaris leonina recovered from the African lion was genetically divided into the general Toxascaris and B. transfuga recovered from the brown bear was genetically divided into the general Baylisascaris which were indicated with blue and red rings, respectively.The general Toxocaris and Ascaris were indicated with yellow and green rings, and the Outgroup was purple, respectively.The numbers along branches indicate bootstrap values derived from different analyses in the order: ML/NJ/MP.Values lower than 50 are shown as "-".Black triangles represent the sequencing results of this experiment.

Table 2 .
Prevalence of intestinal parasites in birds at Zhuyuwan Zoo.

of Positive Samples for Parasite Species Capillaria sp. Amidostomum sp. Eimeria spp.
n = number of samples collected and examined (the same below).

Table 3 .
Prevalence of intestinal parasites in primates at Zhuyuwan Zoo.

Table 4 .
Prevalence of intestinal parasites in non-primate mammals at Zhuyuwan Zoo.

Table 5 .
The overall occurrence of intestinal parasitic infections among various animals at Zhuyuwan Zoo.