Prevalence of gastrointestinal parasitic infections in wild mammals of a safari park and a zoo in Bangladesh

Abstract In safari parks and zoos, wild animals are kept mainly for recreational purposes. Animals in these enclosures are also crucial for the education, research, and conservation aspect. To ensure better management and good health of wild animals in captivity, it is essential to monitor the occurrence of gastrointestinal (GI) parasitic (helminths and protozoa) infections. The current investigation was undertaken to investigate the prevalence of GI parasitic infections in wild mammals at Bangabandhu Sheikh Mujib (BSM) safari park and Chattogram (CTG) zoo of Bangladesh. A total of 72 individual faecal samples were collected from 25 species of wild mammals. Routine qualitative (e.g. direct smear, sedimentation, and flotation) and quantitative (e.g. McMaster technique) tests were performed to identify the eggs or oocysts of helminths and protozoa. Results demonstrated that wild mammals of both BSM safari park and CTG zoo were infected with a total of 17 genera/species of helminths and protozoa. The overall prevalence of GI parasitic infections in wild mammals of both zoological parks was 65.3% (95% confidence interval [CI]: 53.14–76.12), whereas it was 72.4% (95% CI: 52.76–87.27) in the BSM safari park and 60.5% (95% CI: 44.41–75.02) in the CTG zoo. In both zoological parks, infection with nematodes was more frequent compared to other helminth into the wild mammals. The herbivores were more infected with GI parasites than carnivores and omnivores of both BSM safari park and CTG zoo. The mean eggs/oocysts per gram of faeces was the highest in the carnivores compared to herbivores and omnivores of both enclosures. The findings of the current study demonstrated that wild mammals of both BSM safari park and CTG zoo suffered from various GI parasitic infections. Regular monitoring along with proper therapeutic measures may reduce the severe consequences of GI parasitic infections in captive wild animals.


INTRODUCTION
Safari parks and zoos usually differ in their structure and principles. In these premises, wild animals are kept mainly for recreation and preservation of endangered species (Mir et al., 2016;Rahman et al., 2014).
In safari parks, wild animals are allowed to roam freely in their specific territories, having a resemblance to their natural habitats. On the contrary, in zoos, animals are generally kept in enclosures that are entirely different from their natural habitats (Da Silva Barbosa et al., 2019). When wild animals are brought from wild to captivity (e.g. safari parks, zoos), the biology of those animals is altered due to the sudden exposure to an unfavourable and stressful environment. This kind of alteration increases the susceptibility to various infectious diseases (e.g. viral, bacterial, fungal, parasitic), and among those, gastrointestinal (GI) parasitic (helminths and protozoa) infections are the most common in captive wild animals (Adeniyi et al., 2015;Carrera-Játiva et al., 2018;Kolapo & Jegede, 2017;Moudgil et al., 2015). In the natural habitat, animals might have a natural resistance against parasitic infections because of an ecological balance with their parasites. Furthermore, wild animals are less exposed to parasitic infections due to free-roaming in ample areas and low density of animals (Da Silva Barbosa et al., 2019;Mir et al., 2016;Moudgil et al., 2015;Thawait et al., 2014). However, these infections induce negative impacts on the status, behaviour, reproduction, and survival of wild animals (Kvapil et al., 2017;Thawait et al., 2014).
An understanding of GI parasitic infections in captivity is vital for their documentation as these animals were brought from different countries and also collected from the local or wild territories. Furthermore, it is essential to know the mode of transmission and zoonotic potentiality of existing parasites within the wild animals of zoos and safari parks. In Bangladesh, there are two safari parks and six zoos.
Unfortunately, there is no such extensive documentation of parasitic infections of wild mammals in these parks and zoos. Only a few studies have been conducted in zoos (e.g. Rangpur zoo, Bangladesh national zoo) and in Dulahazra safari park to identify the GI parasitic infections (Hossain et al., 2021;Khatun et al., 2014;Rahman et al., 2014;Raja et al., 2014). Therefore, the current investigation was designed to study the occurrence of GI parasitic infections in wild mammals of the most popular Bangabandhu Sheikh Mujib (BSM) safari park and Chattogram (CTG) zoo of Bangladesh.

Ethical approval
All procedures were reviewed and approved by zoo and safari park authorities before conducting the research. The samples in this study were opportunisticall y collected from the territories or cages of wild mammals. Non-invasive method was used during the collection of faecal samples.

Study periods and sites
The study was conducted on wild mammals of BSM safari park,  (Table S2). Zoo enclosures were cleaned regularly, and anthelmintics were provided two to three times in a year.
In both parks, carnivores were fed with beef or chicken, having a daybreak per week; omnivores were fed mainly with fruits, vegetables, beef, eggs, bread, so forth, whereas herbivorous animals were regularly provided with grass, cereal grains, vegetables, wheat bran, gram, so forth (Tables S1 and S2). Animals were monitored by the animal caretakers and treated by veterinarians.

Study animals, sample collection, and preservation
A total of 72 faecal samples were collected from 25 species of wild mammals of both safari park and zoo (Tables S1 and S2). Among those, 29 samples were collected from the wild mammals of the safari park (20 herbivores, seven carnivores, and two omnivores) and 43 from the zoo (22 herbivores, seven carnivores, and 14 omnivores). Approximately 5-10 g of individual faecal samples were collected from each animal species. Samples from carnivores and omnivores were collected in fresh condition, before cleaning the cages in the early morning. Samples from herbivores, housed in open-natural (the BSM safari park) and open-top (the CTG zoo) enclosures, were collected soon after the defecation. Immediately after collection, samples were placed in dry, clean, and labelled plastic sample containers and preserved with 10% formalin. All the samples were then transported to the parasitology laboratory of the department of pathology and parasitology of Chattogram Veterinary and Animal Sciences University, Chattogram, Bangladesh. Samples were kept at 4 • C until laboratory examination.  (Soulsby, 1982;Urquhart, 1996).

Examination of faecal samples
Briefly, 4 g of faeces was mixed thoroughly with 56 ml of saturated salt solution. Then, both chambers of the McMaster slide were filled with the diluted suspension and allowed to stand for 5 min before microscopic examination. The eggs and oocysts were then counted.
The morphological features of the eggs of helminths and oocysts of protozoa were identified using the published literature (Arjun et al., 2017;Berentsen et al., 2012;Rahman et al., 2014). Micrometry was performed to estimate the size of eggs and oocysts (Cable, 1950) ( Figure 1).

Statistical analysis
The raw data were compiled, sorted in a spreadsheet using Microsoft Excel 2016, and then imported into STATA-13 ® (STATA Crop, 4905, Lake Way Drive, College Station, TX, USA) to conduct data analysis.
Descriptive analysis with a 95% confidence interval (CI) was performed to estimate the prevalence of parasitic infections in animals based on their categories. Egg/oocyst per gram (EPG/OPG) of faeces was expressed by mean ± standard errors of the mean (SE). A chi-square test was performed to calculate the presence of parasitic infections with associated risk factors in animal species. Analysis of variance was carried out to compare the parasitic load in faecal samples of animals.
Results were considered significant when p ≤ 0.05.

Overall prevalence of helminth and protozoan infections in animals of both BSM safari park and CTG zoo
The overall prevalence of GI parasitic infections (helminths and protozoa) was 65.3% (95% CI: 53.14-76.12, N = 72) in the wild mammals of both BSM safari park and CTG zoo.

Prevalence of gastrointestinal parasitic infections in the BSM safari park and CTG zoo
Between the two study sites, the overall prevalenc of GI parasitic infections was higher in the BSM safari park (

Gastrointestinal helminth and protozoan infections load in wild mammals of the BSM safari park and CTG zoo
The mean EPG/OPG was measured in animal groups though there were some single samples and the number of the positive cases was one or two. The mean EPG/OPG of GI parasites in the wild mammals of both BSM safari park and CTG zoo was not statistically significant (5385.7 ± 4741.4 and 5326.9 ± 3416.3, respectively) (Tables S3 and S4). The mean EPG/OPG was higher in carnivores (19,785.7 ± 13978.6) followed by herbivores (3124.2 ± 2588.46), and omnivores (1428.6 ± 620.9) (Tables S3 and S4). Between the locations, the mean EPG/OPG in herbivores was higher in the CTG zoo (6466.7 ± 21982.68) than the BSM safari park (338.9 ± 403.13), but it was completely opposite in the carnivores where it was higher in the animals of BSM safari park (35,666.7 ± 55,811.95) than in the animals of the CTG zoo (7875 ± 14751.58) (Tables S3 and S4).

DISCUSSION
Gastrointestinal (GI) parasitic infections have adverse effects on the survival of wild animals and their welfare. It is very important to monitor the GI parasitism (helminths and protozoa) to ensure a better management and good health of animals in captivity. The occurrence of GI parasitic infections has been reported in various zoos and national parks of the world (Lim et al., 2008;Mir et al., 2016;Maske et al., 1990;Opara et al., 2010;Parsani et al., 2001;Rahman et al., 2014;Raja et al., 2014;Singh et al., 2006;Thawait et al., 2014). The current investigation has documented the occurrence of GI parasitic infections in BSM safari park and CTG zoo. The overall prevalence of GI parasitic infections in wild mammals of safari park and zoo was consistent with previously published reports (Da Silva Barbosa et al., 2019; Kolapo & Jegede, 2017). Higher (71%-77%) (Cordón et al., 2008;Holsback et al., 2013;Opara et al., 2010;Rahman et al., 2014) and lower prevalences (40%-60%) (Chakraborty et al., 2016;Lim et al., 2008;Parsani et al., 2001;Thawait et al., 2014) of GI parasitic infections were also recorded in wild mammals in captivity. These differences might be due to the variations in geography, captivity conditions, feed and feeding management, sources of water, use of anthelmintics, and husbandry practices (Fagiolini et al., 2010). In the BSM safari park, most of the animals (e.g. herbivores) shared the same habitat, and it was hard to maintain the effective anthelmintic dose for each individual, and this leads to higher occurrence of GI parasites. In addition, active transmission may also takes place in wild or semi-wild habitats, and regular cleaning and dung removal may not possible on a regular basis. Environmental contamination facilitates the transmission of different stages of parasites through contaminated water or fodder, and even mechanically with the zoo workers (Adetunji, 2014;Mir et al., 2016;Otegbade & Morenikeji, 2014).
Helminth infections were more common than protozoan infections in the animals of both study sites, which was consistent with the prior studies (Adeniyi et al., 2015;Lim et al., 2008;Mir et al., 2016;Opara et al., 2010;Rahman et al., 2014;Raja et al., 2014). This finding suggested that the nature of the life cycle of helminths facilitates their survival in the parks or zoos, contributing to more helminth infections.
Higher helminth infections in safari parks than in the zoo might be due to the favourable climatic conditions that favour the transmission and survival of helminths in the areas (Rahman et al., 2014). In contrast, protozoan infections were higher in zoo animals, which can be explained by the favourable factors required for the completion of life cycle, lower infective doses, and ease of transmission in zoo environment (Cordón et al., 2008;Levecke et al., 2007). Among the helminths, the prevalence of infections with nematodes was higher than other types of helminths in both sites, and this was accordant with previous studies (Rahman et al., 2014;Shemshadi et al., 2015). Higher occurrence of infections caused by nematodes was because of their direct life cycle with no involvement of intermediate hosts and being able to be transmitted through contaminated feed, water, and soil (Mir et al., 2016). On the other hand, trematodes and most of the cestodes require at least one intermediate host to complete their life cycle and for potential transmission. This might be the reason for lower occurrence of trematodes and cestodes infections in all animal groups of both study sites (Atanaskova et al., 2011;Mir et al., 2016).
Of all nematodes, Strongylus spp. had a higher occurrence in herbivores, which was consistent with previous studies (Aviruppola et al., 2016;Garijo et al., 2004;Rahman et al., 2014). This could be explained by its proliferative life cycle and survival capacity in the harsh environment (Aviruppola et al., 2016;Rahman et al., 2014). In both study sites, all the samples were collected from zebra, and horses were found infected by helminths, which indicated the active transmission of the parasite in grouped animals. Strongylus spp. have already been reported in most herbivores, for example, horses (Hinney et al., 2011;Umar et al., 2013;Uslu & Guclu, 2007), zebras (Fagiolini et al., 2010;Wambwa et al., 2004), giraffes (Nosal et al., 2016;Varadharajan & Pythal, 1999) Pythal, 1999). The presence of Strongyloides spp. in zebras, wildebeests, blackbucks, and blesbucks was also reported in previous studies (Mir et al., 2016;Varadharajan & Pythal, 1999). Oesophagostomum spp. and Trichostrongylus spp. were found in giraffes, and this was considered the first report from Bangladesh. Both parasites have been reported most commonly in ruminants and wild herbivores, which might get the infections through grazing in the same pasture along with other livestock of the safari park (Abuessailla et al., 2011;Farooq et al., 2012).
Triodontophorus spp. in zebras were reported by prior studies, and these studies stated that the parasite has a higher prevalence in free-ranging zebras (Wambwa et al., 2004). Trichuris spp. was found in captive porcupines and was not reported in Bangladesh before. The presence of Trichuris spp. in bristle-spined porcupines was reported by Kuniy and Brasileiro (2006). Among carnivores, the presence of T. cati and T. leolina in jungle cats of the zoo and lions of both sites was consistent with previous studies (Acharjyo, 2004;Adeniyi et al., 2015;Fagiolini et al., 2010;Raja et al., 2014;Tabaripour et al., 2018). Carnivores usually get these infections through ingesting the intermediate hosts directly (Sepalage & Rajakaruna, 2020). Some literature indicated the co-occurrence of both Toxocara spp. and T. leonina in canids and felids (Dalimi et al., 2006;Labarthe et al., 2004) and suggested that the occurrence is highly variable and depends on factors such as seasonal variability, age of an individual, climate change, and environmental conditions (Okulewicz et al., 2012). The presence of T. cati in fishing cats, Strongyloides spp. in jungle cats, and Ancylostoma spp. in leopard cats reported previously in several studies (Acharjyo, 2004;Moudgil et al., 2015). Ancylostoma spp. was also detected from gibbons, rhesus macaques, and from large Indian civets of the CTG zoo, and this was also reported by previous studies (Adedokun et al., 2002;Habtamu et al., 2017;Pourrut et al., 2011). Infections by Ancylostoma spp. may occur when infective larval stages are ingested directly by the host or through penetrating the skin (percutaneous route) while coming in direct contact with contaminated soil (Colon & Patton, 2012).
This study found a higher occurrence of Fasciola spp. infections in herbivores, which was consistent with previous studies (Khatun et al., 2014;Rahman et al., 2014). Trematodes found in gayals, blesbucks, nyalas, and wildebeests in our study were supported by findings of the previous studies (Boomker et al., 1991;Rahman et al., 2014). Two species of cestodes were identified, and all Spirometra spp. were more frequently identified from carnivores. Spirometra spp. in lions, jungle cats, and leopard cats in our study was consistent with the findings of previous studies (Berentsen et al., 2012;Khatun et al., 2014;Lim et al., 2008;Moudgil et al., 2015;Raja et al., 2014). Though there was no record of finding Spirometra spp. in Bengal foxes, Scioscia et al. (2014) reported the first finding of Spirometra erinacei in Pampas fox as a new definitive host.
Among the protozoa, Isospora spp. and Eimeria spp. infections were found in carnivores (lions and tigers) of the BSM safari park and herbivores (gayals and porcupines) of the CTG zoo. Isospora spp. in tigers and lions were reported by several prior studies (Berentsen et al., 2012;Bjork et al., 2000;Chauhan et al., 1973;Fagiolini et al., 2010;González et al., 2007;Mahali et al., 2010;Muraleedharan & Iswaraiah, 1984 (Marques et al., 2012;Overgaauw and van Knapen, 2013). Strongyloides spp. has also been reported for cutaneous, intestinal, pulmonary, or disseminated human infections (Fagiolini et al., 2010;Jaleta et al., 2017). Schistosoma spp. causes various diseases in humans including inflammatory and obstructive disease in the urinary system (Schistosoma haematobium) or liver fibrosis, hepatosplenic, and intestinal infections (Schistosoma mansoni and Schistosoma japonicum) (Colley et al., 2014;Gryseels et al., 2006;Kebede et al., 2020). In this study, we did not examine the samples from the animals caretakers which might determine and confirm the potential cross-transmission of parasites to human population. This limitation could be addressed by future studies.

CONCLUSIONS
This study provides the scientific evidence that gastrointestinal helminths and protozoan infections are very common in wild mammals of the BSM safari park and CTG zoos in Bangladesh. Monitoring of these infections is very important for ensuring better health of animals. This is also important from public health aspects. As animal caretakers are in close contact with wild mammals, there are risks of cross-transmission of infections. Furthermore, these animals may act as reservoir hosts for many of such zoonotic parasites which may ultimately result in active transmission to the human population. We recommend further detailed molecular and epidemiological investigation on the occurrence of parasitic infections in the animals of safari parks and zoos in Bangladesh with the identification of their associated risk factors.