Prevalence and morphotype diversity of Trichuris species and other soil-transmitted helminths in captive non-human primates in northern Nigeria

A study to determine the prevalence and morphotype diversity of soil-transmitted helminths in captive non-human primates (NHPs) in northern Nigeria was conducted. Simple flotation and sedimentation methods were used to examine fecal samples. A Morphometric analysis was done on Trichuris spp. eggs to determine the diversity of whipworm circulating in NHPs in the study area. High prevalence (60%) of infection was recorded in captive NHPs; Patas Monkey (n=17), Tantalus Monkey (n=9), Mona Monkey (n=7), Vervet Monkey (n=2), Mangabey Monkey (n=1), Baboon (n=14), and Chimpanzee (n=8) from parks and zoological gardens located in four Nigerian states (Borno, Gombe, Kano, and Plateau) and the Federal Capital Territory (FCT), Abuja. Captive NHPs examined were infected with helminths either as single, double or triple infections. Four zoonotic soil transmitted helminth (STH) genera, Trichuris, Strongyloides, Ancylostoma, and Enterobius were detected in the examined animals. Eggs of Trichuris spp. were the most prevalent with four morphotypes suggesting several morphotypes of whipworm were circulating among the NHPs in this region. Further studies are required to elucidate the epidemiologic and public health implications of these findings.


INTRODUCTION
Non-human primates (NHPs) enclosures in zoological gardens or parks in Nigeria are among the most popular attractions to visitors, especially children; however, the maintenance of wild animals in captivity is fraught with numerous challenges particularly that of parasitic disease conditions due to high contamination of the environment (Rao & Acharjyo 1984;Vanitha et al. 2011). This is exacerbated by lowered immunity of the animals due to the stress of captivity, consequently diminishing their resistance to parasitic diseases (Gracenea et al. 2002;Perez Cordon et al. 2008). Therefore, gastrointestinal parasite infections are among the most common diseases found in non-human primates (Bezjian et al. 2008;Strait et al. 2012). Although the captive animals do not show alarming signs of parasitism, it has been reported that some of the helminth parasites they harbor have zoonotic potential and are, therefore, considered to be a threat to public health Klaus et al. 2017). Soil-transmitted helminths (STHs) such as Ascaris spp., Trichuris spp., Ancylostoma spp., and Strongyloides spp. that can easily be transferred from NHPs to humans and vice versa through contaminated environments are a major concern (Ranglack & Yeager 1986;Bethony et al. 2006;Lynn 2010). Single or mixed infections of zoonotic STHs have often been recorded in NHPs from different countries: Bangladesh (Raja et al. 2014), Central African Republic (Hasegawa et al. 2014), Tanzania (Petrželkova et al. 2010), China (Li et al. 2017), India (Hussain et al. 2013), Sri Lanka (Aviruppola et al. 2016), Malaysia (Klaus et al. 2017), andSpain (Perez Corden et al. 2008). Several studies have been carried out on the prevalence of helminth infection in NHPs in various zoological gardens and/or parks in the southern part of Nigeria: Oyo State (Adedokun et al. 2002;Emikpe et al. 2002;Adetunji 2014), Ondo State (Egbetade et al. 2014), Cross River State (Mbaya & Udendeye 2011), and Imo state (Opara et al. 2010). Comparatively, only a few studies have been conducted on the helminth infections of NHPs in northern Nigeria (Nwosu 1995;Mbaya & Nwosu 2006;Mbaya et al. 2006a,b;Dawet et al. 2013). The aim of this study, therefore, was to determine the prevalence and diversity of helminths in captive NHPs in northern Nigeria and to discuss the public health implications.

Sampling sites
The study was conducted in Zoological Gardens and Parks in four northern states and Abuja (9.076 0 N, 7.398 0 E) the Federal Capital Territory (FCT) of Nigeria. The four states; Borno (11.831 0 N, 13.151 0 E), Gombe (10.279 0 N, 11.173 0 E), Kano (12.002 0 N, 8.592 0 E) and Plateau (9.896 0 N, 8.858 0 E) were selected based on convenience and accessibility of NHPs for sample collection.

Sample collection
Fecal samples were collected opportunistically from individual captive NHP with the help of the caretakers over a period of six months (November 2017 to April 2018). Fresh feces were collected from the ground under the nest of individual NHPs. Approximately 5g of feces was scooped from the surface of each fecal mass using a disposable hand glove and transferred into a screw capped bottle. Each sample was labeled appropriately and transported in a cold box to the laboratory for analysis.

Fecal analysis
Samples were processed and analyzed in the Helminthology Research Laboratory, National Veterinary Research Institute (NVRI), Vom, Plateau State, Nigeria. First, each sample was examined macroscopically for the presence of helminths or taenid segments.

Microscopic analysis
Simple tube flotation and sedimentation: Fecal samples were individually processed by simple tube flotation in saturated sodium chloride solution (SG 1.20) and simple sedimentation techniques (Greiner & McIntosh 2009).
The preparation was then examined using direct light microscope (100X and 400X magnifications) for the presence of parasite eggs. The identification of the parasites was based on egg morphology, shape, size and color according to standard keys (Samuel et al. 2001;Hasegawa et al. 2009).

Morphometry analysis
Helminth eggs were measured (length and width) by using a calibrated light microscope. Mean values of measurements were given in micrometers (µm) ± standard deviations (SD).

Prevalence of helminths in NHPs from northern Nigeria
Helminth eggs were detected in fecal samples of NHPs from zoos or parks in all the states studied. Overall, 60% of the animals studied had helminth eggs in their feces. The highest prevalence (100%, n=4/4) was recorded in samples from the Abuja Children Park and Zoo, followed by (89%, n=8/9) in samples from the Gombe Zoo and the lowest prevalence (33%, n=1/3) was recorded in samples from the Maiduguri Zoological Garden (Table 1). Prevalence according to NHP species was highest (78%) for Tantalus Monkeys Chlorocebus tantalus. The lowest prevalence (47%, n=8/17) was observed in Patas Monkeys while the fecal sample of the only Mangabey Monkey Cercocebus sp. screened in this study was negative for helminth eggs ( Table 1).

Diversity of helminths
Four helminth genera: Trichuris, Ancylostoma, Strongyloides, and Enterobius and one unidentified parasite egg were detected from the feces of NHPs from northern Nigeria. Trichuris spp. eggs were detected with a high prevalence (31/44) across all the NHP species screened in the study. This was followed by Strongyloides spp. (4/44) and Ancylostoma spp. (3/44) both affecting three NHP species each. An unidentified egg that resembled an egg of Opisthorchis sp. was detected in   (Table 2).

Egg morphotypes detected in NHP examined
Four morphotypes of Trichuris spp. eggs (T1-T4) were detected in this study. All the trichurid egg morphotypes were thick-shelled, with prominent bipolar plugs but varied in shape, size, and colour. Egg morphotypes T1 and T2 appear to be more common in the various species of monkeys examined and occur as mixed infections, while T3 and T4 were commonly associated with baboons and chimpanzees. The eggs of other helminth genera, Ancylostoma, Strongyloides, and Enterobius detected in this study occurred as single morphotypes (Table 3).

DISCUSSION
Captive NHPs in Nigerian zoological gardens and parks attract attention due to their agility and playfulness. Thus, their wellbeing and survival is paramount to conservationists, veterinarians and zoo administrators. Parasitic diseases, particularly helminth infections have been reported to constitute a challenge to the health of NHPs (Samuel et al. 2001;Vanitha et al. 2011;Wren et al. 2015). The results from this survey showed that more than half (60%) of the examined NHPs in northern Nigeria were infected with helminth parasites. Four helminth genera of zoonotic importance, viz., Trichuris, Ancylostoma, Strongyloides, and Enterobius were detected in the NHPs examined in this study. These helminths have a high potential for transmission to humans because of their simple life cycles (Li et al. 2017). Thus, they are listed among the major cause of soil-transmitted helminth infections globally (Bethony et al. 2006). The differences in prevalence of these helminths observed in the study locations could be attributed to differences in host species susceptibility to helminth infections and the variations in climatic conditions between the study sites. Such observation on the effects of climate on parasite prevalence have been reported (Cordon et al. 2008;Wren et al. 2015).
Trichuris eggs were the most prevalent (70.5%) in the infected NHPs in this study. This finding is in accord with an earlier survey of NHPs in northern (Dawet et al. 2013) and southern (Mbaya & Udendeye, 2011;Adetunji 2014) parts of Nigeria. High prevalence of Trichuris sp. was also observed in NHPs in Côte d'Ivoire This study provides additional information on the metric details of the trichurid eggs present in the NHPs in northern Nigeria suggesting the diversity of this parasite in the region. It appears that NHPs in northern Nigeria are infected with various Trichuris spp. based on the morphology and dimensions of the eggs detected during this study. A similar observation was earlier made in a study in south west Nigeria but the authors did not provide any metric details of the Trichuris eggs detected (Egbetade et al. 2014). Therefore, our study is the first to provide morphometric analyses of Trichuris eggs infecting NHPs in Nigeria. Four morphotypes (T1-T4) of Trichuris eggs, with mean size ranging from 43-53 x 22-30 µm (length x width) were detected in this study suggesting a diversity of this parasite in the NHPs examined. Indeed, even among the NHP species there is variability in the morphology of Trichuris eggs detected. Similar observations have been reported (Petrzelkova et al. 2010;Klaus et al. 2017). Thus, our findings agree with several reports on morphometric and molecular Table 3

. Morphologic and morphometric analyses of helminth eggs detected in non-human primates in northern Nigeria
Helminth genera

J TT
studies of trichurid eggs in NHPs conducted in areas with different climatic conditions from Nigeria (Hasegawa et al. 1983;Dupain et al. 2009;Ghai et al. 2014;Raja et al. 2014;Cavallero et al. 2015;Klaus et al. 2017;Li et al. 2017). It is noteworthy that the dimensions of some of the eggs of Trichuris spp. detected in this study are similar to those of human T. trichiura, suggesting zoonotic or reverse zoonotic transmissions. This finding has implications for veterinarians, public health workers, and wildlife managers, in terms of the epidemiology of the disease and the choice of treatment and control measures to be adopted (Melfi & Poyser 2007). Therefore, the assumption hitherto, among wildlife parasitologists that all the Trichuris infecting NHPs are the same as T. trichiura of humans should be reconsidered, however, the other three zoonotic helminths genera; Ancylostoma, Strongyloides and Enterobius detected in this study occurred as monotypes each with dimensions similar to those of the species infecting human, suggesting possible circulation of these worms between humans and NHPs in the study area.
Taken together, our results demonstrate the presence of zoonotic helminths and a diversity of Trichuris sp. infection amongst NHPs. Therefore, a comprehensive study to elucidate the genetic diversity of Trichurids infection NHPs in Nigeria is desirable. This will assist to distinguish the species and genotypes of this parasite in NHPs in northern Nigeria and to determine their pathogenicity. Therefore, molecular studies on pinworm diversity in Nigerian NHPs are needed in order to elucidate the species and morphotypes circulating in the country.

CONCLUSION
Captive NHPs in zoological gardens and parks in northern Nigeria are infected with helminths of public health significance. High prevalence of Trichuris spp. coupled with a diversity of their egg morphotypes were observed in the NHPs examined. Further investigation using modern tools like molecular phylogenetics in order to fully understand their epidemiology and zoonotic potentials is warranted. www.threatenedtaxa.org The Journal of Threatened Taxa (JoTT) is dedicated to building evidence for conservation globally by publishing peer-reviewed articles online every month at a reasonably rapid rate at www.threatenedtaxa.org. All articles published in JoTT are registered under Creative Commons Attribution 4.0 International License unless otherwise mentioned. JoTT allows allows unrestricted use, reproduction, and distribution of articles in any medium by providing adequate credit to the author(s) and the source of publication.