Introduction
Captive ungulates represent diverse species housed in zoological parks, wildlife sanctuaries, and conservation centres worldwide (Naz et al., Reference Naz, Arooj, Ali and Farooq2021). Ensuring the optimal health and well-being of these animals is of paramount importance for conservation efforts aimed at maintaining genetic diversity within populations, particularly those under human care and management (Naz et al., Reference Naz, Arooj, Ali and Farooq2021). Unfortunately, some gastrointestinal tract (GIT) nematodes can have significant negative impacts on the health of captive ungulate populations (Phetla et al., Reference Phetla, Chaisi and Malatji2024). These GIT nematode infections lead to diarrhea, behavioral changes affecting feeding and social interactions, severe weight loss, and sometimes mortality, thus significantly constraining ungulate health in captive environments and adversely impacting animal welfare and productivity (Hoberg and Brooks, Reference Hoberg and Brooks2015). As GIT nematodes have evolved to coexist with their hosts, the term “nemabiome” has been coined to describe the intricate ecosystem involving these parasitic nematodes (Avramenko et al., Reference Avramenko, Redman, Lewis, Yazwinski, Wasmuth and Gilleard2015). These complex parasitic communities ultimately play a vital yet understudied role in veterinary health (Beaumelle et al., Reference Beaumelle, Redman, de Rijke, Wit, Benabed, Debias, Duhayer, Pardonnet, Poirel, Capron and Chabot2021). Therefore, there is a need to develop improved techniques for assessing the species composition of gastrointestinal nematode populations in wildlife, both in terms of identifying the constituent species and determining their relative abundance (Avramenko et al., Reference Avramenko, Redman, Lewis, Bichuette, Palmeira, Yazwinski and Gilleard2017). Although these GIT nematode complexes are still poorly understood, strategies utilising next-generation sequencing (NGS) technology, like those used to study the microbiome, demonstrate enormous promise in this respect (Avramenko et al., Reference Avramenko, Redman, Lewis, Yazwinski, Wasmuth and Gilleard2015).
The GIT nematodes that commonly infect ungulates, especially prevalent species like Haemonchus contortus, can significantly impact the host’s immune response, nutrient metabolism, and overall physiological fitness (Sulliotti, Reference Sulliotti2023). Consequently, understanding the composition and dynamics of the intestinal nemabiome is crucial for elucidating the ecological interactions and health implications in ungulate populations (Sulliotti, Reference Sulliotti2023). Traditional microscopic techniques have limited the comprehensive assessment of nemabiome diversity, composition, and dynamics in captive ungulates due to their low resolution and taxonomic identification capabilities. Leveraging the power of NGS technologies presents a transformative opportunity to overcome these limitations and gain unprecedented insights into the complex nemabiome communities inhabiting gastrointestinal tracts of captive ungulates.
This study explores the application of GIT nemabiomes in captive ungulates using an NGS approach in support of SANBI’s preventative medicine monitoring program.
Material and methods
A total of 13 samples were from five ungulate species, namely Roan antelope (n=1), Eland (n=1), Sable antelope (n=9), Arabian oryx (n=1) and Blue duiker (n=1) housed in different enclosures at the SANBI/National Zoological Garden. This was a retrospective study using DNA samples collected from a previous study (Mosala, Reference Mosala2017). A 311- to 331-bp fragment of the rDNA ITS-2 region was polymerase chain reaction (PCR)-amplified from the 13 ungulate samples using primers NC1 and NC2 (Gasser et al., Reference Gasser, Chilton, Hoste and Beveridge1993). Briefly, the PCR mix consisted of 12.5 μL AmpliconRed master mix (Lasec, South Africa), 1 μL (10 μM) of each primer, 2 μL template DNA, and 8.5 μL of nuclease-free water to a total volume of 25 μL. The PCR cycling conditions were initial denaturation at 95 °C for 5 min, 95 °C for 30 sec (denaturation); 54 °C, 30 sec (annealing); 68 °C for 1 min (extension), for 40 cycles, 68 °C for 10 min (final extension). The amplicons were submitted to Inqaba Biotechnical Industries (Pretoria, South Africa) where they were barcoded, quantified, and pooled into a single library to generate a normalised library. The library was sequenced on the Illumina NextSeq platform. The paired-end reads raw data were processed using Qiime2 with default settings (Caporaso et al., Reference Caporaso, Kuczynski, Stombaugh, Bittinger, Bushman, Costello, Fierer, Peña, Goodrich, Gordon and Huttley2010). The DADA2 plugin was used to remove ambiguous bases, low-quality reads, and chimeras. Sequences were clustered to operational taxonomic units (OTUs) using a cut-off of 97% similarity using VSEARCH (Rognes et al., Reference Rognes, Flouri, Nichols, Quince and Mahé2016). The resulting OTUs were compared against the trained nematode ITS2 rDNA (Avramenko et al., Reference Avramenko, Redman, Lewis, Bichuette, Palmeira, Yazwinski and Gilleard2017) for taxonomic assignments. Exploratory analyses were performed in R v.3.5.1 and Bioconductor v.3.0 (Gentleman et al., Reference Gentleman, Carey, Bates, Bolstad, Dettling, Dudoit, Ellis, Gautier, Ge, Gentry and Hornik2004).
Results and discussion
The overall analysis of gastrointestinal nemabiome in 13 samples from five species of captive ungulate revealed informative data of sequences of GIT nematode infections classified to species level. The total number of reads ranged between 19254 and 95540 per sample (Supplementary Table 1), with sable antelope having the lowest and eland having the highest number of reads. The results indicated that wild ungulates at the National Zoological Garden are infected by nematodes of three genera, namely Cooperia, Haemonchus, and Trichostrongylus, along with a substantial proportion of sequences that could not be classified at the genus or species level and were categorized as “unidentified” (Figure 1a). The genus Haemonchus and Trichostrongylus were identified to species level as Haemonchus contortus (H. contortus), and Trichostrongylus colubriformis (T. colubriformis), while Cooperia was not identified to species (Figure 1b). Haemonchus contortus was the most dominant, common nematode species across all samples and species, followed by Trichostrongylus colubriformis (Figure 1b).
Figure 1. (a) Species diversity and composition of gastrointestinal nematodes to genus level of captive wild ungulates from the SANBI National Zoological Gardens. (b) Species diversity and composition of gastrointestinal nematodes to species level of wild ungulates from the SANBI National Zoological Gardens.
GIT nematode infections varied among the studied ungulate species. Haemonchus contortus was identified from all ungulate host species. A study by Zieger et al. (Reference Zieger, Boomker, Cauldwell and Horak1998) in Zambia indicated the Eland was mostly affected by H. contortus, similarly, Phetla et al. (Reference Phetla, Chaisi and Malatji2024) supported the findings that H. contortus was the most dominant GIT nematode species infecting wild ruminants, including wild ungulates, across sub-Saharan African countries. Haemonchus contortus and T. colubriformis have a global distribution and commonly infect various wild ruminants, including Roe deer, Fallow deer, Red deer, and Mouflon (Halvarsson et al., Reference Halvarsson, Baltrušis, Kjellander and Höglund2022). According to Phetla et al. (Reference Phetla, Chaisi and Malatji2024), these nematodes are also common in wild ungulates in sub-Saharan Africa.
ITS-2 rDNA metabarcoding has previously been used to determine the nemabione of wild and domestic ruminants in other parts of the world. According to the study by Queiroz et al. (Reference Queiroz, Levy, Avramenko, Redman, Kearns, Swain, Silas, Uehlinger and Gilleard2020), analysis of the sheep nemabiome from more than 90 flocks across western Canada indicated that H. contortus was the predominant gastrointestinal nematode species, followed by Teladorsagia circumcincta and T. colubriformis. Many of the surveyed flocks had high intensities of these parasitic infections. This is the case with the current study as four ungulates (two Sable antelopes, one Eland, and one Arabian Oryx) were co-infected by T. colubriformis and H. contortus. This is not surprising as these species have a global distribution and have been reported in different domestic and wild hosts (Abuessailla et al., Reference Abuessailla, Ismail and Agab2013).
Cooperia infection was detected to genus level in only one Sable antelope (Sable antelope 3, Figure 1(a)). Cooperia are usually low in both domestic and wildlife (Roos and Grant, Reference Roos and Grant1993). Furthermore, the study by Avramenko et al. (Reference Avramenko, Redman, Lewis, Bichuette, Palmeira, Yazwinski and Gilleard2017) indicated that one of the Cooperia spp. (Cooperia punctata) was generally found in lower intensities in cattle; however, they still formed a significant parasite burden in several eastern Canadian herds. The infection patterns in this study reveal a complex interplay of single and multiple parasitic species across the ungulate populations. This diversity of GIT nematode infections among and within ungulate species underscores the intricacy of wildlife parasitology and its potential implications for animal health and conservation.
In terms of GIT nematode infections, free-ranging wild animals typically exhibit higher parasite species richness than their captive counterparts (Phetla et al., Reference Phetla, Chaisi and Malatji2024). This increased diversity in free-ranging animals can be attributed to their unrestricted movement across various habitats, diverse feeding patterns, frequent contact with different host species, and natural exposure to the environmental stages of parasites. In contrast, captive animals generally show lower parasite species diversity due to controlled conditions, including restricted habitat exposure, managed diets, limited animal contact, regular antiparasitic treatments, and routine veterinary care. This is evident in this study as only three genera (Haemonchus, Trichostronglylus, and Cooperia) were detected across the captive ungulate species housed at the SANBI Zoological Garden. The controlled environment and preventive health measures in captivity effectively reduce the likelihood of animals encountering multiple parasite species, resulting in lower overall parasite species richness.
The distribution of genera in our study could be influenced by various factors, such as the host species, environmental conditions, and management practices in the captive setting (Chaudhari et al., Reference Chaudhari, Madke and Ambore2022).
According to Phetla et al. (Reference Phetla, Chaisi and Malatji2024), nematodes are the most diverse and widespread GIT parasites found across different species of wild ungulates and geographic regions. Wild ungulates can therefore play an important role in the transmission of GIT nematodes, especially Haemonchus, Trichostrongylus, and Cooperia species to domestic animals, particularly where they share grazing areas. These genera have a global presence in Europe and South Africa (Boomker et al., Reference Boomker, Booyse, Watermeyer, De Villiers, Horak and Flamand1996) and are distinguished by elevated temperatures and humidity which provided favorable conditions for the presence and spread of species including Haemonchus contortus, Trichostrongylus colubriformis, and Cooperia spp. The manifestation of these GIT nematodes is due to their well-adapted life cycle involving free-living larval stages in the environment (e.g., grass, soil), increasing their chances of GIT parasite transmission to grazing host animals.
Understanding the diversity and composition of GIT nematodes in captive wildlife is crucial for management, as some nematode species are potentially virulent and infections can result in severe disease or mortality (Hoberg et al., Reference Hoberg, Lichtenfels and Pilitt2015; Pittman et al., Reference Pittman, Shepherd, Thacker and Myers2020). These infections can be transmitted to susceptible animals in shared enclosures. Additionally, the high proportion of unidentified nematode sequences underscores the need for more extensive reference nematode databases for accurate taxonomic identification of nematodes to species level. This could facilitate a better understanding of the nemabiome dynamics and potentially pathogenic or commensal relationships between nematodes and their ungulate hosts (Avramenko et al., Reference Avramenko, Redman, Lewis, Yazwinski, Wasmuth and Gilleard2015). Further investigations, incorporating analysis of larger sample size and additional molecular markers, could provide more insights into the nemabiome composition and the functional roles of the identified and unidentified nematode species in captive ungulate populations (Hoberg et al., Reference Hoberg, Lichtenfels and Pilitt2015; Chaudhari et al., Reference Chaudhari, Madke and Ambore2022).
Conclusion
This study investigated the gastrointestinal nematode communities (nemabiomes) in captive ungulates in the SANBI National Zoological Garden in South Africa. The study revealed a limited diversity of GIT nematode infections, identifying only three nematode genera: Haemonchus, Trichostrongylus, and Cooperia. Among these, Haemonchus contortus showed the highest prevalence, followed by Trichostrongylus colubriformis, while Cooperia was the least abundant. However, these infections can be transmitted to susceptible animals in the zoo, leading to mortality. These findings highlight the need for more comprehensive research to fully characterise the nemabiome composition in captive and free-ranging ungulate populations in South Africa and to determine the risk of transmission to domestic livestock. Moreover, the high proportion of unidentified nematode sequences emphasizes the importance of expanding and improving reference databases and molecular techniques for accurate taxonomic identification of nematodes. This would facilitate a better understanding of the nemabiome dynamics and the potential pathogenic or commensal relationships between nematodes and their ungulate hosts.
Supplementary material
The supplementary material for this article can be found at http://doi.org/10.1017/S0022149X24000804.
Availability of data
The nucleotide sequence data reported are available in the NCBI GenBank databases under the BioSample accessions number (SAMN42574826 – SAMN42574838) and SRA accession number PRJNA1137257.
Acknowledgements
The study was supported by resources from the South African National Biodiversity Institution (SANBI).
Financial support
This project was funded by the National Research Foundation (NRF) Thuthuka grant (TTK 190211415437) awarded to M.C. The financial assistance of the NRF towards this research is acknowledged. Opinions expressed and conclusions arrived at are those of the authors and are not to be attributed to the NRF.
Competing interest
None.
