In total, we collected individual larvae across all host species. To extract DNA, the ethanol was evaporated from each sample using a vacuum centrifuge. The sample was centrifuged briefly, and the larvae were freeze-fractured by placing them at 0 C for 20 minutes, followed by minutes at 60 0 C and 20 minutes at 94 0 C. The ITS locus is known to reliably differentiate closely related nematode species [ 26 — 32 ].
This marker is also popular due to its lower level of intra-species polymorphism compared to mtDNA [ 27 , 33 ].
We further amplified CO1 because this locus undergoes rapid evolution and is good choice for differentiating cryptic parasite species [ 27 ] as well as phylogeographic groups within a single species [ 27 , 33 ]. However, these genetic markers are rarely used together in a single study or for identification of a single nematode species yet when used in combination they enhance identification output and provide more genetic information [ 25 , 26 ]. Gel electrophoresis 1. The products were sequenced in both directions using Dye Terminator sequencing Applied Biosystems.
Sequence data were examined and cleaned using Sequencher software v. The sequences were deposited in the Genbank. We aligned sequences using ClustalX2 software [ 35 ] with the following alignment parameters: gap opening penalty at 15; gap extension at 6. We used MEGA 6. Models with the lowest BIC scores Bayesian Information Criterion were considered to best describe the substitution patterns. All phylogenetic trees were derived by MEGA 6. This research involved non-invasive coprological techniques and fecal samples used in this study did not necessitate the capture or disturbance to the study animals.
We identified a rich diversity of nematodes, trematodes and cestodes by egg morphology Table 1 and molecular techniques. The total number of parasite taxa identified by both techniques was 22 Table 2. Nematodes included Enterobius spp. Fig 1A , Strongyloides spp. Fig 1B , strongylid ova Fig 1C , Primasubulura spp.
Fig 1D , Trichuris spp. Figs 2A and 2D , Spirurina spp. Fig 3A , Streptopharagus spp. The identified trematodes were Paramphistomum spp. Fig 3E and Fasciola spp. Fig 3F. The cestodes were Moniezia expansa Fig 2B and M. Within Moniezia spp.
These included M. With the additional information provided by genetic markers, we found a total of 22 distinct parasite taxa Table 2 , with the highest parasite richness observed in non-human primates, followed by livestock and least in wild ungulates. Baboons exhibited the highest parasite richness; however, this difference was likely because of our greater sampling effort in baboons samples we searched baboon samples per season as compared to 48 samples for the other host species.
Images of nematode eggs A Enterobius spp. Images of helminth eggs A Trichuris spp. Images of helminth eggs A Spirurina spp. B Streptopharagus spp. C Unidentified Spirurid - morphotype A. F Fasciola hepatica. Because of the challenges of genotyping cultured larvae low DNA quantity and quality; see Methods , we only obtained positive PCRs from reactions, and we were only able to obtain clean sequences, suitable for taxonomic identification, for 18 larvae. Molecular analysis revealed that baboons were infected with two species of Strongyloides : S.
Phylogenetic relationships among S. All the sequences retrieved from the GenBank and used for the phylogenetic reconstruction of Fig 4 are in the S1 Table. We also identified several strongylid nematodes in hosts from Amboseli Fig 5.
Our single isolate of Trichostrongylus colubrifomis from Kenyan baboons [GenBank: KT] was distinct from Trichostrongylus sequences found on GenBank, which were sampled from humans and sheep Fig 5. The Oesophagostomum bifurcum we identified from baboons [GenBank: KT] clustered with isolates on GenBank sampled from gorillas, humans, and other primates Fig 5.
Moreover, Cooperia oncophora clustered in a clade that included other isolates of C. Haemonchus contortus had a closer relationship with other nematode species isolated elsewhere from goat sheep and giraffe. All the sequences retrieved from the GenBank and used for the phylogenetic reconstruction of Fig 5 are in S2 Table. The rooted maximum likelihood tree based on mtDNA was derived from bootstrap replicates using Necator spp. Accession numbers for GenBank sequences included in the tree are in the S1 Table. The rooted maximum likelihood tree based on ITS of the rDNA was derived from bootstrap replicates using Strongylus edentatus as the outgroup.
Accession numbers for GenBank sequences included in the tree are in the S2 Table. Combining information from our coprological and molecular methods, we identified distinct helminth taxa across the nine-host species Table 2. All these sympatric host species were infected with at least one type of helminth, but helminth community composition varied across host species Table 2.
Nonhuman Primates in Biomedical Research: Volume 2 : Diseases
Molecular analysis revealed seven taxonomic groups of nematodes Table 3 that could not have been differentiated by egg morphology. The single most dominant gastrointestinal parasite in the host community was Trichuris spp. Moreover, these two primate species shared four different species of helminths Primasubulura spp. This is the first study to assess parasite diversity in a sympatric host community that includes bovids livestock and wild ungulates and non-human primates.
Our results reveal a dynamic parasite-host interaction characterized by parasite sharing among hosts, yet restricted by host evolutionary history. The taxonomic diversity detected in the ungulates and primates from Amboseli includes species that are of veterinary and public health importance and whose epidemiology and phylogeography is less known in Kenya. In addition, we found that seasonality was linked to parasite richness such that hosts had higher diversity in the wet compared to dry season Table 1. This pattern agrees with current scientific understanding of seasonal effects on helminth propagation, environmental persistence and transmission [ 37 — 39 ].
Frontmatter - Flynn's Parasites of Laboratory Animals - Wiley Online Library
Further, in our study, genetic tools enabled us to differentiate closely related nematode species co-circulating within a single host population. The low number of genotyped larvae in this study was probably a consequence of multiple factors, such as loss of larvae during vacuum evaporation of ethanol, and poor DNA quality, which led to a high rate of sequencing errors. Therefore, in our opinion, the results of this study possibly do not represent the entire richness that is present in the nine host species studied in Amboseli. The taxonomic richness of the parasites was highest in non-human primates, followed by livestock and least in wild ungulates Table 2.
Specifically, baboons harbored the highest number of distinct parasite species Table 2 , which is likely due to differences in parasite sampling intensity between hosts. We expected that wild bovids would harbor higher parasite richness compared to livestock because livestock in Amboseli are likely to be treated with anti-helmintics. However, contrary to this expectation, our results indicate that livestock in Amboseli are exposed to and infected by diverse helminth taxa that may be reciprocally transmitted between livestock and wild ruminants.
It is generally expected that animals which occupy large home ranges or range widely should have higher prevalence and parasite species richness because they will likely encounter greater diversity of habitats and host taxa, which predispose them to higher risks of infection with more diverse parasites [ 40 ]. Although, the role of range size on parasite richness is not explicit and often inconclusive, we posit that the practice of transhumance exposes livestock to more diverse parasite infective stages leading to higher parasite diversity.
In a previous study, cattle co-grazed with wildlife in a confined Conservancy in Kenya had lower parasite richness compared to the pastoralist cattle in the present study [ 22 ], which shows that range size used by the animal may influence parasite richness. Most of the wild bovids tend to have restricted range sizes, which could be a risk for higher worm intensity rather than diversity. The presence of Strongyloides spp.
We found that the evolutionary relationships of S. This observation agrees with Hasegawa et al.
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However, evolutionary rates of S. In contrast, rates of evolution of S. Specifically, S. We also observed that the baboon population in Amboseli was simultaneously infected by both S. The phylogeny we reconstructed indicates that S. In addition, we observed a signal of S. For instance, S. Information about S. Since S. In fact, over million people worldwide suffer from Strongyloides spp. People who frequently use or share habitats dominated by non-human primates typically acquire S. In summary, the co-infections of S.
The presence of Oesophagostomum bifurcum in the Amboseli baboon population is also of concern from a public health perspective because of its zoonotic potential. The O. Specifically, the sub-structuring theory posits that populations of O. Recent detection of multiple cryptic Oesophagostomum species that co-infect humans and other non-human primates [ 52 ] supports the existence of zoonotic Oesophagostomum spp.
Baboons in the Amboseli ecosystem were previously found to harbor unidentified species of Trichostrongylus [ 53 ]; however, the genetic evidence in this study confirms that the baboons are infected with T. This nematode species that we identified in baboons was distinct from the rest of the Trichostrongylus spp. Nematodes in the genus Trichostrongylus comprise many species of veterinary importance [ 54 — 56 ], and some species, such as T.
In areas where humans and animal hosts have overlapping habitats, T. It is therefore important to determine actual human occurrence of T.