Novel insight into the genetic diversity of strongylid nematodes infecting South-East and East Asian primates.

With many non-human primates (NHPs) showing continued population decline, there is an ongoing need to better understand their ecology and conservation threats. One such threat is the risk of disease, with various bacterial, viral and parasitic infections previously reported to have damaging consequences for NHP hosts. Strongylid nematodes are one of the most commonly reported parasitic infections in NHPs. Current knowledge of NHP strongylid infections is restricted by their typical occurrence as mixed infections of multiple genera, which are indistinguishable through traditional microscopic approaches. Here, modern metagenomics approaches were applied for insight into the genetic diversity of strongylid infections in South-East and East Asian NHPs. We hypothesized that strongylid nematodes occur in mixed communities of multiple taxa, dominated by Oesophagostomum, matching previous findings using single-specimen genetics. Utilizing the Illumina MiSeq platform, ITS-2 strongylid metabarcoding was applied to 90 samples from various wild NHPs occurring in Malaysian Borneo and Japan. A clear dominance of Oesophagostomum aculeatum was found, with almost all sequences assigned to this species. This study suggests that strongylid communities of Asian NHPs may be less species-rich than those in African NHPs, where multi-genera communities are reported. Such knowledge contributes baseline data, assisting with ongoing monitoring of health threats to NHPs.

First insight into strongylid nematode diversity and anthelmintic treatment effectiveness in beef cattle in the Czech Republic explored by HTS metagenomics.

Parasitic diseases and mitigation of their effects play an important role in the health management of grazing livestock worldwide, with gastrointestinal strongylid nematodes being of prominent importance. These helminths typically occur in complex communities, often composed of species from numerous strongylid genera. Detecting the full diversity of strongylid species in non-invasively collected faecal samples is nearly impossible using conventional methods. In contrast, high-throughput amplicon sequencing (HTS) can effectively identify co-occurring species. During the four-year project, we collected and analysed faecal samples from beef cattle on >120 farms throughout the Czech Republic. Strongylids were the predominant nematodes, detected in 56% of the samples, but at a low level of infection. The apparent limitations in identifying strongylid taxa prompted this pilot study on a representative group of samples testing positive for strongylids using ITS-2 metabarcoding. The most widespread genera parasitizing Czech cattle were Ostertagia (O. ostertagi) and Oesophagostomum spp., followed by Trichostrongylus and Cooperia, while Bunostomum, Nematodirus and Chabertia were present only in a minority. As comparative material, 21 samples of cattle from the Danube Delta in Romania were used, which, in contrast, were dominated by Haemonchus placei. Finally, the effect of ivermectin treatment was tested at two Czech farms. After treatment with the anthelmintic, there was a shift in the strongylid communities, with a dominance of Cooperia and Ostertagia.

Anoplocephalid tapeworms in mountain gorillas (Gorilla beringei beringei) inhabiting the Volcanoes National Park, Rwanda.

Cestodes of the family Anoplocephalidae parasitize a wide range of usually herbivorous hosts including e.g. rodents, ungulates, primates, elephants and hyraxes. While in some hosts, the epidemiology of the infection is well studied, information is lacking in others. In this study of mountain gorillas in the Virunga Massif, an extensive sample set comprising adult cestodes collected via necropsies, proglottids shed in feces, and finally, fecal samples from both night nests and identified individuals were analysed. Anoplocephala gorillae was the dominant cestode species detected in night nest samples and individually known gorillas, of which only 1 individual hosted a Bertiella sp. It was shown that the 2 species can be distinguished through microscopy based on egg morphology and polymerase chain reaction (PCR) assays for diagnostics of both species were provided. Sequences of mitochondrial (cox 1) and nuclear (ITS1, 18S rDNA, 28S rDNA) markers were used to evaluate the phylogenetic position of the 2 cestodes detected in mountain gorillas. Both types of fecal samples, from night nests and from identified individuals, provided comparable information about the prevalence of anoplocephalid cestodes, although the analysis of samples collected from identified gorilla individuals showed significant intra-individual fluctuation of A. gorillae egg shedding within a short period. Therefore, multiple samples should be examined to obtain reliable data for wildlife health management programmes, especially when application of anthelmintic treatment is considered. However, while A. gorillae is apparently a common symbiont of mountain gorillas, it does not seem to impair the health of its host.

Genetic analysis challenges the presence of Ixodes inopinatus in Central Europe: development of a multiplex PCR to distinguish I. inopinatus from I. ricinus.

BACKGROUND: Ixodes ricinus is an important vector of several pathogens, primarily in Europe. Recently, Ixodes inopinatus was described from Spain, Portugal, and North Africa and then reported from several European countries. In this study, a multiplex polymerase chain reaction (PCR) was developed to distinguish I. ricinus from I. inopinatus and used in the surveillance of I. inopinatus in Algeria (ALG) and three regions in the Czech Republic (CZ). METHODS: A multiplex PCR on TROSPA and sequencing of several mitochondrial (16S rDNA, COI) and nuclear markers (TROSPA, ITS2, calreticulin) were used to differentiate these two species and for a subsequent phylogenetic analysis. RESULTS: Sequencing of TROSPA, COI, and ITS2 separated these two species into two subclades, while 16S rDNA and calreticulin could not distinguish I. ricinus from I. inopinatus. Interestingly, 23 nucleotide positions in the TROSPA gene had consistently double peaks in a subset of ticks from CZ. Cloning of these PCR products led to a clear separation of I. ricinus and I. inopinatus indicating hybridization and introgression between these two tick taxa. Based on a multiplex PCR of TROSPA and analysis of sequences of TROSPA, COI, and ITS2, the majority of ticks in CZ were I. ricinus, no I. inopinatus ticks were found, and 10 specimens showed signs of hybridization. In contrast, most ticks in ALG were I. inopinatus, four ticks were I. ricinus, and no signs of hybridization and introgression were detected. CONCLUSIONS: We developed a multiplex PCR method based on the TROSPA gene to differentiate I. ricinus and I. inopinatus. We demonstrate the lack of evidence for the presence of I. inopinatus in Central Europe and propose that previous studies be re-examined. Mitochondrial markers are not suitable for distinguishing I. inopinatus from I. ricinus. Furthermore, our data indicate that I. inopinatus and I. ricinus can hybridize, and the hybrids can survive in Europe.

Identification of potentially zoonotic parasites in captive orangutans and semi-captive mandrills: Phylogeny and morphological comparison.

Cysts and trophozoites of vestibuliferid ciliates and larvae of Strongyloides were found in fecal samples from captive orangutans Pongo pygmaeus and P. abelii from Czech and Slovak zoological gardens. As comparative material, ciliates from semi-captive mandrills Mandrillus sphinx from Gabon were included in the study. Phylogenetic analysis of the detected vestibuliferid ciliates using ITS1-5.8s-rRNA-ITS2 and partial 18S ribosomal deoxyribonucleic acid (rDNA) revealed that the ciliates from orangutans are conspecific with Balantioides coli lineage A, while the ciliates from mandrills clustered with Buxtonella-like ciliates from other primates. Morphological examination of the cysts and trophozoites using light microscopy did not reveal differences robust enough to identify the genera of the ciliates. Phylogenetic analysis of detected L1 larvae of Strongyloides using partial cox1 revealed Strongyloides stercoralis clustering within the cox1 lineage A infecting dogs, humans, and other primates. The sequences of 18S rDNA support these results. As both B. coli and S. stercoralis are zoonotic parasites and the conditions in captive and semi-captive settings may facilitate transmission to humans, prophylactic measures should reflect the findings.

Gastrointestinal symbiont diversity in wild gorilla: A comparison of bacterial and strongylid communities across multiple localities.

Western lowland gorillas (Gorilla gorilla gorilla) are Critically Endangered and show continued population decline. Consequently, pressure is mounting to better understand their conservation threats and ecology. Gastrointestinal symbionts, such as bacterial and eukaryotic communities, are believed to play vital roles in the physiological landscape of the host. Gorillas host a broad spectrum of eucaryotes, so called parasites, with strongylid nematodes being particularly prevalent. While these communities are partially consistent, they are also shaped by various ecological factors, such as diet or habitat type. To investigate gastrointestinal symbionts of wild western lowland gorillas, we analysed 215 faecal samples from individuals in five distinct localities across the Congo Basin, using high-throughput sequencing techniques. We describe the gut bacterial microbiome and genetic diversity of strongylid communities, including strain-level identification of amplicon sequence variants (ASVs). We identified strongylid ASVs from eight genera and bacterial ASVs from 20 phyla. We compared these communities across localities, with reference to varying environmental factors among populations, finding differences in alpha diversity and community compositions of both gastrointestinal components. Moreover, we also investigated covariation between strongylid nematodes and the bacterial microbiome, finding correlations between strongylid taxa and Prevotellaceae and Rikenellaceae ASVs that were consistent across multiple localities. Our research highlights the complexity of the bacterial microbiome and strongylid communities in several gorilla populations and emphasizes potential interactions between these two symbiont communities. This study provides a framework for ongoing research into strongylid nematode diversity, and their interactions with the bacterial microbiome, among great apes.

Further data on the distribution of Dirofilaria spp. in the Czech Republic in dogs.

Based on previously published data, the Czech Republic is regarded an endemic country of the onchocercid nematodes Dirofilaria immitis (Leidy, 1856) and Dirofilaria repens Railliet et Henry, 1911. Nevertheless, while cases of D. repens are commonly reported from dogs in South Moravia, no recent records of D. immitis are available. Therefore, the present study was performed to clarify the occurrence of both species of Dirofilaria Railliet et Henry, 1910. Blood samples of 551 dogs sampled during 2015 and 2016 were analysed microscopically for presence of microfilariae and blood sera were examined by IDEXX SNAP® 4Dx® test (IDEXX, USA). DNA from blood of microscopically positive dogs was extracted and PCR protocol amplifying fragment of cytochrome c oxidase I (COI) gene was performed; PCR products were then sequenced. All dogs from the Bohemian part of the Czech Republic were negative. The prevalence of D. repens in the Moravian region was 5.7 % (27/476). BLAST analyses of obtained sequences confirmed the presence of D. repens (99-100% identical to KX265049). All sampled animals showed a negative result for D. immitis antigen in IDEXX SNAP® 4Dx® test. Our study confirmed the previously reported occurrence of D. repens in South Moravia and revealed its spreading from the epicentre to the north and west. PCR with subsequent sequencing together with negative results for D. immitis antigen in IDEXX SNAP® 4Dx® test revealed only D. repens infection. A previously published autochthonous infection of dogs with D. immitis in South Moravia was not confirmed.

Giardia duodenalis in a clinically healthy population of captive zoo chimpanzees: Rapid antigen testing, diagnostic real-time PCR and faecal microbiota profiling.

Giardia duodenalis is one of the most common intestinal parasites of humans, with a worldwide distribution. Giardia duodenalis has been reported in both wild and captive populations of non-human primates, namely chimpanzees. In this study we investigated an entire troop of clinically healthy chimpanzees (n = 21) for the presence of G. duodenalis and its association with faecal microbiota profile. Faecal samples (n = 26) were collected from the chimpanzee exhibit from a zoo in Sydney, Australia. Diagnosis of G. duodenalis was made using a Rapid Antigen Test (RAT) as a point-of-care-test and compared to a reference standard real-time PCR test. Approximately half of the chimpanzee faecal samples tested positive for G. duodenalis by both RAT (13/26, 50%) and real-time PCR (14/26, 53.85%). The RAT sensitivity was 85.7% (95% CI: 63.8%-96%) and specificity was 91.7% (95% CI: 68.3%-99%) when compared to the in-house real-time PCR. Genotyping of the samples revealed the presence of zoonotic assemblage B. Microscopic analysis revealed the presence of Troglodytella spp. (14/26), Balantioides sp. (syn. Balantidium sp.) (8/26) as well as Entamoeba spp. (3/26). Microbiota profile based on 16S rRNA gene sequencing revealed that the community was significantly different between G. duodenalis positive and negative samples if RAT results were taken into an account, but not real-time PCR diagnostics results. Proteobacteria and Chloroflexi were the significant features in the dataset that separated G. duodenalis positive and negative samples using LEfSe analysis. Being able to rapidly test for G. duodenalis in captive populations of primates assists in point-of-care diagnostics and may better identify animals with subclinical disease. Under the investigated conditions of the zoo setting, however, presence of G. duodenalis either detected by RAT or real-time PCR was not associated with clinically apparent disease in captive chimpanzees.

The winner takes it all: dominance of Calicophoron daubneyi (Digenea: Paramphistomidae) among flukes in Central European beef cattle.

In Europe, paramphistomosis caused by Paramphistomum spp. was historically regarded as being of minor importance. However, Calicophoron daubneyi has recently been recognized as an emerging pathogen in Europe due to its increasing prevalence and negative impact on livestock production. In search for paramphistomid flukes, 5573 beef cattle fecal samples from 115 farms across the whole Czech Republic were examined from March 2019 to June 2021. The eggs of paramphistomid flukes were identified in 29.9% of samples. Internal transcribed spacer 2 sequences from 90 adult flukes and 125 fecal samples collected across Czech Republic confirmed C. daubneyi infection in the Czech beef cattle. Ninety mitochondrial DNA sequences obtained from adult C. daubneyi specimens revealed 13 individual haplotypes, two of them recorded for the first time. Although C. daubneyi is a new parasite in beef cattle herds in the Czechia, it clearly dominates the parasitological findings in the country's beef cattle. The common occurrence of C. daubneyi in most of the beef cattle herds indicates environmental conditions suitable also for the life cycle of Fasciola hepatica and risk of its emergence.

Heterogeneity in patterns of helminth infections across populations of mountain gorillas (Gorilla beringei beringei).

Conservation efforts have led to the recovery of the endangered mountain gorilla populations. Due to their limited potential for spatial expansion, population densities increased, which may alter the epidemiology of infectious diseases. Recently, clinical gastrointestinal illnesses linked to helminth infections have been recorded in both gorilla populations. To understand drivers and patterns of helminth infections we quantified strongylid and tapeworm infections across both Virunga Massif and Bwindi populations using fecal egg counts. We assessed the impact of age, sex, group size, season and spatial differences used as a proxy, which reflects observed variation in the occurrence of gastrointestinal problems, vegetation types, gorilla subpopulation growth and associated social structure on helminth infections. We revealed striking geographic differences in strongylid infections with higher egg counts mostly in areas with high occurrences of gastrointestinal disease. Increased helminth egg counts were also associated with decreasing group size in some areas. Observed spatial differences may reflect mutual effects of variations in subpopulation growth rates, gorilla social structure, and vegetation associated with altitude across mountain gorilla habitat. Helminth infection intensities in Virunga gorillas were lowest in the youngest and the oldest animals. Elucidating parasite infection patterns of endangered species with low genetic diversity is crucial for their conservation management.

Mapping gastrointestinal gene expression patterns in wild primates and humans via fecal RNA-seq.

BACKGROUND: Limited accessibility to intestinal epithelial tissue in wild animals and humans makes it challenging to study patterns of intestinal gene regulation, and hence to monitor physiological status and health in field conditions. To explore solutions to this limitation, we have used a noninvasive approach via fecal RNA-seq, for the quantification of gene expression markers in gastrointestinal cells of free-range primates and a forager human population. Thus, a combination of poly(A) mRNA enrichment and rRNA depletion methods was used in tandem with RNA-seq to quantify and compare gastrointestinal gene expression patterns in fecal samples of wild Gorilla gorilla gorilla (n = 9) and BaAka hunter-gatherers (n = 10) from The Dzanga Sangha Protected Areas, Central African Republic. RESULTS: Although only a small fraction (< 4.9%) of intestinal mRNA signals was recovered, the data was sufficient to detect significant functional differences between gorillas and humans, at the gene and pathway levels. These intestinal gene expression differences were specifically associated with metabolic and immune functions. Additionally, non-host RNA-seq reads were used to gain preliminary insights on the subjects' dietary habits, intestinal microbiomes, and infection prevalence, via identification of fungi, nematode, arthropod and plant RNA. CONCLUSIONS: Overall, the results suggest that fecal RNA-seq, targeting gastrointestinal epithelial cells can be used to evaluate primate intestinal physiology and gut gene regulation, in samples obtained in challenging conditions in situ. The approach used herein may be useful to obtain information on primate intestinal health, while revealing preliminary insights into foraging ecology, microbiome, and diet.

Low diversity of Angiostrongylus cantonensis complete mitochondrial DNA sequences from Australia, Hawaii, French Polynesia and the Canary Islands revealed using whole genome next-generation sequencing.

BACKGROUND: Rats (Rattus spp.) invaded most of the world as stowaways including some that carried the rat lungworm, Angiostrongylus cantonensis, the cause of eosinophilic meningoencephalitis in humans and other warm-blooded animals. A high genetic diversity of A. cantonensis based on short mitochondrial DNA regions is reported from Southeast Asia. However, the identity of invasive A. cantonensis is known for only a minority of countries. The affordability of next-generation sequencing for characterisation of A. cantonensis genomes should enable new insights into rat lung worm invasion and parasite identification in experimental studies. METHODS: Genomic DNA from morphologically verified A. cantonensis (two laboratory-maintained strains and two field isolates) was sequenced using low coverage whole genome sequencing. The complete mitochondrial genome was assembled and compared to published A. cantonensis and Angiostrongylus malaysiensis sequences. To determine if the commonly sequenced partial cox1 can unequivocally identify A. cantonensis genetic lineages, the diversity of cox1 was re-evaluated in the context of the publicly available cox1 sequences and the entire mitochondrial genomes. Published experimental studies available in Web of Science were systematically reviewed to reveal published identities of A. cantonensis used in experimental studies. RESULTS: New A. cantonensis mitochondrial genomes from Sydney (Australia), Hawaii (USA), Canary Islands (Spain) and Fatu Hiva (French Polynesia), were assembled from next-generation sequencing data. Comparison of A. cantonensis mitochondrial genomes from outside of Southeast Asia showed low genetic diversity (0.02-1.03%) within a single lineage of A. cantonensis. Both cox1 and cox2 were considered the preferred markers for A. cantonensis haplotype identification. Systematic review revealed that unequivocal A. cantonensis identification of strains used in experimental studies is hindered by absence of their genetic and geographical identity. CONCLUSIONS: Low coverage whole genome sequencing provides data enabling standardised identification of A. cantonensis laboratory strains and field isolates. The phenotype of invasive A. cantonensis, such as the capacity to establish in new territories, has a strong genetic component, as the A. cantonensis found outside of the original endemic area are genetically uniform. It is imperative that the genotype of A. cantonensis strains maintained in laboratories and used in experimental studies is unequivocally characterised.

Mammomonogamus nematodes in felid carnivores: a minireview and the first molecular characterization.

Five of the 13 known species of Mammomonogamus have been described in members of the family Felidae, including domestic cats, making felids the most frequent hosts of Mammomonogamus. The occurrence of Mammomonogamus in felids is geographically scattered and information on the life cycle and other aspects of infections is lacking. The paucity of data opens the questions on possible conspecificity of some of the described species of Mammomonogamus and on the existence of possible reservoirs for infections in domestic cats in geographically isolated endemic foci of infection. To test such hypotheses, we compared sequences of mitochondrial and nuclear markers obtained from Mammomonogamus adults or eggs collected from domestic cats in three geographically distant localities. Based on morphology, geographic origin and site of infection, the worms examined can be referred to as Mammomonogamus ierei and Mammomonogamus auris. Phylogenetic analyses of both mitochondrial and ribosomal DNA markers showed monophyly of the genus Mammomonogamus and suggested the existence of at least two species in cats. Review of the literature, the existence of several species and the discontinuous geographic distribution of Mammomonogamus infections in domestic cats suggest an historical spillover of infection from wild reservoirs, presumably wild felids.

Diversity of Mammomonogamus (Nematoda: Syngamidae) in large African herbivores.

Four species of Mammomonogamus are known from large African herbivores. A recent study demonstrated that a single Mammomonogamus species was shared by both western lowland gorillas (Gorilla gorilla gorilla) and African forest elephants (Loxodonta cyclotis) in Central African Republic, suggesting lower species diversity than previously described in literature. We examined more than 500 fecal samples collected from sympatric African forest elephants, western lowland gorillas, and African forest buffaloes (Syncerus caffer nanus) at four study sites across Central Africa and examined them by coproscopic methods to detect Mammomonogamus eggs, which were found at three of the study sites. Subsequently, sequences of 18S rDNA, 28S rDNA, and cox1 amplified from individual eggs were analyzed. Phylogenetic analyses of both nuclear and mitochondrial DNA revealed two clades: one formed by sequences originating from Gabonese buffaloes and the other comprising gorillas and elephants. The gorilla-elephant clade was further differentiated depending on the locality. We show the existence of at least two distinct species of Mammomonogamus, M. loxodontis in elephants and gorillas and M. nasicola in buffaloes. The available information on Mammomonogamus in African herbivores is reviewed.

Correction to: Do habituation, host traits and seasonality have an impact on protist and helminth infections of wild western lowland gorillas?

Affiliation of Klára J. Petrzelková was incorrectly assigned as 2, 9, 10 in the original version of this article when in fact it should have been 3, 9, 10. Correct affiliations are presented here.

Do habituation, host traits and seasonality have an impact on protist and helminth infections of wild western lowland gorillas?

Increased anthropogenic activity can result in parasite exchanges and/or general changes in parasite communities, imposing a health risk to great apes. We studied protist and helminth parasites of wild western lowland gorilla groups in different levels of habituation, alongside humans inhabiting Dzanga-Sangha Protected Areas in the Central African Republic. Faeces were collected yearly during November and December from 2007 to 2010 and monthly from November 2010 to October 2011. Protist and helminth infections were compared among gorilla groups habituated, under habituation and unhabituated, and the effect of host traits and seasonality was evaluated. Zoonotic potential of parasites found in humans was assessed. No significant differences in clinically important parasites among the groups in different stages of habituation were found, except for Entamoeba spp. However, humans were infected with four taxa which may overlap with taxa found in gorillas. Females were less infected with spirurids, and adults had higher intensities of infection of Mammomonogamus sp. We found seasonal differences in the prevalence of several parasite taxa, but most importantly, the intensity of infection of unidentified strongylids was higher in the dry season. This study highlights that habituation may not necessarily pose a greater risk of protist and helminth infections in gorilla groups.

Host specificity and basic ecology of Mammomonogamus (Nematoda, Syngamidae) from lowland gorillas and forest elephants in Central African Republic.

Syngamid strongylids of the genus Mammomonogamus undoubtedly belong among the least known nematodes with apparent zoonotic potential and the real diversity of the genus remains hard to evaluate without extensive molecular data. Eggs of Mammomonogamus sp. are frequently found in feces of African forest elephants (Loxodonta cyclotis) and western lowland gorillas (Gorilla gorilla gorilla) in Dzanga-Sangha Protected Areas. Using sedimentation-based coproscopic techniques, we found the eggs of Mammomonogamus in 19·7% elephant and 54·1% gorilla fecal samples with 8-55 and 1-24 eggs per gram of fecal sediment for elephants and gorillas, respectively. We used a combination of light microscopy, scanning electron microscopy and analysis of cytochrome c oxidase subunit I (cox1) and a partial sequence of 18S rDNA isolated from single eggs to test the hypothesis of possible Mammomonogamus conspecificity in gorillas and elephants. Whereas 18S rDNA sequences were identical in both gorillas and elephants, we distinguished seven different haplotypes within the cox1. Two haplotypes were found in both gorillas and elephants suggesting sharing of Mammomonogamus. Assignment of the parasite to M. loxodontis is proposed. Provided sequences represent the first genomic data on Mammomonogamus spp.

Schistosoma mansoni in Gabon: Emerging or Ignored?

Schistosomiasis affects millions of people across Africa. We detected eggs of Schistosoma mansoni in western lowland gorilla and central chimpanzee fecal samples in Loango National Park, Gabon. We analyzed nuclear and mitochondrial DNA, namely internal transcribed spacer and cytochrome c oxidase subunit 1 fragments, and the resulting maximum likelihood phylogenetic analyses and haplotype network of the ITS and COI, respectively, showed that the samples from gorillas and chimpanzees clustered clearly within the S. mansoni clade. This is the first confirmed record of S. mansoni from Gabon, which urges surveillance in the area and prompts questions regarding the extent of zoonotic transmission and the clinical impact.