New Insights on Tools for Detecting ß-Tubulin Polymorphisms in Trichuris trichiura Using rhAmpTM SNP Genotyping.

Soil-transmitted helminth (STH) infections, commonly treated with benzimidazoles, are linked to resistance through single nucleotide polymorphisms (SNPs) at position 167, 198, or 200 in the ß-tubulin isotype 1 gene. The aim of this study was to establish a novel genotyping assay characterized by its rapidity and specificity. This assay was designed to detect the presence of SNPs within the partial ß-tubulin gene of Trichuris trichiura. This was achieved through the biallelic discrimination at codons 167, 198, and 200 by employing the competitive binding of two allele-specific forward primers. The specificity and reliability of this assay were subsequently confirmed using Trichuris samples isolated from captive primates. Furthermore, a molecular study was conducted to substantiate the utility of the ß-tubulin gene as a molecular marker. The assays showed high sensitivity and specificity when applied to field samples. Nevertheless, none of the SNPs within the ß-tubulin gene were detected in any of the adult worms or eggs from the analyzed populations. All specimens consistently displayed an SS genotype. The examination of the ß-tubulin gene further validated the established close relationships between the T. trichiura clade and Trichuris suis clade. This reaffirms its utility as a marker for phylogenetic analysis.

New genetic lineage of whipworm present in Bactrian camel (Camelus bactrianus).

With a global population of around 35 million in 47 countries, camels play a crucial role in the economy of many marginal and desert areas of the world where they survive in harsh conditions. Nonetheless, there is insufficient knowledge regarding camels' parasite fauna which can reduce their milk and meat production. A molecular study for the Trichuris population of Camelus bactrianus from Spain is presented based on sequences of mitochondrial (cox1, cob, rrnL) and ribosomal (ITS1 and ITS2) DNA regions. Bayesian Inference and Maximum Likelihood methods were used to infer phylogenies for (i) each gene separately, (ii) the combined mitochondrial data, and (iii) the combined mitochondrial and ribosomal dataset. Molecular analyses revealed the existence of two different genetic lineages in the Trichuris parasites populations of C. bactrianus. Future studies should focus on whether there is a coevolution process corresponding to the wild or domestic character of C. bactrianus and Camelus dromedarius. Furthermore, it is necessary to increase integrative taxonomic studies on Trichuris spp. based on morphological, biometric, and molecular data, which will inevitably contribute to our knowledge of the etiology of trichuriasis.

Rodentolepis microstoma isolated from different species of Sigmodontinae rodents (Rodentia: Cricetidae) in the Cuenca del Plata, Argentina: Morphological aspects and molecular characterization.

The purpose of this paper was to study specimens of the genus Rodentolepis isolated from eight species of Sigmodontinae rodents (Rodentia: Cricetidae) from six provinces in the Cuenca del Plata, Argentina, based on morphological, morphometric and molecular characteristics (ITS1 rDNA and cox1 mtDNA). The genetic distances among studied specimens and other Hymenolepididae from rodents available in the GenBank were analyzed and phylogenetic inferences were provided. A total of 955 specimens of Sigmodontinae rodents were examined from seven localities of six provinces in the Cuenca del Plata region in Argentina. Tapeworms were removed from the rodents' small intestines. Conventional studies were used for the morphological and molecular analysis. Specimens of R. microstoma were identified. An amended diagnosis and detailed morphological description of this species is provided. The molecular analyses showed that the specimens studied form the same clade as that of R. microstoma previously studied from other hosts and regions. The genetic polymorphisms of R. microstoma observed corresponded to different groups of species hosts and regions. Moreover, eight species of sigmodontine rodents and 33 localities from the Cuenca del Plata region in Argentina constitute new host and geographical records. This study shows the importance of using integrative taxonomic approaches that combine morphological and molecular characters to understand biological diversity. Moreover, the discovery of R. microstoma in humans suggests the importance of further studies on this zoonotic cestode. This study provides important data on the taxonomy and distribution of R. microstoma to advance knowledge of the transmission dynamics of this parasite.

The Use of MALDI-TOF MS as a Diagnostic Tool for Adult Trichuris Species.

Trichuriasis is considered a neglected tropical disease, being the second most common helminthiasis in humans. Detection of Trichuris in routine diagnosis is usually done by microscopic detection of eggs in fecal samples. Other molecular analyses are more reliable and could be used, but these analyses are not routinely available in clinical microbiology laboratories. The use of matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) is increasing since the last decades due to its recent evidence as a potential role for reliable identification of microorganisms and a few nematodes. But, for parasites detection, normalized protocols and the acquisition and introduction of new species to the database are required. We carried out a preliminary study confirming the usefulness of MALDI-TOF MS for the rapid and reliable identification of Trichuris suis used as control and the creation of an internal database. To create main spectra profiles (MSPs), the different parts of five whipworms (esophagus and intestine) were used, developing different tests to verify the repeatability and reproducibility of the spectra. Thus, to validate the new internal database, 20 whipworms, separating the esophagus and intestine, were used, of which 100% were accurately identified as T. suis, but could not distinguish between both parts of the worm. Log score values ranged between 1.84 and 2.36, meaning a high-quality identification. The results confirmed that MALDI-TOF MS was able to identify Trichuris species. Additionally, a MALDI-TOF MS profile of T. suis proteome was carried out to develop the first internal database of spectra for the diagnosis of trichuriasis and other Trichuris spp.

Characterization of trichuris species from porcupine (Hystrix cristata) at zoological garden of Spain.

Population of Trichuris sp. isolated from Hystrix cristata was analyzed based on morphological, biometrical characteristics and mitochondrial (cox1, cob, rrnL) and ribosomal (ITS1) (rDNA) region sequences. Morphological and biometrical results revealed that Trichuris sp. from H. cristata present a high similarity with Trichuris landak from Hystrix javanica and less similarity with other Trichuris species from porcupine species (Trichuris hystricis, Trichuris lenkorani and Trichuris mettami). The lack of molecular data corresponding to Trichuris species that parasitize the porcupine (genus Hystrix and Atelerix) has not allowed a comparative molecular or phylogenetic study. Molecular analyses revealed the existence of two different haplotypes that did not correspond to different morphospecies. Relationships among Trichuris sp. from H. cristata and other Trichuris spp. have been resolved by molecular sequence data in this study. Thus, the combined analysis of one ribosomal and three mitochondrial markers revealed a sister relationship between whipworms parasitizing porcupine and other Trichuris spp. from rodents and canids and separated from the rest of Trichuris spp. from other hosts species. It is necessary accurate information on the possible zoonotic behavior of different Trichuris species for health workers to improve existing control measures. Thus, it is necessary to increase the studies of integrative taxonomy on Trichuris spp. based on morphological, biometrical and molecular data, which will inevitably contribute to our knowledge on the etiology of trichuriasis.

Complete Mitochondrial Genome of Trichuristrichiura from Macaca sylvanus and Papio papio.

Trichuriasis is among the most prevalent worldwide parasitism caused by helminths. For many years, Trichuris spp. have been described with a relatively narrow range of both morphological and biometrical features. The use of the complete mitochondrial genome (mitogenome) is an alternative and powerful molecular method for inferring phylogenies. Here, we present an overview of the contributions of mitogenome for Trichuris spp. from human and non-human primates. In addition, we carry out structural and phylogenetic comparative analyses with genomes of Trichuris species available in public datasets. The complete mt genomes of Trichuris trichiura and Trichuris sp. from Macaca sylvanus and T. trichiura from Papio papio are 14,091 bp, 14,047 bp and 14,089 bp in length, respectively. The three mt genomes are circular and consist of 37 genes-13 PCGs (cox1-3, nad1-6, nad4L, atp6, atp8 and cob), 22 transfer RNA genes (tRNAs), and two rRNAs (rrnL and rrnS). The molecular evidence presented here supports the hypothesis that T. trichiura de M. sylvanus (TMF31) and T. trichiura de P. papio (TPM1) were similar but genetically different with respect to Trichuris sp. from macaques (TMM5). The phylogenetic study also supported the evolution of the different Trichuris species. In conclusion, we suggest the existence of two cryptic species parasitizing M. sylvanus.

Trichuris trichiura isolated from Macaca sylvanus: morphological, biometrical, and molecular study.

BACKGROUND: Recent studies have reported the existence of a Trichuris species complex parasitizing primate. Nevertheless, the genetic and evolutionary relationship between Trichuris spp. parasitizing humans and Non-Human Primates (NHP) is poorly understood. The hypothesised existence of different species of Trichuris in primates opens the possibility to evaluate these primates as reservoir hosts of human trichuriasis and other putative new species of whipworms. RESULTS: In this paper, we carried out a morphological, biometrical and molecular study of Trichuris population parasitizing Macaca sylvanus from Spain based on traditional morpho-biometrical methods, PCA analysis and ribosomal (ITS2) and mitochondrial (cox1 and cob) DNA sequencing. Morphological results revealed that Trichuris sp. from M. sylvanus is Trichuris trichiura. Ribosomal datasets revealed that phylogenetic relationships of populations of Trichuris sp. from M. sylvanus were unresolved. The phylogeny inferred on mitochondrial datasets (partitioned and concatenated) revealed similar topologies; Thus, phylogenetic trees supported the existence of clear molecular differentiation between individuals of Trichuris sp. from M. sylvanus appearing in two different subclades. CONCLUSIONS: Based on morphological parameters, biometrical measurements, and molecular sequence analysis, we conclude that the whipworms isolated from M. sylvanus were T. trichiura. Further, the evolutionary relationship showed that these worms belonged to two genotypes within the T. trichiura lineage. Since T. trichiura is of public health importance, it is important to carry out further studies to improve the understanding of its hosts range, evolution and phylogeography.

Trichuris trichiura (Linnaeus, 1771) From Human and Non-human Primates: Morphology, Biometry, Host Specificity, Molecular Characterization, and Phylogeny.

Human trichuriasis is a Neglected Tropical Disease, which affects hundreds of millions of persons worldwide. Several studies have reported that non-human primates (NHP) represent important reservoirs for several known zoonotic infectious diseases. In this context, Trichuris infections have been found in a range of NHP species living in natural habitats, including colobus monkeys, macaques, baboons, and chimpanzees. To date, the systematics of the genus Trichuris parasitizing humans and NHP is unclear. During many years, Trichuris trichiura was considered as the whipworm present in humans and primates. Subsequently, molecular studies suggested that Trichuris spp. in humans and NHP represent several species that differ in host specificity. This work examines the current knowledge of T. trichiura and its relationship to whipworm parasites in other primate host species. A phylogenetic hypothesis, based on three mitochondrial genes (cytochrome c oxidase subunit 1, cytochrome b, and large subunit rRNA-encoding gene) and two fragments of ribosomal DNA (Internal Transcribed Spacer 1 and 2), allowed us to define a complex of populations of T. trichiura hosting in a large variety of NHP species, in addition to humans. These populations were divided into four phylogenetic groups with a different degree of host specificity. From these data, we carry out a new morphological and biometrical description of the populations of Trichuris based on data cited by other authors as well as those provided in this study. The presence of T. trichiura is analyzed in several NHP species in captivity from different garden zoos as possible reservoir of trichuriasis for humans. This study contributes to clarify questions that lead to identification of new taxa and will determine parasite transmission routes between these primates, allowing the implementation of appropriate control and prevention measures.

Insights into the molecular systematics of Trichuris infecting captive primates based on mitochondrial DNA analysis.

Nematodes belonging to the Trichuris genus are prevalent soil-transmitted helminths with a worldwide distribution in mammals, while humans are mainly affected in areas with insufficient sanitation such as in Africa, Asia and South America. Traditionally, whipworms infecting primates are referred to Trichuris trichiura, but recent molecular and morphological evidence suggests that more than one species may be able to infect humans and non-human primates. Here, we analyzed the genetic diversity and phylogeny of Trichuris infecting five different non-human primate species kept in captivity using sequencing of three mitochondrial genes (cox1, rrnL and cob). Phylogenetic analyses of both single and concatenated datasets suggested the presence of two main evolutionary lineages and several highly supported clades likely existing as separate taxa. The first lineage included Trichuris infecting the mantled guereza (Colobus guereza kikuyensis), the chacma baboon (Papio ursinus) and the green monkeys (Chlorocebus spp.), clustering together with Trichuris suis; the second lineage included Trichuris infecting the Japanese macaque (Macaca fuscata) and the hamadryas baboon (Papio hamadryas), clustering together with Trichuris spp. infecting humans. These results were supported by the genetic distance between samples, which suggested that at least two taxa are able to infect macaques, baboons and humans. The present study improves our understanding of the taxonomy and evolutionary relationships among Trichuris spp. infecting primates. It moreover suggests that multiple Trichuris spp. may circulate among host species and that Trichuris in non human primates (NHPs) may be zoonotic. Further studies are important to better understand the epidemiology of Trichuris in primates and for implementing appropriate control and/or conservation measures.

A new Trichuris species (Nematoda: Trichuridae) from capybaras: Morphological-molecular characterization and phylogenetic relationships.

A new species of Trichuris (Nematoda: Trichuridae) is described from the capybara, Hydrochoeris hydrochaeris (Rodentia: Caviidae), from two localities of Corrientes Province, based on morphological characteristics and ITS1 and ITS2 of nuclear rDNA region sequences. This species present morphological differences with 28 species of the genus described from North and South American rodents. Also, the new species is molecularly characterized and differentiated from those species from Muridae, Cricetidae and Myocastoridae rodents recorded from North and South America, Europe and Africa. Trichuris cutillasae n. sp. constitutes the ninth record from rodents of Argentina, and the second record for Caviidae. The phylogenetic analysis showed that the specimens of new species are in the same clade, and separated from specimens of T. myocastoris (ITS1and ITS2), T. navonae, T. bainae, Trichuris sp. and T. pardinasi (ITS2). Similarly, analysis of concatenated sequences ITS1 and ITS2 added further evidence that T. cutillasae n. sp. is a different species from those previously analyzed.

Evidence of cryptic species in the genus Tinaminyssus (Acari: Rhinonyssidae) based on morphometrical and molecular data.

The study of cryptic species allows to describe and to understand biodiversity, and the evolutionary processes shaping it. Mites of the family Rhinonyssidae are permanent parasites of the nasal cavities of birds, currently including about 500 described species and 12 genera. Here, we tested the hypothesis that mites from five populations of the genus Tinaminyssus-three isolated from European turtle doves (Streptopelia turtur), and two from Eurasian collared doves (Streptopelia decaocto; Aves: Columbiformes)-are, in fact, two cryptic species inhabiting different hosts. First, we performed a morphometrical study on 16 traits. Then, we used the ITS1-5.8S rDNA-ITS2 nuclear region (ITS region), and a fragment of the mitochondrial cytochrome c-oxidase 1 (COI) to carry out phylogenetic and species delimitation analyses on Tinaminyssus species. Morphological analyses revealed a lack of biometric differentiation among Tinaminyssus populations from the two host species. However, molecular analyses indicated a high degree of genetic differentiation between populations of Tinaminyssus sp. from S. turtur and S. decaocto. Overall, results show that they can be considered as different cryptic species, suggesting a case of evolutionary stasis, likely because of the anatomical similarity between closely-related bird host species.

Morphological-molecular characterization and phylogenetic relationships of a new Trichuris species (Nematoda: Trichuridae) parasitic on Holochilus chacarius (Cricetidae: Sigmodontinae) from the Chaco ecoregion (Argentina).

A new Trichuris species isolated from Holochilus chacarius (Cricetidae: Sigmodontinae) from the Chaco ecoregion in Argentina is described based on morphological characteristics and mitochondrial (cox1, cob) and nuclear (ITS2) markers. The new species is distinguished from 27 species of Trichuris from North and South American rodents based on morphological and biometrical features, such as the absence of a spicular tube, presence of a cylindrical spicular sheath, non-protrusive vulva, length of spicule, and proximal and distal cloacal tube. In addition, the results based on three molecular markers of the new species and three Trichuris species previously analysed from sigmodontine rodents: Trichuris pardinasi, Trichuris navonae and Trichuris bainae confirmed that the specimens here studied belong to a different species. Molecular data are further used to discuss the phylogenetic relationships among the Trichuris species of rodents from Argentina. The combined analysis of mitochondrial genes (cox1 and cob) revealed four clades corresponding with four different species of Trichuris. T. navonae (Akodontini rodents) and T. massoiai n. sp. as a sister group related to T. bainae (Oryzomyini rodents) and separated of T. pardinasi (Phyllotini rodents).

Description of a new species, Trichuris ursinus n. sp. (Nematoda: Trichuridae) from Papio ursinus Keer, 1792 from South Africa.

In the present work, we carried out a morphological, biometrical and molecular study of whipworms Trichuris Roederer, 1761 (Nematoda: Trichuridae) parasitizing Papio ursinus Keer 1792 (Chacma baboon). Biometrical and molecular data suggest a new species of Trichuris parasitizing baboons. In addition of main morphological features (spicule, spicule sheath, spicule tube, proximal cloacal tube, distal cloacal tube, vulva, vagina), the mean values of individual variables between Trichuris colobae, Trichuris suis, Trichuris trichiura examined by Student's t tests suggest that T. ursinus n. sp. constitutes a new species. The combined analysis of three markers (cox1, cob and ITS2) revealed a sister relationships between T. colobae and T. ursinus n. sp. Mitochondrial sequences revealed a higher inter-specific similarity between T. ursinus n. sp., T. suis and T. colobae. Phylogenetic hypotheses for both mitochondrial genes strongly supported distinct genetic lineages corresponding to different species of the genus Trichuris associated with certain hosts. Thus, T. suis, T. colobae and T. ursinus n. sp. appeared as a sister group and separated from Trichuris spp. from humans and other species of primates.

Molecular diversification of Trichuris spp. from Sigmodontinae (Cricetidae) rodents from Argentina based on mitochondrial DNA sequences.

A molecular phylogenetic hypothesis is presented for the genus Trichuris based on sequence data from mitochondrial cytochrome c oxidase 1 (cox1) and cytochrome b (cob). The taxa consisted of nine populations of whipworm from five species of Sigmodontinae rodents from Argentina. Bayesian Inference, Maximum Parsimony, and Maximum Likelihood methods were used to infer phylogenies for each gene separately but also for the combined mitochondrial data and the combined mitochondrial and nuclear dataset. Phylogenetic results based on cox1 and cob mitochondrial DNA (mtDNA) revealed three clades strongly resolved corresponding to three different species (Trichuris navonae, Trichuris bainae, and Trichuris pardinasi) showing phylogeographic variation, but relationships among Trichuris species were poorly resolved. Phylogenetic reconstruction based on concatenated sequences had greater phylogenetic resolution for delimiting species and populations intra-specific of Trichuris than those based on partitioned genes. Thus, populations of T. bainae and T. pardinasi could be affected by geographical factors and co-divergence parasite-host.

Nuclear and mitochondrial genes for inferring Trichuris phylogeny.

Nucleotide sequences of the triose phosphate isomerase (TPI) gene (624 bp) and mitochondrial cytochrome b (cob) gene (520 bp) were obtained by PCR and evaluated for utility in inferring the phylogenetic relationships among Trichuris species. Published sequences of one other nuclear gene (18S or SSU rRNA, 1816-1846 bp) and one additional mitochondrial (mtDNA) gene (cytochrome oxidase 1, cox1, 342 bp) were also analyzed. Maximum likelihood and Bayesian inference methods were used to infer phylogenies for each gene separately but also for the combined mitochondrial data (two genes), the combined nuclear data (two genes), and the total evidence (four gene) dataset. Few Trichuris clades were uniformly resolved across separate analyses of individual genes. For the mtDNA, the cob gene trees had greater phylogenetic resolution and tended to have higher support values than the cox1 analyses. For nuclear genes, the SSU gene trees had slightly greater resolution and support values than the TPI analyses, but TPI was the only gene with reliable support for the deepest nodes in the tree. Combined analyses of genes yielded strongly supported clades in most cases, with the exception of the relationship among Trichuris clades 1, 2, and 3, which showed conflicting results between nuclear and mitochondrial genes. Both the TPI and cob genes proved valuable for inferring Trichuris relationships, with greatest resolution and support values achieved through combined analysis of multiple genes. Based on the phylogeny of the combined analysis of nuclear and mitochondrial genes, parsimony mapping of definitive host utilization depicts artiodactyls as the ancestral hosts for these Trichuris, with host-shifts into primates, rodents, and Carnivora.

Morphological and molecular characterization of a new Trichuris species (Nematoda- Trichuridae), and phylogenetic relationships of Trichuris species of Cricetid rodents from Argentina.

Populations of Trichuris spp. isolated from six species of sigmodontine rodents from Argentina were analyzed based on morphological characteristics and ITS2 (rDNA) region sequences. Molecular data provided an opportunity to discuss the phylogenetic relationships among the Trichuris spp. from Noth and South America (mainly from Argentina). Trichuris specimens were identified morphologically as Trichuris pardinasi, T. navonae, Trichuris sp. and Trichuris new species, described in this paper. Sequences analyzed by Maximum Parsimony, Maximum Likelihood and Bayesian inference methods showed four main clades corresponding with the four different species regardless of geographical origin and host species. These four species from sigmodontine rodents clustered together and separated from Trichuris species isolated from murine and arvicoline rodents (outgroup). Different genetic lineages observed among Trichuris species from sigmodontine rodents which supported the proposal of a new species. Moreover, host distribution showed correspondence with the different tribes within the subfamily Sigmodontinae.

Trichuris colobae n. sp. (Nematoda: Trichuridae), a new species of Trichuris from Colobus guereza kikuyensis.

In the present work, a morphological and biometrical study of whipworms Trichuris Roederer, 1761 (Nematoda: Trichuridae) parasitizing Colobus guereza kikuyensis has been carried out. Biometrical and statistical data showed that the mean values of individual variables between Trichuris suis and Trichuris sp. from C. g. kikuyensis differed significantly (P < 0.001) when Student's t test was performed: seven male variables (width of esophageal region of body, maximum width of posterior region of body, width in the place of junction of esophagus and the intestine, length of bacillary stripes, length of spicule, length of ejaculatory duct, and distance between posterior part of testis and tail end of body) and three female variables (width of posterior region of body, length of bacillary stripes, and distance of tail end of body and posterior fold of seminal receptacle). The combination of these characters permitted the discrimination of T. suis with respect to Trichuris sp. from C. g. kikuyensis, suggesting a new species of Trichuris. Furthermore, males of Trichuris sp. from C. g. kikuyensis showed a typical subterminal pericloacal papillae associated to a cluster of small papillae that were absent in males of T. suis, while females of Trichuris from Colobus appeared with a vulval region elevated/over-mounted showing a crater-like appearance. The everted vagina showed typical triangular sharp spines by optical microscopy and SEM. Thus, the existence of a new species of Trichuris parasitizing C. g. kikuyensis has been proposed.

Molecular characterization and phylogeny of whipworm nematodes inferred from DNA sequences of cox1 mtDNA and 18S rDNA.

A molecular phylogenetic hypothesis is presented for the genus Trichuris based on sequence data from the mitochondrial cytochrome c oxidase 1 (cox1) and ribosomal 18S genes. The taxa consisted of different described species and several host-associated isolates (undescribed taxa) of Trichuris collected from hosts from Spain. Sequence data from mitochondrial cox1 (partial gene) and nuclear 18S near-complete gene were analyzed by maximum likelihood and Bayesian inference methods, as separate and combined datasets, to evaluate phylogenetic relationships among taxa. Phylogenetic results based on 18S ribosomal DNA (rDNA) were robust for relationships among species; cox1 sequences delimited species and revealed phylogeographic variation, but most relationships among Trichuris species were poorly resolved by mitochondrial sequences. The phylogenetic hypotheses for both genes strongly supported monophyly of Trichuris, and distinct genetic lineages corresponding to described species or nematodes associated with certain hosts were recognized based on cox1 sequences. Phylogenetic reconstructions based on concatenated sequences of the two loci, cox1 (mitochondrial DNA (mtDNA)) and 18S rDNA, were congruent with the overall topology inferred from 18S and previously published results based on internal transcribed spacer sequences. Our results demonstrate that the 18S rDNA and cox1 mtDNA genes provide resolution at different levels, but together resolve relationships among geographic populations and species in the genus Trichuris.