High-throughput sequencing of Strongyloides stercoralis - a fatal disseminated infection in a dog.

The rhabditid nematode Strongyloides stercoralis is known worldwide as the causative agent of strongyloidiasis in humans. In addition to public health concerns, S. stercoralis also infects dogs, which represent a possible reservoir for potentially zoonotic transmissions. We describe the first confirmed case of fatal disseminated infection in a dog in the Czech Republic. The microscopic and histological results were supported by a complex genotyping approach. Using high-throughput sequencing of the hypervariable region (HVR-IV) of 18S rDNA and Sanger sequencing of the partial cytochrome c oxidase subunit 1 gene (cox1), the potentially zoonotic haplotype/lineage A of S. stercoralis was confirmed, while the solely canine haplotype/lineage B was not found. The development of the disease is mainly associated with immunodeficiency, and in this case, it was triggered by inappropriate treatment, in particular the use of corticosteroids.

Hookworm genomics: dusk or dawn?

Hookworms are parasites, closely related to the model nematode Caenorhabditis elegans, that are a major economic and health burden worldwide. Primarily three hookworm species (Necator americanus, Ancylostoma duodenale, and Ancylostoma ceylanicum) infect humans. Another 100 hookworm species from 19 genera infect primates, ruminants, and carnivores. Genetic data exist for only seven of these species. Genome sequences are available from only four of these species in two genera, leaving 96 others (particularly those parasitizing wildlife) without any genomic data. The most recent hookworm genomes were published 5 years ago, leaving the field in a dusk. However, assembling genomes from single hookworms may bring a new dawn. Here we summarize advances, challenges, and opportunities for studying these neglected but important parasitic nematodes.

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.

Strongyloides questions-a research agenda for the future.

The Strongyloides genus of parasitic nematodes have a fascinating life cycle and biology, but are also important pathogens of people and a World Health Organization-defined neglected tropical disease. Here, a community of Strongyloides researchers have posed thirteen major questions about Strongyloides biology and infection that sets a Strongyloides research agenda for the future. This article is part of the Theo Murphy meeting issue 'Strongyloides: omics to worm-free populations'.

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.

Haemogregarines of the Genera Haemogregarina, Hemolivia, and Hepatozoon Infecting Vietnamese Freshwater Turtles, with Additional Notes on Primer Specificity and Primer-template Mismatches Affecting Diagnostic Success.

The 18S rRNA gene is a widely used molecular marker for haemogregarines. In recent decades, many primers more or less specific to various haemogregarine genera have been designed. This study applied five commonly used primers targeting the 18S rRNA gene of haemogregarines to blood samples from 168 individuals of nine turtle species captured in Northern Vietnam. Three haemogregarine genera, Haemogregarina, Hemolivia, and Hepatozoon, were detected. Selective specificity of primers EF/ER, HemoFN/HemoRN, and Hemo1/Hemo2 to haemogregarine genera was observed and elucidated by primer-template mismatches. In total, 13 out of 168 turtles (prevalence 7.7%) were both microscopically and PCR positive for haemogregarines. Additionally, a single Heosemys grandis turtle was PCR positive but microscopically negative. Numerous turtles carried mixed infections by various haemogregarines; a single turtle was even coinfected by haemogregarines of all three studied genera: Haemogregarina, Hemolivia, and Hepatozoon. Among the detected haemogregarines, some provided sufficient molecular and morphological data for completing their species diagnosis. Two were described as new species: Haemogregarina cyclemydis sp. nov. from Cyclemys pulchristriata and Hemolivia cruciata sp. nov. from Cuora galbinifrons, so far the first Hemolivia from Southeast Asia. Haemogregarina cuorae Chai and Chen, 1990, required a redescription with reassignment to the genus Hepatozoon Miller, 1908.

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.

High Prevalence and Low Diversity of Rickettsia in Dermacentor reticulatus Ticks, Central Europe.

We collected 1,671 Dermacentor reticulatus ticks from 17 locations in the Czech Republic, Slovakia, and Hungary. We found 47.9% overall prevalence of Rickettsia species in ticks over all locations. Sequence analysis confirmed that all tested samples belonged to R. raoultii, the causative agent of tick-borne lymphadenopathy.

Strongyloides genotyping: a review of methods and application in public health and population genetics.

Strongyloidiasis represents a major medical and veterinary helminthic disease. Human infection is caused by Strongyloides stercoralis, Strongyloides fuelleborni fuelleborni and Strongyloides fuelleborni kellyi, with S.stercoralis accounting for the majority of cases. Strongyloides f. fuelleborni likely represents a zoonosis acquired from non-human primates (NHPs), while no animal reservoir for S. f. kellyi infection has been found. Whether S. stercoralis represents a zoonosis acquired from dogs and cats remains unanswered. Over the past two decades various tools have been applied to genotype Strongyloides spp. The most commonly sequenced markers have been the hyper-variable regions I and IV of the 18S rRNA gene and selected portions of the cytochrome c oxidase subunit I gene. These markers have been sequenced and compared in Strongyloides from multiple hosts and geographical regions. More recently, a machine learning algorithm multi-locus sequence typing approach has been applied using these markers, while others have applied whole genome sequencing. Genotyping of Strongyloides from dogs, cats, NHPs and humans has identified that S. stercoralis likely originated in dogs and adapted to human hosts. It has also been demonstrated that S. stercoralis is distinct from S. f. fuelleborni and S. f. kellyi. Two distinct genetic clades of S. stercoralis exist, one restricted to dogs and another infecting humans, NHPs, dogs and cats. Genotyping of S. f. fuelleborni has identified two separate clades, one associated with African isolates and another Indochinese peninsular clade. This review summarises the history and development of genotyping tools for Strongyloides spp. It describes the findings of major studies to date in the context of the epidemiology and evolutionary biology of these helminths, with a specific focus on human-infecting species.