Welcome to the ICOPA 2022 Online Program

Introduction

Subcutaneous dirofilariosis is a vector-borne zoonosis caused by parasitic nematode Dirofilaria repens. Currently, diagnostic methods are based on the occurrence of microfilariae in the bloodstream, ineffective in case of amicrofilaremic infections. Therefore the aim of our study was to develop a new sensitive and specific diagnostic method. Our approach is based on both serological and molecular techniques.

Methods

First, sera samples collected from dogs were classified as infected or non-infected based on: Knott’s test, Dirofilaria repens Somatic Antigen (DrSA) ELISA and Real-Time PCR (qPCR). Sera were used to estimate the diagnostic potential of D. repens recombinant protein ES20/22 and synthetic peptide LH10 selected using phage display technology. Additionally, we designed species-specific primers and MGB-Eclipse probes which enabled the development of a new multiplex qPCR with the use of genomic and cell-free DNA of D. repens.

Results

The LH10 peptide allowed the detection of infected dogs with and without active microfilaremia. Remarkably, results showed a similar pattern to DrSA ELISA and were confirmed by qPCR. Our molecular method enables the detection of fewer than 10 copies of the gene of interest and a fast single-tube differentiation between D. repens and D. immitis and co-infections of these two species.

Conclusions

Our serological method is a first step to developing a new diagnostic tool that may be used for early diagnosis of dirofilariosis. In addition, the multiplex qPCR may be an alternative for quick confirmation and differentiation of infection.

Financial support for this study was provided by The National Centre for Research and Development, Poland (grant LIDER IX 0106/L-9/2017).

References:

1. doi: 10.1038/s41598-022-06116-8

Introduction

Haemonchus contortus is a blood-feeding parasite which is considered one of the greatest health concerns in small ruminants. Available molecular diagnosis are laboratory-dependent and variations of the polymerase chain reaction (PCR). Recombinase polymerase amplification (RPA) is a technique suitable for a point of care diagnosis operating at a temperature of 36°C to 42°C and results within 20-30 minutes. The work presented is a qualitative proof of concept RPA for the detection of H. contortus.

Methods

The ITS2 gene of H. contortus was targeted. The time-temperature conditions were optimised by performing the reaction in combinations in a portable dry block heater using the TwistAmp Basic kit. The amplicons were analysed in 2.5% agarose gel electrophoresis. Additional investigations were conducted to reduce the non-specific amplification and validation of the assay was done.

Results

The sensitivity was recorded up to 100 fg of the serially diluted template DNA. There was no amplification when DNA from Teladorsagia circumcincta, a closely related species, was used. The specificity was validated using a proven single-species ITS2 gene PCR and loop-mediated isothermal amplification (LAMP) assay. Using 0.8M of betaine reduced the non-specific amplification. The best time-temperature factor was at 20 min at 37°C.

Conclusions

Our proof of concept study showed faster assay design by adapting the available PCR primers for RPA with minimal equipment. Our study was aimed at naked-eye detection of results but with the unavailability of the kit in the market, the methodology had to be modified. At the time of writing this abstract, we were working on improving the result detection system with the possibility of quantification for a point of care assay .

Introduction

Parasitic diseases and mitigation of their impact play an important role in health management of grazing cattle worldwide. Among them strongylid nematodes cause mortalities and enormous economic losses every year. They are known to form complex communities within their hosts; these in large herbivores frequently consist of numerous coexisting species with various taxonomic affinities. Identification of all individual strongylid species/haplotypes occurring in noninvasively collected fecal samples using traditional coproscopic methods based on microscopy or molecular analysis is almost impossible. On the contrary, high-throughput amplicon sequencing (HTS) can effectively identify individual strongylid species occurring in the samples.

Methods

During the four-year project we collected and examined almost 7000 fecal samples of beef cattle coming from over 120 farms spread all over the Czech Republic. Strongylids are overall prevalent (53 % in total), however, occur in low infection intensities. We selected samples positive for strongylids and conducted HTS utilising Illumina ITS-2 metabarcoding.

Results

The most widespread genera parasitizing in Czech cattle are Ostertagia and Oesophagostomum, then Trichostrongylus and Cooperia, while Bunostomum, Nematodirus and Chabertia are represented in a very minority. Moreover, we identified shifts in strongylid communities when applying anthelmintic treatment.

Conclusions

Regular and effective screening for parasites detectable in feces prevents the emergence and spread of anthelmintic resistance and remains a pillar for helminth control in extensive pastoral livestock systems with intensified transboundary animal movements.

Introduction

Equine trypanosomosis comprises three diseases caused by protozoa of the subgenus Trypanozoon: Trypanosoma equiperdum (causative agent of dourine), T. brucei (nagana) and T. evansi (surra). Due to the absence of a vaccine and the recurring treatment failure, the development of sensitive and specific diagnostic tests remains crucial for controlling these diseases. In this study, we evaluated the capacity of the 7SL-derived small RNA to serve as a biomarker of active infection, and we developed a microsphere-based immunoassay for equine trypanosomoses diagnosis.

Methods

The 7SL-sRNA detection was performed by a two-step RT-qPCR with a specific stem loop primer-probe. Sera from 6 horses experimentally infected with T. equiperdum (collected every 2 to 4 days post infection) and 63 negative field samples were tested.

The microsphere-based immunoassay was carry out with the xMAP^® technology (Luminex) for which the performance of 8 recombinant antigens (enolase, GM6, PFR1, PFR2, ISG65, VSGat) was evaluated and the selected antigen validated with 349 equine sera.

Results

The 7SL-sRNA signal was detected between 2 and 7 days after horse infections, and the signal remained detectable even when the parasitemia became subpatent.

Among the 8 antigens tested during microsphere-based immunoassay development, GM6 was the most performant showing a sensitivity of 97.9% and a specificity of 96.0%.

Conclusions

These tests remain to be validated with samples from naturally infected horses, but our results demonstrated i) that the detection of the 7SL-sRNA by RT-qPCR could constitute a valuable candidate for the control of Trypanozoon infection of equids and ii) that the GM6 antigen constitutes a suitable target for the diagnosis of equine trypanosomosis using xMAP^® technology.

Introduction

Domestic and wild felids are responsible for faecal dissemination of T. gondii in the environment and for the generation of novel genotypes through genetic recombination during sexual reproduction in the intestine. Besides, the parasite may undergo an extraintestinal multiplication in their tissues like in other intermediate hosts. The aims of this study were to investigate the occurrence and genetic background of T. gondii in the Eurasian lynx (Lynx lynx) population in Switzerland.

Methods

To estimate seroprevalence, 183 blood samples collected from dead lynx between 2002 and 2021 were tested by ELISA and IFAT. Samples with inconclusive results were defined by immunoblot. Tissues from 92 animals were analysed by real-time PCR for T. gondii. Additionally, 176 faecal samples were examined coproscopically for endoparasites. Selected positive DNA samples from oocysts and tissues were further genotyped by a multilocus PCR-RFLP approach.

Results

A total of 150/183 (82.0%, 95%CI 79.1-84.8%) sera tested positive for T. gondii antibodies. T. gondii DNA was detected in tissues (skeletal muscle, brain or heart) from 10/92 lynx. T. gondii oocysts were detected in faeces from 3/176 (1.7%, 95%CI 0.7-2.7%) animals. Two different T. gondii genotypes were identified: a lineage II variant (ToxoDB #3) in three animals and a novel genotype, containing a combination of type III, II and I alleles in a further animal.

Conclusions

The Swiss lynx population is frequently in contact with T. gondii and both intermediate virulent genotypes commonly circulating in Europe (ToxoDB #3), as well as a new genotype were shown to occur. Besides, this is the first report of T. gondii oocysts shedding in the Eurasian lynx, suggesting its involvement in parasite dissemination.

Introduction

Cystic echinococcosis is a zoonotic parasitic disease caused by Echinococcus species. Turkey is one of the highly endemic regions for CE and the disease is one of the major public health problems. The study was aimed to assess the current situation of the CE in sheep and goats in Turkey and also to provide data on circulating genotypes in the country.

Methods

A total of 3319 sheep and 64 goats were farmed in 11 provinces in Turkey and slaughtered in private slaughterhouses in Erzurum province. The cysts collected from organs were cut and examined to indicate a fertile cyst. The germinal membranes and the existed protoscolices were taken for the molecular analysis by targeting the partial fragments of 12S and COI gene regions.

Results

In sheep, 1051 of 3319 had CE infection whereby 50.05% (526) were detected in lungs, 14.65% (154) in livers, and 35.3% (371) both lungs and livers; and 3 of 64 goats had a liver infection. A prevalence of 81.76% fertile cysts in sheep while sterile cysts were revealed in all selected goats. The molecular identification of E. granulosus s.s. genotype (G1-G3) were clarified from the use of Ribosomal ribonucleic acid (rRNA) and Cytochrome c oxidase subunit 1 (cox1) genes. All the obtained sequences showed the E. granulosus s.s. genotype (G1-G3) as the predominant genotype in Turkey and showed sheep potential to transmit CE.

Conclusions

Therefore, to understand more about the molecular epidemiology of Echinococcus spp. in Turkey, we propose a future comprehensive genetic survey on intermediate and definitive hosts of the E. granulosus species.

Introduction

Effective gastrointestinal nematode (GIN) management in livestock industries is becoming increasingly difficult due to the rise of anthelmintic resistance and changes in the temporal and geographical distribution of major GINs. Underpinning the response to these challenges is the need for a fast-tracked diagnostic identification technique, making it easier for livestock producers to make informed GIN management decisions.

Methods

We developed a new diagnostic approach to identify GINs that integrates a remote-location digital faecal egg count platform, FECPAKG2, with internal transcribed spacer 2 nemabiome metabarcoding. The technique involves a quick and simple protocol to harvest concentrated strongyle eggs from the FECPAKG2 cassette utilising a repurposed pipette tip, followed by DNA isolation and Illumina next generation amplicon sequencing.

Results

The GIN compositions and alpha diversity generated by our approach was not significantly different to traditional morphological larval differentiation and nemabiome metabarcoding of larval and faecal samples. We demonstrated that storing FECPAKG2 harvested eggs in either lysis buffer or 80% ethanol had no impact on GIN identification outcomes for at least 60 days; enabling the transport of biological samples from their remote origins to a molecular diagnostic facility, in the absence of a cold chain.

Conclusions

Taking advantage of an already well-established platform such as FECPAKG2, and providing the livestock producers that use it with the option to identify GINs in their samples and contribute to large-scale GIN distribution and/or anthelmintic resistance surveys, is an important future direction for the FECPAKG2 egg nemabiome metabarcoding approach.

Introduction

Toxoplasma gondii has a clonal population structure in Europe. The currently available typing method, based on 15 microsatellite regions, has a limited ability to discriminate between different type II strains. We therefore aimed to establish a next-generation sequencing (NGS)-based method with a higher typing resolution.

Methods

T. gondii isolates were collected from different parts of Europe and assessed by whole genome sequencing (WGS). Highly polymorphic regions (HPRs) were identified. After confirmation by Sanger sequencing, 18 HPRs were used to design a primer panel suitable to establish an Ion AmpliSeq-based typing tool.

Results

In-silico analyses of 81 genome datasets revealed that the HPR-based typing was able to differentiate a larger number of haplotypes than microsatellite typing. Confirmatory experiments applying the new typing tool to DNAs from European field strains covered all 18 regions and almost all the SNPs identified by AmpliSeq corresponded to those expected after WGS analysis.

Conclusions

The results of the study suggest that such NGS-based typing techniques are feasible and that these tools may have very high typing resolution, sufficient to trace infection sources in outbreaks and to detect the introduction of exotic strains.

Funding: This work was done as part of TOXOSOURCES project, supported by funding from the

European Union’s Horizon 2020 Research and Innovation programme under grant agreement No

773830: One Health European Joint Programme.

Introduction

The BoviPar project (Sustainable control of pasture parasites in Norwegian beef and dairy cattle) was established at the Norwegian University of Life Sciences (NMBU) to address the substantial knowledge gap in GIN and F. hepatica status in Norwegian cattle. As part of this project, various extraction techniques and molecular screening platforms will be assessed for their ability to determine parasite load and species distribution.

Methods

While assessing GIN and F. hepatica presence via traditional methods (McMaster and sedimentation) samples will be processed for DNA extraction from four distinct stages of faeces preparation i) direct extraction from the faeces ii) direct extraction post larval culture iii) DNA extraction from Baermanised L3s and iv) purified eggs. These templates were then assessed via quantitative PCR (qPCR) and droplet digital PCR (ddPCR) across a range of targets.

Results

Samples were successfully obtained from 200 first season grazers. From these animals, 80 samples with a wide range in eggs per gram (EPG) (0-1750) of strongylid type eggs, were selected for the various extraction methods (320 samples in total). None of the animals were positive for F. hepatica via sedimentation. DNA has been extracted from all 320 samples with molecular assays to be run imminently. These data will be presented in August in Copenhagen.

Conclusions

This dataset will provide valuable comparative information on the sensitivity and specificity of two widely used molecular platforms, qPCR and ddPCR, from a variety of common faecal processing stages in addition to assessing how well each platform and DNA source performs across a range of known targets. This will be useful to researchers aiming to optimise future study of pasture parasites of ruminants globally.

Abstract Body

Agricultural expansion and urbanization in Southeast Asia have one of the fastest growing rates in the world, and despite of their large-scale footprint on tropical ecosystems, little attention has been given to changes to local biodiversity and ecosystem functioning. Based on field surveys conducted throughout Cambodia between 2014 and 2020, we evaluated how extensive land conversion in the country has driven changes in bat community composition and shaped helminth transmission processes.

Helminth parasites were recovered from the gastrointestinal tract of 451 bat specimens, representing 33 bat species in 14 different habitat types, and categorized morphologically to the species level where possible and to family level where not.

Nematodes were the most prevalent parasite group (84%) followed by cestodes (33%) and trematodes (6.3%). Overall parasite species richness was higher in evergreen and deciduous forest, and lower in disturbed and urban habitats.

This study represents the most extensive survey yet of bat parasites in Cambodia. Our results indicate that bat helminth communities are affected by land conversion and linked to species-specific differences in terms of bat foraging requirements. The lower number of helminth species detected in bats from urban/agricultural areas might be indicative of a reduced capacity of these anthropogenic habitats to support diverse invertebrate assemblages as well. Given the information gap regarding bat-helminth dynamics in the region, our systematic collection of bat parasite data provides valuable information to help in establishing biodiversity baselines, identifying rare taxa, and monitoring changes in parasite biodiversity.

Introduction

The present study provides new insight into suitable microsporidian-hosts associations. It relates regional and continental-wide host specialization in microsporidians infecting amphipods to degraded and recovering habitats across two German river catchments.

Methods

Amphipods were collected in 31 sampling sites with differing degradation and restoration gradients. Specimens were morphologically (hosts) and molecularly identified (host and parasites). Amphipod diversity and abundance, microsporidian diversity, host phylogenetic specificity, and continental-wide β-specificity were investigated and related to each other and/or environmental variables.

Results

14 microsporidian MOTUs, mainly generalist parasites, infecting six amphipods MOTUs were detected, expanding the current knowledge on the host range. There was no difference in microsporidian diversity and host specificity among restored and near-natural streams or between those located in urban and rural areas. Similarly, microsporidian diversity was generally not influenced by water parameters. Host abundances did not influence microsporidian MOTUs richness across restored and near-natural sites. High host turnover across the geographical range suggests that neither environmental conditions nor host diversity plays a significant role in establishing into restored areas.

Conclusions

Host diversity and environmental parameters do not indicate the persistence and dispersal of phylogenetic host generalist microsporidians in environments that experienced anthropogenic disturbance. Instead, these might depend on more complex mechanisms such as the production of resistant spores, host switching, and host dispersal acting individually or conjointly.

Introduction

Agricultural expansion in Southeast Asia has converted most natural landscapes into mosaics of forest interspersed with plantations, dominated by the presence of generalist species that benefit from resource predictability. Dietary shifts resulting from these adaptations can alter the structure of host parasite communities and ultimately impact the fitness and survival of host populations. Our study focuses on the Asian water monitor lizard (Varanus salvator), one of the largest predators in Asian wetlands, as a model species to understand the health consequences of dietary shifts in an oil palm dominated landscape in Sabah, Malaysian Borneo.

Methods

We evaluated the influence of diet diversity on the parasite species richness and prevalence of lizards living in forest patches and oil palm plantation estates.

Results

We observed that lizards feeding on less diverse diets, mostly dominated by rodents in oil palm plantations, hosted less diverse parasite communities with overall higher parasite prevalence. However, parasites with complex transmission modes, such as trematodes, were more prevalent in forested areas.

Conclusions

By working with a widely distributed carnivorous generalist as a model species, we outlined how human-dominated landscapes can pose a negative effect on wildlife species whose diet may be altered by the influence of human activities, such as farming and extensive agriculture.