30th Annual Meeting of the German Society for Parasitology

Prof. Dr.

Winter activity of I. ricinus and D. reticulatus in Germany – results from field studies and a veterinarian submission study

Abstract text Introduction The most important tick species in Germany are Ixodes ricinus, vector of the causative agents of Lyme borreliosis and granulocytic anaplasmosis, rarely found during colder months, and Dermacentor reticulatus, vector of the potentially fatal agent of canine babesiosis, known to be more cold tolerant. Objectives We provide an update on the seasonal activity pattern and possible winter activity of both species to assess whether current national recommendations on tick control in dogs and cats might need adaptations. Materials &amp; methods Tick activity was monitored at nine collection sites via flagging (April 2020-April 2022), as well as in quasi-natural field plots. In a nationwide submission study (May 2020-June 2021), veterinarians sent in ticks from cats and dogs. Results An average of two I. ricinus and 23 D. reticulatus were flagged per 100 m² between December 2020 and February 2021. In the winter 2021/22, one I. ricinus and three D. reticulatus were collected on average. Ixodes ricinus winter activity in tick plots started in February 2021 (4.6%; 16/350), followed by a mean daily winter activity of 1.1% (4/350) in 2021/22. Dermacentor reticulatus showed a mean activity of 20% (20/100) in the winter 2020/21 and 14.7% (22/150) in 2021/22. Of the 19,514 submitted ticks, 211 I. ricinus and 324 D. reticulatus were collected in the winter 2020/21. Conclusion All study approaches demonstrated winter activity of both tick species. To protect dogs and cats with outdoor access from ticks and associated pathogens, an effective year-round tick control is strongly recommended.

Emerging Parasitic Diseases

Veterinary Parasitology

Talk

Cold plasma: a new option for poultry red mite control in laying hen farms?

Abstract text The poultry red mite (PRM), Dermanyssus gallinae, is one of the most common ectoparasites in poultry farming worldwide and can lead to substantial animal health and economic damages. The main problem in control is the rapid development of resistance to a wide range of chemical acaricides. Therefore, research on sustainable and effective control measures is becoming increasingly important. In the present study, the acaricidal effects of cold atmospheric pressure plasma (CAPP) treatment on different development stages of the PRM were investigated under laboratory conditions. For this purpose, mites and mite eggs were exposed to a single plasma pulse generated under different plasma settings, using the ambient air gas mixture at atmospheric pressure. The results showed that all developmental stages of the PRM could be killed by CAPP treatment. The mortality rates showed significant stage-specific differences and increased with progressing time after plasma exposure. The average mortality rate was 99.7% after 12 hours in all plasma-exposed mites. The results show that CAPP has acaricidal effects on all developmental stages of the PRM, including eggs. Further studies are needed to show whether this method can also be used under practical conditions in occupied laying hen houses for efficient control of PRM infestations. Acknowledgement: The project is supported by funds of the German Government´s Special Purpose Fund held at Landwirtschaftliche Rentenbank.

One Health/NTD/Zoonoses

Use of medicinal plants for helminth control – in vitro effect of a hydroethanolic extract from Combretum mucronatum leaves on larvae of gastrointestinal nematodes

Abstract text Combretum mucronatum is a forest liane that is used to treat parasite infections in parts of Western and Central Africa, where these infections are often diagnosed by traditional healers and herbal practitioners. However, anthelmintic properties of C. mucronatum on helminths of human and veterinary importance are insufficiently characterized. To understand whether C. mucronatum leave extract (CME) can be a potential natural resource for the control of gastrointestinal nematodes, its activity against infectious larvae of parasitic nematodes was tested in vitro. To this end, the inhibitory activity of hydroethanolic components of C. mucronatum leaves was evaluated by larval migration assays. Even low concentrations of the CME had an inhibitory effect on the migration of larvae of Ascaris suum (IC50=5.5 µg/ml), Ancylostoma caninum (IC50=19.0 µg/ml) and Trichuris suis (IC50=7.4 µg/ml), whereas inhibition of larvae of Toxocara canis (IC50=310.0 µg/ml) and Toxocara cati (IC50=7249.8 µg/ml) was seen at higher doses only. While larvae of Ostertagia ostertagi (IC50=48.9 µg/ml), Cooperia oncophora (IC50=28.3 µg/ml) and Trichostrongylus colubriformis (IC50=2.1 µg/ml) were highly sensitive to the plant extract, larvae of most other gastrointestinal strongylids were only moderately or slightly affected. The larval migration assays indicated a high activity of CME against larvae of helminths of genera which contain also classical soil-transmitted nematodes of human health concern. Ongoing qPCR-based assays on the transcription of chemo resistance-inducing and detoxification-related genes may unravel mechanisms underlying this activity. Moreover, in vivo experiments with H. contortus-infected goats treated with different dosages of CME will show whether the extract is suitable for the treatment of H. contortus infections.

Drug Development/Target Identification

Investigating the nemabiome using the COI marker region to decipher the cyathostomin diversity in equids Investigating the nemabiome using the COI marker region to decipher the cyathostomin diversity in equids

Abstract text Introduction Cyathostomins are ubiquitous in equids. While the nematode genera of equids have in general low species diversity, the Cyathostominae with 51 accepted species are the exception and represent a highly diverse multi-species complex. They are the most common parasites in the large intestine of equids and can cause severe clinical symptoms due to simultaneous excystation of the encysted larvae into the gut lumen. Anthelmintic resistance in parasitic nematodes is widespread and, therefore, of considerable veterinary and economic importance. Morphological identification and differentiation of these species requires detailed knowledge of their morphology and intensive training. Objective Improved tools for species identification are needed to investigate the interaction and influence of competing species within an ecological niche in horse intestine. The cytochrome c oxidase I (CO1) gene represents an efficient marker to estimate strongyle species richness. Compared to the internal transcribed spacer 2 (ITS-2) marker region, this marker region allows a reliable differentiation between Cylicostephanus calicatus and Coronocyclus coronatus. Materials and methods Therefore, the CO1 marker region was used to perform the Next Generation Sequencing (NGS) approach to detect strongyle population diversity. To compare the nemabiome between hosts with different geographical background and different treatment histories, an average of 10 treated and 10 untreated horses from Brazil, Ukraine, Kentucky, Scotland and Germany were examined and strongyle third stage larvae (L3) were obtained. Using Illumina MiSeq v300 amplicons were sequenced and a bioinformatic data processing pipeline was developed which provides a tool to distinguish between the different cyathostomin species. Results A total of 144 samples were analysed. On average, 38,500 reads could be generated per sample. Using the metabarcoding-dual-indexing approach, as well as bioinformatic pipelines, it was possible to assign the non-overlapping sequences to each individual sample and Strongylidae species could be detected. Conclusions Metabarcoding is a powerful tool which enables molecular characterization and investigation of the diversity and dynamics of strongyle populations within and between different hosts. The evaluation of the NGS data set allows identifying of the different cyathostomin species, per animal and the effects of geographic regions and management practices.

Molecular Parasitology

Dr. med. vet.

Comparison of different diagnostic methods for the detection of Toxocara spp. in faecal samples of cats and dogs

Abstract text Introduction: Toxocara canis and Toxocara cati are parasitic nematodes that occur worldwide. Embryonated Toxocara spp. eggs in the environment pose a zoonotic risk, especially for children. Little is known about the diagnostic performance and comparison of different detection methods of Toxocara spp. in faeces of cats and dogs. Objectives: The aim of this study is to assess the analytical sensitivity of different methods for the detection of Toxocara spp. in faeces of cats and dogs. In addition, a high-throughput method has been developed to facilitate epidemiological studies at the species level. Materials &amp; methods: Two automated DNA extraction methods using mechanical lysis and the King Fisher® flex system were compared regarding their performances. DNA was assessed for parasite-specific genome sequences by TaqMan® real-time PCR. All faecal samples were also examined using the sedimentation-flotation method to allow comparisons between molecular and conventional parasitological methods. Furthermore, a sieving method for the detection, enrichment and purification of parasite eggs in faecal samples was developed and subsequently applied. Results: The analytical sensitivity of real-time PCR in combination with automated DNA extraction methods was higher if King Fisher® in 96-well-plates was applied (limit of detection: &lt; 1 egg/extraction [200 µl] (T. cati), 3 eggs/extraction [200 µl] (T. canis)) as compared to the use of 24-deep-well-plates (limit of detection: 2 eggs/extraction [1 ml] (T. cati), 28 eggs/extraction [1 ml] (T. canis)). The frequently used sedimentation-flotation method had a slightly lower analytical sensitivity (limit of detection: 78 eggs/sample) compared to the new established sieving method (limit of detection: 10 eggs/sample). Conclusion: Real-time PCR in combination with the automated DNA extraction in 96-well-plates and the sieving method had comparable analytical sensitivities. The sieving method was developed to purify and enrich parasite eggs, with a higher detection sensitivity than the sedimentation-flotation method. However, the method based on the automated DNA extraction and subsequent real-time PCR provides a significantly higher sample throughput. Thus, it allows the examination of at least five times more samples at the same time at species level. Therefore, this method is markedly more advantageous than the conventional flotation method from a labour, economic and epidemiological point of view.

Diagnosis and Vaccinatio

Dr. rer. nat.

Prof., Dr.

Typing of European Toxoplasma gondii type II strains by a novel Ion AmpliSeq-based method

Abstract text Introduction Toxoplasma gondii has a clonal population structure in Europe, where the type II clonal lineage prevails. A genotyping method based on 15 microsatellite (MS) regions represents the current reference standard. Objectives We aimed to establish a highly discriminatory next-generation sequencing (NGS)-based typing method for T. gondii to differentiate closely-related type II strains, to trace back infection sources and monitor for recombinant and emerging T. gondii strains. Materials &amp; methods Toxoplasma gondii field isolates (n=110) were collected from different parts of Europe and assessed by whole genome sequencing (WGS). In comparison to ME49 (a type II reference), different highly polymorphic regions (HPRs) were identified, showing a considerable number of single nucleotide polymorphisms (SNPs). After confirmation by Sanger sequencing, 18 HPRs were used to design a primer panel for multiplex PCR in order to establish a multilocus Ion AmpliSeq-based typing method. The isolates analysed by WGS were typed with the novel method, and the sensitivity of the method was tested with serially diluted reference DNA samples. Results Type I, II and III clonal lineages were clearly separated by the 18 HPR Ion AmpliSeq typing technique. Two recombinant strains were correctly identified as type II x III strains. Among the type II strains, the method could differentiate a larger number of haplotypes compared to MS typing. The numbers and identities of SNPs were identical in outbreak isolates, indicating a common source. Furthermore, almost all SNPs identified by the method corresponded to those that were expected based on WGS. By testing serially diluted DNA samples, the technique exhibited similar analytical sensitivity as MS typing. Conclusion The results of our study show that reliable typing of T. gondii strains using an NGS-based multilocus typing method is feasible. The developed 18 HPR-based Ion AmpliSeq method has a high typing resolution, which appears promising for tracing infection sources in outbreaks and for detecting recombinant strains. Funding: This work was done as part of TOXOSOURCES project, supported by funding from the European Union&quot;s Horizon 2020 Research and Innovation programme under grant agreement No 773830: One Health European Joint Programme.

Session II
Veterinary Parasitology & Wildlife Parasites I

Appointment

Chairs

Programme

Session IIVeterinary Parasitology & Wildlife Parasites I

Appointment

Chairs

Programme

Session II
Veterinary Parasitology & Wildlife Parasites I