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Abstract Background There is a higher risk for nematode infections associated with outdoor-reared pigs. Next to Ascaris suum, Oesophagostomum dentatum and Trichuris suis, there is the potential of infections with other nodular worm species, Hyostrongylus rubidus, Stongyloides ransomi and Metastrongylus spp. lungworms. Next-generation sequencing methods describing the nemabiome have not yet been established for porcine nematodes. Methods FLOTAC was used for faecal egg counts of porcine gastrointestinal nematodes and lungworms in piglets, fatteners and adults individually. A nemabiome analyses based on ITS-2 gene region metabarcoding was used to differentiate strongyle species. Additionally, questionnaire data was analysed using mixed-effect regression to identify potential risk factors associated with parasite occurrences and egg shedding intensity. Results On 15 of 17 farms nematode eggs were detected. Ascaris suum, strongyles and T. suis were detected on 82%, 70% and 35% of the 17 farms, respectively. Lungworms were detected on one out of four farms with access to pasture. Strongyloides ransomi was not detected. 32% (CI 28–36%), 27% (24–31%), 5% (4–7%) and 3% (0.9–8%) of the samples where tested positive for strongyles, A. suum, T. suis and lungworms, respectively. The nemabiome analysis revealed three different strongyle species, with O. dentatum being the most common (mean 93.9%), followed by O. quadrispinulatum (5.9%) and the hookworm Globocephalus urosubulatus (0.1%). The bivariate and multivariate risk factor analyses showed among others that cleaning once a week compared to twice a week increased the odds significantly for being infected with A. suum (OR 78.60) and strongyles (2077.59). Access to pasture was associated with higher odds for A. suum (43.83) and strongyles (14.21). Compared to shallow litter systems, deep litter and free range systems resulted in significant higher odds for strongyles (85.74, 215.59, respectively) and T. suis (200.33, 623.08). Conclusions Infections with A. suum, O. dentatum, O. quadrispinulatum, T. suis, Metastrongylus spp. and G. urosubulatus are present in German outdoor-reared pigs. This is the first report of G. urosubulatus in domestic pigs in Europe. Metabarcoding based on the ITS-2 region is a suitable tool to analyse the porcine nemabiome. Furthermore, management practices have the potential of reducing the risk of parasite infections.

Abstract Caenorhabditis elegans is an appealing tool for experimental evolution and for working with antiparasitic drugs, from understanding the molecular mechanisms of drug action and resistance to uncover new drug targets. We present a new methodology for studying the impact of antiparasitic drugs in C. elegans. Viscous medium was initially designed for C. elegans maintenance during long-term evolution experiments. Viscous medium provides a less structured environment than the standard nematode growth media agar, yet the bacteria food source remains suspended. Further, the Viscous medium offers the worm population enough support to move freely, mate, and reproduce at a rate comparable to standard agar cultures. Here, the Viscous medium was adapted for use in antiparasitic research. We observed a similar sensitivity of C. elegans to anthelmintic drugs as in standard liquid media and statistical difference to the standard agar media through a larval development assay. Using Viscous medium in C. elegans studies will considerably improve antiparasitic resistance research, and this medium could be used in studies aimed at understanding long-term multigenerational drug activity.

Anthelmintic resistance in sheep parasitic gastrointestinal nematodes is widespread and a severe health and economic issue but prevalence of resistance and involved parasite species are unknown in Germany. Here, the faecal egg count reduction test (FECRT) was performed on eight farms using fenbendazole, ivermectin and moxidectin and on four farms using only moxidectin. A questionnaire was used to obtain data on management practices to potentially identify risk factors for presence of resistance. All requirements of the recently revised WAAVP guideline for diagnosing anthelmintic resistance using the FECRT were applied. Nematode species composition in pre- and post-treatment samples was analysed with the nemabiome approach. Using the eggCounts statistic package, resistance against fenbendazole, ivermectin and moxidectin was found on 7/8, 8/8 and 8/12 farms, respectively. No formal risk factor analysis was conducted since resistance was present on most farms. Comparison with the bayescount R package results revealed substantial agreement between methods (Cohen's κ = 0.774). In contrast, interpretation of data comparing revised and original WAAVP guidelines resulted in moderate agreement (Cohen's κ = 0.444). The FECR for moxidectin was significantly higher than for ivermectin and fenbendazole. Nemabiome data identified 4 to 12 species in pre-treatment samples and treatments caused a small but significant decrease in species diversity (inverse Simpson index). Non-metric multidimensional scaling and k-means clustering were used to identify common patterns in pre- and post-treatment samples. However, post-treatment samples were scattered among the pre-treatment samples. Resistant parasite species differed between farms. In conclusion, the revised FECRT guideline allows robust detection of anthelmintic resistance. Resistance was widespread and involved multiple parasite species. Resistance against both drug classes on the same farm was common. Further studies including additional drugs (levamisole, monepantel, closantel) should combine sensitive FECRTs with nemabiome data to comprehensively characterise the anthelmintic susceptibility status of sheep nematodes in Germany.

Abstract Background Peritoneal dialysis (PD) remains underutilised in Germany, prompting the initiation of the Sustainable Intensification of Peritoneal Dialysis in Schleswig–Holstein (SKIP-SH) project. The SKIP-SH cohort study aims to demonstrate the presumed benefits of PD, including enhanced quality of life and reduced healthcare personnel requirements, and to generate data to strengthen the use of PD. Methods The prospective SKIP-SH cohort study recruits patients with advanced chronic kidney disease (CKD) and their caregivers. Comprehensive data, including demographic information, medical history, clinical course, laboratory data, and quality-of-life assessments, are collected. Additionally, biomaterials will be obtained. Primary study objectives are documenting the clinical course and complications, time on therapy for new dialysis patients, reasons influencing treatment modality choices, circumstances at the initiation of dialysis, and quality of life for patients with CKD and their caregivers. The collected biomaterials will serve as a basis for further translational research. Secondary objectives include identifying factors impacting disease-related quality of life, clinical complications, and therapy dropout, estimating ecological footprints, and evaluating healthcare costs and labour time for initiating and sustaining PD treatment. Discussion PD is notably underutilised in Germany. The current therapy approach for advanced CKD often lacks emphasis on patient-focused care and quality-of-life considerations. Furthermore, adequate explorative research programs to improve our knowledge of mechanisms leading to disease progression and therapy failure in PD patients are scarce. The overarching goal of the SKIP-SH cohort study is to address the notably low PD prevalence in Germany whilst advocating for a shift towards patient-focused care, quality-of-life considerations, and robust translational research. Trial registration This study was registered with the German trial registry (Deutsches Register klinischer Studien) on November 7, 2023, under trial number DRKS00032983.

L’accessibilité numérique (AN), norme obligatoire, depuis 2009, pour les bibliothèques modifie la conception des interfaces des espaces documentaires numériques. Les institutions des bibliothèques tentent de réfléchir aux évolutions à apporter dans le parcours des usagers. La mission d’« égalité d'accès pour tous » des bibliothèques , conduit à questionner la dimension éthique qu’engage l’AN — recherche du bien-être d’autrui — pour les bibliothécaires (leurs manières d’agir). L’article cherche à étudier ce qui donne du sens à la mise en place de cette norme. Il est question d’interroger l’éthique de l’AN des espaces documentaires, ses enjeux définitionnels, ses effets auprès des professionnels et ses intérêts pour l’institution.

Ascaris spp. undergo extensive migration within the body before establishing patent infections in the small intestinal tract of humans and pigs. However, whether larval migration is critical for inducing efficient type 2 responses remains poorly understood. Therefore, we investigated systemic versus local adaptive immune responses along the hepato-tracheal migration of Ascaris suum during primary, single infections in conventionally raised pigs. Neither the initial invasion of gut tissue nor migration through the liver resulted in discernable Th2 cell responses. In contrast, lung-stage larvae elicited a Th2-biased pulmonary response, which declined after the larvae had left the lungs. In the small intestine, we observed an accumulation of Th2 cells upon the arrival of fourth-stage larvae (L4) to the small intestinal lumen. In parallel, we noticed robust and increasing Th1 responses in circulation, migration-affected organs, and draining lymph nodes. Phenotypic analysis of CD4+ T cells specifically recognizing A. suum antigens in the circulation and lung tissue of infected pigs confirmed that the majority of Ascaris-specific T cells produced IL-4 (Th2) and, to a much lesser extent, IL-4/IFN-g (Th2/1 hybrids) or IFN-g alone (Th1). These data demonstrate that lung-stage but not the early liver-stage larvae lead to a locally restricted Th2 response. Significant Th2 cell accumulation in the small intestine occurs only when L4 complete the body migration. In addition, Th2 immunity seems to be hampered by the concurrent, nonspecific Th1 bias in growing pigs. Together, the late onset of Th2 immunity at the site of infection and the Th1-biased systemic immunity likely enable the establishment of intestinal infections by sufficiently large L4 stages and pre-adult worms, some of which resist expulsion mechanisms.

Introduction: The time from dialysis onset to enrollment on the kidney waiting list (listing time) is a crucial step on the path to receiving a kidney allograft; however, this process has received very little research attention in the Eurotransplant (ET) area. Methods: We retrospectively analyzed data from the German transplantation registry, including patients who were on the waiting list for a first kidney transplant in Germany between 2006 and 2016. Listing time was evaluated using a mixed linear model. The outcomes on the kidney waiting list were assessed using competing risk analyses. Results: We assessed a total of 43,955 patients. Listing occurred at a higher pace in patients receiving living donor transplantations (median 0.4 years from dialysis onset) than in deceased donor transplantations (Eurotransplant Kidney Allocation System [ETKAS] 1.1 years, European Senior Program [ESP] 1.4 years, Acceptable Mismatch program 1.3 years), with 28.5% of living donor transplantations performed preemptively. There was only modest variation in listing time between the transplant centers. Patients with a history of viral infection, high immunization; hemodialysis patients; and patients with a higher body mass index (BMI) had a delayed listing process. Two of 3 patients listed in the ETKAS, excluding those with potential bonus points (pediatric, other organ transplantations), were eventually transplanted. Older patients, male patients, patients with blood type O, and patients with diabetic nephropathy as the underlying renal disease had the highest risk not to proceed to transplantation. Conclusion: Although long waiting times remain the biggest hurdle for transplantation in Germany, there is ample room for improvement of the listing process.

Fasciola hepatica infections lead to severe health problems and production losses in sheep farming, if not treated effectively. Triclabendazole has been used extensively over decades due to its unique efficacy range against all definitive hostfluke stages but published data about the susceptibility of F. hepatica to anthelmintics in Germany are lacking. This study aimed to identify current F. hepatica infections in German sheep flocks by coproscopic examinations and to evaluate the efficacy of anthelmintics with a focus on triclabendazole in a field study conducted from 2020 to 2022. Initial screening included 71 sheep farms, many of them with known history of fasciolosis. In this highly biased sample set, the frequency of F. hepatica infection at individual sheep and farm level were 12.8% and 35.2%, respectively. Additionally, eggs of Paramphistominae were found at frequencies of 4.8% and 15.5% at individual sheep and farm level, respectively. Due to low egg shedding intensity, faecal egg count reduction (FECR) tests could only be conducted on a few farms. The efficacy of triclabendazole was tested on 11 farms and albendazole on one farm, including 3–53 sheep/farm. Individual faecal samples were collected before and two weeks after treatment to evaluate the FECR using the sedimentation or FLUKEFINDER® or a modified FLUKEFINDER® method. On all farms a coproantigen reduction test was conducted in parallel. Lacking efficacy of triclabendazole even at double dosage was shown on one farm associated with a high number of animal losses due to acute fasciolosis. On this farm, the Fasciola miracidium development test was additionally performed, revealing a high in vitro ovicidal activity of albendazole while closantel was effective in vivo. On all other farms, sufficient efficacy of triclabendazole was observed. In conclusion, triclabendazole resistance appears not to be widespread on German sheep farms but, when present, can have serious effects on animal health.

Abstract Background In Europe, canine babesiosis is most frequently caused by Babesia canis and Babesia vogeli, and occasionally by Babesia gibsoni.. In Germany, B. canis is recognized as endemic. The aims of this study were to assess how often Babesia spp. infections were diagnosed in a commercial laboratory in samples from dogs from Germany, and to evaluate potential risk factors for infection. Methods The database of the LABOKLIN laboratory was screened for Babesia spp.-positive polymerase chain reaction (PCR) tests for dogs for the period January 2007–December 2020. Sequencing was performed for positive tests from 2018 and 2019. Binary logistic regression analysis was performed to determine the effects of sex, season, and year of testing. Questionnaires were sent to the submitting veterinarians to obtain information on travel abroad, tick infestation, and ectoparasite prophylaxis of the respective dogs. Fisher’s exact test was used to calculate statistical significance and P

Abstract The diagnostics of ruminant parasites remains one of the cornerstones for parasite control best practices. Field veterinarians have several techniques at their disposal (fecal egg count, coproculture, FAMACHA®, plasma pepsinogen, ELISA-Ostertagia, ELISA-Fasciola, Baermann and ELISA-Lungworm) for the identification and/or quantification of gastrointestinal nematodes, lungworms and liver fluke infecting small ruminants and cattle. Each of these diagnostic tools has its own strengths and weaknesses and is more appropriate for a specific production operation and/or age of the animal (young and adults). This review focuses on the usability and interpretation of the results of these diagnostic tools. The most advanced technical information on sampling, storage, advantages and limitations of each tool for different types of production operations and animal categories is provided. Graphical abstract

Abstract We evaluated the variation in the frequency of benzimidazole (BZ) resistance-associated alleles at codons 200, 167 and 368 (F200Y, F167Y, V368L) of the β-tubulin isotype 1 gene during the patent period in isolates of Haemonchus contortus susceptible and resistant to BZ using pyrosequencing. Four lambs 5–6 months old were infected with 5000–6000 infective third-stage larvae (L3) of the susceptible MHco1 and the multi-resistant MHco4 isolates, respectively. Faecal samples were collected 28-times during 20–90 days post-infection (dpi). Coprocultures were subsequently prepared to produce L3 for genotyping. The frequency of the resistant allele (TAC) at codon 200 in MHco1 was lowest at 43 and 76 dpi with at each time point 0% and highest at 36 dpi with 10.85%, with a mean of 6.47% ± 2.39 and a coefficient of variation of 37.01%. The frequency of the TAC at codon 200 in MHco4 was lowest at 76 dpi with 25.6% and highest at 90 dpi with 49.25%, with a mean of 35.7% ± 4.42 and a coefficient of variation of 12.39%. No resistance alleles were detected in MHco1 at either codon 167 or 368. For MHco4 isolate, resistance alleles were detected only on codon 167 with a mean of 8.00% ± 4.83 and a mean coefficient of variation of 60.40%. Our results demonstrate the considerable variation in the frequency of resistant alleles in the susceptible and resistant isolates during the patent period. This variation should be considered when testing for the presence of BZ resistance in populations of gastrointestinal parasites, especially those with a low frequency of TAC.

Abstract Background Intestinal parasite contamination from infected dogs can place other dogs and humans at risk. A study was initiated to estimate the prevalence of canine intestinal parasitism by collecting fecal samples in cities across Western Europe. Methods Fresh fecal samples were collected from 2469 dogs visiting 164 parks in 33 cities across 12 countries. Each owner responded to a questionnaire focusing on their dog’s signalment and recent anthelmintic treatment history. The collected samples were examined for hookworms, whipworms, ascarids and Giardia using a coproantigen diagnostic immunoassay and microscopy following centrifugal flotation. Results Nematodes or Giardia were detected in at least one sample from 100% of cities and in 93.3% of parks. Nematodes were detected in 57% of parks. Overall, 22.8% of dogs tested positive for an intestinal parasite, with Giardia being the most commonly identified parasites (17.3% of dogs, 83.5% of parks). For nematode infection, 7.6% of all dogs tested positive, with 9.9% of dogs aged 1 month had passed since the previous dose. Conclusions The prevalence estimates of intestinal parasite infections in dogs reported here highlight the need for owner education concerning guidelines for regular testing and treatment, even in older dogs. Failure to adhere to guidelines can result in ongoing transmission of these infections, including those with zoonotic potential. Combining coproantigen immunoassay with centrifugal flotation for diagnostic testing and regular anthelmintic treatment are important measures for ensuring optimal intestinal parasite control. Graphical Abstract

Basic knowledge on the biology and epidemiology of equine strongylid species still needs to be improved to contribute to the design of better parasite control strategies. Nemabiome metabarcoding is a convenient tool to quantify and identify species in bulk samples that could overcome the hurdle that cyathostomin morphological identification represents. To date, this approach has relied on the internal transcribed spacer 2 (ITS-2) of the ribosomal RNA gene, with a limited investigation of its predictive performance for cyathostomin communities. Using DNA pools of single cyathostomin worms, this study aimed to provide the first elements to compare performances of the ITS-2 and a cytochrome c oxidase subunit I (COI) barcode newly developed in this study. Barcode predictive abilities were compared across various mock community compositions of two, five and 11 individuals from distinct species. The amplification bias of each barcode was estimated. Results were also compared between various types of biological samples, i.e., eggs, infective larvae or adults. Bioinformatic parameters were chosen to yield the closest representation of the cyathostomin community for each barcode, underscoring the need for communities of known composition for metabarcoding purposes. Overall, the proposed COI barcode was suboptimal relative to the ITS-2 rDNA region, because of PCR amplification biases, reduced sensitivity and higher divergence from the expected community composition. Metabarcoding yielded consistent community composition across the three sample types. However, imperfect correlations were found between relative abundances from infective larvae and other life-stages for Cylicostephanus species using the ITS-2 barcode. While the results remain limited by the considered biological material, they suggest that additional improvements are needed for both the ITS-2 and COI barcodes.

Abstract Background Canine vector-borne disease (CVBD) has been an area of increasing interest in Europe over the last few decades, and there have been changes in the prevalence and distribution of many of these diseases. Monitoring CVBD infections in Europe is often done by individual countries, but aggregated data for the European countries are helpful to understand the distribution of CVBDs. Methods We used an extensive retrospective database of results from point-of-care rapid enzyme-linked immunosorbent assay (ELISA) tests on dogs across Europe to identify distribution and seropositivity in animals tested for selected CVBDs (Anaplasma spp., Ehrlichia spp., Borrelia burgdorferi, Leishmania spp., and Dirofilaria immitis) from 2016 through 2020. Geographic distribution of positive tests and relative percent positive values were mapped by the Nomenclature of Territorial Units for Statistics classification for regions with sufficient test results for reporting. Results A total of 404,617 samples corresponding to 1,134,648 canine results were available from dogs tested in 35 countries over the 5-year study period. Over this period the number of test results per year increased whereas test positivity decreased. Leishmania spp. had the largest increase in total test results from 25,000 results in 2016 to over 60,000 results in 2020. Test positivity for Leishmania spp. fell from 13.9% in 2016 to 9.4% in 2020. Test positivity fell for Anaplasma spp. (7.3 to 5.3%), Ehrlichia spp. (4.3 to 3.4%), and Borrelia burgdorferi (3.3 to 2.4%). Dirofilaria immitis test positivity trended down with a high of 2.7% in 2016 and low of 1.8% in 2018. Leishmania spp. test positivity was highest in endemic areas and in several non-endemic countries with low numbers of test results. Co-positivity rates were significantly higher than expected for all pathogen test positive pairs except for Ehrlichia spp. with Borrelia burgdorferi and D. immitis with Borrelia burgdorferi. Conclusions This study represents the largest data set on CVBD seropositivity in Europe to date. The increase in the number of test results and decreasing test positivity over the study period may reflect changes in testing behavior and increased screening of healthy animals. The Europe-wide mapping of CVBD provides expected test positivity that can help inform veterinarians’ decisions on screening and improve prevention and identification of these important, sometimes zoonotic, diseases. Graphical Abstract

Abstract Background Due to high prevalence of anthelmintic resistance in equine helminths, selective treatment is increasingly promoted and in some countries a positive infection diagnosis is mandatory before treatment. Selective treatment is typically recommended when the number of worm eggs per gram faeces (epg) exceeds a particular threshold. In the present study we compared the semi-quantitative sedimentation/flotation method with the quantitative methods Mini-FLOTAC and FECPAKG2 in terms of precision, sensitivity, inter-rater reliability and correlation of worm egg counts to improve the choice of optimal diagnostic tools. Methods Using sedimentation/flotation (counting raw egg numbers up to 200), we investigated 1067 horse faecal samples using a modified Mini-FLOTAC approach (multiplication factor of 5 to calculate epgs from raw egg counts) and FECPAKG2 (multiplication factor of 45). Results Five independent analyses of the same faecal sample with all three methods revealed that variance was highest for the sedimentation/flotation method while there were no significant differences between methods regarding the coefficient of variance. Sedimentation/flotation detected the highest number of samples positive for strongyle and Parascaris spp. eggs, followed by Mini-FLOTAC and FECPAKG2. Regarding Anoplocephalidae, no significant difference in frequency of positive samples was observed between Mini-FLOTAC and sedimentation/flotation. Cohen’s κ values comparing individual methods with the combined result of all three methods revealed almost perfect agreement (κ ≥ 0.94) for sedimentation/flotation and strong agreement for Mini-FLOTAC (κ ≥ 0.83) for strongyles and Parascaris spp. For FECPAKG2, moderate and weak agreements were found for the detection of strongyle (κ = 0.62) and Parascaris (κ = 0.51) eggs, respectively. Despite higher sensitivity, the Mini-FLOTAC mean epg was significantly lower than that with FECPAKG2 due to samples with > 200 raw egg counts by sedimentation/flotation, while in samples with lower egg shedding epgs were higher with Mini-FLOTAC than with FECPAKG2. Conclusions For the simple detection of parasite eggs, for example, to treat foals infected with Parascaris spp., sedimentation/flotation is sufficient and more sensitive than the other two quantitative investigared in this study. Mini-FLOTAC is predicted to deliver more precise results in faecal egg count reduction tests due to higher raw egg counts. Finally, to identify animals with a strongyle epg above a certain threshold for treatment, FECPAKG2 delivered results comparable to Mini-FLOTAC. Grpahical Abstract

Anthelmintic resistance (AR) has thus far only rarely been reported for intestinal helminths of dogs and cats, in contrast to parasites of livestock and horses. We highlight possible reasons for this striking and important discrepancy, including ecological, biological and genetic factors and/or intervention regimens of key intestinal helminths concerning both host groups. In view of the current knowledge related to the genetics, mechanisms and principles of AR development, we point at issues which in our view contribute to a comparatively lower risk of AR development in intestinal helminths of dogs and cats. Finally, we specify research needs and provide recommendations by which, based on the available information about AR in ruminant and equine helminths, the development of AR in dog and cat helminths may best be documented, prevented or at least postponed.

Abstract Background Improved knowledge on vector-borne pathogens in wildlife will help determine their effect on host species at the population and individual level and whether these are affected by anthropogenic factors such as global climate change and landscape changes. Here, samples from brown hyenas (Parahyaena brunnea) from Namibia (BHNA) and spotted hyenas (Crocuta crocuta) from Namibia (SHNA) and Tanzania (SHTZ) were screened for vector-borne pathogens to assess the frequency and genetic diversity of pathogens and the effect of ecological conditions and host taxonomy on this diversity. Methods Tissue samples from BHNA (n = 17), SHNA (n = 19) and SHTZ (n = 25) were analysed by PCRs targeting Anaplasmataceae, Rickettsia spp., piroplasms, specifically Babesia lengau-like piroplasms, Hepatozoidae and filarioids. After sequencing, maximum-likelihood phylogenetic analyses were conducted. Results The relative frequency of Anaplasmataceae was significantly higher in BHNA (82.4%) and SHNA (100.0%) than in SHTZ (32.0%). Only Anaplasma phagocytophilum/platys-like and Anaplasma bovis-like sequences were detected. Rickettsia raoultii was found in one BHNA and three SHTZ. This is the first report of R. raoultii from sub-Saharan Africa. Babesia lengau-like piroplasms were found in 70.6% of BHNA, 88.9% of SHNA and 32.0% of SHTZ, showing higher sequence diversity than B. lengau from South African cheetahs (Acinonyx jubatus). In one SHTZ, a Babesia vogeli-like sequence was identified. Hepatozoon felis-like parasites were identified in 64.7% of BHNA, 36.8% of SHNA and 44.0% of SHTZ. Phylogenetic analysis placed the sequences outside the major H. felis cluster originating from wild and domestic felids. Filarioids were detected in 47.1% of BHNA, 47.4% of SHNA and 36.0% of SHTZ. Phylogenetic analysis revealed high genetic diversity and suggested the presence of several undescribed species. Co-infections were frequently detected in SHNA and BHNA (BHNA median 3 pathogens, range 1–4; SHNA median 3 pathogens, range 2–4) and significantly rarer in SHTZ (median 1, range 0–4, 9 individuals uninfected). Conclusions The frequencies of all pathogens groups were high, and except for Rickettsia, multiple species and genotypes were identified for each pathogen group. Ecological conditions explained pathogen identity and diversity better than host taxonomy. Graphic Abstract

Abstract Cyathostomins are important intestinal nematode parasites of equines and include 50 accepted species. Their taxonomy has been frequently revised and the presence of cryptic species suggested. Furthermore, usually molecular- and morphology-based phylogenetic analyses give divergent results. In this study, the nucleotide sequences of the nuclear second internal transcribed spacer (ITS-2) and the mitochondrial partial cytochrome c oxidase subunit I (COI) were determined for adults of six cyathostomin species (Coronocyclus coronatus, Coronocyclus labiatus, Cylicocyclus nassatus, Cylicostephanus calicatus, Cylicostephanus longibursatus, Cylicostephanus minutus) collected from different equine species within two geographic regions. Maximum likelihood trees were calculated for ITS-2, COI, and concatenated data. No obvious differentiation was observed between geographic regions or equine host species. As previously reported, Coronocyclus coronatus and Cylicostephanus calicatus revealed a close relationship. Cryptic species were detected in Cylicostephanus minutus and Cylicostephanus calicatus. Cylicocyclus nassatus and Coronocyclus labiatus showed diverse mitochondrial and nuclear haplotypes occurring in different combinations, while Cylicostephanus longibursatus was comparatively homogenous. In conclusion, a combined analysis of nuclear and mitochondrial haplotypes improved resolution of the phylogeny and should be applied to the remaining cyathostomin species and across additional equine host species and geographic regions.

Abstract Background Benzimidazole (BZ) anthelmintics are widely used to control infections with parasitic nematodes, but BZ resistance is an emerging threat among several nematode species infecting humans and animals. In Sudan, BZ-resistant Haemonchus contortus populations were recently reported in goats in South Darfur State. The objective of this study was to collect data regarding the situation of BZ resistance in cattle parasitic nematodes in South Darfur using phenotypic and molecular approaches, besides providing some epidemiological data on nematodes in cattle. Methods The faecal egg count reduction test and the egg hatch test (EHT) were used to evaluate benzimidazole efficacy in cattle nematodes in five South Darfur study areas: Beleil, Kass, Nyala, Rehed Al-Birdi and Tulus. Genomic DNA was extracted from pools of third-stage larvae (L3) (n = 40) during trials, before and after treatment, and pools of adult male Haemonchus spp. (n = 18) from abattoirs. The polymorphisms F167Y, E198A and F200Y in isotype 1 β-tubulin genes of H. contortus and H. placei were analysed using Sanger and pyrosequencing. Results Prevalence of gastro-intestinal helminths in cattle was 71% (313/443). Reduced albendazole faecal egg count reduction efficacy was detected in three study areas: Nyala (93.7%), Rehed Al-Birdi (89.7%) and Tulus (88.2%). In the EHT, EC50 values of these study areas ranged between 0.032 and 0.037 µg/ml thiabendazole. Genus-specific PCRs detected the genera Haemonchus, Trichostrongylus and Cooperia in L3 samples collected after albendazole treatment. Sanger sequencing followed by pyrosequencing assays did not detect elevated frequencies of known BZ resistance-associated alleles in codon F167Y, E198A and F200Y in isotype 1 β-tubulin gene of H. placei (≤ 11.38%). However, polymorphisms were detected in H. contortus and in samples with mixed infections with H. contortus and H. placei at codon 198, including E198L (16/58), E198V (2/58) and potentially E198Stop (1/58). All pooled L3 samples post-albendazole treatment (n = 13) were identified as H. contortus with an E198L substitution at codon 198. Conclusions To the knowledge of the authors, this is the first report of reduced albendazole efficacy in cattle in Sudan and is the first study describing an E198L substitution in phenotypically BZ-resistant nematodes collected from cattle.

Abstract Background With intensive use of anthelmintic drugs in recent decades, anthelmintic resistance (AR) in horse nematodes is becoming a growing issue in many countries. However, there is little available information about the parasites, treatment practices or AR in the horse population in Lithuania. The aim of this study was to assess the current situation of AR on horse farms in Lithuania. The study was conducted in 25 stables on horses with a strongyle faecal egg count (FEC) of ≥ 200 eggs per gram. A faecal egg count reduction test (FECRT) was performed on each farm after administration of ivermectin (IVM) or pyrantel (PYR). Results The efficacy of IVM was comparatively high, with 98.8% of 250 horses having a zero egg count 14 days after treatment. Two conditions were used to interpret the FECRT results for PYR: firstly, resistance was determined when FECR was

McMaster (McM) method is one of the most widely used techniques for the assessment of faecal parasites shedding in veterinary practices because of its simplicity. However, due to its light sensitivity, recently, the Mini-FLOTAC (MF) has been introduced as a possible alternative for faecal worm egg counts. This study aims to compare the diagnosis performance of MF to McM technique. Faecal samples from 40 animals randomly selected in sheep, goats and rabbits’ farms were collected and examined individually using MF and McM techniques. A statistical difference (p 0.05). MF showed better diagnostic performance in term of the prevalence (MF: 32.5–100% vs McM: 7.5–70%) and the precision values (MF: 85.52–90.44% vs McM: 49.52–63.07%). This study demonstrated that MF appears to be the more reliable alternative technique for veterinary practices.

The infection of horses with strongylid nematodes is highly prevalent, with multi-species infections being the rule. Strongylus spp. and in particular Strongylus vulgaris are amongst the most pathogenic strongyle equine parasites. Presumably due to regular strategic anthelmintic treatments in combination with long prepatencies, prevalence of these worms was severely reduced in past decades. In this study, 484 horses from 48 farms in Berlin/Brandenburg, Germany were sampled between May 2017 and January 2018. Mini-FLOTAC and combined sedimentation/flotation were used to analyse faecal samples and larval cultures were carried out from individual strongyle infected horses for molecular testing for Strongylus spp. infection. Additionally, for Strongylus vulgaris, antibodies against a recombinant larval antigen were quantified in an ELISA. Strongyle type eggs were detected in 66.7% of the individual faecal samples. Nematode DNA was amplifiable from 311 samples and S. vulgaris and Strongylus edentatus were detected in four (1.3%) and 10 (6.3%) of these, respectively, the latter using a novel high-resolution-melt PCR targeting S. edentatus, Strongylus equinus, and Strongylus asini. On the farm level, prevalence for Strongylus spp. by PCR was 12.5%. Applying a conservative cut-off (sensitivity 0.43, specificity 0.96), 21.2% of all serum samples were positive for antibodies against S. vulgaris larvae (83.3% prevalence on farm level). Newly developed pyrosequencing assays to analyse putatively benzimidazole resistance associated polymorphisms in codons 167, 198, and 200 of the isotype 1 β-tubulin gene of S. vulgaris did not detect such polymorphisms in the four positive samples. Low age and increasing access to pasture were risk factors for egg shedding and seropositivity for S. vulgaris. Time since last treatment increased whereas use of moxidectin and ivermectin for the last treatment decreased the risk for strongyle egg shedding. Noteworthy, horses under selective treatment had significantly higher odds to be seropositive for anti-S. vulgaris antibodies than horses treated four times per year (odds ratio 4.4). The serological findings suggest that exposure to S. vulgaris is considerably higher than expected from direct diagnostic approaches. One potential explanation is the contamination of the environment by a few infected horses, leading to the infection of many horses with larvae that never reach maturity due to regular anthelmintic treatments.

Introduction Despite continued efforts, long-term outcomes of kidney transplantation remain unsatisfactory. Kidney graft rejections are independent risk factors for graft failure. At the participating centres of the TRAnsplant BIOpsies study group, a common therapeutic standard has previously been defined for the treatment of graft rejections. The outcomes of this strategy will be assessed in a prospective, observational cohort study.Methods and analysis A total of 800 kidney transplantation patients will be enrolled who undergo a graft biopsy because of deteriorating kidney function. Patients will be stratified according to the Banff classification, and the influence of the treatment strategy on end points will be assessed using regression analysis. Primary end points will be all-cause mortality and graft survival. Secondary end points will be worsening of kidney function (≥30% decline of estimated Glomerular Filtration Rate [eGFR] or new-onset large proteinuria), recurrence of graft rejection and treatment response. Baseline data and detailed histopathology data will be entered into an electronic database on enrolment. During a first follow-up period (within 14 days) and subsequent yearly follow-ups (for 5 years), treatment strategies and clinical course will be recorded. Recruitment at the four participating centres started in September 2016. As of August 2020, 495 patients have been included.Ethics and dissemination Ethical approval for the study has been obtained from the ethics committee of Kiel (AZ B 278/16) and was confirmed by the committees of Munich, Mainz and Stuttgart. The results will be reported in a peer-reviewed journal, according to the Strengthening the Reporting of Observational Studies in Epidemiology criteria.Trial registration number ISRCTN78772632; Pre-results.

Knockdown-resistance (kdr) against pyrethroids in bed bugs (Cimex lectularis) is associated with the presence of several point mutations in the voltage-sensitive sodium channel α-subunit gene and/or an increased metabolic detoxification by cytochrome P450 monooxygenases (CYPs). In the present study, pyrosequencing assays were developed to quantify the presence of the kdr substitutions (V419L or L925I substitution) in bed bugs in Berlin, Germany. In 14 of 17 bed bug field strains, pyrosequencing revealed the presence of the substitution L925I with allele frequencies between 30% and 100%. One field strain additionally carried the substitution V419L with allele frequencies of 40% in males and 96% in females. In seven of the 17 field strains, mRNA levels of four CYP genes were examined using RT-qPCR. Relative to a susceptible laboratory reference strain, five field strains showed significantly higher mRNA levels of cyp397a1 with 7.1 to 56-fold increases. One of these strains additionally showed a 4.9-fold higher mRNA level of cyp398a1 compared to the reference strain, while cyp4cm1 and cyp6dn1 showed no significant differences. Our findings indicate that multiple resistance mechanisms are present in German C. lectularius populations simultaneously.

Abstract Immunity and parasites have been linked to the success of invasive species. Especially lower parasite burden in invasive populations has been suggested to enable a general downregulation of immune investment (Enemy Release and Evolution of Increased Competitive Ability Hypotheses). Simultaneously, keeping high immune competence towards potentially newly acquired parasites in the invasive range is essential to allow population growth. To investigate the variation of immune effectors of invasive species, we compared the mean and variance of multiple immune effectors in the context of parasite prevalence in an invasive and a native Egyptian goose (Alopochen aegyptiacus) population. Three of ten immune effectors measured showed higher variance in the invasive population. Mean levels were higher in the invasive population for three effectors but lower for eosinophil granulocytes. Parasite prevalence depended on the parasite taxa investigated. We suggest that variation of specific immune effectors, which may be important for invasion success, may lead to higher variance and enable invasive species to reduce the overall physiological cost of immunity while maintaining the ability to efficiently defend against novel parasites encountered.

Abstract Background Effective and sustainable worm control in horses would benefit from detailed information about the current regional occurrence of tapeworms. Different diagnostic methods are currently available to detect Anoplocephala spp. infections in horses. However, the format as well as the sensitivity and specificity of the methods vary considerably. Methods A coprological, serological and questionnaire study was conducted to investigate the prevalence and risk factors of tapeworm infections on 48 horse farms in the region of Berlin and Brandenburg, Germany. In total, faecal samples of 484 horses were analysed using the double centrifugation/combined sedimentation-flotation and mini-FLOTAC. Serum (n = 481) and saliva (n = 365) samples were analysed by ELISAs to determine antibody levels against Anoplocephala spp. 12/13 kDa excretory/secretory (E/S) antigens. Results Cestode eggs were detected in 0.6% of faecal samples (farm prevalence 6.3%) without differences between the two methods. In contrast, antibodies against Anoplocephala spp. were detected in 16.2% (farm prevalence 52.1%) and in 29.5% (farm prevalence 75.7%) of the serum and saliva samples, respectively. Both ELISA based methods for detection of tapeworms reported a greater number of infected animals requiring treatment than were positively identified by coproscopy. Logistic regression analysis identified permanent pasture access, large pastures and regular pasture changes and high strongyle egg counts as risk factors for positive serum antibody responses to Anoplocephala spp. while last treatment with praziquantel was protective. Other protective factors were the presence of foals and high numbers of horses on the farm. Daily removal of faeces from the pasture and horse age did not have a significant effect. Conclusions The findings of the present serological investigation indicate that tapeworm prevalence in Berlin/Brandenburg horse farms is much higher than would be anticipated by using conventional/coproscopic analyses. Moreover, the majority of tapeworm-positive horses had not received a cestocidal drug at their last treatment. Considering the already known low sensitivity of the coproscopic detection, the equine veterinary diagnostics can be enhanced by the use of antibody detection methods such as the saliva-based ELISA.

Abstract Background The genus Marshallagia (Family Haemonchidae, subfamily Ostertagiinae) contains multiple species of nematodes parasitising the abomasum (or duodenum) of ruminants, in particular of Caprinae. Male specimens have been described to be polymorphic with the frequent/major morphotype initially described in the genus Marshallagia while the minor/rare morphotype was initially often placed in the genus Grossospicularia. Due to common morphological features, certain pairs of morphotypes were suggested to belong to the same species such as Marshallagia marshalli/M. occidentalis. However, molecular evidence to confirm these pairs of morphotypes belonging to the same species is missing. Methods In the present study, Marshallagia sp. were collected from domestic sheep in Uzbekistan. Male specimens were morphologically described with particular emphasis on the structure of the bursa copulatrix. After DNA isolation from morphologically identified specimens, PCRs targeting the ribosomal internal transcribed spacer 2 (ITS2) and mitochondrial cytochrome c oxidase subunit 1 (cox1) regions were conducted. After Sanger sequencing, maximum likelihood phylogenetic analyses and pairwise identities between sequences were calculated. Results The major morphotypes of M. marshalli, M. schumakovitschi and M. uzbekistanica and the minor morphotypes M. occidentalis, M. trifida and M. sogdiana were identified and their morphology was documented in detail. ITS2 sequences showed little variation and did not allow diagnosing species. In contrast, phylogenetic analysis of cox1 sequences identified highly supported clusters and verified that M. marshalli, M. occidentalis and M. uzbekistanica are different morphotypes of the species M. marshalli while M. schumakovitschi and M. trifida represent distinct morphotypes of M. trifida. For M. sogdiana no corresponding major morphotype could be identified in the present study. Due to a large barcoding gap, comparison of cox1 sequences in terms of percent identity was sufficient to reliably assign the sequences to a particular species without phylogenetic analysis. Conclusions The data presented here create a framework that will allow the classification of other members of the genus in the future and underline that parallel morphological and molecular analysis of specimens is crucial to improve the taxonomy of polymorphic species.

A fragment of a Dracunculus-like worm was extracted from the hind limb of a 2-year-old dog from Toledo, Spain. Cytochrome oxidase I and rRNA sequences confirmed an autochthonous mammalian Dracunculus worm infection in Europe. Sequence analyses suggest close relation to a parasite obtained from a North American opossum.

Abstract Background Benzimidazole (BZ) resistance in gastrointestinal nematodes is a worldwide problem for livestock production, particularly in small ruminants. Assignment of the emergence of resistance using sensitive and reliable methods is required to adopt the correct strategies for control. In Sudan, BZ resistant Haemonchus contortus populations were recently reported in goats in South Darfur. This study aimed to provide additional data regarding albendazole efficacy and to describe the prevailing molecular BZ resistance mechanisms. Methods Faecal egg count reduction and egg hatch tests (EHT) were used to evaluate albendazole efficacy in three different areas of South Darfur using naturally (Rehed Al-Birdi and Tulus) and experimentally infected (Tulus and Um Dafuq) goats. Using samples from Central, East and South Darfur, pyro- and Sanger sequencing were used to detect the polymorphisms F167Y, E198A and F200Y in H. contortus isotype 1 β-tubulin in DNA extracted from pooled third-stage larval (L3) samples (n = 36) on days 0 and 10 during trials, and from pooled adult male H. contortus (treated goats, n = 14; abattoirs, n = 83) including samples from populations previously found to be resistant in South Darfur. Results Albendazole efficacies at 5, 7.5 and 10 mg/kg doses were 73.5–90.2% on day 14 in natural and experimental infections while 12.5 mg/kg showed > 96.6% efficacy. EC50 in the EHT were 0.8 and 0.11 µg/ml thiabendazole in natural and experimental infection trials, respectively. PCRs detected Haemonchus, Trichostrongylus and Cooperia in L3 samples from albendazole-treated goats. Haemonchus contortus allele frequencies in codons 167 and 200 using pyrosequencing assays were ≤ 7.4% while codon 198 assays failed. Sanger sequencing revealed five novel polymorphisms at codon 198. Noteworthy, an E198L substitution was present in 82% of the samples (L3 and adults) including all post-treatment samples. Moreover, E198V, E198K and potentially E198I, and E198Stop were identified in a few samples. Conclusions To our knowledge, this is the first report of E198L in BZ resistant H. contortus and the second where this is the predominant genotype associated with resistance in any strongyle species. Since this variant cannot be quantified using pyrosequencing, the results highlight important limitations in the general applicability of pyrosequencing to quantify BZ resistance genotypes.

Abstract Background In three randomized, controlled laboratory efficacy studies, the efficacy in the prevention of patent infections of a topical combination of imidacloprid 10%/moxidectin 1% (Advocate® spot-on formulation for cats, Bayer Animal Health GmbH) against larval stages and immature adults of Aelurostrongylus abstrusus, as well as the treatment efficacy of a single or three monthly treatments against adult A. abstrusus, were evaluated. Methods Cats were experimentally inoculated with 300–800 third-stage larvae (L3). Each group comprised 8 animals and the treatment dose was 10 mg/kg bodyweight (bw) imidacloprid and 1 mg/kg bw moxidectin in each study. Prevention of the establishment of patent infections was evaluated by two treatments at a monthly interval at three different time points before and after challenge infection. Curative efficacy was tested by one or three treatments after the onset of patency. Worm counts at necropsy were used for efficacy calculations. Results In Study 1, the control group had a geometric mean (GM) of 28.8 adult nematodes and the single treatment group had a GM of 3.4 (efficacy 88.3%). In Study 2, the control group had a GM of 14.3, the prevention group had a GM of 0 (efficacy 100%), while the treatment group had a GM of 0.1 (efficacy 99.4%). In Study 3, the GM worm burden in the control group was 32.6 compared to 0 in all three prevention groups (efficacy 100% for all of those groups). Conclusions The monthly administration of Advocate® reliably eliminated early larval stages and thereby prevented lung damage from and patent infections with A. abstrusus in cats. Regarding treatment, a single application of Advocate® reduced the worm burden, but it did not sufficiently clear the infection. In contrast, three monthly treatments were safe and highly efficacious against A. abstrusus.

The sand flea Tunga penetrans is one of the zoonotic agents of tungiasis, a parasitic skin disease of humans and animals. The dog is one of its main reservoirs. This negatively controlled, randomized, double-masked clinical trial evaluated the therapeutic and residual efficacy of fluralaner for treatment of dogs naturally infested with T. penetrans. Sixty-two dogs from an endemically affected community in Brazil were randomly assigned to either receive oral fluralaner (Bravecto chewable tablets) at a dose of 25 to 56 mg fluralaner/kg body weight, or no treatment (31 dogs per group). Dogs were clinically examined using a severity score for acute canine tungiasis (SCADT), parasitological examinations as defined by the Fortaleza classification, and pictures of lesions on days 0 (inclusion and treatment), 7 ± 2, 14 ± 2, 21 ± 2, 28 ± 2, 60 ± 7, 90 ± 7, 120 ± 7 and 150 ± 7. The percentage of parasite-free dogs after treatment was >90% between days 14 and 90 post-treatment with 100% efficacy on study days 21, 28 and 60. Sand flea counts on fluralaner treated dogs were significantly lower (p 90% on day 7, > 95% on days 14 and 90, and 100% from day 21 to 60, and with a significant difference between groups from day 7 to 120. From day 7 to day 120, mean SCADT scores were significantly reduced in treated dogs with a mean of 0.10 compared to 1.54 on day 120 in untreated dogs. Therefore, a single oral fluralaner administration is effective for treating and achieving long lasting (> 12 weeks) prevention for tungiasis in dogs.