Giardia duodenalis multi-locus genotypes in dogs with different levels of synanthropism and clinical signs.

BACKGROUND: In dogs, infections with Giardia duodenalis are mainly caused by assemblages C and D, but also by the potentially zoonotic assemblages A and B. The aims of this study were to assess differences in assemblages (i) between dogs living mainly in close proximity to humans (synanthropic dogs) versus dogs living mainly among other dogs, (ii) between samples of dogs with or without loose stool, and (iii) related to the amount of cysts shedding. METHODS: One hundred eighty-nine qPCR Giardia positive fecal samples of dogs originating from four groups (household, sheltered, hunting, and dogs for which a veterinarian sent a fecal sample to a diagnostic laboratory) were used for genotyping. For this, multi-locus genotyping of beta-giardin, triose phosphate isomerase, and glutamate dehydrogenase and genotyping of SSU rDNA gene fragments were performed. Fecal consistency was scored (loose or non-loose stool), and cysts per gram of feces were determined with qPCR. RESULTS: Assemblage D was the most prevalent in all groups, followed by the other canid assemblage C. Also, mixed C/D was common. In two (synanthropic) household dogs, the potentially zoonotic assemblage AI was present. Although occurrence of assemblage AI in household dogs was not significantly different from dogs living among other dogs (sheltered and hunting dogs), it was significantly higher compared to dogs for which a sample was sent to a diagnostic laboratory. Dogs with assemblage D shed significantly more cysts than dogs with other assemblages (except for mixed C/D results) or dogs in which no assemblage could be determined. None of the assemblages was significantly associated with loose stool. CONCLUSION: Not only do dogs mainly shed the canid Giardia duodenalis assemblages D and/or C, the numbers of cysts per gram for the canid assemblage D were also higher than for the potential zoonotic assemblage AI. Based on the assemblages shed by dogs, the risk to public health posed by dogs is estimated to be low, even though the dogs that shed AI were synanthropic household dogs. Loose stool in infected dogs was not associated with any particular Giardia assemblage.

Increasing importance of anthelmintic resistance in European livestock: creation and meta-analysis of an open database.

Helminth infections are ubiquitous in grazing ruminant production systems, and are responsible for significant costs and production losses. Anthelmintic Resistance (AR) in parasites is now widespread throughout Europe, although there are still gaps in our knowledge in some regions and countries. AR is a major threat to the sustainability of modern ruminant livestock production, resulting in reduced productivity, compromised animal health and welfare, and increased greenhouse gas emissions through increased parasitism and farm inputs. A better understanding of the extent of AR in Europe is needed to develop and advocate more sustainable parasite control approaches. A database of European published and unpublished AR research on gastrointestinal nematodes (GIN) and liver fluke (Fasciola hepatica) was collated by members of the European COST Action "COMBAR" (Combatting Anthelmintic Resistance in Ruminants), and combined with data from a previous systematic review of AR in GIN. A total of 197 publications on AR in GIN were available for analysis, representing 535 studies in 22 countries and spanning the period 1980-2020. Reports of AR were present throughout the European continent and some reports indicated high within-country prevalence. Heuristic sample size-weighted estimates of European AR prevalence over the whole study period, stratified by anthelmintic class, varied between 0 and 48%. Estimated regional (country) prevalence was highly heterogeneous, ranging between 0% and 100% depending on livestock sector and anthelmintic class, and generally increased with increasing research effort in a country. In the few countries with adequate longitudinal data, there was a tendency towards increasing AR over time for all anthelmintic classes in GIN: aggregated results in sheep and goats since 2010 reveal an average prevalence of resistance to benzimidazoles (BZ) of 86%, macrocyclic lactones except moxidectin (ML) 52%, levamisole (LEV) 48%, and moxidectin (MOX) 21%. All major GIN genera survived treatment in various studies. In cattle, prevalence of AR varied between anthelmintic classes from 0-100% (BZ and ML), 0-17% (LEV) and 0-73% (MOX), and both Cooperia and Ostertagia survived treatment. Suspected AR in F. hepatica was reported in 21 studies spanning 6 countries. For GIN and particularly F. hepatica, there was a bias towards preferential sampling of individual farms with suspected AR, and research effort was biased towards Western Europe and particularly the United Kingdom. Ongoing capture of future results in the live database, efforts to avoid bias in farm recruitment, more accurate tests for AR, and stronger appreciation of the importance of AR among the agricultural industry and policy makers, will support more sophisticated analyses of factors contributing to AR and effective strategies to slow its spread.

TITLE: Importance croissante de la résistance aux anthelminthiques chez les ruminants européens : création et méta-analyse d'une base de données ouverte. ABSTRACT: Les helminthes sont omniprésents dans les systèmes de production de ruminants au pâturage et sont responsables de coûts et de pertes de production importants. La résistance aux anthelminthiques (RA) des parasites est maintenant répandue dans toute l'Europe, bien qu'il existe encore des lacunes dans nos connaissances dans certaines régions et certains pays. La RA est une menace majeure pour la pérennité de la production moderne de ruminants, en diminuant la productivité, en compromettant la santé et le bien-être des animaux, et en augmentant les émissions de gaz à effet de serre au travers d'une augmentation du parasitisme et des intrants agricoles. Une meilleure compréhension de l'étendue de la RA en Europe est nécessaire pour développer et préconiser des approches de lutte antiparasitaire plus durables. Une base de données intégrant des informations publiées et non publiées en Europe concernant la RA des nématodes gastro-intestinaux (NGI) et de la douve du foie (Fasciola hepatica) a été compilée par les membres de l'action européenne COST « COMBAR ¼ (« Combattre la résistance aux anthelminthiques chez les ruminants ¼) et combinée avec les données d'une précédente étude systématique concernant la RA des NGI. Au total, 197 publications sur la RA des NGI étaient disponibles pour analyse, représentant 535 études dans 22 pays et couvrant la période 1980­2020. Des signalements de RA étaient présents sur tout le continent européen et certains rapports indiquaient une forte prévalence nationale. Les estimations heuristiques pondérées par la taille de l'échantillon de la prévalence de la RA en Europe sur toute la période d'étude, stratifiées par classe d'anthelminthiques, variaient de 0 à 48 %. La prévalence régionale (nationale) estimée était très hétérogène, variant entre 0 % et 100 % selon le secteur de l'élevage et la classe d'anthelminthique, et augmentait généralement avec les efforts de recherche dans le pays. Dans les quelques pays disposant de données longitudinales adéquates, il y avait une tendance à l'augmentation de la RA au fil du temps pour toutes les classes d'anthelminthiques des NGI : les résultats agrégés chez les ovins et caprins depuis 2010 révèlent une prévalence moyenne de résistance aux benzimidazoles (BZ) de 86 %, aux lactones macrocycliques sauf moxidectine (ML) de 52 %, au lévamisole (LEV) de 48 % et à la moxidectine (MOX) de 21 %. Tous les genres principaux de NGI ont survécu au traitement dans diverses études. Chez les bovins, la prévalence de la RA variait selon les classes d'anthelminthiques de 0 à 100 % (BZ et ML), 0 à 17 % (LEV) et 0 à 73 % (MOX), et Cooperia et Ostertagia ont survécu aux traitements. Une RA suspectée chez F. hepatica a été signalée dans 21 études portant sur 6 pays. Pour les NGI, et encore plus pour F. hepatica, il y avait un biais d'échantillonnage en faveur des exploitations individuelles suspectées de RA, et l'effort de recherche était biaisé vers l'Europe occidentale et en particulier le Royaume-Uni. La capture continue des résultats futurs dans la base de données, en direct, les efforts pour éviter les biais dans le recrutement des exploitations, des tests plus précis pour la RA et une meilleure appréciation de l'importance de la RA parmi l'industrie agricole et les décideurs politiques, soutiendront des analyses plus sophistiquées des facteurs contribuant à la RA, et des stratégies efficaces pour ralentir sa propagation.

Mapping QTL influencing gastrointestinal nematode burden in Dutch Holstein-Friesian dairy cattle.

BACKGROUND: Parasitic gastroenteritis caused by nematodes is only second to mastitis in terms of health costs to dairy farmers in developed countries. Sustainable control strategies complementing anthelmintics are desired, including selective breeding for enhanced resistance. RESULTS AND CONCLUSION: To quantify and characterize the genetic contribution to variation in resistance to gastro-intestinal parasites, we measured the heritability of faecal egg and larval counts in the Dutch Holstein-Friesian dairy cattle population. The heritability of faecal egg counts ranged from 7 to 21% and was generally higher than for larval counts. We performed a whole genome scan in 12 paternal half-daughter groups for a total of 768 cows, corresponding to the approximately 10% most and least infected daughters within each family (selective genotyping). Two genome-wide significant QTL were identified in an across-family analysis, respectively on chromosomes 9 and 19, coinciding with previous findings in orthologous chromosomal regions in sheep. We identified six more suggestive QTL by within-family analysis. An additional 73 informative SNPs were genotyped on chromosome 19 and the ensuing high density map used in a variance component approach to simultaneously exploit linkage and linkage disequilibrium in an initial inconclusive attempt to refine the QTL map position.

Autochthonous Angiostrongylus vasorum infection in dogs in The Netherlands.

Until recently, autochthonous infections with Angiostrongylus vasorum were not thought to occur in The Netherlands. However, in 2007 four dogs were diagnosed with angiostrongylosis. Three of them had never left the country. Subsequently, faecal samples from 485 dogs living in the same region where the positive dogs had been diagnosed in 2007 were collected through veterinarians, hunting associations and kennels between January and April 2008. A. vasorum larvae were found in faecal samples of four dogs. Three of those had never been outside the country. Though only few samples were found positive, A. vasorum is now considered autochthonous in The Netherlands.

Host factors associated with Giardia duodenalis infection in dogs across multiple diagnostic tests.

BACKGROUND: The aim of this study was to assess potential associations between Giardia duodenalis infection in dogs, as determined by three diagnostic tests, and dog's group of origin, fecal consistency, age, sex, neuter status, and co-infections with other gastrointestinal parasites. METHODS: Fecal samples from 1291 dogs from four groups (household, shelter, hunting and clinical dogs) were tested with qPCR, rapid enzyme immunochromatographic assay (IDEXX SNAP® Giardia), and direct immunofluorescence (DFA, Merifluor) for presence of G. duodenalis. Moreover, fecal samples were tested with centrifugation sedimentation flotation (CSF) coproscopical analysis for presence of gastrointestinal parasites. Associations were expressed as odds ratios (ORs). RESULTS: Several significant associations were found, of which a few were consistent for all three tests and Giardia positivity in general (positive with at least one of these tests). Dogs older than one year were significantly less likely to test positive for Giardia than younger dogs. Group-housed dogs, especially hunting dogs, were significantly more likely to test positive for Giardia compared to household and clinical dogs. A consistently significant association with Trichuris appeared to be driven by the high prevalence in hunting dogs. Although there was no significant association between loose stool and Giardia infection in the overall population, household dogs were significantly more likely to test Giardia-positive when having loose stool. Overall, Giardia-positive dogs with loose stool shed significantly more cysts, both determined semi-quantitatively with CSF and quantitatively by qPCR, than positive dogs with no loose stool. When other gastrointestinal parasites were present, significantly fewer cysts were detected with CSF, but this was not confirmed with qPCR. CONCLUSION: Giardia is the most common gastrointestinal parasite in Dutch dogs, except for hunting dogs, in which Trichuris and strongyle-type eggs (hookworms) prevailed. Giardia infection was not significantly associated with loose stool, except for household dogs. Young dogs and group-housed dogs were significantly more often Giardia-positive. These associations were consistent across diagnostic tests. Young dogs, clinical dogs and dogs with loose stool shed Giardia cysts in the highest numbers. If another gastrointestinal parasite was present lower numbers of cysts were observed by microscope (CSF), but not with a molecular method (qPCR).

100 Questions in Livestock Helminthology Research.

An elicitation exercise was conducted to collect and identify pressing questions concerning the study of helminths in livestock, to help guide research priorities. Questions were invited from the research community in an inclusive way. Of 385 questions submitted, 100 were chosen by online vote, with priority given to open questions in important areas that are specific enough to permit investigation within a focused project or programme of research. The final list of questions was divided into ten themes. We present the questions and set them briefly in the context of the current state of knowledge. Although subjective, the results provide a snapshot of current concerns and perceived priorities in the field of livestock helminthology, and we hope that they will stimulate ongoing or new research efforts.

Comparing four diagnostic tests for Giardia duodenalis in dogs using latent class analysis.

BACKGROUND: To accurately diagnose giardiosis in dogs, knowledge of diagnostic test characteristics and expected prevalence are required. The aim of this work was to estimate test characteristics (sensitivity and specificity) of four commonly used diagnostic tests for detection of Giardia duodenalis in dogs. METHODS: Fecal samples from 573 dogs originating from four populations (household dogs, shelter dogs, hunting dogs and clinical dogs) were examined with centrifugation sedimentation flotation (CSF) coproscopical analysis, direct immunofluorescence assay (DFA, Merifluor Cryptosporidium/Giardia®), a rapid enzyme immunochromatographic assay (IDEXX SNAP Giardia®) and qPCR (SSU rDNA) for presence of G. duodenalis. Bayesian latent class analysis was used to determine test performance characteristics and to estimate G. duodenalis prevalence of each of the four dog populations. RESULTS: All tests were highly specific. IDEXX SNAP Giardia® showed the highest specificity (99.6%) and qPCR the lowest (85.6%). The sensitivities were much more variable, with qPCR showing the highest (97.0%) and CSF the lowest (48.2%) sensitivity. DFA was more sensitive than IDEXX SNAP Giardia®, but slightly less specific. Prevalences of G. duodenalis differed substantially between populations, with the hunting dogs showing the highest G. duodenalis prevalence (64.9%) and the household dogs the lowest (7.9%). CONCLUSIONS: This study identifies qPCR as a valuable screening tool because of its high sensitivity, whereas methods using microscopy for cyst identification or cyst wall detection should be used in situations where high specificity is required. G. duodenalis is a prevalent gastro-intestinal parasite in Dutch dogs, especially in dogs living in groups (hunting and shelter dogs) and clinical dogs.

Recurrent patent infections with Toxocara canis in household dogs older than six months: a prospective study.

BACKGROUND: To reduce environmental contamination with Toxocara canis eggs, the current general advice is to deworm all dogs older than six months on average four times a year. However, only a small proportion of non-juvenile household dogs actually shed T. canis eggs, and some dogs shed eggs more frequently than others. The identification of these frequent shedders and the associated risk factors is an important cornerstone for constructing evidence-based deworming regimens. The purpose of this study is to identify risk factors associated with recurrence of periods of shedding Toxocara eggs in a cohort of household dogs older than six months. METHODS: We performed a prospective study (July 2011 to October 2014) on shedding Toxocara eggs in a cohort of 938 household dogs older than six months from all over the Netherlands. The median follow-up time was 14 months. Monthly, owners sent faecal samples of their dogs for Toxocara testing and completed a questionnaire. Dogs were dewormed only after diagnosis of a patent infection (PI). Survival analysis was used to assess factors influencing the time to first diagnosed PIs (FPI) and the time to recurrent PIs (RPI). RESULTS: The overall prevalence of PIs was 4.5 %, resulting in an estimated average incidence of 0.54 PIs/dog/year. No PI was diagnosed in 67.9 % of the dogs, 17.5 % of the dogs went through only one PI and 14.6 % had > 1 PI. Prevalence of PIs always peaked during wintertime. Increased hazards for first diagnosed PIs were associated with coprophagy, geophagy, walking off-leash for ≥ 80 % of walking time, reported worms in the faeces, feeding a commercial diet and suffering from urologic or respiratory conditions. Median time to reinfection was nine months. Factors associated with increased hazards for recurrent PIs were taking corticosteroids, changing dog's main purpose, and proxies for veterinary care-seeking behaviours. CONCLUSIONS: We concluded that targeted anthelmintic treatments in household dogs may be feasible as PIs tend to (re)occur in specific periods and in groups of dogs at high risk. Moreover, recurrent PIs appear to be influenced more by factors related to impaired immunity than environmental exposure to Toxocara eggs.

Environmental contamination with Toxocara eggs: a quantitative approach to estimate the relative contributions of dogs, cats and foxes, and to assess the efficacy of advised interventions in dogs.

BACKGROUND: Environmental contamination with Toxocara eggs is considered the main source of human toxocariasis. The contribution of different groups of hosts to this contamination is largely unknown. Current deworming advices focus mainly on dogs. However, controversy exists about blind deworming regimens for >6-month-old dogs, as most of them do not actually shed Toxocara eggs. We aim to estimate the contribution of different non-juvenile hosts to the environmental Toxocara egg contamination and to assess the effects of different Toxocara-reducing interventions for dogs. METHODS: A stochastic model was developed to quantify the relative contribution to the environmental contamination with Toxocara eggs of household dogs, household cats, stray cats, and foxes, all older than 6 months in areas with varying urbanization degrees. The model was built upon an existing model developed by Morgan et al. (2013). We used both original and published data on host density, prevalence and intensity of infection, coprophagic behaviour, faeces disposal by owners, and cats' outdoor access. Scenario analyses were performed to assess the expected reduction in dogs' egg output according to different deworming regimens and faeces clean-up compliances. Estimates referred to the Netherlands, a country free of stray dogs. RESULTS: Household dogs accounted for 39% of the overall egg output of >6-month-old hosts in the Netherlands, followed by stray cats (27%), household cats (19%), and foxes (15%). In urban areas, egg output was dominated by stray cats (81%). Intervention scenarios revealed that only with a high compliance (90%) to the four times a year deworming advice, dogs' contribution would drop from 39 to 28%. Alternatively, when 50% of owners would always remove their dogs' faeces, dogs' contribution would drop to 20%. CONCLUSION: Among final hosts of Toxocara older than 6 months, dogs are the main contributors to the environmental egg contamination, though cats in total (i.e. both owned and stray) transcend this contribution. A higher than expected compliance to deworming advice is necessary to reduce dogs' egg output meaningfully. Actions focusing solely on household dogs and cats are unlikely to sufficiently reduce environmental contamination with eggs, as stray cats and foxes are also important contributors.

Dictyocaulus viviparus: re-emerging or never been away?

For over 40 years a highly effective vaccine against the bovine lungworm has been commercially available. The use of it successfully reduced the number of outbreaks in calves. However, the past decade has seen a dramatic increase in lungworm outbreaks in adult cows in the UK. This might indicate that Dictyocaulus viviparus is re-emerging as a significant parasite in the dairy cattle industry. Much is still unknown, and here the most important aspects requiring urgent attention are put into perspective.

Cytochrome oxidase c subunit 1 polymorphisms show significant differences in distribution between a laboratory maintained population and a field isolate of Cooperia oncophora.

A 474 bp fragment of the mitochondrial cytochrome oxidase c subunit 1 (cox1) of Cooperia oncophora was cloned and sequenced. The overall nucleotide diversity of the cox1 fragment varied from 0.5 to 2.0% between individuals. Two nucleotide substitutions were found within two RsaI endonuclease restriction sites and were used in a PCR-based restriction fragment length polymorphism (PCR-RFLP) assay to asses the intra-population variation of C. oncophora. Testing 816 individuals revealed the existence of three different haplotypes, having either both (type I) or only one (types II and III) RsaI site. Laboratory maintained individuals obtained at different time points after infection showed no significant difference in the distribution of the three haplotypes. Neither was there a difference in the distribution between male and female worms, confirming that the mitochondrial genome of C. oncophora is also maternally inherited. Nevertheless, there was a significant difference in the prevalence of the RsaI point mutation in the cox1 gene between the laboratory maintained population of C. oncophora and a Dutch field isolate, indicating that these RFLPs can be used to study genetic variation within or among C. oncophora populations.

Path analysis of subsistence farmers' use of veterinary services in Zimbabwe.

We used factor and path analyses to identify the causal paths or relationships between variables affecting the demand and use by subsistence farmers of the various services offered by government-employed veterinary livestock technicians (VLTs) in Zimbabwe in 1996. We examined whether the farmer had implemented the VLT-recommended livestock-management procedures. Various factors that have been reported or theorized as important in the demand for veterinary services by subsistence farmers are described. The important factors positively affecting whether farmers had sought veterinary assistance in the last 24 months included familiarity of the farmer with the VLT, if situated closer to the VLT's base, and if the farmer received further training in agriculture (either through courses or from attendance at demonstrations on animal husbandry conducted by the VLT). Factors affecting whether farmers implemented the recommended management practices that had a monetary cost included familiarity with the VLT, proximity to the VLT's premises, farmer literacy, club membership, and farmer's wealth. There was a difference in the paths generated for preventive measures that had a monetary cost versus those measures that involved only a labour or effort costs. For the former practices, the VLT played an important role informing farmers by extension and demonstrations, and farmer's membership of a club increased their performance.

A simple, robust and semi-automated parasite egg isolation protocol.

Large-scale parasite quantification is required for improving our understanding of the epidemiology and genetics of host-parasite interactions. We describe a protocol that uses a low-density salt solution for flotation and centrifugation of nematode eggs. Subsequently, sucrose flotation and precipitation are used to obtain clear egg preparations. Most traditional quantification protocols such as the McMaster technique are unsuited for the standardized processing of large numbers of samples and the analysis of large amounts of feces per sample. Consequently, they are suited only for small-scale surveys. Our protocol, which can be used to analyze up to 6 g of feces, results in clear egg preparations that are concentrated in wells of a microtiter plate and that are suited for digital recording and automated counting. Starting from a fecal suspension in the first flotation solution to a digital recording requires approximately 40 min per 24 samples.

Antibodies elicited by the bovine lungworm, Dictyocaulus viviparus, cross-react with platelet-activating factor.

Parasite N-glycans may play an important role in helminth infections. As antibodies from Dictyocaulus viviparus-infected calves strongly react with N-glycans, we investigated the characteristics of the major immunodominant glycoprotein (GP300) of this parasite. Probing of worm extracts with various lectins demonstrated unique binding of GP300 to wheat germ agglutinin. Analysis of lectin-purified GP300 revealed that the glycan was substituted with phosphorylcholine and reacted with the phosphorylcholine-specific antibody TEPC-15. Competitive enzyme-linked immunosorbent assay with GP300-coated plates and GP300-specific immunoglobulin G (IgG) in conjunction with free phosphorylcholine or TEPC-15 demonstrated that antibodies from infected calves recognized phosphorylcholine on GP300. Additional assays showed that these antibodies cross-reacted with the phosphorylcholine moiety present on platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine), a proinflammatory mediator of the host. Heavily infected calves contained high levels of serum GP300-specific IgG1 but low levels of IgA and IgG2 and showed a reduced influx of eosinophils in the lungs, all consistent with a neutralization of PAF activity. In conclusion, we demonstrated that D. viviparus infection elicits GP300-specific antibodies that cross-react with PAF and may neutralize PAF function, thus limiting the development of a protective response as well as parasite-induced host pathology.