Genomic introgression mapping of field-derived multiple-anthelmintic resistance in Teladorsagia circumcincta.

Preventive chemotherapy has long been practiced against nematode parasites of livestock, leading to widespread drug resistance, and is increasingly being adopted for eradication of human parasitic nematodes even though it is similarly likely to lead to drug resistance. Given that the genetic architecture of resistance is poorly understood for any nematode, we have analyzed multidrug resistant Teladorsagia circumcincta, a major parasite of sheep, as a model for analysis of resistance selection. We introgressed a field-derived multiresistant genotype into a partially inbred susceptible genetic background (through repeated backcrossing and drug selection) and performed genome-wide scans in the backcross progeny and drug-selected F2 populations to identify the major genes responsible for the multidrug resistance. We identified variation linking candidate resistance genes to each drug class. Putative mechanisms included target site polymorphism, changes in likely regulatory regions and copy number variation in efflux transporters. This work elucidates the genetic architecture of multiple anthelmintic resistance in a parasitic nematode for the first time and establishes a framework for future studies of anthelmintic resistance in nematode parasites of humans.

Explaining patterns of infection in free-living populations using laboratory immune experiments.

The host response to different helminth species can vary and have different consequences for helminth persistence. Often these differences are generated by changes in the dynamics and intensity of the immune components against parasites with distinct life history strategies. We examined the immune response of rabbits to primary infections of the gastrointestinal nematodes Trichostrongylus retortaeformis and Graphidium strigosum under controlled conditions for 120 days post-challenge. Results showed that rabbits developed a robust and effective immune response against T. retortaeformis and abundance quickly decreased in the duodenum and was completely cleared in the remaining sections of the small intestine within 4 months. Infected individuals exhibited an initial strong inflammatory response (IFN-γ), IL-4 expression also increased and was coupled to a rapid serum and mucus IgG and IgA and eosinophilia. Strong IL-4, serum IgA and IgG responses and eosinophilia were also observed against G. strigosum. However, parasite abundance remained consistently high throughout the infection, and this was associated with relatively low mucus antibodies. These findings suggest that immunity plays a key role in affecting the abundance of these nematodes, and different immune mechanisms are involved in regulating the dynamics of each infection and their long-term persistence in free-living host populations.

The dynamics of macroparasite host-self-infection: a study of the patterns and processes of pinworm (Oxyuridae) aggregation.

OBJECTIVES: Among parasites, Taylor's power law identifies a tight relationship in aggregation of macroparasite infection intensity with few exceptions; notably, the nematode family Oxyuridae tends to have higher than expected aggregation. Oxyuridae infect a wide range of mammalian hosts and have a unique reproductive strategy that involves conventional horizontal transmission, as well as re-infection of an already infected host. We asked the question, do the unique aspects of pinworm life-history explain an exception to the widely observed patterns of aggregation of parasite populations? METHODS: We empirically examined the differences among Oxyuridae (genus: Syphacia) compared with other helminth (genus: Heligmosomoides) parasite aggregations in 2 rodent hosts with similar ecology: the yellow-necked mouse (Apodemus flavicollis) from Trento, Italy and the white-footed mouse (Peromyscus leucopus) from Pennsylvania, USA. To investigate the effects of pinworm life-history characteristics on generating aggregation, we present a stochastic model that explores aggregation under a range of host-self-infection, parasite death, and transmission scenarios. RESULTS: Oxyuridae parasites had consistently greater aggregation compared to other nematodes regardless of host or parasite species identity, and pinworm aggregation exceeded the range of macroparasite aggregation described previously. CONCLUSIONS: Our simulations demonstrate that host-self-infection, on its own, is sufficient to generate aggregation values greater than the predicted values.

Genetic variation in resistance to mixed, predominantly Teladorsagia circumcincta nematode infections of sheep: from heritabilities to gene identification.

In cool temperate areas, such as Scotland, sheep are infected by a variety of nematodes but the dominant nematode is Teladorsagia circumcincta. Resistant animals have one or more of the following features: fewer adult nematodes, more inhibited larvae, shorter adult nematodes and decreased production of nematode eggs. In lambs at the end of the first grazing season, the heritability of adult worm length is very strong, whereas the heritability of egg production is moderate. The heritability of worm number is low while there is no detectable genetic variation in the number of inhibited larvae. The major mechanisms underlying resistance to T. circumcincta appear to be the IgA mediated suppression of worm growth and the mast cell mediated regulation of worm number. Mast cell responses are slow to develop, possibly because they are responsible for protein loss and reduced growth of the host. Two genes have been repeatedly associated with resistance to T. Circumcincta: the MHC class II DRB1 locus on chromosome 20 and the interferon-gamma locus on chromosome 3. Although the causative mutations are still unknown both genes are plausible candidates.

A further increase in the prevalence and intensity of infection with Ashworthius sidemi nematodes in red deer in the Lower Silesian Wilderness

The nematode Ashworthius sidemi is a typical parasite of Asiatic deer, particularly sika deer (Cervus nippon). It was probably first carried into Poland by migrating red deer. The aim of the present study was to determine the prevalence and intensity of A. sidemi infection in roe deer and red deer in the Lower Silesian Wilderness in the winter season 2015/2016. Parasitological necropsy of 34 abomasa of red deer from Forest District Ruszów was performed. The prevalence of A. sidemi infection was found to be 91.1%, and the intensity to range from 10 to 2680 nematodes, with a mean intensity of 394 parasites. This prevalence of ashworthiosis and maximum intensity of infection were respectively 17.2% and 38% higher than observed in the previous season (2013/2014). The mean intensity was also slightly higher than reported previously.

Hidden in plain sight - Multiple resistant species within a strongyle community.

Ovine parasitic gastroenteritis is a complex disease routinely treated using anthelmintics. Although many different strongyle species may contribute to parasitic gastroenteritis, not all are equally pathogenic: in temperate regions, the primary pathogen is Teladorsagia circumcincta. In this study we investigated benzimidazole and ivermectin resistance on a commercial sheep farm in southeast Scotland. We assessed the impact of species diversity on the diagnosis of resistance using the faecal egg count reduction test and in vitro bioassays, and correlated the results with the frequency of benzimidazole resistance-associated genotypes measured in the T. circumcincta population by pyrosequencing of the ß-tubulin isotype-1 gene. Faecal egg count reduction test results showed efficacies of 65% for albendazole and 77% for ivermectin, indicating moderate resistance levels on the farm. However, PCR speciation of the same populations pre- and post-treatment revealed that removal of susceptible species had masked the presence of a highly resistant population of T. circumcincta. Less than 25% of individuals in the pre-treatment populations were T. circumcincta, the remainder consisting of Cooperia curticei, Chabertia ovina, Oesophagostomum venulosum and Trichostrongylus spp. In contrast, post-treatment with albendazole or ivermectin, the majority (88% and 100% respectively) of the populations consisted of T. circumcincta. The egg hatch test for benzimidazole resistance and the larval development test for ivermectin resistance were carried out using eggs obtained from the same populations and the results were broadly consistent with the faecal egg count reduction test. Thirty individual T. circumcincta from each sampling time point were assessed for benzimidazole resistance by pyrosequencing, revealing a high frequency and diversity of resistance-associated mutations, including within the population sampled post-ivermectin treatment. These results highlight the potential diversity of parasite species present on UK farms, and their importance in the diagnosis of anthelmintic resistance. On this particular farm, we demonstrate the presence of a highly dual-resistant population of T. circumcincta, which was strongly selected by treatment with either benzimidazoles or ivermectin, while other potentially less pathogenic species were removed.

Production of eosinophil chemoattractant activity by ovine gastrointestinal nematodes.

Eosinophilia is a well documented feature of helminth infections but the precise nature of the interaction between parasite and eosinophil remains an enigma. This paper describes experiments demonstrating that ruminant gastrointestinal trichostrongyles produce potent chemoattractant activity for ovine bone marrow-derived eosinophils in vitro. This activity was initially identified as a constituent of whole worm extracts of third and fourth larval (L3, L4), and adult stages of Teladorsagia circumcincta, and adult Haemonchus contortus. Similar activity was detected in excretory/secretory (E/S) material derived from live T. circumcincta L3. Subsequently, by adapting the assay technique to incorporate live worms directly into the system, it was shown that L3 of both T. circumcincta and H. contortus produced eosinophil chemoattractant activity. In contrast, neither whole worm extracts, or E/S preparations from mixed stages of the free-living nematode Caenorhabditis elegans contained eosinophil chemoattractant activity, and there was no evidence of chemoattractant production by live C. elegans. The results described are challenging to the traditional dogma that eosinophils are host-protective effector cells, and raise the intriguing possibility that ovine nematodes actively encourage recruitment of eosinophils. Local eosinophil-mediated mucosal damage, comparable to that seen in the asthmatic lung, may then provide a permissive local microenvironment for the parasite. Moreover, if they prove important for pathogenicity, nematode chemoattractants could offer future potential as novel therapeutic targets.

Cryopreservation of the first-stage larvae of trichostrongylid nematode parasites.

First stage (L1) larvae of Haemonchus contortus, Trichostrongylus colubriformis and Ostertagia circumcincta can be cryopreserved in the presence of DMSO using a two-step freezing protocol involving an initial period at -80 degrees C prior to transfer to liquid nitrogen. Thawed L1 larvae continue development in vitro producing third stage (L3) larvae that are infective to sheep when dosed per os. Establishment rates for L3 larvae grown from thawed L1 larvae were 40 and 80% for H. contortus and T. colubriformis, respectively. There was no difference in survival or infectivity between benzimidazole (BZ)-susceptible and BZ-resistant H. contortus isolates and cryopreservation caused no shift in their BZ-resistance status as indicated in an in vitro larval development assay. Cryopreservation also had no effect on the sensitivity of these isolates to the avermectins or levamisole in vitro. High survival rates (60-70%), good levels of establishment and the stability of anthelmintic resistance status of isolates indicate that little if any selection occurs during the cryopreservation process. L1 larvae of all 3 species have been successfully recovered after 16 months storage in liquid nitrogen, cultured to the L3 stage and established in sheep.

The pathogenicity of haemonchosis in sheep and goats in Iraq: 1. Clinical, parasitological and haematological findings.

The response of Awassi sheep and the local breed of Iraqi goats to primary infection with a prevalent trichostrongylid nematode, Haemonchus contortus, was compared by examining their worm burden and the pathogenic effects of the parasite on these hosts following experimental infection. The results showed that the goats had lower worm burdens and had suffered less severe clinical and haematological disturbances than the Awassi sheep. The worm counts in sheep were higher than in goats but the count of the mucosal phase of the larvae was higher in goats. These differences are possibly immunologic in origin though a genetic resistance may operate, primarily at the level of worm establishment. The pathophysiological changes are correlated with parasitological and clinical observations in the two host species.

Some aspects of inhibited development of trichostrongylids in ruminants.

Inhibited development is an important aspect of the biology of some gastrointestinal nematodes of ruminants, particularly species of the subfamily Ostertagiinae and Haemonchus spp. There is a seasonality in the occurrence of the phenomenon. It tends to occur predominantly during the unfavourable season for the free-living stages. Thus 'winter' and 'summer-dry season' patterns of inhibition can be observed. Furthermore, the phenomenon is influenced by immunity of the host and by management factors. Different studies show conflicting results on the phenomenon of inhibition. This is examplified by studies related to H. contortus in small ruminants. Other studies indicate that large differences in propensity for inhibition can indeed occur in a single region. However, it has to be stressed that inadequate necropsy techniques can easily result in underestimation of the proportion of inhibited larvae, particularly in Haemonchus spp. in cattle. The macrocyclic lactones are highly effective against inhibited larvae of gastrointestinal nematodes. Treatment may even prevent establishment of inhibited larvae of Ostertagia and Trichostrongylus axei up to 1 month after treatment. The efficacy of benzimidazoles seems to be lower in the middle of the 'inhibition' period than at the beginning or the end. Some studies indicated that the presence of inhibited stages may be important for the build up of immunity. However, removal of high inhibited O. ostertagi burdens at housing does not impair development of immunity. Very little progress has been made on the molecular mechanisms of inhibited development.

Experimental infection of sheep by the camel stomach worm, Haemonchus longistipes.

An attempt has been made to infect sheep by Haemonchus longistipes with the objective of developing a relatively cheap and manageable model of camel haemonchosis. A large proportion of inoculated animals (28/34) developed patent infections; the prepatent period was short, lasting for 3 to 4 weeks in the majority of infected animals. Worm burdens were low and so was the total egg output. Most of the recovered worms were reduced in size.

Infectivity and pathogenicity of three isolates of Ostertagia ostertagi in cattle.

Two laboratory-maintained isolates of Ostertagia ostertagi and 1 recently isolated from grazing cattle, were compared for their infectivity and pathogenicity in Holstein-Friesian calves. Calves were inoculated with 200 000 and 300 000 infective larvae in 2 experiments. Calves in both experiments developed anorexia and diarrhea; generally calves were more severely affected by the larger inoculum. Patent infections and clinical signs developed later in the calves inoculated with the field isolate. Despite decades of laboratory passage of 2 of the isolates and the geographic diversity of all 3 isolates studied, there were no significant differences among them in their infectivity as measured by numbers of nematodes and percentage of arrested development at necropsy and by pathogenicity in calves.

Development of Ostertagia leptospicularis in cattle, and the differentiation of infective larvae and female adults from those of O. ostertagi.

The development of Ostertagia leptospicularis was observed in helminthologically-naive calves. Infective larvae and parasitic stages of the species were also examined in an endeavour to find a reliable means of distinguishing these from comparable stages of O. ostertagi. In the case of infective larvae, it was found that the distance from the tip of the larval tail to the tip of the sheath ("tail length") provided the most simple means of distinguishing between the species (reliability of approximately 97%). The ratio of "tail length" to total body length may be used to provide still greater reliability where this is essential. The rate of development and morphology of the early parasitic stages of O. leptospicularis in calves were found to be similar to those described for O. ostertagi by Rose (1969) and with the exception of late 4th stage males there appeared to be no simple morphological means by which parasitic larval stages of the 2 species could be distinguished. However, adult (5th stage) female O. leptospicularis were found to be distinguishable from those of O. ostertagi (reliability of approximately 98%) on the basis of the position of the cervical papillae in relation to a junction between 2 regions of the central oesophagus. Males, from the late 4th stage, were readily distinguishable on the basis of the spicule structure.

Study on the pathogenicity of ostertagiosis due to Ostertagia circumcincta in sheep, in Iraq.

The response of Awassi sheep to ostertagia circumcincta, the most prevalent ovine strongylid species in Iraq, was studied. A dose of 100000 larvae of O. circumcincta (Iraqi strain) induced moderate clinical symptoms of ostertagiosis. These symptoms were correlated with elevated pH of the abomasal fluid and increased plasma pepsinogen levels. There was no evidence of larval inhibition since the majority of the fourth stage larvae (L4) continued their development. No appreciable loss of worm population was observed and most of the parasites survived and exhibited a prolific egg-laying potential six months after initial infection. Assessments of the pathophysiological changes were performed and correlated with parasitological and clinical observations. The Awassi breed of sheep, the most prevalent in the Arab Middle East, seems to be more susceptible to ostertagiosis than other breeds and there was no spontaneous self-cure in this breed.

Variation among Merino sheep in susceptibilty to lice (Bovicola ovis) and association with susceptibility to trichostrongylid gastrointestinal parasites.

Sheep of two bloodlines of Merino were artificially infested with equal numbers of lice (Bovicola ovis) and the resulting louse populations were monitored over the following 20 months. The sheep were shorn 6 and 17 months after infestation and, for analysis, the louse counts considered in 3 years separated by shearings. Nematode faecal egg counts (FECs) were assessed on faecal samples collected on five occasions, three times following natural challenge and twice after artificial challenge with 40,000 trichostrongyloid larvae (84% Trichostrongylus vitrinus). In addition, blood samples were collected and measured for B. ovis-specific immunoglobulins (predominantly IgG), B. ovis-specific IgE and serum total IgE. Bloodlines differed significantly in the size of louse populations at the end of year 2, FEC after both natural and artificial challenge and in serum levels of all three antibodies (p<0.05). There were also large variations in louse counts and FEC among sheep within bloodlines. Louse counts at inspections after louse populations had been allowed to build up were highly repeatable, both between and within years. However, correlations with counts at inspections soon after initial infestation and following shearing were lower. FEC after natural challenge was correlated with louse counts in year 2 (r=0.45, p<0.01) and year 3 (r=0.38, p<0.05), but the correlation with counts in year 1 was not significant (r=0.25, p>0.05). FEC following artificial challenge was significantly correlated with louse counts in year 3 (r=0.36, p<0.05), but not in year 2 (r=0.25, p>0.05) or year 1 (r=0.04, p>0.05). Louse counts in the 3 years were significantly correlated with anti-B. ovis antibody concentration (r=0.60, 0.48, 0.36), but not with levels of either anti-B. ovis or total serum IgE. These results suggest that sheep with greater resistance to gastrointestinal parasites also tend to be less susceptible to lice. Whether this is due to interaction of the effects of the parasites or to correlation in underlying resistance mechanisms requires clarification.

The influence of relative resistance and urea-supplementation on deliberate infection with Teladorsagia circumcincta during winter.

The consequences for lambs of infection over the winter with Teladorsagia circumcincta were quantified by deliberate, trickle infection of selected animals at 7 months of age. Infected and control uninfected animals were each allocated into four groups, relatively resistant animals on a normal diet, relatively resistant animals on an isocaloric diet supplemented with urea, and relatively susceptible animals on the same two diets. Resistance and susceptibility was assessed by faecal egg counts following natural infection during the summer preceding the deliberate infection. During the deliberate infection egg counts remained low and most parasites recovered at necropsy were inhibited larvae. Nonetheless, infection reduced weight gain, decreased albumin and fructosamine concentrations and provoked a noticeable pepsinogen and eosinophil response. As most larvae were inhibited these responses may have been largely a consequence of immuno-inflammatory responses in the host rather than the direct action of parasites themselves. Relatively resistant animals on the supplemented diet allowed fewer larvae to establish and had higher fructosamine concentrations, higher albumin concentrations and decreased pepsinogen responses. Therefore, a combination of relatively resistant sheep and nutritional supplementation appears most efficient at controlling infection.

Parenterally administered gastrointestinal nematode infective larvae viable after more than 15 years in liquid nitrogen.

After cryopreservation for 13.3-15.8 years, the viability of the infective larvae (L3) of Trichostrongylus axei, T. colubriformis, Oesophagostomum columbianum, Haemonchus contortus, Ostertagia circumcincta, T. falculatus, Nematodirus spathiger, Chabertia ovina and Dictyocaulus filaria was assessed in sheep, by being deposited at their predilection sites. D. filaria was, however, an exception, in that the L3 were injected into the jugular vein. The mean development of all the species was 22.8%, but if three species (O. columbianum, C. ovina and D. filaria), that developed poorly are disregarded, then the mean development was 33.4%, similar to previous tests after shorter periods of cryopreservation. The L3 of some of the species appeared sluggish when examined 10-15 min after being thawed, and in the case of H. contortus practically all the larvae of the original batch tested in the previous trials of the series appeared dead when thawed for use in the present trial, and were replaced by another batch of L3 of the same species. When re-examined after about 8 h, however, a high percentage of the L3 of the original batch appeared to have become revitalised, and their viability was tested in a trial reported elsewhere. The intestinal cells of the majority of the L3 of N. spathiger, O. circumcincta and C. ovina were vesiculated when they were thawed. Nevertheless, the degree of development of the former two species was of the highest in the trial, and it can be concluded that this phenomenon does not necessarily impede the viability of larvae.

Trichostrongylus colubriformis: cultivation of free-living stages.

Successful cultivation of Trichostrongylus colubriformis from hatched first-stage to third-stage larvae was achieved in media containing NCTC 135, chick embryo extract, fetal calf serum, and either lactalbumin hydrolysate or freshly prepared baker's yeast extract. Medium Tc5, containing yeast extract, was the simplest medium with optimal results. Cultured third-stage larvae were able to produce patent infections in guinea pigs.

Comparative aspects of plasma antioxidant status in sheep and goats, and the influence of experimental abomasal nematode infection.

This paper provides, for the first time, comparative data on the plasma antioxidant status of two ruminant species, namely sheep and goats. In addition, the influence of experimental infection with Teladorsagia circumcincta on antioxidant status in the same two species is compared and contrasted. In general terms, antioxidant status was significantly higher in uninfected kids than in lambs. Differences in protein sulphydryl groups and vitamin E concentrations were particularly noteworthy; trends were similar, however, for albumin, vitamin A and total antioxidant capacity (TAC). Parasitological results, based on worm burden, faecal egg counts and peripheral blood eosinophil numbers, confirmed that goat kids were more susceptible than lambs to experimental T. circumcincta infection. "Trickle infection" had a variable impact on both total and individual antioxidant status; particularly during the early weeks, the trend was for reduced values in lambs and increased values in kids, as compared with uninfected controls. Subsequent challenge infection was associated with a transient decrease in TAC and albumin in trickle-infected animals of both species, and in appropriate control animals. The observed differences in plasma antioxidant capacity between sheep and goats may have important implications in terms of the comparative resilience of sheep and goats to parasite infection.

Field study on the epidemiology and pathogenicity of different isolates of bovine Ostertagia spp.

The epidemiological features of three different isolates of bovine Ostertagia spp under similar initial levels of larval challenge were compared in the field. Two of the isolates, consisting mainly of Ostertagia ostertagi, and a low proportion of Skrjabinagia lyrata conformed in epidemiological behaviour with those investigated by previous workers, though the worm burdens which established did not give rise to the expected clinical signs. The third isolate behaved in a different way, yielding very high faecal egg counts which were followed by high pasture larval counts, heavy worm burdens and severe clinical disease. This isolate, while consisting mainly of O ostertagi and a few S lyrata, also contained a proportion of O leptospicularis, and it is suggested that this species may influence the dynamics of the host-parasite relationship in bovine ostertagiasis.