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.

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.

Immuno-epidemiology of chronic bacterial and helminth co-infections: observations from the field and evidence from the laboratory.

Co-infections can alter the host immune responses and modify the intensity and dynamics of concurrent parasitic species. The extent of this effect depends on the properties of the system and the mechanisms of host-parasite and parasite-parasite interactions. We examined the immuno-epidemiology of a chronic co-infection to reveal the immune mediated relationships between two parasites colonising independent organs, and the within-host molecular processes influencing the dynamics of infection at the host population level. The respiratory bacterium, Bordetella bronchiseptica, and the gastrointestinal helminth, Graphidium strigosum, were studied in the European rabbit (Oryctolagus cuniculus), using long-term field data and a laboratory experiment. We found that 65% of the rabbit population was co-infected with the two parasites; prevalence and intensity of co-infection increased with rabbit age and exhibited a strong seasonal pattern with the lowest values recorded during host breeding (from April to July) and the highest in the winter months. Laboratory infections showed no significant immune-mediated effects of the helminth on bacterial intensity in the lower respiratory tract but a higher abundance was observed in the nasal cavity during the chronic phase of the infection, compared with single bacterial infections. In contrast, B. bronchiseptica enhanced helminth intensity and this was consistent throughout the 4-month trial. These patterns were associated with changes in the immune profiles between singly and co-infected individuals for both parasites. This study confirmed the general observation that co-infections alter the host immune responses but also highlighted the often ignored role of bacterial infection in helminth dynamics. Additionally, we showed that G. strigosum had contrasting effects on B. bronchiseptica colonising different parts of the respiratory tract. At the host population level our findings suggest that B. bronchiseptica facilitates G. strigosum infection, and re-infection with G. strigosum assists in maintaining bacterial infection in the upper respiratory tract and thus long-term persistence.

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.

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.

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.

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.

Investigations on the seasonal patterns of strongyle infections in grazing lambs, and the occurrence of anthelmintic resistance on sheep and goat farms in western Anatolia, Turkey.

The seasonal patterns of strongyle infections in untreated, weaned lambs were determined on four governmental farms during a grazing season. In three farms, the infection level (predominantly Teladorsagia spp. and Trichostrongylus spp.) measured by egg counts or worm burdens remained low throughout the study; higher egg counts mainly caused by Haemonchus contortus were transiently recorded on the fourth farm. Significant body weight gains were observed in all groups, but they varied between farms irrespective of the level of strongyle infections, suggesting that the economic effectiveness of anthelmintic treatments of weaned lambs is doubtful under the extensive grazing conditions and the hot, dry climate in the region. In the second part of the study, faecal egg count reduction tests were performed for albendazole, thiabendazole, tetramisole and ivermectin on 12 sheep and goat farms to provide first information on anthelmintic resistance in trichostrongyles of small ruminants in Turkey. There was no hint of benzimidazole resistance, and unequivocal evidence of ivermectin resistance was missing. In contrast, tetramisole resistance was detected on one sheep farm.

Helminths in a giraffe (Giraffa camelopardalis xgiraffa) from a zoo in Spain.

A pregnant female Cape giraffe (Giraffa camelopardalis giraffa) died from an unknown cause in the Aitana Zoo, Alicante, Spain. Neither clinical signs nor macroscopic lesions were observed at necropsy. The alimentary tract was removed and examined for parasites. A total of 2 724 nematodes were found, including Camelostrongylus mentulatus, Trichostrongylus axei, Ostertagia ostertagi, Teladorsagia circumcincta, Teladorsagia trifurcata, Marshallagia marshalli, Trichostrongylus vitrinus, Trichostrongylus colubriformis, Spiculopteragia asymmetrica and Trichuris giraffae. Only T. giraffae and C. mentulatus have been previously reported from giraffes. The other nematodes are common in mouflons, fallow and red deer, which can usually be found in the same paddock as the giraffes in Aitana Zoo. Although its occurrence is unusual in this host, C. mentulatus was the most abundant nematode in our giraffe. This parasite has been related to disease, and even death, in several wild ruminants.

Trichostrongylid infections in sheep after rainfall during summer in southern Australia.

OBJECTIVE: To relate trichostrongylid infections acquired by sheep during summer to prevailing weather conditions. PROCEDURE: Groups of worm-free 'tracer' sheep were put onto pastures, previously contaminated with trichostrongylid eggs, for successive periods of 2 weeks from December to March. After grazing the sheep were housed for 6 weeks. Weekly worm egg counts and worm counts were used to estimate the numbers of worms acquired and related to weather conditions during the grazing period. RESULTS: No worm eggs were detected in the faeces of sheep that grazed at the end of January when only 7 mm of rainfall was recorded. At other times rainfall between 12 and 24 mm occurred and strongyle egg counts were generally either < 50 or > 150 eggs per g (epg). Mean counts of 1,100 Ostertagia and Trichostrongylus adults gave rise to mean counts of about 350 epg whereas about 6,000 Nematodirus spp were associated with mean egg counts of about 200 Nematodirus spp epg. CONCLUSIONS: Rainfall events during summer determine the numbers of trichostrongylid larvae acquired by sheep in summer but further studies are necessary before the implications for strategic control programs in southern Australia can be fully assessed.

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.

[Libyostrongylus douglassii in ostriches (Struthio camelus ssp.) in the Netherlands: case report and review]. / Libyostrongylus douglassii bij struisvogels (Struthio camelus ssp.) in Nederland: case report en literatuuroverzicht.

Several types of worms can parasitize ostriches, the most pathogenic of which is the wire worm (Libyostrongylus douglassii). This worm, which occurs in the stomach, can cause severe lesions which can lead to a high mortality, especially among young birds. This article describes mortality due to this parasite on an ostrich farm and discusses relevant literature on L. douglassii.

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.

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.

Effects of mixed grazing of first- and second-year calves on trichostrongylid infections in Lithuania.

The objective of this study was to examine whether susceptible calves grazing together with second-year resistant heifers are less exposed to trichostrongylid infection than are calves grazing on their own. Two groups of animals representing each age category were turned out onto pasture on 24 May 1997 and grazed at comparable stocking rates. The grazing of calves and heifers together was compared to groups of each age category grazing separately. The results indicated that herbage larval counts were significantly reduced in the second part of the grazing season on the plot grazed by the mixed group compared to the plot grazed by the first-season calves only. The mixed grazing strategy protected the young calves and no clinical signs were observed in this group, while most of the calves that grazed alone exhibited clinical signs. The availability of herbage was reduced towards the end of the season, with subsequent competition for the grass forcing all the animals to graze the tufts around the faecal pats, where the quality of the grass is poor and the numbers of infective larvae are high. The effect of this was visible in the form of increased parasite burdens in the calves that were grazed together with the heifers, confirmed by increased blood serum pepsinogen concentrations and reduced daily weight gains in the second part of the grazing season. The lower numbers of infective larvae on the pasture were probably achieved through the heifers ingesting many of the larvae but subsequently depositing relatively few eggs, since they had acquired some degree of resistance against trichostrongylid infections during their first grazing season. Thus they did not suffer any parasitological ill-effects during mixed grazing with first-season calves.

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.

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.