Anthelmintic resistance among gastrointestinal nematodes of cattle on dairy calf to beef farms in Ireland.

BACKGROUND: The control of gastrointestinal nematodes (GIN) of cattle in pasture-based production systems such as Ireland is highly dependent on the availability of efficacious anthelmintics. There is very little information available on the efficacy of the broad-spectrum anthelmintics against GIN of cattle in Ireland and the aim of this study was to determine the prevalence of anthelmintic resistance on dairy calf to beef farms. RESULTS: GIN burden was monitored on thirty-six recruited farms by performing herd level faecal egg counts (FEC) every 2 weeks. Of these, nine farms were lost from the study as calves were treated with an anthelmintic for Dictyocaulus viviparus, two were lost as they treated for GIN, one dropped out of the study and on one the herd FEC did not reach the threshold for carrying out the Faecal Egg Count Reduction Test (FECRT). On the remaining 23 farms, once the herd FEC reached 100 eggs per gram, a FECRT was carried out. Pre and post-treatment larval cultures were also performed to identify the GIN to genus level. The efficacy of fenbendazole, levamisole, ivermectin and moxidectin was evaluated on 15, 11, 16 and 11 farms respectively. Resistance to fenbendazole was identified on 9 farms (60%) with resistance suspected on a further farm. Resistance to levamisole, ivermectin and moxidectin was detected on 2 (18%), 16 (100%) and 8 (73%) farms respectively. The predominant genera detected pre and post-treatment were Cooperia and Ostertagia with both genera detected post-treatment with fenbendazole and ivermectin. Due to the low proportion of Ostertagia spp. pre-treatment, the efficacy of levamisole or moxidectin against this genus could not be reliably established. CONCLUSIONS: Anthelmintic resistance was widespread on the sampled dairy calf to beef farms in Ireland with resistance to benzimidazole, levamisole, ivermectin and moxidectin detected.

Epidemiological investigation of a severe rumen fluke outbreak on an Irish dairy farm.

Although the rumen fluke, Calicophoron daubneyi is now very common and widespread throughout Western Europe, reports of clinical cases are still rare. This study explores the epidemiological background to a severe rumen fluke outbreak in 6-month-old heifers on a dairy farm in Ireland. Sequence analysis of the cytochrome oxidase subunit 1 (Cox1) gene of the rumen fluke metacercariae on pasture failed to identify predominant, possibly pathogenic subtypes. However, estimates of metacercarial load indicated that the animals were exposed to a daily dose of about 5334 C. daubneyi metacercariae for a period of 3 weeks resulting in the build-up of very large numbers of immature worms in the small intestine. It is hypothesized that specific environmental conditions may favour this parasite over its competitor, the liver fluke, Fasciola hepatica, possibly by allowing it to emerge earlier. The possibility that C. daubneyi may be better adapted to the Irish climate than F. hepatica together with the fact that selective treatment against F. hepatica effectively frees the niche for C. daubneyi, may result in the gradual replacement of F. hepatica by C. daubneyi.

Genetic basis of benzimidazole resistance in Teladorsagia circumcincta in Ireland.

Resistance to benzimidazole (BZ) anthelmintics is common in ovine nematodes of economic importance. Single nucleotide polymorphisms (SNP) at three positions in the isotype 1 ß- tubulin gene have been associated with BZ resistance and molecular tests for the detection of BZ resistance have been developed. In order to determine if such tests are practicable in Ireland the polymorphisms associated with BZ resistance must be identified. To this end, BZ-resistant nematodes were recovered from four farms in Ireland. Resistant Teladorsagia circumcincta, Cooperia curticei and Trichostrongylus colubriformis were recovered, with resistant T. circumcincta the most common and the only species studied further. Sequencing of the isotype 1 ß-tubulin gene from resistant T. circumcincta identified a T - A transition, resulting in an F200Y substitution known to be responsible for BZ-resistance, on three of the farms. However, on the fourth farm the frequency of the resistant A allele was only 0.33 indicating another BZ resistance mechanism may be present on this farm. An additional polymorphism resulting in a substitution of glutamate for leucine (E198L) was also found on this farm at low frequency (0.17). No polymorphisms at position 167 were identified on any farm. Therefore, molecular tests to detect BZ resistance in T. circumcincta in Ireland could prove useful; however, they may result in some instances of resistance remaining undetected.

A nationwide survey of anthelmintic treatment failure on sheep farms in Ireland.

BACKGROUND: Between 2013 and 2015 the Department of Agriculture, Food and the Marine (DAFM) administered a sheep technology adoption programme (STAP), with the aim of increasing profitability on Irish sheep farms by encouraging the adoption of best management practices. One of the options available to STAP participants was to test the efficacy of the anthelmintic treatment (benzimadazole, levamisole or macrocyclic lactone) used in their flocks by means of a drench test, which is a modification of the faecal egg count reduction test; individual faecal samples were collected from the same group of lambs before and after anthelmintic treatment, the number of eggs present pre and post treatment was subsequently determined from a pooled sample. RESULTS: In total, 4211 drench tests were undertaken by farmers during the 3 years of the programme. Information on the anthelmintic product used was available for 3771 of these tests; anthelmintics from the classes benzimidazole (BZ), levamisole (LV) and macrocyclic lactone (ML) (avermectins (AVM) plus moxidectin (MOX)) were used in 42.0%, 23.4% and 32.5% of tests, respectively. The remaining 2.1% of tests involved an inappropriate product. The efficacy of treatment against 'other trichostrongyles' (excluding Nematodirus spp and Strongyloides papillosus.) could be established for 1446 tests, and 51% of these tests were considered effective (i.e. a reduction of faecal egg count (FEC) ≥ 95%). There was a significant difference among the drug groups in efficacy; 31.5%, 51.9%, 62.5% and 84% of treatments were considered effective for BZ, LV, AVM, MOX, respectively. The efficacy of treatment against Nematodirus spp. could be established for 338 tests and the overall efficacy was 96%. CONCLUSIONS: Due to the significant difference among the anthelmintic classes for efficacy against 'other trichostrongyles' along with the high level of efficacy against Nematodirus spp., a genus for which anthelmintic resistance is rarely reported, it is concluded that anthelmintic resistance was responsible for the majority of the anthelmintic treatment failures observed.

Ticks and Tick-borne diseases in Ireland.

Throughout Europe interest in tick-borne agents is increasing, particularly with regard to those that can cause human disease. The reason for this is the apparent rise in the incidence of many tick-borne diseases (TBD's). While there has never been a national survey of ticks or TBD's in Ireland, the trend here appears to be the reverse with a decline in the incidence of some agents seemingly associated with decreasing tick numbers particularly on agricultural land. In the absence of robust baseline data, however, this development cannot be confirmed. This review collates the limited information available from several dated published records on tick species and a small number of studies focused on certain TBD's. Some pilot data on tick density and TBD agents collected in 2016 are also presented. The aim is to explore the particular situation in Ireland with regard to ticks and TBD's and to provide a reference for future workers in the field.

Breed differences in humoral and cellular responses of lambs to experimental infection with the gastrointestinal nematode Teladorsagia circumcincta.

While Texel lambs have increased resistance to infection with the gastrointestinal nematode Teladorsagia circumcincta compared to Suffolk lambs, the underlying resistance mechanisms are still unknown. The aim of this study was to compare parasitological, humoral and cellular responses of Texel and Suffolk lambs over time following a single experimental infection with T. circumcincta. Gastrointestinal nematode free (but not naïve) lambs received a single oral dose of 3 × 104 infective T. circumcincta larvae. The variables examined included worm burden, mucosal and serum IgA, abomasal mast cells and eosinophils, haematological parameters and plasma pepsinogen. Texel lambs had significantly lower worm burden on day 14 and lower plasma pepsinogen concentration from day 14 onwards than Suffolks and their response in mucosal IgA to infection occurred earlier. The results from the study suggest that an earlier local IgA response in the Texel contributes to the resistant characteristics of the breed, while the increased level of plasma pepsinogen in the Suffolk lambs implies greater abomasal tissue damage arising from the nematode infection.

Determining the Prevalence and Seasonality of Fasciola hepatica in Pasture-based Dairy herds in Ireland using a Bulk Tank Milk ELISA.

BACKGROUND: Fasciola hepatica is a helminth parasite of global importance in livestock, with major economic impact. However information on F. hepatica infections in Irish pasture-based dairy herds is limited. Therefore this study was conducted in order to determine the prevalence, seasonality and management factors associated with F. hepatica. A total of 319 Irish dairy herds were selected for this study. Bulk tank milk (BTM) samples were collected from 290 dairy farms on a quarter year basis, while from a further 29 dairy farms BTM samples were collected on a monthly basis to provide a more detailed pattern of F. hepatica exposure in Irish herds. BTM samples were analysed using a commercially available F. hepatica antibody detection ELISA. Furthermore, within-herd prevalence of F. hepatica was assessed in a subset of these 29 herds (n = 17); both individual serum samples and bulk tank milk samples were collected. RESULTS: A within-herd prevalence of ≤ 50 % was found for herds with negative bulk tank milk samples. The mean prevalence of the 290 study herds was 75.4 % (Range 52 %-75.1 %), with the highest prevalence being observed in November (75.1 %). The seasonal pattern of F. hepatica shows elevated antibodies as the grazing season progressed, reaching a peak in January. A significant association was found between F. hepatica and age at first calving. CONCLUSION: This study demonstrates that F. hepatica is present in a large proportion of Irish dairy herds and provides a basis on which control practices, particularly in adult dairy cows, can be reviewed.

High level of treatment failure with commonly used anthelmintics on Irish sheep farms.

BACKGROUND: In 2013 a Technology Adoption Program for sheep farmers was established to encourage the implementation of best management practices on sheep farms in Ireland. There were 4,500 participants in this programme in 2013. As part of this programme, farmers had the option to carry out a drench test to establish the efficacy of their anthelmintic treatment. RESULTS: Flock faecal samples were collected before and after treatment administration and gastrointestinal nematode eggs enumerated. In total there were 1,893 participants in the task, however only 1,585 included both a pre- and post-treatment faecal sample. Of those, 1,308 provided information on the anthelmintic product that they used with 46%, 23% and 28% using a benzimidazole (BZ), levamisole (LEV) and macrocyclic lactone (ML) product respectively. The remaining farmers used a product inapplicable for inclusion in the task such as a flukicide or BZ/LEV combination product. Samples were included for analysis of drench efficacy if the pre-treatment flock egg count was ≥200 eggs per gram and the interval post-sampling was 10-14 days for BZ products, 4-7 days for LEV products and 14-18 days for ML products. These criteria reduced the number of valid tests to 369, 19.5% of all tests conducted. If the reduction post-treatment was ≥95% the treatment was considered effective. Only 51% of treatments were considered effective using this criterion. There was a significant difference in efficacy between the anthelmintic drug classes with BZ effective in only 30% of treatments, LEV effective in 52% of cases and ML effective in 76% of cases. CONCLUSIONS: Gastrointestinal nematode anthelmintic treatments, as practiced on Irish farms, have a high failure rate. There was a significant difference between the efficacies of the anthelmintic classes with BZ the least effective and ML the most effective.

A differential interplay between the expression of Th1/Th2/Treg related cytokine genes in Teladorsagia circumcincta infected DRB1*1101 carrier lambs.

Substantial debate exists on whether the immune response between sheep resistant and susceptible to gastrointestinal nematodes can be differentiated into a Th1 and Th2 phenotype. The present study addresses the hypothesis that variation in resistance to Teladorsagia circumcincta between DRB1*1101 (associated with reduced faecal egg count and worm burden) carriers and non-carriers is due to a differential interplay in the expression of Th1/Th2 and regulatory T (Treg) related cytokine genes. Lambs from each genotype were either slaughtered at day 0 (un-infected control) or infected with 3 × 10(4) Teladorsagia circumcincta L3 and slaughtered at 3, 7, 21, and 35 days later. Lambs carrying the DRB1*1101 allele had a significantly lower worm burden (P < 0.05) compared to the non-carriers. Abomasal mucosal cytokine gene expression was evaluated by quantitative real-time PCR and comparison made for time and genotype effects. The response generated varied through the course of infection and was affected by genotype. DRB1*1101 carriers had an up-regulated expression of the Th1-related cytokine genes (IL-1ß, TNFα, and IFN-γ) at day 3, but this was replaced by an up-regulated expression of Th2-related cytokine genes (IL-10 and IL-13) and Treg-related cytokine genes (IL-2RA-CD25, TGFα, TGFß, Arg2, MIF and FOXP3) by day 7. Conversely, in the non-carriers these changes in gene expression were delayed until days 7 and 21 post infection (pi), respectively. It is concluded that resistance to Teladorsagia circumcincta in animals carrying the DRB1*1101 allele is influenced by an earlier interplay between Th1, Th2 and T regulatory immune response genes.

The dynamic influence of the DRB1*1101 allele on the resistance of sheep to experimental Teladorsagia circumcincta infection.

Suffolk sheep carrying the DRB1*1101 (previously referred to as-DRB1*0203 or G2) allele have been reported to show increased resistance to natural Teladorsagia circumcincta infection compared to non-carriers. The objective of this study was to compare the biochemical and physiological responses of DRB1*1101 carrier and non-carrier twin lambs to an experimental infection with 3 × 10(4) L3 Teladorsagia circumcincta. The variables studied included worm burden, faecal egg count, abomasal mast cells, IgA, IgE, IgG1 plus IgG2 and haematological parameters at 0, 3, 7, 21 and 35 days post infection (dpi), and duodenal smooth muscle contractility at 0 and 35 dpi. DRB1*1101 carrier lambs had significantly lower worm burden, higher mast cell and plasma platelet counts than the DRB1*1101 non-carriers (P < 0.05). Before infection, the non-carrier lambs exhibited significantly higher mucosal levels of all antibody isotypes measured compared to the carriers; these levels remained relatively stable over the course of infection in the non-carriers while there was a slow build up of these antibodies in the carriers up to day 21 post infection (pi). The DRB1*1101 non-carrier lambs had a significantly higher plasma lymphocyte count, and produced greater duodenal contractile force relative to the carrier lambs (P < 0.05). There was no significant difference between genotypes in the level of plasma eosinophils, monocytes, neutrophils or FEC. This evidence suggests that resistance conferred by DRB1*1101 is acquired rather than innate, depends on worm expulsion rather than fecundity and is dependent on mucosal mast cell proliferation, platelet activation, and IgA and IgE antibody responses.

Update on the presence of Ixodes ricinus at the western limit of its range and the prevalence of Borrelia burgdorferi sensu lato.

It is often suggested that due to climate and environmental policy changes, the risk from tick-borne disease is increasing, particularly at the geographical limits of the vector distribution. Our project aimed to determine whether this was true for the risk of Lyme borreliosis in Ireland which is the western-most limit of Ixodes ricinus, the European vector of Borrelia burgdorferi sensu lato. The availability of a historical data set of tick infection rates compiled in the 1990s represented a unique opportunity as it provided a baseline against which current data could be compared. Following construction of a spatial predictive model for the presence and absence of I. ricinus based on data from 491 GPS locations visited between 2016 and 2019, 1404 questing nymphs from 27 sites were screened for the presence of Borrelia spp. using a TaqMan PCR aimed at the 23S rRNA gene sequence. All positive ticks were further analysed by nested PCR amplification and sequence analysis of the 5 S-23 S intergenic spacer. The model indicated that areas with the highest probability of tick presence were mostly located along the western seaboard and the Shannon and Erne river catchments, coinciding with historical high incidence areas of bovine babesiosis, while the infection rate of questing nymphs with B. burgdorferi s.l. and the prevalence of the various genospecies have remained surprisingly stable over the last 3 decades. Clear communication of the potential disease risk arising from a tick bite is essential in order to allay undue concerns over tick-borne diseases among the general public.

Live weight as a basis for targeted selective treatment of lambs post-weaning.

Targeted selective treatment (TST) has been proposed as a sustainable method of gastrointestinal nematode control that reduces the number of anthelmintic treatments administered, thereby preserving a susceptible nematode population in refugia. In order to minimise the impact of withholding treatment on animal performance, animals that would benefit most should be selected for treatment. However, the most suitable criteria for selecting which animals to treat remain a subject of research. The impact of implementing a TST strategy based on lamb live weight was investigated, and whether heavy lambs were more resilient than light lambs if left untreated. The study was conducted using weaned lambs on 3 sheep farms, and over 2 years. On each farm lambs were weighed and divided into heavy, medium and light weight classes. Within the heavy (n = 225) and light (n = 218) weight classes lambs were randomly allocated to two treatments; anthelmintic treatment or no anthelmintic treatment. All lambs in the medium weight class were treated. Animal performance and parasitological parameters were assessed over a 28-day period. Anthelmintic treatment had a significant effect (P < 0.01) on faecal egg count, average daily weight gain, body condition score and dag score; there was no treatment by weight class interaction. Anthelmintic treatment had no effect on plasma pepsinogen concentration or on blood leukocyte numbers. Withholding anthelmintic treatment from lambs therefore had a negative effect on worm egg count and animal performance with no evidence that heavy lambs were more resilient than light lambs when left untreated.

Spatial patterns of Fasciola hepatica and Calicophoron daubneyi infections in ruminants in Ireland and modelling of C. daubneyi infection.

BACKGROUND: Fasciola hepatica has always represented a threat to Irish livestock because the Irish climate is highly suitable for the main local intermediate host of the parasite, the snail Galba truncatula. The recent clinical emergence of infections due to Calicophoron daubneyi has raised the question of whether the two parasites, which share a niche during part of their life-cycles, interact in some way. Here, we used geographical information systems (GIS) to analyse the distribution of both parasites in cattle and sheep. We also developed the first predictive model of paramphistomosis in Ireland. RESULTS: Our results indicated that, in cattle, liver fluke infection is less common than rumen fluke infection and does not exhibit the same seasonal fluctuations. Overall, we found that cattle had a higher likelihood of being infected with rumen fluke than sheep (OR = 3.134, P < 0.01). In addition, infection with one parasite increased the odds of infection with the other in both host species. Rumen fluke in cattle showed the highest spatial density of infection. Environmental variables such as soil drainage, land cover and habitat appeared to be the most important risk factors for C. daubneyi infection, followed by rainfall and vegetation. Overall the risk of infection with this parasite was predicted to be higher in the west of the country. CONCLUSIONS: This study shows differences between the infection rates and spatial patterns of bovine and ovine infections with F. hepatica and C. daubneyi in Ireland. Whether the reasons for this are due to susceptibility, exposure and/or management factors is yet to be determined. Furthermore, the rumen fluke model indicates distinct risk factors and predicted distribution to those of F. hepatica, suggesting potential biological differences between both parasite species.

Can the amount of digestible undegraded protein offered to ewes during late pregnancy affect the performance and immune response of their offspring to gastrointestinal nematodes?

Maternal nutrition during pregnancy is a major environmental influence on foetal development with consequent effects on postnatal performance. We hypothesised that the level of intake of digestible undegraded protein (DUP) by the dam in late pregnancy would impact on the effectiveness of the immune response by offspring to gastrointestinal nematode infection. Eighty-five twin/triplet-bearing ewes, which were indoors from mid-pregnancy, were randomly assigned to 4 treatment groups for the final 6 weeks of pregnancy. Treatments were silage plus one of two iso-energetic and iso-nitrogenous concentrates (differing in DUP concentration; 29 and 94g/kg DM) offered at one of two feed levels (18/30 and 24/35kg in total for twin/triplet-bearing ewes, respectively). Ewes with triplets had one lamb removed at birth so that all ewes nursed 2 lambs when put to pasture as one flock in a 5-paddock rotational grazing system; all lambs were slaughtered after 29 weeks. Faecal egg count (FEC) and levels of serum IgA and IgE specific for Teladorsagia circumcincta were assessed for all lambs at various time points between 10 weeks of age and slaughter. Animal performance (live weight, live-weight gain, carcass weight) was recorded for all lambs. Worm burden at slaughter was determined for a sample of 12 lambs from each treatment. Nematodirus spp. FEC, 'other strongyles' FEC, and serum IgA and IgE specific for T. circumcincta were unaffected either by the concentration of DUP in the concentrate or by the level of concentrate offered to ewes in late pregnancy (P>0.1). Likewise, the dietary regime of the dams had no effect on lamb performance (P>0.1). It is concluded that increasing the DUP intake of ewes in late pregnancy had no effect on the immune response of their offspring to gastrointestinal nematode infection acquired through grazing naturally infected pasture.

Can the amount of digestible undegraded protein offered to ewes in late pregnancy affect the periparturient change in resistance to gastrointestinal nematodes?

Ewes experience a temporary decline in resistance to gastrointestinal nematodes (GIN) during the periparturient period, characterised by a rise in faecal egg count (FEC) that represents a major source of pasture contamination for naïve progeny. The aim of this study was to assess the effect of level of supplementation with digestible undegraded protein (DUP) during the last 6 weeks of pregnancy on periparturient FEC and the performance of ewes with a naturally acquired parasite infection. Eighty-five Belclare and Belclare x Scottish Blackface twin/triplet-bearing ewes were allocated to 1 of 4 dietary groups representing the combination of 2 concentrates (DUP concentration 29 and 94g/kg dry matter) with 2 levels of concentrate during the final 6 weeks of gestation (18 and 30kg in total for ewes with twins; 24 and 35kg for ewes with triplets). All ewes were housed during the pre-partum feeding period and offered grass silage ad libitum; food intake was recorded daily. The intake of DUP varied from 26 to 72g/d among treatments and was reflected in variation of 0.76 to 1.20 in metabolizable protein supply as a proportion of requirements. After lambing, ewes and lambs grazed on permanent sheep pasture, without concentrate supplementation, until weaning (14 weeks post lambing). The variables studied, from week 6 pre-lambing up to week 10 post-lambing, included: FEC, serum pepsinogen concentration, body weight (BW) and body condition score (BCS). The effect of week (relative to lambing date) on FEC was highly significant (P<0.001). However, diet did not influence FEC (P>0.05) at any stage either pre- or post-partum. Pepsinogen concentration also varied with time but was not influenced by dietary treatment (P>0.05). The changes in BW and BCS from 6 weeks before lambing to weaning were not affected by the concentration of DUP in the supplement but ewes on treatments involving the higher level of supplementation lost less BW and BCS (P<0.001). The results of this study indicate that the level of DUP supplementation during the last 6 weeks of pregnancy does not affect FEC, BW or BCS of housed ewes with a naturally acquired GIN infection.

Transcriptional profiling of the ovine abomasal lymph node reveals a role for timing of the immune response in gastrointestinal nematode resistance.

Gastrointestinal nematodes are a serious cause of morbidity and mortality in grazing ruminants. The major ovine defence mechanism is acquired immunity, with protective immunity developing over time in response to infection. Nematode resistance varies both within and between breeds and is moderately heritable. A detailed understanding of the genes and mechanisms involved in protective immunity, and the factors that regulate this response, is required to aid both future breeding strategies and the development of effective and sustainable nematode control methods. The aim of this study was to compare the abomasal lymph node transcriptome of resistant and susceptible lambs in order to determine biological processes differentially expressed between resistant and susceptible individuals. Scottish Blackface lambs, with divergent phenotypes for resistance, were challenged with 30,000 Teladorsagia circumcincta larvae (L3), and abomasal lymph nodes recovered at 7 and 14days post-infection (dpi). High-throughput sequencing of cDNA from the abomasal lymph node was used to quantitatively sample the transcriptome with an average of 32 million reads per sample. A total of 194 and 144 genes were differentially expressed between resistant and susceptible lambs at 7 and 14 dpi respectively. Differentially expressed networks and biological processes were identified using Ingenuity Pathway Analysis. Genes involved in the inflammatory response, attraction of T lymphocytes and binding of leukocytes were more highly expressed in resistant animals at 7 dpi and in susceptible animals at 14 dpi indicating that resistant animals respond to infection earlier than susceptible animals. Twenty-four Single Nucleotide Polymorphisms (SNP) within 11 differentially expressed genes, were tested for association with gastrointestinal nematode resistance in the Scottish Blackface lambs. Four SNP, in 2 genes (SLC30A2 and ALB), were suggestively associated with faecal egg count. In conclusion, a large number of genes were differentially expressed in the abomasal lymph node of resistant and susceptible lambs responding to gastrointestinal nematode challenge. Resistant Scottish Blackface lambs appear to generate an earlier immune response to T. circumcincta. In susceptible lambs this response appears to be delayed. SNP in 2 differentially expressed genes were suggestively associated with faecal egg count indicating that differentially expressed genes may be considered candidate loci for mediating nematode resistance.