Rumen fluke in Irish sheep: prevalence, risk factors and molecular identification of two paramphistome species

peer-reviewedBackground Rumen flukes are trematode parasites found globally; in tropical and sub-tropical climates, infection can result in paramphistomosis, which can have a deleterious impact on livestock. In Europe, rumen fluke is not regarded as a clinically significant parasite, recently however, the prevalence of rumen fluke has sharply increased and several outbreaks of clinical paramphistomosis have been reported. Gaining a better understanding of rumen fluke transmission and identification of risk factors is crucial to improve the control of this parasitic disease. In this regard, a national prevalence study of rumen fluke infection and an investigation of associated risk factors were conducted in Irish sheep flocks between November 2014 and January 2015. In addition, a molecular identification of the rumen fluke species present in Ireland was carried out using an isolation method of individual eggs from faecal material coupled with a PCR. After the DNA extraction of 54 individual eggs, the nuclear fragment ITS-2 was amplified and sequenced using the same primers. Results An apparent herd prevalence of 77.3 % was determined. Several risk factors were identified including type of pasture grazed, regional variation, and sharing of the paddocks with other livestock species. A novel relationship between the Suffolk breed and higher FEC was reported for the first time. The predominant rumen fluke species found was C. daubneyi. Nevertheless, P. leydeni was unexpectedly identified infecting sheep in Ireland for the first time. Conclusions An exceptionally high prevalence of rumen fluke among Irish sheep flocks has been highlighted in this study and a more thorough investigation is necessary to analyse its economic impact. The isolation of individual eggs coupled with the PCR technique used here has proven a reliable tool for discrimination of Paramphistomum spp. This technique may facilitate forthcoming studies of the effects of paramphistomosis on livestock production. The most noteworthy finding was the identification of P. leydeni affecting sheep in Ireland, however further studies are required to clarify its implications. Also, a significant relationship between Suffolk breed and a heavier infection was found, which can be used as a starting point for future research on control strategies of rumen fluke infection.This study was funded by Irish Department of Agriculture, Food and the Marine research stimulus funding; project reference 13/ S/405

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

This study was financed by both the Irish dairy levy and Merial.peer-reviewedBackground 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.MerialIrish Dairy Lev

Gastrointestinal nematode control practices on lowland sheep farms in Ireland with reference to selection for anthelmintic resistance

Gastrointestinal parasitism is a widely recognised problem in sheep production, particularly for lambs. While anthelmintics have a pivotal role in controlling the effects of parasites, there is a paucity of data on how farmers use anthelmintics. A representative sample of Irish lowland farmers were surveyed regarding their parasite control practices and risk factors that may contribute to the development of anthelmintic resistance. Questionnaires were distributed to 166 lowland Irish sheep producers. The vast majority of respondents treated their sheep with anthelmintics. Lambs were the cohort treated most frequently, the majority of farmers followed a set programme as opposed to treating at sign of disease. A substantial proportion (61%) administered four or more treatments to lambs in a 'normal' year. Departures from best practice in anthelmintic administration that would encourage the development of anthelmintic resistance were observed. In conclusion, in the light of anthelmintic resistance, there is a need for a greater awareness of the principles that underpin the sustainable use of anthelmintics and practices that preserve anthelmintic efficacy should be given a very high priority in the design of helminth control programmes on each farm. To this end, given that veterinary practitioners and agricultural advisors were considered to be the farmer's most popular information resource, the capacity of these professions to communicate information relating to best practice in parasite control should be targeted

Comparison of four commercially available ELISA kits for diagnosis of Fasciola hepatica in Irish cattle

peer-reviewedBackground Fasciola hepatica is a liver parasite of mammals and it results in poor welfare outcomes and economic losses in ruminants. While faecal egg count is the test most commonly used for diagnosis, it does not indicate presence of migrating immature stages. Serological techniques increase sensitivity at all stages of the liver fluke infection. The aim of this study was to compare four commercially available ELISA tests for the diagnosis of F. hepatica. For this purpose, we tested three sample types; (i) known F. hepatica status sera from an experimental infection for the comparison of sensitivities and specificities, (ii) sera from pre- and post-flukicide-treated (albendazole, closantel, nitroxynil and triclabendazole) beef cattle to contrast the differences of seropositivity before and after treatment, and (iii) bulk tank milk samples from dairy herds sampled during high and low F. hepatica exposure periods for assessing seasonal variations with the four tests available. Samples were tested using ELISA kits supplied by four manufacturers (Ildana Biotech, IDEXX, Svanova, and Bio-X). Samples were analysed simultaneously and in duplicate. Results In the control population Ildana, IDEXX and Bio-X presented 100% sensitivity (Se) and specificity (Sp), Svanovir presented a Se of 59% and a Sp of 96%. In flukicide-treated beef cattle, kits highlighted decreasing antibody levels 90 days post-treatment in variable degrees. Finally, bulk milk showed a significant decrease in ELISA value between high and low fluke exposure periods with all tests studied. Conclusions Se and Sp found in the present study, confirm that Ildana, IDEXX and Bio-X are accurate for the detection of F. hepatica exposure in Irish cattle. Svanovir Se and Sp in this population, indicate that a larger study is necessary to confirm this test characteristic in Irish herds. In post-treatment use, Bio-X showed a consistent and significant decrease of ELISA value in all groups treated, denoting to be a reliable tool for assessing treatment effect at 90 days post-treatment. Finally, all tests showed to be a reliable tool for the F. hepatica monitoring of high and low exposure seasons, using bulk tank milk samples

Tegument Glycoproteins and Cathepsins of Newly Excysted Juvenile Fasciola hepatica Carry Mannosidic and Paucimannosidic N-glycans

Recently, the prevalence of Fasciola hepatica in some areas has increased considerably and the availability of a vaccine to protect livestock from infection would represent a major advance in tools available for controlling this disease. To date, most vaccine-target discovery research on this parasite has concentrated on proteomic and transcriptomic approaches whereas little work has been carried out on glycosylation. As the F. hepatica tegument (Teg) may contain glycans potentially relevant to vaccine development and the Newly Excysted Juvenile (NEJ) is the first lifecycle stage in contact with the definitive host, our work has focused on assessing the glycosylation of the NEJTeg and identifying the NEJTeg glycoprotein repertoire. After in vitro excystation, NEJ were fixed and NEJTeg was extracted. Matrix-assisted laser desorption ionisation-time of flight-mass spectrometry (MALDI-TOF-MS) analysis of released N-glycans revealed that oligomannose and core-fucosylated truncated N-glycans were the most dominant glycan types. By lectin binding studies these glycans were identified mainly on the NEJ surface, together with the oral and ventral suckers. NEJTeg glycoproteins were affinity purified after targeted biotinylation of the glycans and identified using liquid chromatography and tandem mass spectrometry (LC-MS/MS). From the total set of proteins previously identified in NEJTeg, eighteen were also detected in the glycosylated fraction, including the F. hepatica Cathepsin B3 (FhCB3) and two of the Cathepsin L3 (FhCL3) proteins, among others. To confirm glycosylation of cathepsins, analysis at the glycopeptide level by LC-ESI-ion-trap-MS/MS with collision-induced dissociation (CID) and electron-transfer dissociation (ETD) was carried out. We established that cathepsin B1 (FhCB1) on position N80, and FhCL3 (BN1106_s10139B000014, scaffold10139) on position N153, carry unusual paucimannosidic Man2GlcNAc2 glycans. To our knowledge, this is the first description of F. hepatica NEJ glycosylation and the first report of N-glycosylation of F. hepatica cathepsins. The significance of these findings for immunological studies and vaccine development is discussed

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

peer-reviewedBackground 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.This work was supported by the European Union Horizon 2020 programme (PARAGONE: vaccines for animal parasites. H2020-EU.3.2. SOCIETAL CHALLENGES, under grant agreement No 635408), who provided funding for training and software access used in the analysis. The Department of Agriculture Food and the Marine - Research Stimulus Fund though the Flukeless Research Project (under project no 13/S/405), provided data employed in the modelling process

Removal of adult cyathostomins alters faecal microbiota and promotes an inflammatory phenotype in horses

peer-reviewedThe interactions between parasitic helminths and gut microbiota are considered to be an important, although as yet incompletely understood, factor in the regulation of immunity, inflammation and a range of diseases. Infection with intestinal helminths is ubiquitous in grazing horses, with cyathostomins (about 50 species of which are recorded) predominating. Consequences of infection include both chronic effects, and an acute inflammatory syndrome, acute larval cyathostominosis, which sometimes follows removal of adult helminths by administration of anthelmintic drugs. The presence of cyathostomins as a resident helminth population of the equine gut (the “helminthome”) provides an opportunity to investigate the effect helminth infection, and its perturbation, has on both the immune system and bacterial microbiome of the gut, as well as to determine the specific mechanisms of pathophysiology involved in equine acute larval cyathostominosis. We studied changes in the faecal microbiota of two groups of horses following treatment with anthelmintics (fenbendazole or moxidectin). We found decreases in both alpha diversity and beta diversity of the faecal microbiota at Day 7 post-treatment, which were reversed by Day 14. These changes were accompanied by increases in inflammatory biomarkers. The general pattern of faecal microbiota detected was similar to that seen in the relatively few equine gut microbiome studies reported to date. We conclude that interplay between resident cyathostomin populations and the bacterial microbiota of the equine large intestine is important in maintaining homeostasis and that disturbance of this ecology can lead to gut dysbiosis and play a role in the aetiology of inflammatory conditions in the horse, including acute larval cyathostominosis

A Multiomic Approach to Investigate the Effects of a Weight Loss Program on the Intestinal Health of Overweight Horses

Obesity is endemic in human populations in the western society, and with mounting evidence that the intestinal ecological environment plays a major role in its pathogenesis, identification of therapies based on intestinal microbiota modulation are gaining attention. Obesity in companion animals is also a common clinical problem. We set out using a multidimensional approach, to determine the effectiveness and safety of a weight loss program for horses incorporating diet restriction and exercise. In addition, we aimed to investigate the effect of this program on the overall intestinal health of overweight sedentary horses. The investigation comprised of a randomized, controlled, 6-week study of 14 overweight sedentary horses and ponies who were blocked for age, gender, and breed (controls n = 7, treatment n = 7). The treatment group were fed a restricted diet (1.4% of body weight dry matter intake) and the control group a maintenance diet (2% of body weight as dry matter intake) over the study period. The treatment group were subjected to a prescribed exercise regime, while the control group were exercised to mimic foraging conditions. Several clinical measurements were taken at the start and end of the study, including morphological parameters, ultrasound measurements of subcutaneous fat, and blood pressure. Fecal microbiota analysis was performed using 16S rRNA gene sequence analysis, and fecal metabolome was analyzed using NMR spectroscopy, on samples taken at weeks 1, 3, and 6 of the study. All horses completed the study period successfully. However, two of the treatment group had to have modified exercise regimes. The treatment group showed significant weight loss (p &lt; 0.00001) and an associated decrease in waste circumference (p &lt; 0.0001) when compared with the control group. The alpha-diversity of the fecal microbiota in the treatment group showed a significant increase from the start to the end of the study period (p &lt; 0.05); however, there was no significant difference between groups at any sampling point. There were significant changes (p &lt; 0.05) in the metabolome in both groups between the start and end of the study, but not between groups at any sampling point. Finally, the resting blood pressure of all horses was significantly lower by the end of the study

Liver fluke in Irish sheep: prevalence and associations with management practices and co-infection with rumen fluke

peer-reviewedBackground: The present study aimed to identify the national prevalence of Fasciola hepatica in Irish sheep and to conduct a risk analysis assessment based on management and treatment practices in participating focks. Also, co-infection with rumen fuke was quantifed and its association with liver fuke and management practices was assessed. Methods: A total of 305 sheep focks were selected ensuring even national representation of the sheep population. Participating farms were asked to complete a survey questionnaire on farm management practices and submit faecal samples during the winter of 2014–2015. Pooled faecal samples were analysed for the presence of F. hepatica and coinfection with rumen fuke. Apparent and true prevalence were calculated, additionally, the rate of co-infection with rumen fuke was also obtained. Correlation and regression analyses were used for assessing associations between management practices, liver fuke infection and co-infection with rumen fuke. Results: The national true prevalence of F. hepatica was 50.4% (n=305). Regional prevalence varied from 41% in the east to 52% in the south. Co-infection with rumen fuke was observed in 40% of the studied population and corre‑ lated with increased F. hepatica egg counts (OR=2.9; P≤0.001). Predominant breeds were Sufolk, Texel and Horned Mountain breeds. Beef cattle were the most frequent type of other livestock present on farms and mixed species grazing was frequently reported (73%). More than half of the focks reported a mid-to-late lambing period (MarchApril). Use of mountain land for grazing was of 32%. Flukicides were most commonly used twice over the autumnwinter period. Regression analyses highlighted signifcant association of F. hepatica status, with the presence of other livestock on farm, frequency of fukicides used during the winter and clinical presentation of liver fuke. A signifcant increase in eggs per gram of faeces was observed in Charollais sheep in comparison with all other breeds. Co-infec‑ tion with F. hepatica and Calicophoron daubneyi was also signifcantly associated with the presence of other livestock on the farm, type of fukicide used and clinical fasciolosis. Conclusions: The present study provides up-to-date information on the prevalence of F. hepatica in Irish sheep and adds insight to the epidemiology of the disease. These fndings will be useful for designing new holistic control meas‑ ures for F. hepatica infection

Validation of a serum ELISA test for cyathostomin infection in equines

Cyathostomins are ubiquitous equine nematodes. Infection can result in larval cyathostominosis due to mass larval emergence. Although faecal egg count (FEC) tests provide estimates of egg shedding, these correlate poorly with burden and provide no information on mucosal/luminal larvae. Previous studies describe a serum IgG(T)-based ELISA (CT3) that exhibits utility for detection of mucosal/luminal cyathostomins. Here, this ELISA is optimised/validated for commercial application using sera from horses for which burden data were available. Optimisation included addition of total IgG-based calibrators to provide standard curves for quantification of antigen-specific IgG(T) used to generate a CT3-specific 'serum score' for each horse. Validation dataset results were then used to assess the optimised test's performance and select serum score cut-off values for diagnosis of burdens above 1,000, 5,000 and 10,000 cyathostomins. The test demonstrated excellent performance (Receiver Operating Characteristic Area Under the Curve values >0.9) in diagnosing infection, with >90% sensitivity and >70% specificity at the selected serum score cut-off values. CT3-specific serum IgG(T) profiles in equines in different settings were assessed to provide information for commercial test use. These studies demonstrated maternal transfer of CT3-specific IgG(T) in colostrum to newborns, levels of which declined before increasing as foals consumed contaminated pasture. Studies in geographically distinct populations demonstrated that the proportion of horses that reported as test positive at a 14.37 CT3 serum score (1,000-cyathostomin threshold) was associated with parasite transmission risk. Based on the results, inclusion criteria for commercial use were developed. Logistic regression models were developed to predict probabilities that burdens of individuals are above defined thresholds based on the reported serum score. The models performed at a similar level to the serum score cut-off approach. In conclusion, the CT3 test provides an option for veterinarians to obtain evidence of low cyathostomin burdens that do not require anthelmintic treatment and to support diagnosis of infection