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

Resistance to crown gall disease in transgenic grapevine rootstocks containing truncated virE2 of Agrobacterium

A truncated form of the Ti-plasmid virE2 gene from Agrobacterium tumefaciens strains C58 and A6, and A. vitis strain CG450 was transferred and expressed in somatic embryos of grapevine rootstocks 110 Richter (Vitis rupestris×V. berlandieri), 3309 Couderc (V. rupestris×V. riparia) and Teleki 5C (V. berlandieri×V. riparia) via Agrobacterium-mediated transformation to confer resistance to crown gall disease. Transformation was confirmed in 98% of the 322 lines by enzyme-linked immunosorbent assay for the neomycin phosphotransferase II protein and 97% of 295 lines by polymerase chain reaction for the truncated virE2 transgene. Southern blot analysis revealed the insertion of truncated virE2 at one to three loci in a subset of seven transgenic 110 Richter lines. In vitro resistance screening assays based on inoculations of shoot internode sections showed reduced tumorigenicity and very small galls in 23 of 154 transgenic lines. Non-transformed controls had a 100% tumorigenicity rate with very large galls. Disease resistance assay at the whole plant level in the greenhouse revealed seven transgenic lines (3 lines of 110 Richter, 2 lines of 3309 Couderc and 2 lines of Teleki 5C) were resistant to A. tumefaciens strain C58 and A. vitis strains TM4 and CG450 with a substantially reduced percentage of inoculation sites showing gall as compared to controls. No association was found between the level of resistance to crown gall disease and the source Agrobacterium strain of virE2. Taken together, our data showed that resistance to crown gall disease can be achieved by expressing a truncated form of virE2 in grapevine

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

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

Characteristics of Stem Cells Derived from the Degenerated Human Intervertebral Disc Cartilage Endplate

Mesenchymal stem cells (MSCs) derived from adult tissues are an important candidate for cell-based therapies and regenerative medicine due to their multipotential differentiation capability. MSCs have been identified in many adult tissues but have not reported in the human intervertebral disc cartilage endplate (CEP). The initial purpose of this study was to determine whether MSCs exist in the degenerated human CEP. Next, the morphology, proliferation capacity, cell cycle, cell surface epitope profile and differentiation capacity of these CEP-derived stem cells (CESCs) were compared with bone-marrow MSCs (BM-MSCs). Lastly, whether CESCs are a suitable candidate for BM-MSCs was evaluated. Isolated cells from degenerated human CEP were seeded in an agarose suspension culture system to screen the proliferative cell clusters. Cell clusters were chosen and expanded in vitro and were compared with BM-MSCs derived from the same patient. The morphology, proliferation rate, cell cycle, immunophenotype and stem cell gene expression of the CESCs were similar to BM-MSCs. In addition, the CESCs could be induced into osteoblasts, adipocytes, chondrocytes, and are superior to BM-MSCs in terms of osteogenesis and chondrogenesis. This study is first to demonstrate the presence of stem cells in the human degenerated CEP. These results may improve our understanding of intervertebral disc (IVD) pathophysiology and the degeneration process, and could provide cell candidates for cell-based regenerative medicine and tissue engineering

Bulk milk ELISA and the diagnosis of parasite infections in dairy herds: A review

The bulk milk enzyme-linked immune sorbent assay (ELISA) is a rapid and inexpensive method of assessing herd exposure to pathogens that is increasingly being used for the diagnosis of parasite infections in dairy herds. In this paper, with the dairy herd health veterinarian in mind, we review the principles of the assay and the recent literature on the potential role of bulk milk ELISA for the diagnosis of ostertagiosis, fasciolosis, parasitic bronchitis due to cattle lung worm and neosporosis. It is generally accepted that assay results reflect exposure to the parasite rather than the presence of active infection. Bulk milk ELISA can be a useful tool for the veterinary practitioner as a component of a herd health monitoring programme or in the context of a herd health investigation. It can also play a role in regional or national surveillance programmes. However, the results need to be interpreted within the context of the herd-specific health management, the milk production pattern and the parasite life cycle

Thioredoxin Peroxidase Secreted by Fasciola hepatica Induces the Alternative Activation of Macrophages

Alternatively activated macrophages (AAMφ) are primarily associated with the chronic stages of parasitic infections and the development of a polarized Th2 response. We have shown that Fasciola hepatica infection of BALB/c mice induces a polarized Th2 response during both the latent and chronic stage of disease. The activation status of macrophages was analyzed in this model of helminth infection by evaluating the expression of genetic markers of alternative activation, namely, Fizz1, Ym1, and Arg1. AAMφ were recruited to the peritoneum of mice within 24 h of F. hepatica infection and after intraperitoneal injection of parasite excretory-secretory (ES) products. Administration of a recombinant antioxidant thioredoxin peroxidase (TPx), which is contained within the ES products, also induced the recruitment of AAMφ to the peritoneum. In vitro studies showed that this recombinant TPx directly converts RAW 264.7 macrophages to an alternatively activated phenotype characterized by the production of high levels of interleukin-10 (IL-10), prostaglandin E(2), corresponding with low levels of IL-12. Our data suggest that the Th2 responses induced by the helminth F. hepatica are mediated through the secretion of molecules, one of which is TPx, that induce the recruitment and alternative activation of macrophages

Biochemical characterisation of the recombinant peroxiredoxin (FhePrx) of the liver fluke, Fasciola hepatica

The parasitic helminth Fasciola hepatica secretes a 2-Cys peroxiredoxin (Prx) that may play important functions in host-parasite interaction. Recombinant peroxiredoxin (FhePrx) prevented metal-catalyzed oxidative nicking of plasmid DNA and detoxified hydrogen peroxide when coupled with Escherichia coli thioredoxin and thioredoxin reductase (kcat/Km = 5.2 × 105 M-1 s-1). Enzyme kinetic analysis revealed that the catalytic efficiency of FhePrx is similar to other 2-Cys peroxiredoxins; the enzyme displayed saturable enzyme Michaelis-Menten type kinetics with hydrogen peroxide, cumene hydroperoxide and t-butyl hydroperoxide, and is sensitive to concentrations of hydrogen peroxide above 0.5 mM. Like the 2-Cys peroxiredoxins from a related helminth, Schistosoma mansoni, steady-state kinetics indicate that FhePrx exhibits a saturable, single displacement-like reaction mechanism rather than non-saturable double displacement (ping-pong) enzyme substitution mechanism common to other peroxiredoxins. However, unlike the schistosome Prxs, FhePrx could not utilise reducing equivalents supplied by glutathione or glutathione reductase. © 2006 Federation of European Biochemical Societies