Development of an egg hatch assay for the diagnosis of triclabendazole resistance in Fasciola hepatica: proof of concept.

The aim of this study was to develop an Egg Hatch Assay (EHA) test for the detection of triclabendazole (TCBZ) resistance in Fasciola hepatica. A number of fluke isolates were used, of differing sensitivity to TCBZ. Eggs were exposed to solutions of triclabendazole sulphoxide (TCBZ.SO) for 14 days, then triggered to hatch. Egg development was divided into 6 distinct and easily identifiable stages: dead, empty, unembryonated, cell division, eye spot and hatched. The number of eggs reaching those stages was recorded. Initially, the discriminating dose (1% hatch) was determined for the Cullompton isolate, used as TCBZ-susceptible (TCBZ-S) standard. Once this concentration had been resolved, the response of different isolates to this concentration was examined. The hatch rate of the Fairhurst isolate was not significantly different from that of the Cullompton isolate, confirming its TCBZ-S status. The Patagonia isolate has not been exposed to TCBZ in the field and should be TCBZ-S: the results of the EHA supported this. The egg hatch response of the Oberon and Dutch isolates differed significantly from that of the Cullompton isolate; the former isolates are regarded as TCBZ-resistant (TCBZ-R) and the results confirmed this. Another isolate, the Leon isolate, was originally described as being TCBZ-R, but has since been shown to be TCBZ-S. There was no difference in its response to TCBZ.SO in the EHA from the Cullompton (and Fairhurst and Patagonia) isolate(s), further indicating its TCBZ-S status. The impact of TCBZ.SO treatment on the component stages of egg development was determined and revealed differences between the isolates. In conclusion, the results of the study have shown that it is possible to discriminate between TCBZ-S and TCBZ-R isolates of F. hepatica on the basis of the response of their eggs to an EHA and the test could be used to evaluate the TCBZ sensitivity of unknown field isolates

Survey of the anticoccidial feed additive nicarbazin (as dinitrocarbanilide residues) in poultry and eggs

A survey was carried out on the occurrence of dinitrocarbanilide (DNC), the marker residue for nicarbazin, in poultry produced in Ireland during 2002-2004. Liver (n = 736) and breast muscle samples (n = 342) were tested. DNC residues were found in 40 and 26% of liver and breast muscle samples at levels greater than 12.5 and 5 mu g kg(-1), respectively. DNC residues were found at >200 mu g kg(-1) in 12 and 0% of liver and muscle samples, respectively. Samples of breast muscle (n = 217) imported from 11 countries were also tested for DNC residues. A lower incidence of DNC residues (6%) was found in imported breast muscle. Egg samples (n = 546) were tested and DNC residues were found in nine samples, with levels ranging between 14 and 122 mu g kg(-1). Analysis of poultry, carried out as part of official food inspection in the period 2004-2006, indicated a reduction in the number of broiler liver samples containing DNC at >200 mu g kg(-1), to approximately 7%. Low levels of DNC residues continue to be found in

Enzyme immunoassay for semicarbazide - The nitrofuran metabolite and food contaminant

Semicarbazide (SEM), the marker residue for the banned nitrofuran veterinary antibiotic nitrofurazone (NFZ), has been detected regularly in foods (47% of recent nitrofuran EU Rapid Alerts involve SEM). However, the validity of SEM as a definitive marker for NFZ has been undermined by SEM arising from other sources including azodicarbonamide, a plastics blowing agent and flour treatment additive. An inexpensive screening test for SEM in food matrices is needed-all SEM testing currently uses expensive LC-MS/MS instrumentation. We now report the first production of antibodies against derivatised SEM. A novel carboxyphenyl SEM derivative was used to raise a polyclonal antibody that has been incorporated into a semi-quantitative microtitre plate ELISA, validated according to the criteria set out in Commission Decision 2002/657/EC, for use with chicken muscle. The antibody is highly specific for derivatised SEM, cross-reactivity being 1.7% with NFZ and negligible with a wide range of other nitrofurans and poultry drugs. Samples are derivatised with o-nitrobenzaldehyde and simultaneously protease digested before extraction by cation exchange SPE. The ELISA has a SEM detection capability (CC beta) of 0.25 mu g kg(-1) when a threshold of 0.21 mu g kg(-1) is applied to the selection of samples for confirmation (lowest observed 0.25 mu g kg(-1) fortified sample, n = 20), thus satisfying the EU nitrofurans' minimum required performance limit of 1 mu g kg(-1). N-FZ-incurred muscles (12) containing SEM at 0.5-5.0 mu g kg(-1) by LC-MS/MS, all screened positive by this ELISA protocol which is also applicable to egg and chicken liver. (C) 2007 Elsevier BN. All rights reserved.

An all-in-one dry chemistry immunoassay for the screening of coccidiostat nicarbazin in poultry eggs and liver

An automated immunoassay for the detection of nicarbazin residues in poultry eggs and liver was developed. The assay was based on a novel all-in-one dry chemistry concept and time-resolved fluorometry. The analyte specific antibody was immobilized into a single microtiter well and covered with an insulation layer, on top of which the label was dried in a small volume. The extracted sample was added automatically to the dry microtiter well, and the result was available within 18 min. Due to the rapidity and simplicity, the quantitative immunoassay could also be used as a high throughput screening method. The analytical limit of detection for the assay was calculated as 0.1 ng mL(-1) (n = 12) and the functional limit of detection as 3.2 ng g(-1) for egg (n = 6) and 11.3 ng g(-1) for liver (n = 6) samples. The sample recovery varied from 97.3 to 115.6%. Typically, the intra-assay variations were less than 10%, and interassay variations ranged between 8.1 and 13.6%.

COMPARATIVE ANALYSES OF THE NEUROPEPTIDE-F (NPF)-RELATED AND FMRFAMIDE-RELATED PEPTIDE (FARP)-IMMUNOREACTIVITIES IN FASCIOLA-HEPATICA AND SCHISTOSOMA SPP

Immunochemical techniques were used to determine the distribution, chemical characteristics and relative abundance of immunoreactivity (IR) to two native platyhelminth neuropeptides, neuropeptide F (NPF) (Moniezia expansa) and the FMRFamide-related peptide (FaRP), GNFFRFamide, in the trematodes, Fasciola hepatica and Schistosoma mansoni; the larger S. margrebowiei was used in the chemical analysis. Extensive immunostaining for the two peptides was demonstrated throughout the nervous systems of both F. hepatica and S. mansoni, with strong IR also in the innervation of muscular structures, including those associated with the egg-forming apparatus. The patterns of immunostaining were similar to those previously described for the vertebrate neuropeptide Y superfamily of peptides and for FMRFamide. Ultrastructurally, gold labelling of NPF- and GNFFRFamide-IRs was localized exclusively to the contents of secretory vesicles in the axons and somatic cytoplasm of neurones. Double-labelling experiments showed an apparent homogeneity of antigenic sites, in all probability due to the demonstrated cross-reactivity of the FaRP antiserum with NPF. Radioimmunoassay of acid-ethanol extracts of the worms detected 8.3 pmol/g and 4.7 pmol/g equivalents of NPF- and FMRFamide-IRs, respectively, for F. hepatica, and corresponding values of 4.9 pmol/g and 4.3 pmol/g equivalents for S. margrebowiei. Gel-permeation chromatography resolved IR to both peptides in discrete peaks and these eluted in similar positions to synthetic NPF (M. expansa) and GNFFRFamide, respectively.

Gastric emptying in patients with flatulent dyspepsia, with and without gallbladder disease

It has been suggested that the symptoms of flatulent dyspepsia are caused by a functional disturbance of the upper gastrointestinal tract. The aim of this study was to investigate delayed gastric emptying as the basis of symptoms in patients with and without gallbladder disease and after cholecystectomy. There were 13 dyspeptic patients with gallbladder disease, 12 with normal gallbladders, and 13 post-cholecystectomy patients. Gastric emptying was measured by means of a 99mTc-labelled scrambled egg meal and external scintillation counter. The rate of emptying in the symptomatic groups was compared with that in 24 asymptomatic normal control subjects and 12 non-dyspeptic patients with gallbladder disease. Delayed gastric emptying tended to occur in patients with gallbladder disease with and without dyspepsia and was not specifically associated with symptoms.

Role of Predalors, Habitat Attributes, and Spatial Autocorrelation on the Distribution of Eggs in the Northern Spectacled Salamander (Salamandrina perspicillata)

We predicted that the probability of egg occurrence of salamander Salamandrina perspicillata depended on stream features and predation by native crayfish Austropotamobius fulcisianus and the introduced trout Salmo trutta. We assessed the presence of S. perspicillata at 54 sites within a natural reserve of southern Tuscany, Italy. Generalized linear models with binomial errors were constructed using egg presence/absence and altitude, stream mean size and slope, electrical conductivity, water pH and temperature, and a predation factor, defined according to the presence/absence of crayfish and trout. Some competing models also included an autocovariate term, which estimated how much the response variable at any one sampling point reflected response values at surrounding points. The resulting models were compared using Akaike's information criterion. Model selection led to a subset of 14 models with Delta AIC(c) <7 (i.e., models ranging from substantial support to considerably less support), and all but one of these included an effect of predation. Models with the autocovariate term had considerably more support than those without the term. According to multimodel inference, the presence of trout and crayfish reduced the probability of egg occurrence from a mean level of 0.90 (SE limits: 0.98-0.55) to 0.12 (SE limits: 0.34-0.04). The presence of crayfish alone had no detectable effects (SE limits: 0.86-0.39). The results suggest that introduced trout have a detrimental effect on the reproductive output of S. perspicillata and confirm the fundamental importance of distinguishing the roles of endogenous and exogenous forces that act on population distribution.

The size and shape of shells used by hermit crabs: A multivariate analysis of Clibanarius erythropus

Shell attributes Such as weight and shape affect the reproduction, growth, predator avoidance and behaviour of several hermit crab species. Although the importance of these attributes has been extensively investigated, it is still difficult to assess the relative role of size and shape. Multivariate techniques allow concise and efficient quantitative analysis of these multidimensional properties, and this paper aims to understand their role in determining patterns of hermit crab shell use. To this end, a multivariate approach based on a combination of size-unconstrained (shape) PCA and RDA ordination was used to model the biometrics of southern Mediterranean Clibanarius erythropus Populations and their shells. Patterns of shell utilization and morphological gradients demonstrate that size is more important than shape, probably due to the limited availability of empty shells in the environment. The shape (e.g. the degree of shell elongation) and weight of inhabited shells vary considerably in both female and male crabs. However, these variations are clearly accounted for by crab biometrics in males only. Oil the basis of statistical evidence and findings from past studies. it is hypothesized that larger males of adequate size and strength have access to the larger, heavier and relatively more available shells of the globose Osilinus turbinatus, which cannot be used by average-sized males or by females investing energy in egg production. This greater availability allows larger males to select more Suitable Shapes. (C) 2009 Elsevier Masson SAS. All rights reserved.

The effects of climate change on ovine parasitic gastroenteritis determined using veterinary surveillance and meteorological data for Northern Ireland over the period 1999-2009

While the influence of temperature and moisture on the free-living stages of gastrointestinal nematodes have been described in detail, and evidence for global climate change is mounting, there have been only a few attempts to relate altered incidence or seasonal patterns of disease to climate change. Studies of this type have been completed for England Scotland and Wales, but not for Northern Ireland (NI). Here we present an analysis of veterinary diagnostic data that relates three categories of gastrointestinal nematode infection in sheep to historical meteorological data for NI. The infections are: trichostrongylosis/teladorsagiosis (Teladorsagia/Trichostrongylus), strongyloidosis and nematodirosis. This study aims to provide a baseline for future climate change analyses and to provide basic information for the development of nematode control programmes. After identifying and evaluating possible sources of bias, climate change was found to be the most likely explanation for the observed patterns of change in parasite epidemiology, although other hypotheses could not be refuted. Seasonal rates of diagnosis showed a uniform year-round distribution for Teladorsagia and Trichostrongylus infections, suggesting consistent levels of larval survival throughout the year and extension of the traditionally expected seasonal transmission windows. Nematodirosis showed a higher level of autumn than Spring infection, suggesting that suitable conditions for egg and larval development occurred after the Spring infection period. Differences between regions within the Province were shown for strongyloidosis, with peaks of infection falling in the period September-November. For all three-infection categories (trichostrongylosis/teladorsagiosis, strongyloidosis and nematodirosis), significant differences in the rates of diagnosis, and in the seasonality of disease, were identified between regions. (C) 2012 Elsevier B.V. All rights reserved.

Single-laboratory validation of a multiplex flow cytometric immunoassay for the simultaneous detection of coccidiostats in eggs and feed

Coccidiostats are authorized in the European Union (EU) to be used as poultry feed additives. Maximum (residue) levels (M(R)Ls) have been set within the EU for consumer and animal protection against unintended carry-over, and monitoring is compulsory. This paper describes the single-laboratory validation of a previously developed multiplex flow cytometric immunoassay (FCIA) as screening method for coccidiostats in eggs and feed and provides and compares different approaches for the calculation of the cut-off levels which are not described in detail within Commission Decision 2002/657/EC. Comparable results were obtained between the statistical (reference) approach and the rapid approaches. With the most rapid approach, the cut-off levels for narasin/salinomycin, lasalocid, diclazuril, nicarbazin (DNC) and monensin in egg, calculated as percentages of inhibition (%B/B0), were 60, 32, 76, 80 and 84, respectively. In feed, the cut-off levels for narasin/salinomycin, lasalocid, nicarbazin (DNC) and monensin were 70, 64, 72 and 78, respectively, and could not be determined for diclazuril. For all analytes, except for diclazuril in feed, the rate of false positives (false non-compliant) in blank samples was lower than 1 %, and the rate of false negatives (false compliant) at the M(R)Ls was below 5 %. Additionally, very good correlations (r ranging from 0.994 to 0.9994) were observed between two different analysers, a sophisticated flow cytometer (FlexMAP 3D(®)) and a more cost-efficient and transportable planar imaging detector (MAGPIX(®)), hence demonstrating adequate transferability.

Patterns of synchrony in natterjack toad breeding activity and reproductive success at local and regional scales

Empirical studies of the spatiotemporal dynamics of populations are required to better understand natural fluctuations in abundance and reproductive success, and to better target conservation and monitoring programmes. In particular, spatial synchrony in amphibian populations remains little studied. We used data from a comprehensive three year study of natterjack toad Bufo calamita populations breeding at 36 ponds to assess whether there was spatial synchrony in the toad breeding activity (start and length of breeding season, total number of egg strings) and reproductive success (premetamorphic survival and production of metamorphs). We defined a novel approach to assess the importance of short-term synchrony at both local and regional scales. The approach employs similarity indices and quantifies the interaction between the temporal and spatial components of populations using mixed effects models. There was no synchrony in the toad breeding activity and reproductive success at the local scale, suggesting that populations function as individual clusters independent of each other. Regional synchrony was apparent in the commencement and duration of the breeding season and in the number of egg strings laid (indicative of female population size). Regional synchrony in both rainfall and temperature are likely to explain the patterns observed (e.g. Moran effect). There was no evidence supporting regional synchrony in reproductive success, most likely due to spatial variability in the environmental conditions at the breeding ponds, and to differences in local population fitness (e.g. fecundity). The small scale asynchronous dynamics and regional synchronous dynamics in the number of breeding females indicate that it is best to monitor several populations within a subset of regions. Importantly, variations in the toad breeding activity and reproductive success are not synchronous, and it is thus important to consider them both when assessing the conservation status of pond-breeding amphibians. © 2012 The Authors. Ecography © 2012 Nordic Society Oikos.

The Fasciola hepatica genome: gene duplication and polymorphism reveals adaptation to the host environment and the capacity for rapid evolution

BACKGROUND: The liver fluke Fasciola hepatica is a major pathogen of livestock worldwide, causing huge economic losses to agriculture, as well as 2.4 million human infections annually.

RESULTS: Here we provide a draft genome for F. hepatica, which we find to be among the largest known pathogen genomes at 1.3 Gb. This size cannot be explained by genome duplication or expansion of a single repeat element, and remains a paradox given the burden it may impose on egg production necessary to transmit infection. Despite the potential for inbreeding by facultative self-fertilisation, substantial levels of polymorphism were found, which highlights the evolutionary potential for rapid adaptation to changes in host availability, climate change or to drug or vaccine interventions. Non-synonymous polymorphisms were elevated in genes shared with parasitic taxa, which may be particularly relevant for the ability of the parasite to adapt to a broad range of definitive mammalian and intermediate molluscan hosts. Large-scale transcriptional changes, particularly within expanded protease and tubulin families, were found as the parasite migrated from the gut, across the peritoneum and through the liver to mature in the bile ducts. We identify novel members of anti-oxidant and detoxification pathways and defined their differential expression through infection, which may explain the stage-specific efficacy of different anthelmintic drugs.

CONCLUSIONS: The genome analysis described here provides new insights into the evolution of this important pathogen, its adaptation to the host environment and external selection pressures. This analysis also provides a platform for research into novel drugs and vaccines.

Calmodulin disruption impacts growth and motility in juvenile liver fluke

Background: Deficiencies in effective flukicide options and growing issues with drug resistance make current strategies for liver fluke control unsustainable, thereby promoting the need to identify and validate new control targets in Fasciola spp. parasites. Calmodulins (CaMs) are small calcium-sensing proteins with ubiquitous expression in all eukaryotic organisms and generally use fluctuations in intracellular calcium levels to modulate cell signalling events. CaMs are essential for fundamental processes including the phosphorylation of protein kinases, gene transcription, calcium transport and smooth muscle contraction. In the blood fluke Schistosoma mansoni, calmodulins have been implicated in egg hatching, miracidial transformation and larval development. Previously, CaMs have been identified amongst liver fluke excretory-secretory products and three CaM-like proteins have been characterised biochemically from adult Fasciola hepatica, although their functions remain unknown.

Methods: In this study, we set out to investigate the biological function and control target potential of F. hepatica CaMs (FhCaMs) using RNAi methodology alongside novel in vitro bioassays.

Results: Our results reveal that: (i) FhCaMs are widely expressed in parenchymal cells throughout the forebody region of juvenile fluke; (ii) significant transcriptional knockdown of FhCaM1-3 was inducible by exposure to either long (~200 nt) double stranded (ds) RNAs or 27 nt short interfering (si) RNAs, although siRNAs were less effective than long dsRNAs; (iii) transient long dsRNA exposure-induced RNA interference (RNAi) of FhCaMs triggered transcript knockdown that persisted for ≥ 21 days, and led to detectable suppression of FhCaM proteins; (iv) FhCaM RNAi significantly reduced the growth of juvenile flukes maintained in vitro; (v) FhCaM RNAi juveniles also displayed hyperactivity encompassing significantly increased migration; (vi) both the reduced growth and increased motility phenotypes were recapitulated in juvenile fluke using the CaM inhibitor trifluoperazine hydrochloride, supporting phenotype specificity.

Conclusions: These data indicate that the Ca(2+)-modulating functions of FhCaMs are important for juvenile fluke growth and movement and provide the first functional genomics-based example of a growth-defect resulting from gene silencing in liver fluke. Whilst the phenotypic impacts of FhCaM silencing on fluke behaviour do not strongly support their candidature as new flukicide targets, the growth impacts encourage further consideration, especially in light of the speed of juvenile fluke growth in vivo.