Experimental infection of Black Belly and INRA 401 straight and crossbred sheep with trichostrongyle nematode parasites

Compared to INRA 401 lambs reared in France, Black Belly (BB) lambs reared in Guadeloupe (F.W.I.) were highly resistant to both primary and secondary experimental infection with Haemonchus contortus. To investigate this huge inter-breed difference, a nucleus flock of BB was constituted, and experiments were conducted to: (i) confirm this difference in lambs born in France, (ii) check whether it was similar for Trichostrongylus colubriformis and Teladorsagia circumcincta, and (iii) find out whether this difference was age-related. Forty BB lambs, 84 F1 lambs (BB siresxINRA 401 ewes) and 88 INRA 401 lambs born in two cohorts were used in an experimental design involving three host breeds, both genders and two age-groups (3.5- and 7-month-old when first infected). The limited availability of BB lambs made the study incomplete. Infection consisted of the administration of two doses of 10,000 infective larvae of one of the nematode species, separated by an anthelmintic treatment and an interval of 1 week before the second dose was administered. Fecal egg counts (FECs) were done on Days 28 and 35 after each infection; ewe lambs of the INRA 401 and F1 breeds were necropsied, the worm burden was established, the length of the female worms measured and the eggs in utero counted. For H. contortus and T. colubriformis, the FEC was lower in the BB than in the INRA 401 lambs, and the FEC found for the F1 lambs was intermediate. For T. circumcincta, only data for the F1 and INRA 401 lambs were available, and the FECs for these breeds were the same. In all three breeds, the FEC determined after the second dose was significantly lower than that found after the first dose in the ewe lambs, but not in the ram lambs. Infecting the lambs when they were 7- instead of 3.5-month-old significantly lowered egg excretion of both parasites in the F1 (P<0.0001), but not the INRA 401 lambs. Worm numbers in the F1 and in the INRA 401 ewe lambs confirmed the FEC data, many F1 lambs being free of T. colubriformis. The H. contortus female worms were shorter and had fewer eggs in utero in the F1 than in the INRA 401 lambs. A higher proportion of T. circumcincta was at the fourth larval stage in the F1 lambs. In conclusion, the BB breed is much more resistant than INRA 401 to H. contortus, with the F1 lambs being more like the BBs after the second dose. This difference was also found for T. colubriformis and, to a lesser extent, for T. circumcincta.     

Rating and mapping the suitability of the climate for pest risk analysis*

The assessment of the suitability of the climate for pest establishment is an important part of pest risk analysis (PRA). This paper describes the work undertaken by the EU 7th Framework project PRATIQUE (Enhancements of Pest Risk Analysis Techniques) to develop guidance for this component of PRA. Firstly, there is a guide to rating the suitability of the climate in the PRA area using qualitative methods. Secondly, a Decision‐support scheme (DSS) has been created to assist analysts in deciding whether to map climatic suitability, and to guide the selection of the most appropriate method from the large number available. The process of selecting a climatic mapping method is based on a review of the pest’s climatic responses and distribution. A spreadsheet provides a comparison of the potential problems that can arise, depending on the mapping method and on the amount and quality of available data. Diagrams are provided to help choose the location data category that best represents the possible biases in the known distribution of the pest. A second spreadsheet provides general information on the differences and similarities of each method in terms of categories such as functionality, ease of use and quality assurance. A variety of data, tools and supporting documents are available as appendices to the DSS. All of the tools and guides are freely available online.     

Development of a real-time PCR method for the differential detection and quantification of four solanaceae in GMO analysis: potato (Solanum tuberosum), tomato (Solanum lycopersicum), eggplant (Solanum melongena), and pepper (Capsicum annuum)

The labeling of products containing genetically modified organisms (GMO) is linked to their quantification since a threshold for the presence of fortuitous GMOs in food has been established. This threshold is calculated from a combination of two absolute quantification values: one for the specific GMO target and the second for an endogenous reference gene specific to the taxon. Thus, the development of reliable methods to quantify GMOs using endogenous reference genes in complex matrixes such as food and feed is needed. Plant identification can be difficult in the case of closely related taxa, which moreover are subject to introgression events. Based on the homology of beta-fructosidase sequences obtained from public databases, two couples of consensus primers were designed for the detection, quantification, and differentiation of four Solanaceae: potato (Solanum tuberosum), tomato (Solanum lycopersicum), pepper (Capsicum annuum), and eggplant (Solanum melongena). Sequence variability was studied first using lines and cultivars (intraspecies sequence variability), then using taxa involved in gene introgressions, and finally, using taxonomically close taxa (interspecies sequence variability). This study allowed us to design four highly specific TaqMan-MGB probes. A duplex real time PCR assay was developed for simultaneous quantification of tomato and potato. For eggplant and pepper, only simplex real time PCR tests were developed. The results demonstrated the high specificity and sensitivity of the assays. We therefore conclude that beta-fructosidase can be used as an endogenous reference gene for GMO analysis.     

Improved estimation of trawling tracks using cubic Hermite spline interpolation of position registration data

For control and enforcement purposes, all fishing vessels operating in European waters are equipped with satellite-based Vessel Monitoring by Satellite systems (VMS) recording their position at regular time intervals. VMS data are increasingly used by scientists to study spatial and temporal patterns of fishing activity and thus fishing impact (e.g. surface of sea bed trawled during a fishing trip). However, due to their low resolution (2 h basis), these data may provide a biased perception of fishing impact. We present here a method aiming at interpolating vessel trajectories from VMS data points to obtain higher-resolution data on vessel trajectories which in turn should provide improved estimates of the spatial and temporal patterns of fishing activity and hence fishing impact. This method is based on a spline interpolation technique, the cubic Hermite spline (cHs), using position, heading and speed to interpolate the trawl track of a vessel between two succeeding VMS data points. To take uncertainty of the interpolated track into account, the method also determines a confidence interval, which represents the spatial distribution of vessel presence probability between two successive VMS positions. The cHs method was compared to the straight line interpolation technique using a reference data set with intervals of 6 min which was assumed to represent the real trawl tracks. The results showed that the cHs method approximates the real trawl track markedly better than a straight line interpolation. The cHs method should therefore be preferred to the conventional straight line approach to interpolate vessel tracks in studies aiming at estimating fishing impact from VMS data.     

Haemonchus contortus (Nematoda: Trichostrongylidae) infection in lambs elicits an unequivocal Th2 immune response

Selection of resistant animals and host immunization have been proposed as alternative methods for the control of gastrointestinal nematode parasites. However, a better knowledge of the mechanisms involved in protective immunity against these parasites is required for the development of optimal strategies. In this study, 3 month old INRA 401 lambs (n = 81) were allocated into three groups: uninfected control, challenged either once (primary-infected animals) or twice (previously-infected animals) with 10,000 Haemonchus contortus L3. Uninfected control and challenged animals were sequentially sacrificed at 3, 7, 15 and 28 days post challenge. In both challenged groups, a clear Th2-oriented immune response was observed in the abomasal lymph node and mucosa. IL-4 and IL-13 mRNA over-expression, recruitment of eosinophils, mast cells and globule leukocytes and production of specific systemic IgG and mucosal IgA were observed earlier in previously-infected animals than in primary-infected ones. At 28 days post infection, no differences between intensities of these responses were observed between the challenged groups. Worm establishment rates were similar in previously-infected and primary-infected lambs. However, reductions of worm development, female fecundity and fecal egg output were observed in previously-infected sheep. We conclude that H. contortus infection in young INRA 401 lambs induced an unequivocal Th2 immune response resulting in the regulation of worm egg production without affecting their establishment.     

A decision‐support scheme for mapping endangered areas in pest risk analysis*

This paper describes a decision‐support scheme (DSS) for mapping the area where economically important loss is likely to occur (the endangered area). It has been designed by the PRATIQUE project to help pest risk analysts address the numerous risk mapping challenges and decide on the most suitable methods to follow. The introduction to the DSS indicates the time and expertise that is needed, the data requirements and the situations when mapping the endangered areas is most useful. The DSS itself has four stages. In stage 1, the key factors that influence the endangered area are identified, the data are assembled and, where appropriate, maps of the key factors are produced listing any significant assumptions. In stage 2, methods for combining these maps to identify the area of potential establishment and the area at highest risk from pest impacts are described, documenting any assumptions and combination rules utilised. When possible and appropriate, Stage 3 can then be followed to show whether economic loss will occur in the area at highest risk and to identify the endangered area. As required, Stage 4, described elsewhere, provides techniques for producing a dynamic picture of the invasion process using a suite of spread models. To illustrate how the DSS functions, a maize pest, Diabrotica virgifera virgifera, and a freshwater invasive alien plant, Eichhornia crassipes, have been used as examples.     

Consistency in pest risk analysis – how can it be achieved and what are the benefits?*

This paper describes the efforts in the PRATIQUE EU project to increase consistency that were integrated into the revised EPPO Decision‐support scheme (DSS) for Pest Risk Analysis (PRA). The establishment section is used here to illustrate the changes and improvements of the DSS, as it has undergone the most comprehensive changes with reformulation and restructuring of many questions and detailed rating guidance. When revising the establishment section of the EPPO DSS for PRA, there were three principal objectives that influenced the revision: (i) to focus the assessment on the most relevant factors influencing establishment; (ii) to identify the area where establishment is possible before assessing the suitability of that area, and (iii) to enhance the consistency of the establishment section by providing rating guidance with sub‐questions and examples. To simplify the process, some questions were rephrased as closed questions (answer is either yes or no). Clearer notes were provided to explain what information is needed to answer the questions. Furthermore, consistency has been enhanced by developing tools to help assessors when deciding on an appropriate overall risk rating and uncertainty score by providing a visual summary of the risk ratings and uncertainty scores (‘Visualizer’) and a method for integrating all the responses to the questions to provide a summary score for each major section and an overall assessment of risk (‘Rule based matrix model’).     

Evolution of nematode community in grazing sheep selected for resistance and susceptibility to Teladorsagia circumcincta and Trichostrongylus colubriformis: a 4-year experiment

Field trials were undertaken to compare nematode population dynamics in two experimental flocks of rams selected for high and for low faecal egg counts (FECs) following two doses with 20000 infective larvae, 12000 Teladorsagia circumcincta (Tcirc) and 8000 Trichostrongylus colubriformis (Tcol) separated by a treatment. The selection was done by dosing 200 ram lambs (INRA 401 breed) and FECs proceeded with egg identification on days 28 and 35 post-each-infection. The 30 lambs with the lowest FECs constituted the group of permanent resistant rams (R), the 30 with the highest values the group of susceptible ones (S). Each group grazed separated pastures from April to November, this during 4 consecutive years. The contamination (number of eggs deposited) was estimated every 3 weeks on each pasture. The larval populations was measured by successive groups of tracer lambs. The infection of the permanent rams was done by slaughtering eight R and S rams on mid and end of each grazing season. Each autumn, new R and S ram lambs were selected to complete the R and S groups before turning out in spring. On third year, a third species (Haemonchus contortus, Hcon) was added to compare its behaviour with the two others.The regulation of parasite populations were studied by comparing what happens in R rams and pasture to S ones. The selected R ram lambs had a FEC of Tcirc 50% lower than S ones. This induced a similar lowered pasture contamination, a 25% decrease of infective larval population in years 2 and 3, and the worm burdens in R rams were the half of that in S rams after 2 years. The FEC of Tcol was 75% lower at the selection and induced similar difference in pasture contamination, but 65 and 96% reduction in L3 population, respectively, after 1 and 2 years. The worm burden was reduced by 99%. Adding Hcon species during the third year, in year 4, the L3 population was half in R pasture compared with S one, and R rams had 60% less worms of this species than S ones.This selection without any drench prevent outbreaks observed in young tracer lambs on S pasture. The comparison of selected resistant rams to selected susceptible ones and not to unselected controls gave more rapid information about the tendency of the regulation of the communities of nematode parasites.