Christine Sauvé

Relationship between helminth species diversity, intensity of infection and breeding management in dairy goats

Helminth infection, species diversity (proportion of each species in the community), species number, intensity of infection and anthelmintic resistance were investigated in 16 dairy-goat farms of south-western France. The aim of the study was to estimate which breeding management factors may affect diversity and intensity of infection. Anthelmintic resistance was investigated because this factor may modify composition of nematode communities. Farm history and breeding management were recorded by means of a questionnaire. Natural and experimental infections were compared to determine which strategy was the best to estimate diversity index and number of species from nematode community of a whole herd. A total of 17 species of helminths, among which 14 nematodes, one cestode (Moniezia spp.) and two trematodes (Paramphistomum daubneyi and Dicrocoelium lanceolatum) were recovered in the 26 necropsied culled goats. One culled goat worm burden represented conveniently species number but not species diversity (Shannon index) of parasite community harboured in the whole herd. Experimental sheep infection with larvae from pooled faeces of 15 goats was the best strategy to estimate species diversity in farms. Diversity could be predicted from the number of farms from which goats originated at constitution of the herd, the duration of goat winter withdrawal from pastures, and intensity of infection. Anthelmintic resistance was correlated with the number of farms of origin, area of permanent pastures and intensity of infection. The strategy adopted for the herd constitution was a critical step as species diversity and risk of anthelmintic resistance increased with the number of farms of origin. Intensity of infection was negatively correlated with species diversity. This suggested that more diverse communities were better controlled by the host, in agreement with the widespread statement that diversity would beget stability.

Experimental and modeling approaches to evaluate different aspects of the efficacy of Targeted Selective Treatment of anthelmintics against sheep parasite nematodes.

Maintaining a refugia i.e. a proportion of the population that is not exposed to selection by treatments has been suggested as an alternative to mass treatment in the management of nematode parasites of sheep. Three refugia have been identified: nematodes in untreated hosts, encysted larvae and free-living stages on pastures. Here we tested whether Targeted Selective Treatments (TST) could be relevant in controlling nematode infections and delaying anthelmintic resistance selection. We first conducted a one grazing season experiment to compare all flock treatment (the whole flock was treated monthly) and TST based on monthly excretion eggs and daily weight gain. Nematode burden was higher in TST group, but anthelmintic susceptibility of nematodes was preserved. We then used an individual-based model to evaluate the sustainability of TST on a longer period. The simulation approach indicated that TST20% and TST30% of the flock were efficient both at maintaining resistance at a low level and controlling nematode parasite infections on a mid-term period (i.e. simulation of five grazing seasons). However for an efficient TST, these percentages of treated hosts should be adapted to flock size since the number of nematode parasites exposed to selection increases in large flocks. Our results also showed a high dependence on the timing of treatment i.e. on the size of the refugia constituted by the free-living stages on the pasture.

Contrasting genetic structures of two parasitic nematodes, determined on the basis of neutral microsatellite markers and selected anthelmintic resistance markers.

For the first time, the neutral genetic relatedness of natural populations of Trichostrongylid nematodes was investigated in relation to polymorphism of the β‐tubulin gene, which is selected for anthelminthic treatments. The aim of the study was to assess the contribution of several evolutionary processes: migration and genetic drift by neutral genetic markers and selection by anthelminthic treatments on the presence of resistance alleles at β‐tubulin. We studied two nematode species (Teladorsagia circumcincta and Haemonchus contortus) common in temperate climatic zones; these species are characterized by contrasting life history traits. We studied 10 isolated populations of goat nematode parasites: no infected adult goat had been exchanged after the herds were established. Beta‐tubulin polymorphism was similar in these two species. One and two β‐tubulin alleles from T. circumcincta and H. contortus respectively were shared by several populations. Most of the β‐tubulin alleles were ‘private’ alleles. No recombination between alleles was detected in BZ‐resistant alleles from T. circumcincta and H. contortus. The T. circumcincta populations have not diverged much since their isolation (FST <0.08), whereas H. contortus displayed marked local genetic differentiation (FST ranging from 0.08 to 0.18). These findings suggest that there are severe bottlenecks in the H. contortus populations, possibly because of their reduced abundance during unfavourable periods and their high reproductive rate, which allows the species to persist even after severe population reduction. Overall, the data reported contradict the hypothesis of the origin of β‐tubulin resistance alleles in these populations from a single mutational event, but two other hypotheses (recurrent mutation generating new alleles in isolated populations and the introduction of existing alleles) emerge as equally likely.

A genome scan for QTL affecting resistance to Haemonchus contortus in sheep.

Gastrointestinal nematodes are one of the main health issues in sheep breeding. To identify loci affecting the resistance to Haemonchus contortus, a genome scan was carried out using 1,275 Romane × Martinik Black Belly backcross lambs. The entire population was challenged with Haemonchus contortus in 2 consecutive experimental infections, and fecal egg counts (FEC) and packed cell volumes were measured. A subgroup of 332 lambs with extreme FEC was necropsied to determine the total worm burden, length of female worms, sex ratio in the worm population, abomasal pH, and serum and mucosal G immunoglobulins (IgG) responses. Pepsinogen concentration was measured in another subset of 229 lambs. For QTL detection, 160 microsatellite markers were used as well as the Illumina OvineSNP50 BeadChip that provided 42,469 SNP markers after quality control. Linkage, association, and joint linkage and association analyses were performed with the QTLMAP software. Linkage disequilibrium (LD) was estimated within each pure breed, and association analyses were carried out either considering or not the breed origin of the haplotypes. Four QTL regions on sheep chromosomes (OAR)5, 12, 13, and 21 were identified as key players among many other QTL with small to moderate effects. A QTL on OAR21 affecting pepsinogen concentration exactly matched the pepsinogen (PGA5) locus. A 10-Mbp region affecting FEC after the 1st and 2nd infections was found on OAR12. The SNP markers outperformed microsatellites in the linkage analysis. Taking advantage of the LD helped to refine the locations of the QTL mapped on OAR5 and 13.

Benzimidazole resistance in Trichostrongylus axei in sheep: long-term monitoring of affected sheep and genotypic evaluation of the parasite.

This is the first report of benzimidazole (BZ) resistance in the nematode Trichostrongylus axei in sheep. Trichostrongylus axei infects several species of herbivores including sheep, cattle and horses, and the emergence of anthelmintic resistance could lead to significant problems in its control. Benzimidazole resistance in two sheep flocks in central France was detected by post-treatment worm counts. The sequencing of a central region of the isotype 1 beta-tubulin gene from adult T. axei recovered post-mortem revealed only one, non-synonymous single nucleotide polymorphism at position 200 (Phe200Tyr), which had already been reported for other nematodes. Seven years after BZ treatment ceased, T. axei helminths present were still resistant to BZ suggesting these parasites do not revert to susceptibility to this anthelmintic, even when the selection pressure had been removed for many years. The findings also highlight major changes in the make-up of the nematode burden in sheep flocks that accompanies the emergence of BZ resistance.

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.

Effects of irrigation on appearance and survival of infective larvae of goat gastro-intestinal nematodes in guadeloupe (French West Indies)

In Guadeloupe (French West Indies), faeces from naturally infected goats were deposited during the dry season on three plots, irrigated with long (plot A) or short herbage (B) and non-irrigated with long herbage (C). Microclimatic data and the evolution of L3 population size in faeces, on soil surface and on herbage were followed over a period of 26 days. The initial nematode egg population was comprised of 58% Haemonchus contortus (HC), 25% Trichostrongylus colubriformis (TC) and 17% Oesophagostomum columbianum (OC). Temperature and water content varied in time and space (soil, faeces, herbage) from homogeneous in A to very heterogeneous in C. In A and B, population dynamics were similar with higher values of maxima in A. Larval peaks occurred on day 9 after deposition in faeces in plot A: 23.1, 39.1 and 17.2 L3/100 eggs, respectively for HC, TC and OC; the same day in soil: 1.9, 0.6 and 3.1 L3/100 eggs. On day 26 it remained less than 1 L3/100 eggs in both soil and herbage for the three species. In C, only TC larvae were observed coming, after rain, from eggs in which hatching had been delayed. It was difficult to separate the respective effects of temperature and water content on the development of the eggs, but irrigation gave favourable conditions for all eggs to develop into larvae. Pasture rotation with 28-35 days of regrowth should minimize the increased risk of infection for the goats due to irrigation.

Comparative susceptibility of romanov and lacaune sheep to gastrointestinal nematodes and small lungworms

The susceptibility of Romanov and Lacaune sheep to nematode parasites was studied in a permanent flock grazing an extensive pasture. The output of strongyle eggs and protostrongyle larvae was higher in Romanov ewes. Faecal samples of the Romanov ewes more often harboured Nematodirus eggs while the larvae recovered from cultures of these samples contained a higher percentage of Teladorsagia. Romanov tracer lambs had a significantly heavier adult Teladorsagia burden. This higher natural susceptibility of the Romanov breed was confirmed by experimental infections with T. circumcincta and N. spathiger but not with Trichostrongylus colubriformis. In infections with Muellerius capillaris and Protostrongylus rufescens, the worm burdens were similar but parasite fertility (larvae per adult) seemed to be higher in Romanov tracer lambs.

Studies on mutlispecific resistance of gastrointestinal nematodes to benzimidazoles on dairy-goat farms

Multispecific resistance to benzimidazoles was studied in three selected farms. These farms had bred dairy goats for more than 15 years. The helminths were introduced with the goats at the establishment of the farms which afterwards remained isolated. Nematode resistance could then be related to their own management practices. Faecal egg count tests and egg hatch assays were performed to assess intensity of resistance. The generic (infective larvae in faecal cultures) and specific richness (adult worms) were assessed. The resistant species were Trichostrongylus colubriformis, Teladorsagia circumcincta, Haemonchus contortus and Oesophagostomum venulosum. Faecal egg count reduction tests and egg-hatch assays did not match exactly. Faecal larval counts after treatments gave a distorted picture of multispecific resistance: Haemonchus and Oesophagostomum were very largely over represented. The number of species found in the three farms was relatively low compared with other reports in goat farms of the area. This reduction of diversity might also be due in part to characteristics of breeding management and history (use of permanent pasture and introduction of goats at the establishment of farm).

A short-term divergent selection for resistance to Teladorsagia circumcincta in Romanov sheep using natural or artificial challenge

This experiment was conducted to assess the efficiency of selection on the basis of response to artificial challenges in order to breed sheep resistant to natural infection. A short-term divergent selection process was designed to estimate the genetic parameters of these two traits. Two flocks, including 100 Romanov ram lambs each, were challenged in 1990 when they were 6 months old. One flock received three artificial infections with 20 000 third-stage Teladorsagia circumcincta larvae, at intervals of 7 weeks. Faecal egg counts (FEC) were performed on Days 22, 25 and 28 post infection (p.i.) and the animals were drenched on Day 28 p.i. The other flock was grazed for 5 months on a pasture contaminated with the same species. Faecal samples were taken from the lambs at similar ages. About 5 rams with the lowest FEC and 5 with the highest FEC were selected in each flock and mated with unselected ewes. Their offspring (200 animals) were challenged in 1992, half in the same way as their sires, and the other half by the other method. Because of a drought in the summer of 1990, it was necessary to repeat part of the experiment, and in 1992 the 5 and 8 rams with the lowest and highest FEC, respectively, were selected from the offspring challenged on the pasture in 1992 and were mated with unselected ewes. Their progeny (about 80 animals) were challenged in 1994, half by natural infection, half by artificial infection. The mean FEC of the flock increased from the first to the third artificial infection. The natural infection was highly variable in different years, reflecting the difficulty of assessing resistance using this mode of challenge. Genetic parameters were estimated using animal models and REML solutions. The repeatabilities of the FEC following artificial and natural infection were 0.49 and 0.70 respectively within a period of one week, and 0.22 and 0.41 respectively for periods separated by intervals of 7 weeks; the heritabilities of the single egg count were 0.22 and 0.38 respectively. The genetic correlation was 0.87: the FEC recorded under natural or artificial infection appear to depend on the same genetic potential.