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The distribution of lymphocyte subpopulations (TCD4+ , TCD8+, T C D 4 3 + and l g + cells), macrophages and eosinophils were analysed in the inflammatory infiltrates associated with hepatic lesions and in hepatic lymph nodes (HLN) from rats experimentally infected with F. hepatica and necropsied 1, 2, 3, 4, 6 and 8 week post infection (WPI). W e also investigated the fixation of immunoglobulin isotypes on migrating flukes in the liver. As early as 1WPI, portal tract areas surrounding migratory tunnels were infiltrated with immune and inflammatory cells. The dominant cells were eosinophils and to lesser extent, macrophages and lymphocytes (TCD4+, T C D 8 + and B). Most of the inflammatory and immune cells reached the posterior part of flukes, whereas in front of the parasites these cells were fewer in number. Except for eosinophils, no immune cells penetrated through granuloma consisting of hepatic necrotic cells. As early as 1 W P I , IgM could be detected in the liver, and to a lesser extent IgA, lgG2 a and lgG2 b . At 2 W P I , IgE and IgG, began being detected. lgG2 c was detectable at 3 W P I . In HLN, we observed numerous microscopic follicles in the cortical zone with proliferation of germinal centres and medullary cords. The protective role of infiltrating cell populations and immunoglobulin isotypes and possible mechanisms of immune evasion by the parasite are discussed., L'objectif de cette étude était de caractériser la distribution des différentes cellules immunitaires et inflammatoires. Les souspopulations lymphocytaires (TCD4+, TCD8+, TCD43+ et les cellules lg+), ainsi que les macrophages et les éosinophiles, associés aux lésions hépatiques et dans les ganglions lymphatiques ont été étudiés chez des rats infestés expérimentalement par Fasciola hepatica. Nous avons recherché la fixation des différentes isotypes d'immunoglobulines sur les douves en migration et comparé ces résultats avec la réponse humorale systémique. Dès la première semaine post infestation (SPI), les espaces portes hépatiques sont infiltrés par des cellules inflammatoires et immunitaires. Les cellules dominantes sont des éosinophiles et dans une moindre part, des macrophages et des lymphocytes (TCD4+, TCD8+ et B). La plupart de ces cellules n'atteignent que la partie postérieure du parasite, alors qu'elles sont moins nombreuses devant celui-ci. A l'exception des éosinophiles, aucune autre cellule ne pénètre dans le granulome constitué de nombreuses cellules hépatiques nécrotiques. On détecte les IgM dans le foie dès la 1SPI, et avec une moindre intensité les IgA, lgG2a et les lgG2b. A 2SPI les IgE et les lgG1 deviennent détectables, et enfin à la 3SPI les lgG2c. Au niveau des ganglions lymphatiques hépatiques, nous observons dans la zone corticale plusieurs follicules lymphoïdes microscopiques avec une prolifération des centres germinatifs et des cordons medullaires. Le rôle protecteur des différentes cellules infiltrées et les différentes isotypes d'immunoglobulines, et les éventuels mécanismes d'échappement parasitaires vis-à-vis du système immunitaire sont discutés.

The present study was designed to determine whether IFN gamma and IL-10 were secreted by peripheral blood mononuclear cells (PBMC) and hepatic lymph node mononuclear cells (HLNMC) from 17 Fasciola hepatica infected sheep after in vitro stimulation by Fasciola hepatica excretory-secretory products (FhESP) during the first six weeks of two different infections. The IFN gamma concentration in mononuclear cells (MC) culture supernatants was determined by sandwich ELISA. The presence of IL-10 in MC culture supernatants was determined by the capacity of these to inhibit, with or without monoclonal antibodies to IL-10, the NO production of recombinant bovine IFN gamma (rBoIFN gamma) activated monocytes. FhESP-stimulated PBMC and HLNMC produced IFN gamma in culture supernatants from DPI 7 (day post-infestation 7) to DPI 14. Culture supernatants of PBMC stimulated with FhESP first induced an increase in the NO production of rBoIFN gamma activated monocytes. After 2 or 3 weeks of infection, MC culture supernatant reduced the NO production of rBoIFN gamma activated monocytes. From DPI 35, anti-IL-10 antibodies addition restored or enhanced NO production. During sheep fasciolosis, IFN gamma production increased only until the first two weeks post infection. IL-10 could be secreted throughout the first six weeks of infection and could inhibit monocytes from DPI 35.

The cDNAs of hypodermins, enzymes secreted by the larvae of the parasitic fly Hypoderma lineatum , were sequenced. Four cDNA clones were isolated, one encoding hypodermin A (HA), one encoding hypodermin C (HC), and the two others encoding proteins related to hypodermin B (HB). The amino acid sequences deduced from the nucleotide sequences confirmed that these enzymes are serine proteases. HA and one of the HB proteins had potential N -linked glycosylation sites. Analysis of hypodermin protein, RNA and DNA at different larval stages indicated that protein overexpression is regulated transcriptionally for HA and HB, and by transcriptional and DNA amplification for HC.

International audience; Bovine hypodermosis affecting livestock performance and the leather industry was still widespread in France up to the nineties despite successive directives from the Ministry of Agriculture since 1941, encouraging livestock owners to treat, on a voluntary basis. In 1969 the French Ministry of Agriculture, asked the national Farmers’ Animal Health Organisation (FAHO), to plan a durable hypodermosis control programme and a working group including all the partners in cattle production was set up, under the coordination of the national FAHO. Improved systems of hypodermosis control, including new treatment and surveillance methods were developed. Among the main benefits obtained from this original work, were (i) the identification of scientific data which allowed inexpensive and accurate immuno-surveillance procedures, and a highly effective low-cost treatment, Ivomec1, administered at the micro dose rate (2 mg/kg), which is environmentally non-threatening, and (ii) the development of a new strategy to manage the control programmes progressively, on a regional basis, in two to three concentric zones over three successive years at a maximum. The current programme, coordinated at the National level since 1998, has been implemented in each region of France. The compulsory systemic winter treatments directed against the endo-parasitic stage, carried out by technicians and veterinarians involved the entire bovine population in controlled zones. As each zone reached a hypodermosis herd prevalence of under 5%, usually after two years, the treatments were suspended. However treatments of the infected farms and contiguous farms were maintained. An immuno-survey was carried out, each winter, to evaluate the prevalence of the disease and detect any residual foci or re-infestations. Since 2002, bovine hypodermosis in France is under control with immuno-surveillance maintained at a very low cost. In 2006 hypodermosis became a notifiable disease.

The aims were to identify the parasite communities and to relate the kinetics of infection to environmental factors during summer transhumance. A survey of parasitism of a sheep flock from the South-East of France was conducted during its summer transhumance in the Southern Alps. The flock was composed of 700 young Merino of Arles ewes. The 152 days of transhumance were spent grazing 3 pastures at an altitude of 1200 m, 4 pastures at 1700 m, and then spending 83 days between 2000 and 2500 m before returning back down to the same groups of lower pastures. The species of parasites present were identified in 5 tracer lambs collected 4 times during the survey. In addition, grass and faecal pellets on the pastures and on 28 alpine grasslands in the upper valley were sampled every month to look for the infective larvae of the gastrointestinal nematodes. Data for these grasslands and their use by the flock were available from the previous year. The main parasite species detected were the nematodes Teladorsagia circumcincta and Nematodirus spp.; the tracers collected about 4000 worms, mainly on the pre-alpine pastures. In the higher valley, considerable differences between the different areas were observed with regards to grass availability, pellet deposition and flock frequency. The pellets acted as a reservoir for over-wintering larvae. The most contaminated areas were those that had been grazed during August of the previous year. These findings were used to optimise flock management so as to limit infection and to prevent deterioration of the higher grasslands due to over-grazing and erosion. sheep / transhumance / nematode parasite / epidemiology / mountain pasture / animal behaviour

International audience

A large-scale serological surveillance was conducted for hypodermosis in the north of China. A total of 4175 sera samples of yaks and cattle were collected from five provinces in north China that included Xinjiang, Inner Mongolia, Heilongjiang, Jiling and Gansu provinces, between 2001 and 2002, and were examined for anti-hypoderma antibody by ELISA. The results indicate that the naturally infested positive rates of Hypoderma spp. in the above provinces were 51.77%, 27.02%, 13.00%, 6.03% and 44.41%, respectively. These results suggest a significant warble fly population in the north of China. Positive sera were collected from infested cattle in Pingliang county of the Gansu province monthly between August 2001 and July 2002. These sera were used to evaluate the seasonal kinetics of anti-hypoderma antibody. The kinetics demonstrated that anti-hypoderma antibody was elevated from October to December. Thus, serological surveillances associated with low infestation rates and chemical therapy would best be performed from October to December in the region.

313 sheep were examined in 1996 to assess the importance and seasonal evolution of Oestrus ovis infection in the Algerian region of El-Tarf. Prevalence was found to be 67.4 %. The larval burden was 18 larvae by infected sheep. The prevalence was higher in older sheep than in lambs; intensity was similar. The different larval stages were found all along the year in sheep with prevalence ranging from 33.1 to 80.5 % for L1 , 9.7 to 43.9 % for L2 and 8.4 to 23.0 % for L3. The sheep were the least infected in winter (prevalence from 35.7 to 44 % and intensity seven to ten larvae per sheep). The highest infection was found during the warm season (spring to autumn, prevalence from 62 to 90 % and intensity ranging from 15 to 25). This larval evolution profile suggested the existence of one long cycle (November-April) and possibly two shorts cycles (May-October). This epidemiological pattern is similar to that in Morocco but was slightly different from the situation in Tunisia where the winter cycle was apparently of lesser importance., 313 têtes d'ovins de la région d'EI-Tarf en Algérie ont été examinées en 1996 en vue d'étudier l'infestation des ovins par Oestrus ovis. La prévalence est de 67,4 %. L'intensité d'infestation atteint une moyenne de 18 larves par ovin. La prévalence était plus forte chez les animaux de plus d'un an que chez les agneaux, mais l'intensité était similaire. Les différents stades larvaires ont été retrouvés tout au long de l'année avec des prévalences de 33, 1 à 80,5 % pour les L1, 9,7 à 43,9 % pour les L2 et 8,4 à 23,0 % pour les L3. Une période d'infestation minimale des ovins est mise en évidence en hiver avec des prévalences de 35,7 à 44 % et des intensités de sept à dix larves par ovin infesté. Une période d'infestation maximale est enregistrée pendant la saison chaude (printemps, été, automne) avec une prévalence de 62 à 90 % et des intensités de 15 à 25 larves par ovin infesté. Le profil d'évolution de l'infestation des ovins par Oestrus ovis suggère l'existence d'un cycle long (novembre-avril) en période froide et vraisemblablement de deux cycles courts en période chaude (mai-octobre). La situation épidémiologique est similaire à celle du Maroc, mais diffère un peu de celle décrite en Tunisie, qui présente un cycle d'hiver long de moindre importance

International audience; Experimental infection by F. hepatica was performed on rats. Early recruitment of the peritoneal cell population was observed and revealed transient parasite-killing activity, preceded and followed by a state of total unresponsiveness. The activation peaked at seven days post-infection (dpi) and was characterised by a massive peritoneal cell recruitment, a strong superoxide anion and nitric oxide (NO) production, that were coincident with the fasciolicide activity of these cells, as monitored by an in vitro decrease of juvenile fluke viability in a conditioned medium. The addition of L-NG-monomethyl arginine (LNMMA) to cell cultures abrogated both fasciolicide activity and NO production. Parasites started to die when NO production exceeded 25 $\mu$M and all juvenile flukes were killed by a 90 $\mu$M NO exposition (Lethal Dose 50 between 45.8 and 50.3 $\mu$M, 95% fiducial limits). However, when rat peritoneal cells were cultured in the presence of either infected or control rat serum, juvenile flukes were much more resistant to the oxidative burst, despite a massive attachment of rat peritoneal cells to the parasite tegument. These data suggest that a transient control of fasciolosis may take place in the peritoneum following the parasite intrusion but that the parasite efficiently scavenges the host cellular response to avoid destruction.

A chemotherapy trial was conducted to determine the lowest dosage of injectable preparation of ivermectin against Hypoderma spp. infestation in yaks in Tibetan areas in Tianzhu county, Gansu province, in northwest of China. One hundred and sixty yaks were randomly divided into four groups of 40 yaks for the trial. The first three groups were treated by subcutaneous injection in the neck with 0.1% ivermectin (respectively, 1, 5, 10 microg/kg body weight). The fourth group was not treated and considered as control group. All the experiments were performed in November 2000 and the animals were examined for the presence of warbles in the next March and May. The results indicated that there was no warbles found on the back of treated animal while third stage larvae were palpated on back of some of the yaks in control group. It is concluded that dosage of 1 microg/kg ivermectin injectable was sufficient to kill or stop development of larvae of Hypoderma spp. in naturally infected yaks if administrated in November.

Hypodermin A (HA) is a serine protease secreted by first-instar Hypoderma lineatum larvae (Oestridae, Diptera). It plays a crucial role in induced immunosuppression during hypodermosis. This report describes the production of recombinant HA in Drosophila melanogaster Schneider 2 cells, its purification and its characterization, and compares it with the protease extracted form parasite larvae. The recombinant protein and the native HA have similar biochemical and biological features. Activity of the recombinant protease on the lymphocyte proliferation inhibition and on membrane antigen cleavage was tested and shown to be similar to the native one. Tunicamycin treatment of the recombinant HA shows that the two putative glycosylation sites carry glycan residues. Unglycosylated recombinant HA has the same enzymatic activity as the fully glycosylated protein, indicating that glycosylation is not important for the protease activity of HA.

The prophylactic efficacy of microdoses of injectable and pour-on ivermectin formulations against larval stages of Przhevalskiana silenus was assessed in naturally infected goats in the region of Calabria (southern Italy).Sixty-eight goats from two goat farms were divided into five groups: one group remained untreated, while the other four groups were treated with microdoses of ivermectin (5 and 10 microg/kg injectable formulation and 10 and 20 microg/kg pour-on formulation). The microdoses of ivermectin were fully effective in the treatment of goat warble fly infestation (GWFI) as no larvae emerged from the warbles in the treated groups, while all the larvae emerged in the control groups. Irrespective of the type of formulation used, the difference between the treated groups and the control group was statistically significant (P< 0.001). By contrast, no statistical differences were found between the goats treated with the injectable formulation and those receiving the pour-on applications, and between the two doses of the injectable and pour-on formulations used. Given the plasma concentrations it attains at its lowest dose (0.052 - 0.042 ng/ml for the injectable formulation and 0.030 ng/ml for the pour-on) the injectable formulation seems to offer the most reliable route for the administration of ivermectin microdoses and it is acceptable for milk consumption. The introduction of ivermectin in the early eighties and the use of microdoses in some cases have made it possible to control cattle hypodermosis in large areas of Europe. As with cattle hypodermosis, the administration of ivermectin microdoses in goats is particularly interesting because of the low costs involved and the low levels of residues found in goat milk; it may thus constitute the basis for GWFI control campaigns in areas where the disease is prevalent.

International audience; The yak (Bos grunniens) belongs to the cattle family Bovidae and lives in the mountains of China and adjacent areas. Due to the physiological adaptations of yak to its environment and the lack of data, the ivermectin pharmacokinetic was studied following a single subcutaneous dose at the recommended dose for cattle (0.2 mg kg(-1)). The observed peak plasma concentration (C-max) was 48.93 ng ml(-1) and the time to reach C-max (T-max) was 0.73 day. These results show a faster rate of absorption than in cattle. The values for the absorption half-life (t(1/2a)), the distribution half-life (t(1/2alpha)) and the terminal half-life (t(1/2beta)) were 0.31, 0.74 and 4.82 days, respectively. The calculated area under the concentration-time curve (AUC) was 146.2 ng day ml(-1) and the mean residence time (MRT) was 3.57 days. The availability of ivermectin appears low in yaks in comparison to cattle but equivalent to that reported in horses and is likely to be due to physiological characteristics of this species.

We investigated the phenotype of the T cells (CD4 + and CD8 + ) that produced Th1 (IFN-γ ) and Th2 cytokines (IL-4 and IL-10) during the first two weeks of experimental fasciolosis in rats. We also followed the kinetics of the cytokine and proliferative responses of hepatic mononuclear cells (HMNC) over the same period. We found that HMNC were more numerous in the infected ani- mals than in the controls. The percentage of CD4 + cells increased significantly after infection, whereas the percentage of CD8 + cells did not change. Moreover, the frequency of the cells producing (CP) cytokine changed after infection. The frequency of CP IFN-γ on 7 days postinfection (pi) was similar to that in control animals. However, the frequency of CP IFN-γ was clearly lower on day 14 pi, whereas the frequency of CP IL-4 and CP IL-10 had increased. The CP IL-10-were mostly CD4 + . Mitogenic stimulation (phorbol myristate acetate/ionomycin) of HMNC led to an increase in the amounts of the Th2 cytokines in the supernatant on days 7 and 14 pi, with the increase more pro- nounced on day 14. In contrast, IFN-γ levels also increased by day 7 pi but then decreased to below control levels by day 14. In addition, HMNC proliferation in response to mitogen followed a similar pattern to IFN-γ production. These findings suggested that, during the first 2 weeks of infection, F. hepatica induced a transient Th0 cytokine profile followed by downregulation of the cellular re- sponse and the induction of a Th2 cytokine profile. Fasciola hepatica / rat / cytokine / liver / lymphocytes

Cattle hypodermosis, due to insect larvae, is widely spread over the northern hemisphere. Very efficient insecticides are available and their use in most countries are done on an individual level but never cover the whole cattle population of a country. Untreated animals remain the reservoir of the disease and annually re-infest the cattle population. The economic effects of this disease on animal production (meat, milk and the leather industry) but also on the general cattle health status, have led many European countries to launch organised control programs. The first example of definitive hypodermosis control goes back one hundred years ago when Danish farmers eradicated hypodermosis from the Danish islands by manual elimination of the warbles. Since then, more and more European countries have considered the feasibility and economic returns of such programs. The various factors which foster these programs are related to (i) biological factors, (parasite cattle specificity, synchronous biological cycles of both species of insects involved), (ii) the development of more and more efficient insecticides used only once a year by systemic application, with high efficiency at very low dosages against the first larval stage of Hypoderma spp., (iii) the development of acute techniques of detection of the disease for the monitoring of hypodermosis free countries and (iv) the durable successful results obtained in more and more European countries. Although the programs were imposed by different partners of the livestock channel production (farmers, dairy industry, leather industry) and have been engaged within the last 50 years in many European countries (Denmark, The Netherlands, Ireland, the United Kingdom, the Czech Republic, Germany, France and Switzerland) common features have emerged among these different eradication programs. They all need a preliminary statement of the economic impact of this pest and the farmers' awareness of the economic returns of such programs. The programs' efficacy depends: (i) on a good knowledge ofthe epidemiology of the parasites, (ii) on the simultaneous implementation of the control program on the whole national cattle population whatever the structure monitoring the treatments (veterinary services, farmers association), (iii) on a national Warble fly legislation making the treatments compulsory and (iv) on an acute epidemiological survey as soon as the status of a hypodermosis free country is reached and the treatments are suspended. The sanitary and financial returns of such programs are a benefit to all the partners of livestock production, to the quality of the environment and to the consumers.

We studied the development of the cellular response, particularly with respect to Th1 and Th2 cytokine mRNA levels, in rat liver during the first 14 days of experimental infection with Fasciola hepatica. We analysed the panel of cytokines involved in initiation of the inflammatory and immune response. The levels of various mRNAs, particularly those primarily associated with the acute inflammatory response, and those commonly associated with T-cell proliferation and differentiation, were assessed by reverse transcription-polymerase chain reaction (RT-PCR) in liver samples. We also investigated the immune and inflammatory mediators balance in the liver, draining lymph node and spleen, by RT-competitive PCR quantification of mRNA levels for IL-4, IL-10 and IFN-gamma. Our data provide the first evidence that, in the early phase of infection, the inflammatory response in the liver of infected animals is transiently depressed or delayed. A Th0 profile was initially observed in the liver and hepatic lymph node, which developed into a Th2 profile 2 weeks after infection in the liver only. In the spleen, cytokine down-regulation was initiated and maintained during this period, suggesting that the parasite acts differently locally and in the periphery.

International audience; The response to Fasciola hepatica was studied in sheep infected with 5, 30, 150 metacercariae. The animals were necropsied 12 weeks post-infection (p-i) for counting and measuring flukes. Cellular and humoral responses were detected by peripheral eosinophil count, peripheral blood lymphocyte proliferation with excretory-secretory products (FhESP) and ELISA. All sheep were infected at necropsy except one sheep which was infected with 5 metacercariae. Mean parasitic intensities were 40% , 44% and 27% of the infection dose in sheep infected with 5, 30, 150 metacercariae respectively. FhESP-specific lymphocyte responses of the 3 infected groups were significantly enhanced in weeks 3 and 4 p-i ($p < 0.05$). The kinetics of the specific humoral response were similar for the 3 infected groups but the antibody level was significantly lower in animals infected with 5 metacercariae than in the 2 other infected groups from week 5 p-i to week 12 p-i ($p < 0.05$). Peripheral eosinophil count was significantly enhanced ($p < 0.05$) in infected groups. The numbers of peripheral eosinophils were significantly different between the 3 infected groups in week 3, 4 and 6 p-i and were related to infection level. These results confirm that sheep are highly susceptible to F. hepatica infection, even when infection pressure is very low. Peripheral eosinophilia was dependent of the infection level. The immune response was similar in sheep infected with various numbers of flukes.; Réponses du mouton à différents niveaux d'infestation par Fasciola hepatica. La réponse à Fasciola hepatica a été étudiée chez des moutons infestés par 5, 30 ou 150 métacercaires. Les animaux ont été autopsiés 12 semaines post-infestation (p-i) et les douves ont été dénombrées et mesurées. Les réponses cellulaire et humorale ont été évaluées par dénombrement des éosinophiles circulants, prolifération des lymphocytes circulants avec des produits d'excrétion-sécrétion de F. hepatica (FhESP) et ELISA. À l'autopsie, tous les moutons étaient infestés sauf un mouton infesté par 5 métacercaires. L'intensité parasitaire moyenne étaient de 40% , 44% et 27% de la dose infestante chez les moutons infestés respectivement par 5, 30 et 150 métacercaires. La réponse lymphocytaire spécifique des FhESP était significativement augmentée en semaines 3 et 4 p-i ($p < 0,05$). Les cinétiques de réponse humorale étaient similaires pour les 3 groupes infestés mais le taux d'anticorps était significativement inférieur chez les animaux infestés par 5 métacercaires de la semaine 5 à la semaine 12 p-i ($p < 0,05$). L'éosinophilie périphérique était significativement augmentée ($p < 0,05$) dans les groupes infestés mais le nombre d'éosinophiles était significativement différent entre les 3 groupes infectés en semaine 3, 4 et 6 p-i en fonction du niveau d'infestation. Ces résultats confirment que le mouton est très sensible à l'infestation par F. hepatica, même quand la pression d'infestation est très limitée. L'éosinophilie périphérique était dépendante du niveau d'infestation. La réponse immunitaire était comparable chez les moutons infestés avec un nombre variable de douves.

Two fasciolicide preparations have been compared in 130 rats experimentally infected with Fasciola hepatica. Parasitological, immunological, and biochemical parameters have been followed to monitor the efficacy of the treatments. While Fascinex (triclabendazole) efficiently cured both male and female rats when administered as soon as 4 weeks postinfection, treatment with Ivomec-D (clorsulon + ivermectin) displayed a low efficacy on either male or female rats at this time point (54 and 0%, respectively). Moreover, when administered 8 weeks postinfection, the Ivomec-D treatment proved highly efficient on male rats while it displayed little effect on the female population (100 and 53%, respectively). This unexpected result has been related to an overexpression of a P4503A isoform that is observed only in females that have been treated with Ivomec-D. The influence of this P4503A cytochrome on drug metabolism and the need for the incorporation of both genders in clinical trials are discussed.

The activity of drug-metabolising enzymes was compared in liver and kidneys of adult sheep given single or two-fold fluke infection. Fascioliasis was induced by oral administration of 200 metacercariae of Fasciola hepatica to female sheep either 10 or 20 weeks (mono-infections) or 10 and 20 weeks (bi-infection) before killing. The parasitic pathology was ascertained at autopsy and by clinical observation of animals. In the liver of both mono- and bi-infected animals, significant decreases (P

Parasites are capable of rapid evolutionary changes relative to their hosts, due to short life cycle, short generation time, and high fecundity. The direction of the evolution of parasite virulence can be studied in cross-transfer experiments, combining hosts and parasites from different localities, and comparing the outcome of established (sympatric and potentially locally adapted) and novel (allopatric) combinations of hosts and parasites. We aimed to compare the compatibility with snails hosts, the infectivity of metacercariae in rabbits and rats, and the fitness among different combinations (French-FF and Spanish-SS sympatries and allopatry-FS). The first isolate of Fasciola hepatica and its corresponding intermediate host, Lymnaea truncatula originated from Lugo's northwestern Spain. The second isolate of parasite and snail was collected in the Limoges area in central France. The Spanish snails were more susceptible to their sympatric trematode than the French snails. The Spanish flukes were more infective to intermediate hosts (snails) than the French flukes, but subsequent definitive hosts (rats or rabbits) infections remained similar. The estimated fitness was low in sympatric infections and highly similar (from 4.7 to 5.3). The fitness similarity corresponds, however, to different variations in life-history traits that could represent different strategies among the host-parasite local combinations. The infection rate in snails, metacercarial productivity, metacercarial infectivity, and the estimated fitness were better for allopatric combination (FS). The susceptibility data showed a higher efficiency of flukes in the allopatric snail population than in their local snail population. However, our results were obtained after one generation and from a single isolate and it remains to be determined if all allopatric fluke-snail isolates may present a better fitness. Nevertheless our results indicate that introduction of liver fluke-infected cattle should be monitored carefully, as it could result in the introduction of more efficient parasites.

Cross-reactivity between Hypoderma lineatum antigen and anti-Przhevalskiana silenus antibodies has been demonstrated by an ELISA technique. To evaluate the applicability of a commercial ELISA kit for the immunodiagnosis of goat warble fly infestation, different dilutions of serum and conjugate were tested, the development of antibody to P silenus in naturally infested goats was studied, and the results were compared with an ELISA technique using an antigen extracted from the first instar larvae of H lineatum. The best results were obtained with a serum dilution of 1:50; with both techniques the highest antibody concentration were recorded in October, November and December. In view of the confirmed cross-reactivity between H lineatum antigen and anti-P silenus antibodies, and the simplicity and rapidity of the assay, the commercial ELISA kit can be considered as a useful tool for the diagnosis of goat warble fly infestation.

The aim of the present study was to compare the sensitivity, specificity and usefulness of the DIG-ELISA, DOT-ELISA and Indirect ELISA tests for determining the seroprevalence of fasciolosis in cattle under tropical conditions in Mexico. To standardize the tests, positive and negative sera to F. hepatica from 88 Holstein Freisian adult cows located in an enzootic area of fascioliosis and 88 crossbred adult cattle from a fluke-free area were used. For the epidemiological study, 85 crossbred cattle between 1 to 7 years of age were used. Animals were bled every two months, from March 1995 to September 1996 and the sera obtained were stored at −70°C, until used. Indirect ELISA showed a sensitivity of 96.5% and a specificity of 98.8%, DIG-ELISA 97.5% and 80.0% and DOT-ELISA 93.1% and 95.4%, respectively. During 1995, Indirect ELISA yielded the highest levels of IgG anti-F. hepatica antibodies. However, in 1996, after animal treatment with triclabendazole, DIG-ELISA tended to show higher percentages of antibody-positive animals, but it was not significantly different (p>0.05) from the other tests. Comparisons made in parallel to the faecal sedimentation test demonstrated that all serological tests detected higher percentages of positive animals. Only one serum out of ten (10%) of Paramphistomum spp. cross-reacted with the DOT-ELISA test, but no cross-reaction was observed with sera from animals with other parasites. All ELISA tests were highly sensitive and specific; they may be recommended for use in seroepidemiological surveys for F. hepatica.

The protease hypodermin A (HA) is produced by the parasitic warble-fly larva and is implicated in the modulation of the bovine immune system. This study examines the effect of this enzyme on the cell surface markers of bovine lymphocytes. HA interfered with the binding of all anti-lymphocyte receptor antibodies tested. Anti-BoCD2 and CD5 staining was completely abolished. But the mean fluorescence intensity (MFI) only was diminished for antibodies against BoCD4, CD8 and CD18. On the contrary, the MFI for anti-MHC Cl I molecules staining was increased. This effect of HA began as early as one h, and was reversed by removal of HA. Heating or PMSF treatment, which both inhibit protease activity, abolished the action of HA on the surface antigens. The HA concentrations (100 micrograms/ml) needed to alter antibody binding were similar to those that inhibited phytohaemagglutinin (PHA)-induced proliferation. These results show that enzymatic activity of HA on lymphocyte surface markers may be implicated in the inhibition of lymphocyte proliferation.

A cattle herd from the experimental farm of INRA in Nouzilly has been treated for hypodermosis since October 1990. Additionally, a regional eradication scheme has been implemented in this area since autumn 1992. Bi-monthly warble counts were performed between March and July each year on an average of 200 animals. No warble was recorded in this herd from 1991 to 1994 with the exception of two dairy cows in 1993. In autumn 1994, therapeutic control measures were stopped. Serological surveys were performed in the autumn of each year from 1991 until 1995. Anti-Hypoderma antibodies were found in 25%, 27.2%, 4.3%, 3.2% and 0% of the animals respectively. An experimental low infestation was conducted in the summer of 1994. During the spring 1994, third instars of Hypoderma bovis were collected from naturally infested animals. From a total of 13 pupae, six adults (four males and two females) emerged and were released in the herd of Nouzilly on 24 June and 4 July. In October 1994 serological investigations revealed two animals seropositive for hypodermosis. This number increased to six in January 1995. The antibody kinetics of these six animals remained parallel throughout the next 6 months: the titres increased up to April and started to fall in May to return to negative values in August. Manual examinations of the animals at weekly intervals between April and July revealed the presence of four warbled animals with one, one, two and three warbles respectively. The two other seropositive animals remained warble free. One other animal showed antibody titre fluctuations between negative and low positive values, but was warble free in the spring. In October 1995 all the animals of the herd were seronegative. The interpretation and the value of a sensitive immunodiagnosis in a large eradication programme are discussed and compared with warble counts, especially in the case of a low level of infestation.

Macrophages, eosinophils, neutrophils and lymphocyte subpopulations (OvCD5+, OvCD4+, OvCD8+, OvWC1+ and lg+) were identified in sections of hepatic tissue and hepatic lymph nodes from sheep experimentally infected with F. hepatica and necropsied 14, 28, 42 or 56 days post infection. The migratory tunnels produced by juvenile flukes appeared as local areas of necrosis surrounded by infiltrating inflammatory cells, particularly numerous macrophages, eosinophils and OvCD4+ lymphocytes n addition, B lymphocyte proliferation was observed in hepatic lymph nodes and in hepatic portal tracts. Only three juvenile flukes were identified in the sections. They where partially in contact with healthy tissue and partially with neutrophils, macrophages and eosinophils; they were covered by IgM Host-parasite interactions resulting from immune response regulation by helper T lymphocytes and from immune evasion by the parasite are discussed.

Blood leukocyte changes, serum hepatic enzyme levels, lymphocyte proliferation in response to Concanavalin A (ConA) and to parasitic excretory-secretory products (FhESP), and antibody (IgG and IgM) responses (ELISA and Western blot) were studied in sheep, the natural susceptible host of F. hepatica, during the first 3 months of an experimental primary or secondary infection. The proportion of flukes established was similar in once- and twice-infected groups, but the flukes originating from the secondary infection migrated more rapidly to the bile ducts. Primary infection induced a marked peripheral eosinophilia from 3 to 13 weeks post-primary infection (PPIW). FhESP-specific IgM were produced from PPIW 2 with peaks in PPIW 3 and 9–10; FhESP-specific IgG increased from PPIW 2 to 6 and became stable afterwards. Western blotting revealed 12 major antigenic fractions in FhESP from 12, 15, 20, 24, 27, 28.5, 30, 41, 51, 56, 69 and 156 kDa; some non-specific ones have been characterized. A sequential recognition of higher then lower molecular weight antigens was observed. FhESP-specific lymphocyte proliferation was marked from PPIW 2 to 5. In contrast, ConA stimulation of lymphocytes was decreased. After secondary infection in PPIW 6, immune responses were modified. The ConA-induced lymphocyte proliferation was transitorily increased. In contrast, the humoral response, in particular against the early recognized antigens, and the level and the duration of the FhESP-specific lymphocyte proliferative response, were reduced.

The activity of drug-metabolizing enzymes was compared in liver, kidneys and lungs of rats given single or repetitive fluke infections. Fascioliasis was induced by oral administration of 20 metacercariae of F. hepatica to rats, either 6, or 12 and 6, or 12, 9 and 6 weeks before sacrifice. In the liver of mono-infected rats, significant reductions (24–67%) were observed in microsomal content of cytochrome P450 and all P450-dependent monooxygenases investigated. Conjugations to glutathione or acetate were lowered by 34–50% in these animals. In multiply infected animals, a majority of specific enzymatic activities were unchanged, while some monooxygenase activities such as aminopyrine demethylation or benzo (a)pyrene hydroxylation were increased by 26–76% in the liver of tri-infected rats. A renal compensatory process occurred in all infected groups, since cytochrome P450, benzphetamine demethylation and glutathione conjugation were significantly increased. By contrast, dealkylation of benzphetamine and pentoxyresorufin were decreased in the lungs of monoinfected rats. The development of parasite resistance would account for the recovery of liver drug metabolizing capabilities in multi-infected animals.

The action of Fasciola hepatica on rat complement was evaluated by measuring the haemolytic activity. Extracts from metacercariae and eggs and the excretory and secretory products of adults flukes reduced the classical pathway of complement-mediated lysis by normal rat sera in a protein dose-dependent manner. Whole metacercaria produced the same effect. In vitro incubation of newly excysted juveniles and intact eggs with normal rat serum had no detectable effect on serum complement activity by either the alternative or classical pathway. During the course of rat fasciolosis a depletion in the classical pathway of complement-mediated lysis was demonstrated particularly 6–9 weeks post-infection; the alternative pathway of complement-mediated lysis was not affected.

The effect of stimulating an inflammatory reaction by injecting rats with either Freund's complete adjuvant or turpentine oil shortly before or after they were infected experimentally with Fasciola hepatica on the establishment of the parasite was studied. Freund's complete adjuvant, given either four hours before or four hours after infection with F hepatica and turpentine oil given four hours before infection resulted in a 40 to 50 per cent decrease in the rats' fluke burden. The study of markers of inflammation (acute phase proteins, leucocytes and platelets) demonstrated that the fluke had a modulating effect on the rat's inflammatory response. During the first few days after infection, F hepatica stimulated a transitory inflammatory reaction, as suggested by changes in the level of acute phase proteins (APP) in the serum. No further change in the APP titre occurred until three weeks after infection, when, together with an increase in the extent of the liver lesions, the change in the titre of APP indicated that a systemic and chronic inflammatory process had taken place.

The effects of adult fluke excretions-secretions (ES) on two major functions of circulating bovine polymorphonuclear (PMN) cells were investigated. Under agarose, PMN chemotaxis was not affected by the ES. ES preincubated with PMN for 15, 30 or 60 min at 37°C, before a chemoluminescence assay, inhibited phagocytosis and/or free radical generation in a dose and time dependent manner.

Antibody responses, blood leucocyte and splenic lymphocyte subset patterns were studied during a primary infection with Fasciola hepatica in the rat. The infection induced parasite-specific IgM by 2 weeks after infection. High levels of IgM antibodies were maintained for many weeks. The IgE response was biphasic with peaks at 5 and 9 weeks after infection which were correlated with different phases in the development of F. hepatica in the rat. Both IgG2a and IgG1 antibodies were detected but the titre of IgG2a augmented slightly and rose more slowly than did that of IgG1. There was a rise in neutrophil and eosinophil numbers. Neutrophils did not increase before the fourth week but eosinophil numbers were raised by the second week after infection and remained high during the whole migratory phase of the parasite. In the spleen, the percentage of B lymphocytes increased and there was a decrease in the percentages of CD4+ and CD8+-like T lymphocytes. These results suggested that, in the rat infested with F. hepatica, TH2-like lymphocytes could be preferentially stimulated, as has been reported in murine schistosomiasis.

A survey of helminth infection was conducted in a flock of 290 ewes distributed into Romanov (R), Merinos d'Arles (M) and Romanov × Merinos (R × M) genetic types, grazing irrigated pasture in the south of France. Faecal egg and larval counts were done seven times per year from 1981 to 1984 on homogeneous groups of ewes and then individually once to four times every autumn from 1985 to 1988. Helminth fauna was diverse and more abundant during autumn. High levels of strongyle infection occurred in the ewes that remained on the same irrigated pastures during summer. Moving to Alpian pastures during the summer lowered autumnal infection. Significant differences between genotypes in intensity of infection were observed in the order R > R × M . M for strongyles ( Teladorsagia circumcincta and/or Trichoslrongylus vitrinus , Chahertia ovina and/ or Oesophagostomum venulosum, Nematodirus spp.), Momezia spp. and Dictyocauius filaria . The hierarchy was reversed for Fasciola hepatica infection and not consistent from one year to another for protostrongylid infections. The effect of lactation intensity on the postparturient rise was studied by equilibrating number of ewes according to reproductive status (zero, one or two lambs in lactation). Merino ewes with two lambs in lactation, as well as primiparous Romanov ewes, had significantly higher strongyle infections than the others. The repeatabilities of the larval and egg counts between the four trial years were 0.24, 0.23 and 0.16, respectively, for protostrongyles, Nematodirus and strongyles, with higher intra-annual values for protostrongyles and inconsistently significant results for strongyles due to the presence of several species.

The antibody and lymphocyte responses to Fasciola hepatica were studied in rats. Infested rats were shown to produce antibodies against excretory-secretory (ES) products of adult flukes as early as the first week after infestation. Immunoblotting revealed fractions of ES products of adult flukes to which antibodies were progressively produced during the course of the infestation. Proliferation of peripheral blood lymphocytes, splenocytes and thymocytes when incubated with different mitogens (Concanavalin A (ConA) or Pokeweed mitogen (PWM) or different liver fluke antigens (metacercariae antigen (EM) or ES products of adult flukes) have been studied. In response to these mitogens or antigens, splenocytes were stimulated on the second and fourth weeks after infestation. Thymocytes were significantly activated by PWM on the second week but peripheral blood lymphocytes did not show any statistically significant response. Results obtained in antibody production, immunoblotting and lymphocyte proliferation suggested sequential releases of F. hepatica substances and the existence of common proteins between adult and juvenile parasite stages. Cellular and humoral responses observed in this work did not seem to confer a complete resistance to liver fluke primary infestation on the rat.

Chabaudie N. , Villejoubert C. and Boulard C. 1991. The response of cattle vaccinated with hypodermin A to a natural infestation of Hypoderma bovis and Hypoderma lineatum . International Journal for Parasitology 21 : 859–862. In this study, we have determined whether immunization with hypodermin A (HA), associated with various adjuvants, could provide protective immunity for calves when challenged with a natural hypoderma infestation. Groups of naive calves were vaccinated with HA antigen alone or with adjuvants [Freund's incomplete adjuvant (FIA) or alumina phosphate (AP)]. Subcutaneous injection with HA antigen with or without adjuvant did not significantly protect calves against a natural hypodermosis infestation. The humoral response during the infestation period was evaluated by ELISA. A significant earlier and greater response was induced in groups vaccinated with HA alone and HA combined with FIA. These results indicate that HA, in this vaccination protocol, induces a very incomplete protection in calves exposed to a natural infestation.

An enzyme-linked immunosorbent assay (ELISA) was used on pooled serum and milk samples to determine whether hypodermosis could be detected where a larger sero-epidemiological survey was required. This study was undertaken to assess the potential of this assay for testing sera on milk samples, pooled from 10 cows, and determining the period of the year when detection was optimal. The sensitivity of the assay was determined by increasingly diluting a positive serum with pooled negative sera, from 1:10 to 1:100. The diagnostic lower limit of the assay requires at least two serological reactors within a herd of 100. The kinetic development and depletion of anti-Hypoderma antibody of individual and pooled sera or milk from 30 cows was evaluated from November to July. Anti-Hypoderma antibody levels of two groups of 8 calves, one control and one teated with ivermectin (Ivomec), were tested from October to June. These preliminary results indicate that an ELISA assay on serum or milk samples pooled from 10 cows can be used between February and April to evaluate the prevalence of hypodermosis within cattle herds in France, demonstrating the feasibility of using pooled serum already collected for bovine leucosis testing.

Purified enzymes of Hypoderma lineatum (Insecta, Oestridae), were assayed for their proteolytic activity on bovine C3 in normal cattle sera. The products of cleavage by these serine proteases (hypodermins A, B, and C), were analysed by electrophoresis in SDS polyacrylamide gels followed by immunoblotting. The enzymatic attack was initially directed at the alpha polypeptide chain by hypodermin A at a concentration of 1 micrograms/ml of serum and by hypodermin B at 5 micrograms/ml. The generated peptides differed in their molecular size from those produced during natural degradation of C3 in a control serum by physiologically relevant enzymes. Hypodermin A, at a concentration of 1 microgram/ml, also caused a cleavage of the beta chain. At 5 micrograms/ml, hypodermin A induced total degradation of the C3 molecule. Hypodermin B (5 micrograms/ml) starts splitting C3 near cleavage sites of factor I. Bovine C3 appears to be highly sensitive to hypodermins A and B in normal sera. Apparent molecular sizes and alignment of the bovine C3 cleavage products are presented schematically. Hypodermin C, a collagenolytic enzyme, had no effect on C3 in normal sera. The biological consequences for the immunopathological reactions associated with hypodermosis are discussed.

1. 1. An enzyme with a collagenolytic activity has been isolated by chromatography on SE Sephadex from the first instar larvae of Hypoderma lineatum. 2. 2. The purified collagenase of molecular weight around 16,000 and of isoelectric point pH 3.5, cleaves the collagen molecule in two fragments of 3 4 1 4 at neutral pH. 3. 3. This enzyme is slightly inhibited by diisopropylfluorophosphate but specific trypsin or chymotrypsin inhibitors have no effects, neither do EDTA, cyanide, or p-chloromercuribenzoate.

In France, farm workers are occasionally infested with the larvae of Hypoderma lineatum and H. bovis, which are primirily parasites of cattle. A specific serological test for diagnosing human cases is described here. The immunological responses in cattle are directed mainly against the proteolytic secretion of the first instar larvae. This secretion was used as an antigen for the diagnostic immuno-electrophoretic test in man. The antigen was highly specific and produced negative results to human sera from 108 patients with other forms of parasitoses and 23 non- parasitized controls. We report here on 13 cases of Hypoderma infestation in man. Ten of these were positive and three negative to the test. In the negative cases, the serum was collected 1–2 months after the emergence of the larvae. The immunological response disappeared rapidly after larval emergence; in one case within 9 days.

The hemolytic activity of bovine complement activated via the classical or alternative pathways was studied employing as target cells, erythrocytes of sheep, rabbit, guinea-pig and chicken. A spontaneous activation of bovine complement via the alternative pathway by rabbit and guinea-pig erythrocytes at a high level and chicken erythrocytes at a lower degree, was observed. Consequently hemolytic activity following activation via the classical pathway reflects activation via the two pathways when erythrocytes of these three species were used. In spite of a low degree of hemolysis, sheep erythrocytes have been chosen for their high specificity in the evaluation of bovine complement activation via the classical pathway. Activation via the alternative pathway can be performed by the erythrocytes of the three other species studied.

Three serine proteinases (hypodermin A, B and C) of the first instar larvae of Hypoderma lineatum have been assayed for their ability to deplete seric complement of naive or immune cattle. In naive cattle complement consumption is initiated by hypodermin B through the sequence C1-C3 at a concentration of 5 micrograms/ml of serum, and by hypodermin A through the sequence of C3-C9 at a higher concentration of 150 micrograms. The third enzyme presenting a collagenolytic activity has no anti-complementary activity even on C1q. In immune cattle a 70% complement depletion through the classical pathway is observed with 15 micrograms of hypodermin B per ml of serum. The two enzymes A and C appear to play a minor role in the complement depletion via the classical pathway. The biological role of each of these enzymes in the parasite-host interrelationships is discussed. The participation of these enzymes in the immediate hypersensitive reactions following systemic treatment of cattle infested by this endoparasite is considered.

Studies have been made of the effect of treatment with an organophosphorus insecticide on calves with or without Hypoderma infestation. A close relationship has been demonstrated between variations of circulating antibodies and blood histamine levels. It is suggested that histamine is released following an immunological reaction due to the formation of complexes between antigenic proteins of the dying larvae and circulating antibodies. Other etiologic agents such as intrinsic larval toxicity were also examined. The small variations of histamine levels suggest that histamine plays only a minor part in the mediation of symptoms of anaphylaxis often observed in parasitised cattle after preventive treatment against hypodermosis.

The collagenase from the larvae Hypoderma lineatum, with a molecular weight of 24 000 and isoelectric point of 4.1, was obtained in homogeneous form by ion-exchange chromatography. It is stoichiometrically inhibited by diisopropylfluorophosphate. On the other hand it is unaffected by ethylenediaminetetraacetate, p-chloromercuribenzoate, dithiothreitol, N-tosyllysine chloromethyl ketone, N-tosylphenylalanine chloromethyl ketone and ovomucoid trypsin inhibitor. The enzyme which degrades native collagen in its helical parts, has a specific activity on thermally reconstituted collagen fibrils of 150 micrograms collagen degraded x min-1 x (mg enzyme)-1 at 37 degrees C. It hydrolyses casein but has no esterolytic activity characteristic of trypsin, chymotrypsin nor elastase. It has no action on the synthetic peptide 4-phenylazobenzyloxycarbonyl-L-prolyl-L-leucyl-L-glycyl-L-prolyl-D-arginine. The amino acid composition of Hypoderma collagenase indicates a distinct similarity with the serine proteinases of the trypsin family and with another athropode serine collagenase, that of the fiddler crab Uca pugilator. This suggests that eucaryotic collagenases with digestive rather than morphogenic function represent a new category of members of the trypsin family.

The efficacy of pour-on or orally administered decamethrin against bovine hypodermosis was investigated on naturally infested yearlings. Treatment at dose rates of 1, 2 or 10 mg per kg body weight were ineffective, but the highest dose had a long-term lethal effect on warbles. The severe side effects which followed the high dose suggest that this drug is unsatisfactory for systemic grub control. Investigations on the dynamics of circulating anti-Hypoderma antibodies in the post-treatment period showed that there is some sub-lethal activity of decamethrin on the larval physiology even at the lowest doses.

Summary The abnormal fibrinogen « metz , characterized by an inclottability after thrombin addition, shows an abnormality in the structure of the α(A) chain, observed by electrophoresis in polyacrylamide gel. The abnormal clottability is due to an absence of release of fibrinopeptide A from the abnormal chain.

1. The pharmacokinetics of i.v. antipyrine (25 mg/kg) used as a model compound, were determined in young male sheep, before and each month after an oral infestation by 150 metacercariae of Fasciola hepatica, and 8 weeks following a flukicidal treatment. 2. The parasitic pathology was ascertained by the clinical observation of animals and the increase in plasma antibodies directed against liver flukes. 3. A significant decrease in the total plasma clearance of the drug occurred by week 4 to 16, and a 1.7 fold increase in mean residence time occurred by week 12 post-infection. 4. Urinary excretion of antipyrine metabolites was determined before and 8 weeks following the infestation. 4-Hydroxyantipyrine was the major urinary metabolite and its excretion was decreased by 30% in infected sheep, whereas there was no change in the excretion of norantipyrine, 3-hydroxymethylantipyrine or unmetabolized drug. 5. It is concluded that the impairment of antipyrine clearance in the course of fascioliasis could be related to the decrease in liver microsomal cytochrome P-450-dependent mono-oxygenases observed in sheep with a similar parasitic burden.

Boulard Chantal and Weintraub J. 1973. Immunological responses of rabbits artificially infested with the cattle grubs Hypoderma bovis (L.) and H. lineatum (De Vill.) (Diptera : Oestridae). International Journal for Parasitology3: 379–386. Immunological responses against first-instar larvae of Hypoderma bovis (L.) and H. lineatum (De Vill.) were traced in artificially infested rabbits in which larvae grew normally in the first instar but did not survive to the second instar. Passive haemagglutination demonstrated a rapid rise in antibody titres during the first 2 months to maximum levels that persisted until about 200 days of the infestation. Immunoelectrophoretic analyses of the sera demonstrated that larval metabolic products were more important than breakdown products of dead larvae in stimulating the production of antibodies. Larval collagenase, which seemed to induce the most significant immune response, was inhibited by homologous antibodies in the serum of a hyperimmunized rabbit. The implications of these results for infestations in the normal bovine host were discussed, with emphasis on the need to identify antigenic fractions other than collagenase, which by itself had not produced total control of larvae in previous vaccination tests.