A successful, sustainable and low cost control-programme for bovine hypodermosis in France.

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, Ivomec, administered at the micro dose rate (2 microg/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.

Sero-epidemiological surveillance of hypodermosis in yaks and cattle in north China by ELISA.

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.

Early and transient cytotoxic response of peritoneal cells from Fasciola hepatica-infected rats.

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 microM and all juvenile flukes were killed by a 90 microM NO exposition (Lethal Dose 50 between 45.8 and 50.3 microM, 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.

Efficacy of different dosages of ivermectin injectable against the Hypoderma spp. in yaks.

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.

Efficacy of injectable and pour-on microdose ivermectin in the treatment of goat warble fly infestation by Przhevalskiana silenus (Diptera, Oestridae).

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.

Expression of functional hypodermin A, a serine protease from Hypoderma lineatum (Diptera, Oestridae), in Schneider 2 cells.

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.

Durably controlling bovine hypodermosis.

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.

Early hepatic immune response in rats infected with Fasciola hepatica.

We investigated the phenotype of the T cells (CD4+ and CD8+) that produced Th1 (IFN-gamma) and Th2 cytokines (IL-4 and IL-10) during the firsttwo 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 animals 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-gamma on 7 days postinfection (pi) was similar to that in control animals. However, the frequency of CP IFN-gamma 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 pronounced on day 14. In contrast, IFN-gamma 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-gamma production. These findings suggested that, during the first 2 weeks of infection, F hepatica induced a transient ThO cytokine profile followed by downregulation of the cellular response and the induction of a Th2 cytokine profile.

Evaluation of the hepatic NK cell response during the early phase of Fasciola hepatica infection in rats.

The in situ distribution of NK cells in rat liver during the first 28 days of an experimental infection with F hepatica was investigated. NK cells were distributed homogeneously throughout the hepatic parenchyma in uninfected animals. The total number of hepatic mononuclear cells increased significantly following infection, but the proportion of NK cells did not change. After infection, these cells were found around the portal space, around the centrolobular vein, in the periportal fibrosis and in the band of collagen. However, no NK cells could be detected in or around the granuloma during infection. The frequency of both I L-2- and IFNgamma-producing NK cells was higher on day 7 postinfection (pi) but only the percentage of IFNgamma -CD161+ subsets remained elevated thereafter, whereas the percentage of both IL-2+CD161+ and IL-4+CD161+ subsets returned to the baseline. The number of CD161+IL10+ cells did not change significantly. These results suggest that NK cells could be another source for the early production of IFNgamma but provide no evidence that these cells are involved in early events associated with granuloma formation.

DIG-ELISA: Estandarización y evaluación serodiagnóstica en fasciolosis bovina experimental y natural / DGI-ELISA: Standardization and serodiagnostic evaluation in experimental and natural bovine fascioliasis

Los objetivos del presente estudio fueron: Determinar la sensibilidad y especificidad de DIG-ELISA y establecer el valor de corte para realizar diagnóstico de fasciolosis bovina; determinar la capacidad diagnóstica de la prueba en bovinos infectados en forma experimental; valorar la seroprevalencia bimensual y durante un año de los títulos IgG anti-Fasciola hepatica en suero de bovinos infectados en forma natural; y comparar la variación con la presencia de huevos del trematodo identificados en el análisis coproparasitoscópico. Con el propósito de estandarizar la prueba, además de las correspondientes titulaciones de sueros, antígeno y conjugado, se utilizaron 80 sueros de bovinos, de estos últimos 40 pertenecían a animales provenientes del estado de Sonora (zona libre de fasciolosis) y 40 del estado de Hidalgo (zona enzoótica de fasciolosis). La prueba mostró una sensibilidad de 97.5 por ciento y una especificidad de 80.0 por ciento, obteniendo un valor de corte de 7 mm. En otro estudio, se infectaron 15 bovinos jóvenes de sexo indistinto, cada uno con 600 metacercarias del Fasciola hepatica. Los animales fueron sangrados semanalmente y durante 14 ocasiones para obtención de suero. La prueba fue capaz de detectar anticuerpos anti-F. Hepatica desde la segunda semana posinfección, logrando sus mayores incrementos expresados en diámetro de la zona de reacción (DZR) entre la séptima y décima semanas. En otro experimentos se utilizaron 85 bovinos criollos, hembras, de un rancho ubicado en un área enzoótica de fasciolosis en Nautla, Veracruz, en donde se colectó sangre para obtención de suero, así como heces de cada uno de los bovinos. Los animales fueron muestreados bimensualmente y durante un año para correr los cueros con DIG-ELISA y las heces para realizar análisis por sedimentación. Los porcentajes de seroprevalencia fueron del 58 por ciento, 73 por ciento, 80 por ciento, 100 por ciento, 96 por ciento y 79 por ciento para los meses de marzo, mayo, julio, septiembre, enero y marzo, respectivamente, mostrando porcentajes de positividad similares a aquellos obtenidos en el análisis coproparasitoscópico (P>0.01). Se concluye que DIG-ELISA mostró alta sensibilidad y fácil montaje para utilizarse bajo condiciones de campo en el diagnóstico temprano de fasciolisis bovina, generando resultados reproducibles que pueden ser de gran utilidad para estudios seroepidemiológicos