The Production of Polyclonal Antibodies in Laboratory Animals. The Report and Recommendations of ECVAM Workshop 35.

This is the report of the thirty-fifth of a series of workshops organised by the European Centre for the Validation of Alternative Methods (ECVAM). ECVAM's main goal, as defined in 1993 by its Scientific Advisory Committee, is to promote the scientific and regulatory acceptance of alternative methods which are of importance to the biosciences and which reduce, refine or replace the use of laboratory animals. One of the first priorities set by ECVAM was the implementation of procedures which would enable it to become well informed about the state-of-the-art of non-animal test development and validation, and the potential for the possible incorporation of alternative tests into regulatory procedures. It was decided that this would be best achieved by the organisation of ECVAM workshops on specific topics, at which small groups of invited experts would review the current status of various types of in vitro tests and their potential uses, and make recommendations about the best ways forward (1). This joint ECVAM/FELASA (Federation of European Laboratory Animal Science Associations) workshop on The Immunisation of Laboratory Animals for the Production of Polyclonal Antibodies was held in Utrecht (The Netherlands), on 20-22 March 1998, under the co-chairmanship of Coenraad Hendriksen (RIVM, Bilthoven, The Netherlands) and Wim de Leeuw (Inspectorate for Health Protection, The Netherlands). The participants, all experts in the fields of immunology, laboratory animal science, or regulation, came from universities, industry and regulatory bodies. The aims of the workshop were: a) to discuss and evaluate current immunisation procedures for the production of polyclonal antibodies (including route of injection, animal species and adjuvant ); and b) to draft recommendations and guidelines to improve the immunisation procedures, with regard both to animal welfare and to the optimisation of immunisation protocols. This report summarises the outcome of the discussions and includes a number of recommendations and a set of draft guidelines (included in Appendix 1).

An experimental field study on the build up of lungworm infections in cattle.

The build up of lungworm infections was studied in four groups of calves. Calves of Group 1 were infected experimentally with 6 x 10 larvae during the first 3 weeks after turnout. The pasture of Group 2 was contaminated with approximately 35,000 larvae in June and the pasture of Group 3 with approximately 1.3 million larvae in August. Group 4 served as the helminth free control group for challenge infections with 5,000 larvae in October. In Group 1 faecal larval counts increased 5 weeks after the beginning of patency and decreased after another 3 weeks, indicating the development of immunity after the second lungworm generation. In contrast, the development of immunity in Groups 2 and 3 occurred after the first lungworm generation as maximal faecal larval counts were seen within 3.5 weeks after the beginning of patency. Infection levels were highest in Group 3 which was the only group showing clinical signs. These signs became worse after oxfendazole treatment.

Comparison of three enzyme immunoassays for diagnosis of Dictyocaulus viviparus infection.

Three enzyme-linked immunosorbent assays (ELISA) that detect antibodies against Dictyocaulus viviparus in cattle were compared for sensitivity, specificity and seroconversion after primary infection. These assays were (i) an indirect ELISA with crude somatic antigens from adult D. viviparus (ca-ELISA), (ii) an indirect ELISA with purified antigens (sa-ELISA) isolated from somatic antigens of adult D. viviparus and (iii) a competition ELISA with antigen purified with anion chromatography in combination with monoclonal antibodies against D. viviparus. Sera from helminth-naïve calves and sera from calves monospecifically infected with Ostertagia ostertagi, Cooperia oncophora, Nematodirus helvetianus, Ascaris suum or Fasciola hepatica were used to determine the specificity of the assays. Sera from calves and milk cows experimentally or naturally infected with D. viviparus, and from vaccinated calves, were used to test the sensitivity of the assays and to determine when the ELISAs detected seroconversion. The specificity of the competition and the sa-ELISA was 97%, whereas the specificity of the ca-ELISA was 67%. The sensitivities of the sa-ELISA, the competition ELISA and the ca-ELISA were 97, 73 and 99%, respectively. All three assays detected seroconversion between 4 and 6 weeks after primary infection. None of the assays detected seroconversion in calves receiving lungworm vaccination. We conclude that of these three tests, the sa-ELISA can be used most beneficially to diagnose lungworm disease.

The effect of rotational grazing for periods of one or two weeks on the build-up of lungworm and gastro-intestinal nematode infections in calves.

An experiment was carried out with three groups of grazing calves and one housed control group to study the effect of rotational grazing for periods of 1 and 2 weeks on the build up of lungworm and gastro-intestinal nematode infections respectively. The experiment demonstrated that rotational grazing for periods of 1 week on six plots prevented the build-up of heavy lungworm infections. A build up of heavier lungworm infections was observed in a group that was rotationally grazed for periods of 2 weeks on three plots and a group which remained on one plot throughout the grazing season; there was no difference between these two groups. In all three situations, there was an adequate development of immunity against D. viviparus, as measured by worm recovery after challenge infection at the end of the experiment in comparison with worm recovery of the similarly challenged control group. Neither rotational grazing scheme protected the calves against gastrointestinal helminthiasis, because tracer calves, which grazed for 4 days only in August or October, acquired infections which would have resulted in severe illness or even death if necropsy had been postponed for a week.

An epidemiological study of Fasciola hepatica in The Netherlands.

Transmission of F. hepatica under natural conditions was analysed in a three year programme. The variables used were the indirect haemagglutination (IHA) technique, worm establishment in tracer lambs and the population dynamics, infection rate and shedding pattern of Lymnaea truncatula. It is concluded that fluke eggs, infected snails and metacercariae on herbage can survive the winter in the Netherlands. Metacercarial availability was positively correlated to the amount of rainfall in the grazing period. The role developed eggs that survive the winter is important, because this results in earlier infections in the herd. The use of the serological diagnosis method IHA is important to detect F. hepatica infection in an early stage. Use of cellophane paper on floats is a useful method for determining the shedding pattern of cercariae from L. truncatula. It is concluded that collection of metacercariae on cellophane floats, inventarization of L. truncatula and its infection level are useful tools for the prediction of liverfluke infections.

Comparison of an indirect haemagglutination assay and an ELISA for diagnosing Fasciola hepatica in experimentally and naturally infected sheep.

An enzyme-linked immunosorbent assay (ELISA) with somatic (S) or excretory-secretory antigens (ES) was compared with an indirect haemagglutination assay (IHA) for ability to detect antibodies against Fasciola hepatica in sheep. The specificity of both assays was determined by testing sera collected from sheep experimentally or naturally mono-infected with Fasciola hepatica, Haemonchus contortus, Ostertagia circumcincta, Cooperia curticei, Taenia ovis, Eimeria spp., Trichostrongylus vitrinus, Trichostrongylus colubriformis or Nematodirus battus respectively. With S or ES antigens the specificity of the ELISA was 98% or 95% respectively, whereas the specificity of the IHA was 86%. Antibodies directed against Fasciola hepatica were detected by the ELISA with S or ES antigens from 2 weeks after infection until the end of the experiment, whereas the IHA detected antibodies from week 3. We conclude that the ELISA with S antigens compares favourably with the IHA and can be used for the serodiagnosis of ovine fasciolosis in the Netherlands.

[Ascaris suum infection as a cause of clinical problems in cattle?]. / Ascaris suum-infecties als oorzaak van klinische problemen bij runderen?

A sudden decrease in milk yield, increased respiratory rate and occasional coughing were observed in dairy cows on two farms in spring 1991. Pigs were also kept on these farms, and pastures grazed by the cattle had been fertilised with pig slurry. Laboratory investigations of some of the cattle showed eosinophilia and high ELISA titres of antibodies against Ascaris suum. On one farm the clinical symptoms disappeared after the animals had been treated with oxfendazole and on the other farm the symptoms disappeared spontaneously with time.

Dictyocaulus viviparus in calves: effect of rotational grazing on the development of infections.

A study was made of the possibility of reducing lungworm infections in young grazing calves by rotational grazing for weekly periods on six paddocks. For this purpose three groups of four calves each were grazed on separate pastures in 1989, whereas a fourth group served as a permanently housed control group. Two groups of calves were infected experimentally with six doses of 10 larvae of Dictyocaulus viviparus during the first 3 weeks on pasture. In the third group, low natural infections with overwintered larvae occurred. One of the experimentally infected groups was rotationally grazed for weekly periods on six small plots while both other groups were set-stocked. Faecal larval counts and worm counts in tracer calves demonstrated lower lungworm infections in the rotationally grazed group than in both set-stocked groups. However, the numbers of worms found after challenge infection and subsequent necropsy were relatively high in the rotationally grazed group, indicating that development of immunity was less than in both other groups. Owing to the dry weather conditions in the summer of 1989, no serious clinical signs of husk developed in any of the three groups. These dry conditions, however, did not prevent the build-up of heavy pasture infectivity with gastrointestinal nematodes resulting in heavy worm burdens and serious clinical signs in tracer calves grazing for 4 days in August and September-October, respectively. This implies that rotational grazing did not have a clear effect on build-up of gastrointestinal nematode infections.

Identification and isolation of a specific antigen with diagnostic potential from Dictyocaulus viviparus.

The purpose of the investigation was to isolate and identify a specific antigen of Dictyocaulus viviparus that can be used to diagnose lungworm infections in cattle. Somatic, excretion and secretion antigens of adult D. viviparus and somatic antigens of L3 larvae were examined in an indirect enzyme-linked immunosorbent assay (ELISA) to determine whether they cross-reacted with sera collected from calves with mono-infections of Fasciola hepatica. Ostertagia ostertagi, Ascaris suum, or Cooperia oncophora. Serum samples containing antibodies directed against F. hepatica, A. suum, and O. ostertagi cross-reacted with somatic antigens of adult D. viviparus; these sera cross-reacted less with excretion and secretion antigens. When somatic antigens of adult D. viviparus were analysed in a Western blot, a 17-kDa protein that did not react with the heterologous sera was detected. This protein was isolated by ultrafiltration and anion chromatography. Sera collected from calves infected with D. viviparus was tested in indirect ELISAs with either somatic antigens of adult D. viviparus or with a low molecular antigen fraction of this preparation containing the 17-kDa protein. The extinction values that were measured in both assays correlated well. We conclude that the 17-kDa protein isolated from somatic antigens of adult D. viviparus may be useful in developing an improved immunoassay to diagnose lungworm infections in cattle.

Comparison of a levamisole sustained-release bolus and ivermectin treatment to prevent bovine lungworm infection.

The efficacy of a levamisole sustained-release bolus to prevent parasitic bronchitis in calves in their first grazing season was compared to ivermectin treatment at three, eight and thirteen weeks after turn out. Contamination of the pasture was established by experimentally infected seeder calves. Twenty calves were split into two groups. Ten calves of one group received a bolus at the start of the experiment. In the other group the calves were treated with ivermectin at 21, 56 and 91 days. Two principal calves from each group were killed during the experiment to study histopathological changes. Pairs of tracer calves were introduced on both pastures at intervals of four weeks throughout the grazing period. The permanent calves were challenged with lungworm larvae at housing and slaughtered four weeks later. Both systems prevented parasitic bronchitis. Larval output was completely reduced in the ivermectin-treated calves while all bolus-treated calves excreted larvae at certain times. The highest group average was 4 larvae per gram faeces. Eosinophilia, ELISA-titres and histopathological changes confirmed the differences in larval uptake. Challenge infection was not successful in either group and no worms were found at slaughter. Weight gain was significantly different at housing in favour of the ivermectin-treated calves, but after challenge this was reduced due to a higher weight gain in the bolus-treated calves. The practical consequences of the results have been discussed.

[Coccidiosis in lambs: observations in the preventive use of an amprolium-containing medicated feed]. / Coccidiose bij lammeren: waarnemingen bij het preventieve gebruik van een amprolium bevattend gemedicineerd krachtvoer.

The efficacy of preventive in-feed medication with amprolium (2000 ppm) was studied on a farm where clinical coccidiosis in unweaned lambs at pasture has been a problem for the past seven years. Both treated and untreated control lambs had access to the concentrates through creep feeding. In this clinical trial neither the treated group (15-17 mg of amprolium per kg body weight per day for three weeks) nor the control group showed clinical symptoms of coccidiosis. It seems likely that this is attributable to the feeding of concentrates. Nevertheless, the excretion of oocysts by the animals of the treated group was significantly lower than that of the control group. An outbreak of clinical coccidiosis in another group of lambs on this farm was successfully controlled by single drenching, 50 mg.kg-1, followed by the medicated feed. The pharmaceutical availability of amprolium in the concentrates was 95 +/- 1% immediately after preparation and the stability during storage under field conditions for two months was 100% +/- 2%.

A comparison of the efficacy of four different long-acting boluses to prevent infections with Dictyocaulus viviparus in calves.

A field study of calves in their first grazing season tested the efficacy of four long-acting devices--a morantel sustained-release bolus, a levamisole sustained-release bolus, an oxfendazole interval bolus, and an albendazole interval bolus--against Dictyocaulus viviparus. The pasture had been previously contaminated by four calves orally inoculated with infective lungworm larvae. The calves were grazed together with four bolus-treated groups, each comprising four calves. Lungworm infection became patent in the experimentally inoculated calves between 22 and 26 days. Infection in the bolus-treated groups became patent after 54 days. The morantel bolus group excreted the most larvae, followed by the albendazole bolus group, and the levamisole bolus group. The oxfendazole bolus group excreted by far the least larvae. Eosinophil curves and ELISA titres showed that treated groups had essentially the same course of infection. The heavy infection to which the treated calves were exposed produced complete immunity in all groups. Challenge infection of 10,000 larvae at housing did not change any of the test parameters. Post-mortem examination showed only one positive calf with few worms. We concluded that when pastures are heavily infested with lungworm larvae, all boluses prevent severe clinical signs and allow build up of solid immunity, although none completely prevent excretion of larvae.