Immunisation of calves with Ostertagia ostertagi fourth stage larval antigens failed to protect calves from infection.

Excretory-secretory and somatic antigens of Ostertagia ostertagi fourth stage larvae, emulsified with complete Freund's adjuvant, were intraperitoneally administered to calves on three occasions. Two weeks after the last immunisation all calves were infected with a single dose of 130,000 O. ostertagi third stage larvae. All animals were necropsied 25 days after infection. The immunisation procedure resulted in IgG1 and IgG2 antibodies, however no protective immunity was induced as O. ostertagi worm burdens and worm length were similar in all groups.

Identification and isolation of a 19.7 kDa Ostertagia ostertagi specific antigen and evaluation of its potential for immunodiagnosis.

Ostertagia ostertagi adult worm extracts were analysed by Western blotting using sera from calves experimentally infected with O. ostertagi and Cooperia oncophora. Strong differences in antigen recognition were noticed, even between animals from the same group. Two Ostertagia specific antigens with apparent molecular mass of 19.7 (OoA19.7) and 20.7 kDa (OoA20.7) were identified. One of them (OoA19.7) was purified by three subsequent chromatographic steps, i.e. gelfiltration, ion-exchange and reversed phased chromatography. It was demonstrated that this antigen does not show any cross-reactions with heterologous sera from C. oncophora, Dictyocaulus viviparus, Nematodirus helvetianus and Fasciola hepatica-infected animals. It was found that only IgG1 antibodies reacted against OoA19.7. The application of this antigen in an ELISA resulted in a highly species-specific test when compared to crude worm extracts. However, strong individual differences in anti-OoA19.7 response could be noticed between calves which received the same number of O. ostertagi larvae. These individual differences can hinder the application of the ELISA as a diagnostic tool, since the anti-OoA19.7 response does not seem to reflect the level of exposure to L3 larvae. This was supported by the absence of a clear infection-dose-related effect. It was shown that the anti-OoA19.7 response started from week 6 to 8, and reached its highest level at week 15.

Identification and purification of Cooperia oncophora-specific antigens to improve serological diagnosis.

Cooperia oncophora total adult extracts were examined by Western blotting with sera from C. oncophora- and O. ostertagi-infected calves to determine species-specific antigens. It was shown that two antigens with apparent molecular weights of 14.2 and 14.9 kDa were only recognized by calves which received a Cooperia infection and not by Ostertagia mono-infected calves or parasite-naive animals. The partial purification of these two antigens was achieved by gel filtration and ion-exchange chromatography. An enzyme-linked immunosorbent assay (ELISA) was developed based on the fractions containing these two antigens and no cross-reactivity could be noticed with serum from Ostertagia mono-infected calves. In contrast, the ELISA with total worm extracts showed strong cross-reactivity with heterologous serum. It was concluded that the 14.2 and 14.9 kDa Cooperia adult antigens have diagnostic potential, at least to differentiate C. oncophora and O. ostertagi.

Abomasal parasitism in dairy cows in Belgium.

Over a period of one year, from March 1984 to April 1985, the abomasa of 157 dairy cows in East Flanders (Belgium) were collected and examined for the presence of nematodes. Faeces and blood were also collected from the animals. No worms were recovered from 18 cows (11.5%), 118 cows (75%) had a low to moderate worm burden (10-10,000) and 21 cows (13.5%) a high worm burden (greater than 10,000). The geometric mean total number was 2171. Ostertagia ostertagi and Trichostrongylus axei were the main species involved, with the former accounting for 90% of all trichostrongyles recovered. For both worms a seasonal pattern was evident, with the highest worm counts in December-January. Between December and February greater than 97% of the Ostertagia spp. worm burden were EL4. For all animals the average pepsinogen level was 1391 +/- 494 mU tyrosine with no statistically significant relationship between pepsinogen levels and the total number of gastric nematodes. Only 45% of the faeces samples were positive for strongylid eggs with a mean of 43 eggs per gram. The percentage generic composition of L3 larvae collected from faeces was 62% Ostertagia, 18% Trichostrongylus, 6% Cooperia, 4% Oesophagostomum and 0.2% Bunostomum.

Evaluation of pepsinogen, gastrin and antibody response in diagnosing ostertagiasis.

Ostertagia ostertagi is widely distributed and is one of the most important parasites affecting young bovine livestock. There is, therefore, a substantial need for sensitive and specific parameters in support of diagnosis of ostertagiasis, especially for subclinical disease related to production losses. In this review, the value and application of pepsinogen, gastrin and antibody response as diagnostic tools are discussed. These three parameters are useful and comparable for confirming clinical disease in calves during their first grazing season. However, their value for detecting subclinical parasitism is questionable. Differences in the course of gastrin and pepsinogen late in the grazing season can be correlated with larval inhibition and the possibility of ostertagiasis Type II. Relatively few serological methods have been developed for the immunodiagnosis of Ostertagia and until now the indirect antibody-detecting enzyme-linked immunosorbent assay (ELISA) has been the method of choice. Antibody measuring methods have several disadvantages, most notably a lack of sensitivity and specificity, which limits their use in longitudinal epidemiological studies. Considering the necessity of cost effectiveness and ease of use, it is anticipated that additional work will result in the enhancement and quality of current immunodiagnostic methods.

Efficacy of the morantel sustained release trilaminate bolus against gastrointestinal nematodes and its influence on immunity in calves.

An experiment was conducted in calves to investigate the efficacy of a morantel sustained release trilaminate bolus (MSRT) to control gastrointestinal parasitism and to assess the development of immunity during the use of MSRT. Two groups (M and U) of four calves each were infected three times a week with a mixed Ostertagia ostertagi and Cooperia oncophora infection for 12 weeks. Calves of Group M received an MSRT at the start of the experiment. Twenty weeks after the start of the experiment, all animals, including a previously uninfected control group (C), received a challenge with 100,000 Ostertagia and 100,000 Cooperia. After a further 4 weeks all calves were necropsied for worm counts. During the trial calves were weighed and faecal egg counts, larval differentiation and pepsinogen concentrations were determined. The results demonstrated the high level of efficacy of the MSRT in reducing the faecal egg output and preventing parasitic gastroenteritis under conditions of a continuous high rate of infection. Efficacy of treatment was higher for Cooperia than for Ostertagia. Post-mortem worm counts suggested a partially impaired immunity build-up in Group M, at least for Cooperia.

Weight gain and the course of some estimators of gastrointestinal nematode infection in calves during winter housing in relation to the level of exposure during the previous grazing season.

In two experiments groups of calves were exposed to different levels and patterns of infection with Ostertagia spp. and Cooperia spp. The experimental design simulated the stereotypic pattern of herbage infestation, including a normal or a delayed midsummer increase, under conditions of set-stocking. After this simulated 'first grazing season', calves were monitored throughout the subsequent winter housing period. No continuing negative effects of previous infection on growth performance were observed. Calves in all groups gained on average over 0.7 kg day-1, irrespective of previous level of exposure. Differences between the experiments with respect to either level or pattern of infection during the preceding 'first grazing season' were all, to a greater or lesser extent, reflected in faecal egg counts, pepsinogen values, gastrin values and antibody titres against Cooperia spp. or Ostertagia spp. Depending on the time of sampling, pepsinogen values and antibody titres against Ostertagia spp. particularly were useful variables for assessing differences in levels of infection to which groups of calves had been exposed.

The presence of an early L4 larvae population in relation to the immune response of calves against Ostertagia ostertagi.

The influence of different levels of infection with Ostertagia ostertagi on the development of a protective immune response in calves was investigated. Four groups of calves were infected with either 5000 (Group A), 10,000 (Group B), 20,000 (Group C) or 40,000 (Group D) infective larvae (O. ostertagi L3) weekly until treatment began. Group E functioned as controls. All animals were treated with oxfendazole (9 mg ml-1) at Week 17 (Groups A, B and E) or Week 18 (Groups C and D). Sixteen days post-treatment all calves received a challenge infection of 150,000 O. ostertagi L3 spread over 10 consecutive days. Faeces and blood were collected weekly for egg counts and to assess levels of pepsinogen, gastrin and IgG1 and IgG2 Ostertagia antibodies. All calves were necropsied 31 days post-challenge for worm counts. Egg counts and pepsinogen levels were proportional to the infection level during the first few weeks of the experiment. Only in the high-dosed Group D was a gastrin response evoked. Ostertagia IgG1 antibodies increased between Day 25 and Day 95, and in the non-infected control group an antibody rise was observed from Day 67 onwards. All measured parameters except Ostertagia antibodies showed a gradual decrease from Day 70 until the day of treatment. At necropsy there was no significant difference between the groups in the total worm populations. Only the composition of the worm populations differed, with 35% early L4 (EL4) larvae in the previously infected Groups A, B, C and D and only 5% in the control Group E. The results indicate a slow immune response against O. ostertagi in cattle and question the possible role of the EL4 stage in developing immunity.

Quantitative estimation of the level of exposure to gastrointestinal nematode infection in first-year calves.

In two experiments groups of calves were exposed to different levels and patterns of infection with Ostertagia spp. and Cooperia spp. The experimental design simulated the stereotypic pattern of herbage infestation, including a normal or a delayed midsummer increase, under conditions of set-stocking. The purpose of the experiments was to investigate the accuracy of egg counts, pepsinogen and gastrin values and antibody titres as estimators of the level of exposure to infection. Faecal egg counts significantly reflected levels of exposure during the first half of the simulated grazing season. Antibody titres and pepsinogen values reflected levels of exposure best during August and September, partly depending on the pattern and range of levels of exposure. Antibody titres against Cooperia spp. were particularly useful when levels of exposure to gastrointestinal nematode infection were low. Gastrin values were elevated only at high levels of exposure, which caused large weight gain reductions, in the later part of the simulated first grazing season. It is suggested that antibody titres and pepsinogen values can be used for prognostic diagnosis, indicating whether or not control measures should be taken. Both estimators of infection correlated significantly with the realised weight gain at the end of the simulated grazing season. Egg counts in the second month after the initial infection (turnout) also may be of significant value to support decisions concerning control measures. Comparisons with data from field trials and experiments conducted by others under various conditions suggested that the conclusions of the present experiments are also valid under field conditions. Furthermore, the results supported the conclusions drawn from previous field work, that levels of exposure are often very low on commercial farms in the Netherlands.

Production of dairy replacement stock in relation to level of exposure to gastrointestinal nematode infection in the first grazing season: second-year calves and heifers.

In two experiments, groups of calves which had been exposed to different levels and patterns of infection with Ostertagia and Cooperia spp. in a simulated first grazing season (FGS), were followed throughout a natural second grazing season (SGS). Milk yields in the subsequent first lactation period were also recorded. The results suggest that although there had been differences in immune status among groups, which had been infected in the FGS, prior to the SGS, weight gain among these groups was not significantly different during the SGS. Apparently, resistance to the pathogenic effects of reinfection had developed more strongly and at lower levels of exposure to infection than resistance against establishment of larvae as shown after an experimental challenge. The groups of calves not infected during the FGS did gain less than all other groups during the SGS. Further, infection-induced differences in weight gain among the infected groups in the FGS appeared to be permanent, at least up to the end of the SGS. Finally, first lactation yield was positively correlated with body weight at calving. On average, approximately 10 kg less milk was produced for each kg of lower body weight at calving. With respect to the implications for preventive control strategies in the FGS, it is suggested that parasite control should not be applied beyond a level at which weight gain reduction is prevented.

Experimental infection of pigs with Oesophagostomum dentatum: pathogenesis and parasitology of repeated mass infection.

Pigs receiving a limited ration of 1 kg commercial feed per day were infected daily with 50,000 Oesophagostomum dentatum larvae. The animals exhibited serious diarrhoea and anorexia. Although there was neither anaemia nor hypoproteinaemia, there was a significant decrease in plasma sodium and an increase in blood urea nitrogen at the end of the experiment. Large numbers of third and fourth stage larvae were found in the ileal, caecal and colonic mucosae. Only fourth stage larvae, never adults, were observed in the lumen. A continual expulsion of large quantities of third and fourth stage larvae were demonstrated in the faeces beginning with the appearance of diarrhoea. Neither Vibrio coli, Salmonella spp nor Balantidium coli contributed to the course of the enteritis.

Characterisation of Ostertagia ostertagi antigens by the different bovine immunoglobulin isotypes.

Antigenic differences between the developmental stages of Ostertagia ostertagi were studied by SDS-PAGE and Western blotting. Gel electrophoresis showed a complex protein pattern different for every stage with the O ostertagi fourth stage larvae (L4) showing an intermediate protein pattern between the third stage larvae (L3) and the adult stage. Immunoblotting showed that IgG1, IgG2 and IgM immunoglobulins present in serum from uninfected calves identified several O ostertagi antigens at every stage. When using serum from O ostertagi infected calves, O ostertagi specific IgG1 was the predominant bovine immunoglobulin. Specific IgG2 and IgM responses were also observed, while specific IgA antibodies were hardly detectable. Severe IgG1 cross reactivity was demonstrated when using anti-Cooperia oncophora serum.

Antigenic differences between the life cycle stages of Cooperia oncophora.

The antigenic differences among the life cycle stages of Cooperia oncophora were studied by SDS-gel electrophoresis and Western blotting. Sodium dodecylsulphate polyacrylamide gel electrophoresis of the different life cycle stages of C oncophora revealed a complex protein pattern with a decreasing number of protein bands towards the adult stages. Several bands of the fourth stage larvae were in common with both the third stage and the adult nematode. Western blotting with sera from C oncophora monoinfected calves showed that the antigens of the fourth stage larvae were recognised predominantly and the presence of stage specific antigens in all stages. Strong cross reactivity was demonstrated when serum from Ostertagia ostertagi infected calves was used.

Evaluation of a simplified blood pepsinogen assay.

Blood pepsinogen concentrations in cattle were determined, using a simple, direct method based on the hydrolysing effect of serum on buffered bovine albumin substrate (2 g/dl). The amount of tyrosine released was measured at 680 nm, using the Folin and Ciocalteu's colorimetric method. To ensure an optimal digestion in vitro, a glycine-hydrochloric acid buffer (0.1M) was evaluated at various pH. Serum samples with medium (2,591 mU of tyrosine) and high (6,222 mU of tyrosine) pepsinogen concentrations had the highest proteolytic activity at pH 2.5. Substituting albumin by hemoglobin substrate resulted in almost double the amount of hydrolyzed products. For preparation of standard pepsin or pepsinogen curves, the incorporation of serum (activated or inactivated) diminished the tyrosine released to only half the amount obtained without serum. The use of serum or plasma did not significantly affect the results of the test, nor did prolonged storage at -70 C result in marked differences in the results.

Possible resistance of small strongyles from female ponies in The Netherlands against albendazole.

To determine resistance of small strongyles to albendazole, 3 female ponies (group 1) were grazed on a pasture from May to November 1985 and were treated with 7.5 mg of albendazole/kg of body weight, PO, 2 days before turnout in May and again in June and in July. Three other female ponies (group 2) grazed on a similar pasture from May to July, were treated with 7.5 mg of albendazole/kg, and were removed to another pasture until November. In December, ponies from both groups were treated with 7.5 mg of albendazole/kg, and 8 days later, they were euthanatized and necropsied for a critical test. Worm egg counts in the ponies' feces revealed that the May treatment of group 1 and the July treatment of group 2 were more effective than were later treatments. Numbers of small strongyles were higher in group 1 than in group 2. Efficacy of treatment against all developmental stages of small strongyles was higher in group 2 than in group 1. Efficacy was low in both groups against parasitic 3rd- and 4th-stage larvae. Fifteen species of small strongyles were identified at necropsy. Efficacy was limited against adult Cyathostomum coronatum, Cya labratum, Cylicostephanus calicatus, and Cyl poculatus in both groups; Cylicocyclus nassatus, Cyl minutus, and Cyl longibursatus in group 1; and Cya labiatum in group 2. Efficacy was 100% against Cya catinatum, Cyl goldi, and 5 other species that were found in low numbers.

Some observations on the use of the morantel sustained-release bolus in first season-grazing calves on a Belgian dairy cattle farm.

The efficacy of the morantel sustained-release bolus (MSRB) in controlling gastrointestinal parasites in first-season grazing calves was evaluated on a dairy cattle farm in Belgium. The calves grazed a pasture which had been used by bolus-treated animals in the three previous years. The effect of bolus administration was determined with respect to live weight gain, faecal egg shedding, herbage larval counts, serum pepsinogen levels and ELISA antibody titres. In spite of an incomplete reduction of faecal egg shedding during the first months of the grazing season, bolus administration resulted in the prevention of parasitic gastro-enteritis in the calves. A weight gain advantage of 35,2 kg of the bolus-treated animals over the controls was noted already at two months after turnout. This weight gain advantage was maintained until housing. The usefulness of serum pepsinogen values and ELISA antibody titres as parameters in prevention experiments is stressed. Both serological parameters gave more information concerning infection level than did the faecal egg output and the herbage larval counts.

Baby pig diarrhea caused by coccidiosis.

An outbreak of coccidiosis on two Belgian farms is described. Diarrhea started in piglets at 9 or 10 days of life. Zero to three pigs died per litter. The morbidity rate varied from 70 to 90 per cent. Histological examination of the intestines revealed shortening of villi and different stages of the life cycle of coccidia were seen in the enterocytes. Virological examination was negative for corona-, corona-like, and rotavirus. A haemolytic E. coli strain was isolated in one case. As for treatment, good results were obtained by the adding of 1 kg amproleum pre-mix per ton sow feed. Scouring pigs were treated orally with 2 cc of an amprol solution once a day. The diarrhea stopped one day after treatment.

Efficacy of early season anthelmintic treatment against gastrointestinal nematodes.

The efficacy of levamisole and ivermectin in multiple-dose regimes for the control of parasitic gastroenteritis in first-season grazing calves was evaluated on a dairy cattle farm in Belgium. Thirty-nine female Holstein crossbred calves were randomly divided into three groups. Paddock 1 was used for the controls, paddock 2 for the levamisole group (dosed at 3, 6 and 9 weeks after the start of grazing) and paddock 3 for the ivermectin group (dosed at 3 and 8 weeks after turn-out). The treatments were evaluated on the basis of live weight, faecal egg output, and serum pepsinogen levels. The impact of the therapeutic dosing at timed intervals during the first months of the grazing season was remarkable; egg output in the levamisole and ivermectin groups between June and early October was substantially lower. The treatments seem to adequately control Ostertagia, because serum pepsinogen values were much lower from August onwards. Better weight gains were observed in both the treatment groups. The experiment also illustrated the advantage of early housing of calves.

Field evaluation of the efficacy of the fenbendazole slow-release bolus in the control of gastrointestinal nematodes of first-season grazing cattle.

The ability of fenbendazole slow release bolus (Panacur SR Bolus, Hoechst) to control gastrointestinal parasitism in calves during their first grazing season at pasture was evaluated in two field trials. The infection level on both investigated farms was low and the control animals did not develop parasitic gastroenteritis. However, it was possible to demonstrate significant differences in the parasitological and biochemical parameters between the control and treated groups during the grazing season. Faecal egg counts and blood pepsinogen levels in the control cattle at both trials sites were significantly higher than those of the bolus-treated cattle.

Use of an oxfendazole pulse release bolus in the control of parasitic gastroenteritis and parasitic bronchitis in first-season grazing calves.

The efficacy of the oxfendazole pulse release bolus system for the control of parasitic gastroenteritis and parasitic bronchitis in first-season grazing calves was evaluated in Belgium. Twenty-two calves were allocated to two groups. The calves in one group received a bolus at the time of turn out, while the other group remained untreated. The efficacy of the bolus was assessed by comparison of faecal worm egg counts, plasma pepsinogen concentrations, the antibody response to Ostertagia, Cooperia and Dictyocaulus species total plasma protein and albumin concentrations, and weight gains throughout the grazing season and the housing period. The oxfendazole pulse release bolus provided good control of parasitic gastroenteritis dominated by ostertagia. The effects of parasitic gastritis were greatly reduced as shown by the significantly lower values of serum pepsinogen and ostertagia antibody titres. The use of the bolus further reduced the adverse effects of parasitism as indicated by better liveweight gains and normal total plasma protein and albumin concentrations whereas in the untreated control group hypoproteinaemia and hypoalbuminaemia were observed. Most animals exhibited clinical signs of parasitic bronchitis at the end of the grazing season, and the bolus may not adequately control parasitic bronchitis in all cases at all times.