Response of cattle treated with fenbendazole slow release bolus to challenge from nematodes the following season

ELSEVIER Veterinary Parasitology 62 (1996) 107-118 veterinary_ parasitology Response of cattle treated with a fenbendazole slow release bolus to challenge from nematodes the following season D. Kerboeuf a,*, H. Hoste a, j. Hubert a, j.p. Le Stang b a INRA, Station de Pathologie Aviaire et de Parasitologie, Unitd d'Helminthologie, F-37380 Nouzilly, France b Institut de l'Elevage, 13 rue du Champ de Courses, F-27300 Bernay, France Received 17 October 1994; accepted 6 June 1995 Abstract Nematode infection of cattle treated in their first year at pasture with the fenbendazole slow release bolus ('Bolus group') was compared during the second year with that of untreated cattle. Ostertagia was the most prevalent parasite associated with Cooperia. Except for the Dictyocaulus spp. which caused clinical signs of bronchitis in the 'Bolus' group, the infection during the second year resulted in a moderate response of the cattle whatever the group. Rises in both pepsinogen and gastrin levels were correlated with the number of Ostertagia L 3 on herbage. Damages in the abomasal mucosa were more frequent and severe in the 'Bolus' group where more inflammatory signs were observed in spite of a smaller number of worms. Nevertheless, the differences in total weight gains were not significant thanks to a compensatory effect during the second part of the grazing season in the 'Bolus' group, Hypotheses related to a minimum threshold of infection during the first year necessary to develop high enough protection during the second year are discussed. The pathological effects of gastrointestinal nematodes seem to vary more according to the inflammatory response than to the number of worms. Keywords: Controlled release technology; Fenbendazole; Cattle-Nematoda; lmmunity-Nematoda 1. Introduction The successful prophylaxis of helminth infections in cattle includes strategic early season treatments with anthelmintics or the use of intra-ruminal devices with either continuous or pulse release of anthelmintic. " Corresponding author. 0304-4017/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0304-4017(95)00861-6 108 D, Kerboeuf et al. / Veterinary Parasitology 62 (1996) 107-118 Nevertheless, the calves have to be exposed to sufficient antigenic stimulation to induce immunity against gastrointestinal helminthosis and parasitic bronchitis. Several authors have pointed out that, in some circumstances, calves treated in their first year with long acting systems accumulate heavy worm burdens in their second grazing season (see review by Vercruysse et al., 1994). As a consequence, prevention of nematode infection in first year grazing calves should be a careful balance between reducing production loss and allowing immunity build-up by a moderate nematode infection. Eysker et al. (1990) showed that an oxfendazole pulse release bolus may successfully control parasitic bronchitis provided that the pasture larval infections are sufficient to allow the development of immunity. As no information was available as far as the development of immunity is concerned for the fenbendazole slow release bolus, the present experiment was undertaken to assess the response of cattle treated in their first year at pasture (Le Stang and Hubert, 1995) and the possible consequences on weight gains in the second grazing season. 2. Materials and methods 2.1. Experimental design During a first pasture season, 40 male Montb~liard calves were divided into two groups. The first group received a fenbendazole slow release bolus (FSRB, Panacur SR Hoechst) at turnout (mid-April). The FSRB releases 80 mg per animal per day of fenbendazole on average over a 150 day period. The calves of the second group (Control) were treated once in mid-July with fenbendazole oral drench (7.5 mg kg - l , Panacur 2.5% N.D., Distrivet) when pasture contamination was maximum. The two groups were set stocked on separate pastures until mid-October. They were both treated against warble infestation with fenthion (Tiguvon N.D., 2% Pour-On, 25 ml per 100 kg, Bayer). During this first grazing season, the worm burdens were moderate but the differences between the two groups were high, the 'Control' calves being more infected than the 'Bolus' calves except for Nematodirus where the number in the 'Bolus' calves was about ten times that of the 'Control' group (Le Stang and Hubert, 1995). Dictyocaulus was only detected in the 'Control' group, both on the plot and at the post mortem examinations. The present experiment was conducted on 26 of these calves allocated in two groups of 13 during the second grazing season. These 26 calves were turned out at the end of March on a pasture known to be contaminated with gastrointestinal nematodes and lungworms. They were now 16 months old and weighed 385 kg on average. No treatment, anthelmintic or other, was given during the whole trial. In mid-June, about 80 days after turnout, four animals selected from each group were housed for 3 weeks and slaughtered for worm counts and histological examinations. This date was chosen to get information on response to nematodes before the reinfection, due to the second generation of parasites on the pasture, was too high. The animals were representative of each group taking into account the weight, the pepsinogen levels and the results of the last faecal egg counts (10 June). D. K erboeuf et al. / Veterinary Parasitology 62 (1996) 107-118 109 The remaining animals were observed for eventual clinical signs until the end of the season in mid-October. 2.2. Parasitological procedures Faecal, grass and blood samples were taken on 25 March, 30 April, 27 May, 10 June, 23 July, 20 August, 16 September and 14 October. Egg counts were carried out using the technique of McMaster modified by Raynaud (1970) and using magnesium sulphate for flotation. Dictyocaulus spp. larvae were counted according to the Baermann technique (1917). The number of larvae per kg of dry grass was determined using the technique described by Gruner and Raynaud (1980). At the slaughter house, the lungs, the abomasa and small intestines were separated immediately after death. The contents and washings were collected, the volumes were made up to 4 1 (abomasum) or 6 1 (small intestine) and eight 25-ml aliquots were taken. Worm counts were carded out from four aliquots if the number of parasites exceed 100; otherwise the eight aliquots were analyzed. The species and stages of development of the worms were identified. As soon as the contents were collected, the abomasal mucosae were frozen for a further examination according to the technique described by Hubert (1980). Before examination, the mucosae were thawed and a peptic digestion was performed for 4 h at 37°C. The digesta were poured through two wire mesh screens (0.3 mm and 0.032 mm). The screens were washed with water and the washings were made up to 200 ml. Then, four 5-ml aliquots were examined after iodine staining. Dictyocaulus viviparus were recovered by flushing the lungs with water according to the procedure of Inderbitzen (1976) but 40 l of water were used instead of 10 1 of Tyrode's solution. The water was passed through the lungs at a pressure of 100 Pa and the washings were passed over a 32/zm aperture sieve. All the worms were counted and their stage of development was identified. 2.3. Serological measurements Pepsinogen levels were determined using the technique described by Kerboeuf (1979). Blood gastrin levels were assayed using a commercially available human radioimmunoassay kit (Diagnostic Product Corporation, Humbeek, Belgium) which had been validated in cattle (Berghen et al., 1993). The serodiagnosis of parasitic bronchitis (Dictyocaulosis) was carried out using an ELISA test with crude adult worms and larvae as antigen. 2.4. Histology Tissue samples were taken from the abomasum of the eight slaughtered calves. Both fundic and pyloric regions were examined. The samples from each region were divided in two parts and fixed either in 10% buffered formalin (pH 7.3) or in Carnoy's fluid. Following dehydration and embedding in paraffin wax, the tissues were sectioned 5/xm thick. The histological slides, fixed with formalin, were stained with haematoxylin and 110 D. Kerboeuf et al./ Veterinary Parasitology 62 (1996) 107-118 eosin and used to assess the pathological changes of the mucosae and to count eosinophils. The slides fixed with Carnoy's were stained with Alcian Blue (0,5% solution in 0.5 N hydrochlorhydric acid) and safranin acetate (0.125% solution), and were used to count the mucosal mast cells. Mast cell and eosinophil counts in the fundus and the pylorus were performed using a square reticule. Because of the heterogeneous distribution of the inflammatory cells within the tissue, the upper and the lower parts of the fundic and pyloric mucosae were examined separately. For each cell type and each mucosal region, 12 reticule fields were counted (magnif. 400 X ). 2.5. Weight measurements The calves were weighed monthly. 2.6. Statistical analyses The results from the two groups were compared using analysis of variance (ANOVA) and the Newman-Keuls' test. The results of worm counts were transformed [Log (x + 1)]. Linear regression analyses were performed for gastrin and pepsinogen mea- surements. STATITCF (ITCF, Paris) was used. 3. Results 3.1. Clinical signs No clinical signs of parasitic gastroenteritis were observed in any animal of either group. However, clinical signs of parasitic bronchitis (coughing, tachypnoea or dysp- noea, nasal discharge) were recorded from the beginning of August until early Septem- ber in the 'Bolus' group. Nevertheless, no anthelmintic nor antibiotic treatment was carried out. 3.2. Faecal egg counts The results of faecal egg counts showed a very low egg production that never exceeded 50 eggs per g of faeces on average (Fig. IA). Although the values were very similar, the two groups were significantly different (P < 0.05) at the beginning of the grazing season ('Control' > 'Bolus') and in August ('Bolus' > 'Control'). No Dicty- ocaulus larvae were detected. 3.3. Pasture larval counts Fig. 1B shows the total number of larvae per kg of dry grass. The overwintering larval population was high (April). Low pasture larval counts were recorded in May-June followed by an increase from July up to the end of the pasture season. Ostertagia was the most prevalent parasite (Fig. 1C), associated with Cooperia in smaller numbers (Fig. 1D). Other parasites were occasionally observed in small numbers: Nematodirus (164 larvae in April and 66 in October), Dictyocaulus (150 larvae in July) and Trichostrongy- lus (21 larvae in May). 3.4. Pepsinogen measurements 50 At turnout, the mean levels of pepsinogen in both groups were low and close to normal values (Fig. 2A). Nevertheless, the mean values in the 'Control' group were significantly higher (P < 0.001). A 40- 30 8, ,H 20- 10 D. Kerboeuf et al./ Veterinary Parasitology 62 (1996) 107-118 111 Mar Apr May Jun Jul Aug Sep Oct Dates of sampling 8000 B 6000 "~ 4000 ._J 2000 Mar Apr May Jun Jul Aug Sep Oct Dates of sampling Fig. 1. Results of egg counts and grass analyses. (A) Mean number of eggs per g. - + - , 'Bolus group'; - • - , 'Control group'. (B) Mean total number o fL 3 per kg dry grass. (C) Mean number of Cooperia species per kg dry grass. (D) Mean number of Ostertagia species per kg dry grass, (B, C, D) - • - , near dung pats; - * - , I m from dung pats. 112 D. Kerboeuf et al. / Veterinary Parasitology 62 (1996) 107-118 cO ._J 03 o) t~ co _J t~ Q. O O o 8000 / 6000] 4000- 2000- 8000 6000 i 4000 2000 ! 0 j C M'ar Apt May Jun Jul Aug Sep O'ct Dates of sampling D A / I f - - ~ - -~ M'ar Apr May Jun Jul Aug Sep Oct Dates of sampling Fig. l (continued). In both groups, two rises, corresponding to the variations in the number of Osterta- gia, were observed during the grazing season: one from April to the end of July with a maximum in June (> 1600 mU on average) and the other from August to mid-October with a maximum in September (> 1800 mU on average). No significant differences were seen between the two groups. 3.5. Gastrin measurements No significant differences in the gastrin levels were detected and variations were similar in both groups (Fig. 2B). Two months after the animals turned out, the gastrin concentrations peaked to 400 pg ml- 1, and then dropped slightly to stabilize around 300 pg ml- ~. In the last part of the season a slight divergence was observed between both groups of calves as the gastrin values tend to decrease in the 'Bolus' group whereas they slightly increased in the 'Control' group. D. Kerboeuf et al. / Veterinary Parasitology 62 (1996) 107-118 113 2500- 2000- ~ 1500- v r - 0 c 1000 &. 500- Mar Apr May dun Jul Aug sop Oct Dates of sampling 500- B 400- ~ j E "~ 300- .=_ 200- (5 100~ Mar AI3r May Jun Jul Aug Sep Oct Dates of sampling Fig. 2. Results of pepsinogen (A) and gastrin (B) analyses. - + -, 'Bolus group'; - • -, 'Control group'. The variations in the gastrin concentrations during the grazing season were also similar to the changes observed in the pepsinogen levels and a positive and significant correlation ( r= 0.60, df= 14, P < 0.02) was found between the gastrin and the pepsinogen values. 3.6. EL1SA tests (Dictyocaulosis) The high values of the coefficients of variation in both groups expressed a large heterogeneity of the individual responses (Fig. 3). When compared with the antibody levels of similar aged cattle never infected with Dictyocaulus (mean optical density 0.277), these animals could be considered negative when the experiment started. The antibody levels quickly rose thereafter and remained at a relatively stable level until the end of the experiment. No significant differences were recorded between the two groups at any date. 114 D. Kerboeuf et al. / Veterinary Parasitology 62 (1996) 107-118 0.45 . . . . 0.4 J - - i - -+ "u 0.354 : J - " -2~- ! ~- ! ,re" © , 0.31 / / o.2 1_ "/, - - , - - - - - T . . . . . . . . . r r - -~ Mar Apr May Jun Jul Aug Sep O'ct Dates of sampling Fig. 3. Results of ELISA tests (Dictyocaulus). - + - , 'Bolas group'; - • - , 'Control group'. 3.7. Histology The main mucosal lesions were found in the fundic region of the abomasum in both groups of calves and only minor differences were detected in mucosal histopathology between groups (Table 1). The damage to the surface epithelium and the fundic glands appeared more frequent and more severe in the calves from the 'Bolus' group. In two animals of this group, an apparent reduction in the density of parietal cells was also noticed by comparison with the other group. Additionally, signs of an inflammatory reaction (oedema and cellular infiltration of the mucosa) were also more pronounced in the animals previously treated with the bolus. The density of mast cells in the fundic and in the pyloric mucosa was comparable in both groups of animals and no statistically significant differences were detected. The number of eosinophils in the two parts of the fundus in the 'Bolus' group was nearly twice the values observed in the 'Control' one. However again the difference was not Table 1 Mean number of mastocytes per mm 2 of mucosae area ( * ) and mean number of eosinophils per mm 2 of mucosae area ( * * ), four calves per group Groups Fundus Pylorus Anterior Posterior Anterior Posterier part part part part Control * 3.18 (1.0) 5.78 (1.65) 2.58 (0.73) 3.63 (1.63) • * 1.48 (1.18) 6.00 (3.63) 4.48 (2.83) 6.43 (1.58) Bolus * 2.25 (0.78) 5.43 (0.80) 1.68 (1.35) 3.18 (1.20) ' " 2.88 (3.10) 10.1 (5.35) 2.75 (0.88) 6.58 (2.83) Standard deviations are given in parentheses. D. Kerboeuf et al. / Veterinary Parasitology 62 (1996) 107-118 Table 2 Mean worm burdens for the two groups of calves (four calves per group) 115 Nematodes Control group Bolus group No. of worms No. of worms Dictyocaulus viviparus Adults 0.5 (0.2) 16 (4) 4th stages 3 (3) 12 (3) Ostertagia spp. Adults 30482 (11015) 20805 (4866) 4th stages 7647 (2488) 4297 (625) Cooperia spp. Adults 0 127 (85) 4th stages 0 637 (495) Nematodirus helvetianus Adults 0 0 4th stages 7 (7) 0 Mean of worms 38141 (12913) 25896 (5691) Standard errors given in parentheses. significant. In the other regions of the abomasal mucosa the data were similar for both groups. 3.8. Worm counts The gastrointestinal worm burdens in both groups (Table 2) were high with no significant differences between the two groups. Ostertagia was the most prevalent parasite. No inhibited larvae were found. 110- _.z 90- ._> _~ 70- E o 50 .=- 30- t - ._~ 10- -10 Mar Apr May Jun Jul Aug Sep Oct Dates of sampling Fig. 4. Cumulative weight gains (kg). - + - , 'Bolus group'; - • - , 'Control group'. 116 D. Kerboeuf et al. / Veterinary Parasitology 62 (1996) 107-118 Small numbers of Dictyocaulus were found in both groups. Nevertheless, the number of worms in the 'Bolus' group was significantly higher (P < 0.01 for the adult worms and P < 0.05 for the immature parasites). 3.9. Weight During the first part of the grazing season, from the end of May to the end of July, the average weight gains in the 'Control' group were significantly higher than in the 'Bolus' group (P < 0.01). The main difference (41 kg vs. 26 kg) was observed in June-July (Fig. 4). From the end of August to the end of the trial, the differences were reversed, the weight gains of the 'Bolus' group becoming higher, although not significantly different, than that of the 'Control' group. The total weight gains of the 'Control' group were slightly higher than that of the 'Bolus' group (93 kg vs. 89 kg) but the difference was not statistically significant. 4. Discussion During the two grazing seasons, the animals were infected with the same parasites except for Trichostrongylus which were not found during the first year, and Nema- todirus found in very small numbers in the second year and only in the 'Control' group. Thus, the animals must have had a previous antigenic stimulation at least with Ostertagia and Cooperia. For Dictyocaulus, it is highly probable that the 'Bolus' group was not infected during the first season (Le Stang and Hubert, 1995). The levels of infection during the first season differed between the groups. If a threshold of antigenic stimulation does exist, as suggested by Ploeger et al. (1990), then the response can differ from one parasite to another between the two groups. The results of post mortem examinations effectively showed that the antigenic threshold was probably reached during the first year for some species of parasites provided that the treatment did not decrease the infection to too low a level. The level of Ostertagia exposure was probably not high enough in either of the two groups as all the calves were infected, albeit at a modest level, during the second year. The results were different for Cooperia: the worm burden of the 'Bolus' calves during the first year were very low while in the 'Control' ones it exceeded 6000 worms (Le Stang and Hubert, 1995). As a consequence, this previous infection of the 'Control' group probably allowed them to develop immunity during Year 2 whereas the opposite was observed in the 'Bolus' group where infection was present only during the second year. Beside the worm burdens, the pathological effects of parasites even in small number can differ according to the inflammatory response. Several parameters have been used to assess the damages due to parasites. Those in relation to gastrointestinal parasitism were in good agreement with the results of the post mortem examinations. When the animals were turned out in Year 2, the pepsinogen values correlated with the infection at the end of the previous year. The values in the 'Control' group, although close to normal values, were significantly higher than in the 'Bolus' group. The gastrin concentrations were D, Kerboeuf et al./ Veterinary Parasitology 62 (1996) 107-118 117 comparable with those usually described in 1-year-old parasite naive calves (Yasuda et al., 1986; Berghen et al., 1993). Thereafter, the gastrin levels were similar to that observed by Berghen et al. (1990) in second season cattle and appeared indicative of a subclinical level of parasitism (Hilderson et al., 1992). The gastrin concentrations were similar in both groups during most of the study except for the end of the grazing season when gastrin level decreased in the 'Bolus' group whereas it slightly increased in the 'Control' group. As the variations in gastrin levels are mainly related to the presence of adult worms (Fox et al., 1987), the results suggest a more rapid elimination of Ostertagia in the 'Bolus' group. A relationship between gastrin and pepsinogen values has already been described in first season calves (Berghen et al., 1989, 1993). The present results confirm that the relationship also exists in second season cattle. The main parasitological effects in the 'Bolus' group were due to Dictyocaulus against which these cattle were less protected due to the non-existent or only low infection during the first grazing season. The lung infection was responsible for the clinical signs and, probably, for the slowing in weight gains in the middle of the season. ELISA tests showed that the infection was early as the rise in the antibody levels was observed as soon as April-May. Nevertheless, this increase is more correlated with the infection of cattle than with the immune response (Duncan et al., 1992). The response to Dictyocaulus is different from what is observed with other nematodes: few worms allow immunity to develop as well as clinical signs to appear. In other trials, where the bolus treated calves had presumably greater exposure, they developed sufficient immunity to protect them from clinical disease when given a heavy experimental challenge in Year 2 (Downey et al., 1993). In fact, only slight differences were observed between the two groups. The 'Control' group was more infected, at least in the first part of the season. The 'Bolus' group, more naive at the beginning of the second season, developed the heaviest reactions (clinical signs, histological damages, inflammatory cells). Nevertheless, especially at the end of the season, the functional consequences in this group looked less intense, as shown by lower pepsinogen and gastrin levels. The weight gains were in accordance with the response of the animals to parasitic infection. The 'Bolus' group, of which growth was slowed in the middle of grazing season, gained on the 'Control' group at the end of the trial, thanks to compensatory weight gains. Acknowledgements Special thanks are due to Distrivet Company (Dr. J.P. Willemart) for financial support of the experiment, to Dr. E. Abbott (Hoechst Company) for careful examination of the manuscript, to Professor J. Duncan and Dr. J. Mc Keand from the University of Glasgow for the ELISA tests, to Professor J. Vercruysse and Dr. P. Berghen who provide advice and references for the dosage of gastrin, to Professor J.A. Nicolas and G. Dubost from the Veterinary Laboratories in Limoges for the post mortem examinations. We also wish to thank M.D. Andr6 and G. 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