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The influence of a Cooperia oncophora priming on a concurrent challenge with Ostertagia ostertagi and C. oncophora in calves
veterinary parasitology ELSEVIER
Veterinary Parasitology70 (1997) 143-151
The influence of a Cooperia oncophora priming on a concurrent challenge with Ostertagia ostertagi and C. oncophora in calves P. Domy a,*, E. Claerebout a, j. Vercruysse a, H. Hilderson a,l, J.F. Huntley b a Department of Parasitology, Faculty of Veterinary Medicine, Salisburylaan 133, B9820 Merelbeke, Belgium b Moredun Research Institute, 408 Gilmerton Road, Edinburgh EH17 7JH, UK Received 23 August 1996; accepted 14 November 1996
Abstract The development of immunity to Ostertagia ostertagi and Cooperia oncophora and interactions between these species were investigated in experimentally infected calves. Parasitological, serological and histological parameters were used for assessing immune responses. No conclusive evidence of an effect of C. oncophora on the course of an O. ostertagi infection in calves could be shown. Following a challenge with C. oncophora and O. ostertagi of C. oncophora primed calves, no significant reductions in establishment rate, faecal egg counts, worm length or the percentage of early fourth stage larvae could be demonstrated. Results also confirmed earlier work showing the very different degrees of immunity conferred following immunisation with either C. oncophora or O. ostertagi. While a protective immunity was generated in the case of C. oncophora, continuous infection of calves with 420000 L 3 of O. ostertagi during almost 5 months induced immune reactions which affected growth and fecundity of the worms but not the establishment rate.
Keywords: Ostertagia ostertagi; Cooperia oncophora; Cattle-Nematoda;Immunity-Nematoda
1. Introduction Ruminants on pasture are exposed to gastrointestinal nematodes of several species. Interactions, either positive or negative, immunological a n d / o r pathophysiological, have
* Correspondingauthor. i Present address: Hoechst Belgium N.V., Charleroisesteenweg 111-113, B1060 Brussels, Belgium. 0304-4017/97/$17.00 Copyright © 1997 Elsevier ScienceB.V. All rights reserved. PH S0304-4017(96)01142-9
144
P. Dorny et al. / Veterinao' Parasitology 70 (1997) 143-151
been observed between many parasite species (Turner et al., 1962; Dineen et al., 1977; Kloosterman et al., 1984, 1989; Coop et al., 1988; Dobson et al., 1992; Emery et al., 1993). In temperate regions the most common nematodes of cattle are Ostertagia ostertagi and Cooperia oncophora, which live in the abomasum and the small intestine, respectively. They generally occur concurrently but differ in fecundity, pathogenicity and the immunological relationship with their host. Kloosterman et al. (1984) demonstrated a reciprocal negative interaction between these nematode species. It was suggested that this interaction is immune mediated and caused by antigenic substances shared by both species (Frankena, 1987). Satrija and Nansen (1993) and Hilderson et al. (1995) failed to show any protective immunity towards O. ostertagi in calves which had previously been infected with C. oncophora during 17 and 19 weeks, respectively. However, the latter authors noticed that priming with C. oncophora induced the presence of globular leukocytes in the abomasal mucosa. This cell type has been associated with resistance to gastrointestinal nematodes in sheep (Miller, 1984; Douch et al., 1986). The aim of the present experiment was to investigate whether a challenge with C. oncophora in C. oncophora primed calves would elicit immunological reactions which might affect a challenge with O. ostertagi, given concurrently. This was studied using parasitological, serological and histological parameters.
2. Materials and methods
Twenty-four Holstein bull calves, 5-7 months old and reared under worm-free conditions were selected for this experiment. They were individually housed and fed maize silage and soy based concentrate. The calves were allocated to four similar groups of six animals on the basis of age (groups C-C, C-CO, N-O and O-O). During the priming period, calves in groups C-C and C-CO received 20000 third stage larvae ( L 3) week i of C. oncophora, group O-O calves were infected with 20000 L 3 week-1 of O. ostertagi, and group N-O remained uninfected. The weekly infections were spread equally over 3 days (Monday, Wednesday, Friday) and the priming period lasted 21 weeks (Days 1-144). Following the last infection on Day 144, all calves were treated with oxfendazole at 9.0 mg kg -1 for 3 consecutive days (Days 147-149). From Days 154 to 165, calves of all groups were challenged with a total of 156000 L 3 of C. oncophora for group C-C, 156000 L 3 of O. ostertagi for groups N-O and O-O or 156000 L 3 of each for group C-CO. The challenge was administered daily as follows: 4000 L 3 o n Days 154-156, 8000 L 3 o n Days 157-159, 16000 L 3 o n Days 160-162 and 24000 L 3 o n Days 163-165. Faecal and blood samples were taken every week. Twenty-four days following the last challenge infection (Day 189) all calves were necropsied for worm counts. The strains of O. ostertagi and C. oncophora were initially isolated from a commercial dairy farm in 1987 and 1990, respectively, and were maintained in the laboratory by routine passage through nematode-free calves. Third-stage larvae of O. ostertagi and C. oncophora were collected from faecal culture by baermannisation and stored at 10°C until further use. Larvae administered to the calves were less than 10 weeks old.
P. Dorny et al. / Veterinary Parasitology 70 (1997) 143-151
145
Faecal egg counts were estimated using a modified McMaster technique (Thienpont et al., 1979). Necropsy, abomasal and intestinal washings and abomasal digests (HC1pepsin) were done according to standard techniques (Ritchie et al., 1966; Anonymous, 1986). In total, 2% of the worm burden was counted. The lengths of 50 adult Ostertagia from each calf were measured. Serum pepsinogen concentration was determined according to Berghen et al. (1987) and expressed as mU tyrosine ml-1 O. ostertagi and C. oncophora IgG antibodies were determined with an antibody enzyme-linked immunosorbent assay (ELISA) (Canals and Gasbarre, 1990), using crude adult worm soluble extract for O. ostertagi and crude fourth stage soluble extract for C. oncophora. One dilution was used (1/400) for each serum in duplicate. Antibody concentrations are expressed as optical density (OD) at 492 nm. Tissues from the abomasal and small intestinal wall were taken at necropsy before washing and fixed in 4% paraformaldehyde and in Carnoy's fixative. For the quantitative observation of mucosal mast cells a toluidine-blue staining technique (pH 0.5) was used. Eosinophils and globular leucocytes were observed on haematoxylin-eosin stained sections. The number of each cell type was estimated on a total surface of 2 mm 2. Egg counts and worm burdens are expressed as geometric means after transformation to In (count + 1). Significance of differences between groups was calculated on In transformed data using ANOVA. Tukey's HSD multiple range test was used as post hoc test. The Kruskal-Wallis test was used for analysis of cell counts. Probability values of P < 0.05 were considered to indicate significant differences.
3. Results The weekly mean faecal egg counts of the four groups are shown in Fig. I(A). Three weeks post-infection all calves of groups C-C, C-CO and O-O were shedding strongyle eggs in their faeces. The egg count profiles of these groups were similar in the first half of the priming period: the highest faecal egg counts were observed in the three groups
Table 1
Ostertagia ostertagi burdens and measurements of groups C-CO, N-O and O-O Group
O. ostertagi burden a
N-O
92335a (66850-124850) 75436a (61450-103750) 67035a (45300-107900)
Establishment rate
% L4
(%)
C-CO O-O
59.2a
41.4a
48.4a
50.4a
43.0a
73.0b
Length b (mm) Female
Male
7.06a (+0.18) 6.95a (+0.19) 6.45b ( -t-0.23)
5.94b (+0.16) 5.77ab (+0.10) 5.63a ( + 0.22)
a Range in parentheses. b Mean 5: SD. Means in the same column followed by different letters are significantly different at P < 0.05.
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P. Dorny et al. / Veterinary. Parasitology 70 (1997) 143-151 1800
_~--C-C
1600
-.
•
~ - -
-- N-O
C-CO
•
O-O
A
1400 1200
1000 W
8OO 800 4OO 2OO 0
r
4500 4000 3500 3000 2500 2000 1500 1000 5OO 0
-10
I
!
I
~
10
30
50
70
I
f
I
!
I
I
90 110 130 150 170 190 Days Tr
i n f e cDt0-144 i o n sP ~rm i Jary~ l ~ ' ~ a l l e n g e " "
Fig. 1. Geometric mean faecal egg counts (A) and mean serum pepsinogen concentrations (B) in four groups of calves.
on Day 50, after which they decreased in all groups. In the second half, geometric mean egg counts of the Cooperia primed groups (C-C, C-CO) were less than 50 eggs per gram (EPG), whereas the Ostertagia infected group (O-O) fluctuated around 100 EPG. Following anthelmintic treatment all counts became zero. After challenge faecal egg counts rose steadily to reach by Day 189, geometric means o f 1670 (range 6 0 0 - 3 2 5 0 ) EPG and 1168 (550-2350) EPG in groups N-O and C-CO, respectively. In the homologous challenged groups, faecal egg output post-challenge was low, 4 ( 0 - 2 5 ) EPG in the C-C and 60 ( 2 5 - 1 5 0 ) EPG in the O-O groups on Day 189. Statistical analysis of the Ostertagia-challenged groups showed significant differences in egg
147
P. Dorny et al. / Veterinary Parasitology 70 (1997) 143-151
counts on Day 189 ( P < 0.004) and of the post-challenge cumulative egg counts ( P < 0.001) between the three groups. However, no significant differences between the C-CO and N-O groups were found. Data on worm burdens at slaughter and worm measurements are given in Table 1. The C. oncophora burdens are not shown because they were very low (less than 500), in groups C-C and C-CO. No significant differences could be shown in the total Ostertagia population and in the establishment rate. However, the percentage of early fourth stage larvae (EL 4) ( P < 0.01) and adult male ( P < 0.05) and female worm length ( P < 0.01) were significantly affected by the priming history, the O-O group showing a significantly higher percentage of L 4 and shortened worms compared with the C-CO and N-O groups.
----o~C-C
- ~ - -
C-CO
--
-N-O
•
-
O-O
1,8
A
1,6
1,4 1,2
1 0,8 0,6 0,4 0,2 I
I
I
I
I
I -----
I
I
I
I
----4
I= 1,4 1,2
O 1 0,8 0,6 0,4 0,2 0
-10
I
I
I
I
I
10
30
50
70
90
Days
I
~
I
110 130 150 170 190 Tr
0-144 Primary infections ~ l ~ a l l e n g e Fig. 2. Mean serum antibody concentrations of four groups of calves to soluble extract of fourth stage C. oncophora (A) and soluble extract of adult O. ostertagi (B) in an ELISA.
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P. Dorny et aL / Veterina~ Parasitology 70 (1997) 143-151
Mean serum pepsinogen concentrations of the four groups are presented in Fig. I(B). Concentrations rose quickly to high values (over 4000 mU tyrosine) during priming with Ostertagia in group O-O and decreased after treatment. In the Cooperia-primed groups (C-C and C-CO) a slight increase of pepsinogen concentration was noted. After challenge all Ostertagia-challenged groups showed a quick increase to 4000-4500 mU tyrosine. Antibody concentrations to Ostertagia and Cooperia are shown in Fig. 2(A)Fig. 2(B), respectively. Specific antibody responses were found in both the Cooperia and Ostertagia primed groups, reaching a plateau approximately 3 months after the first infection. Slight cross-reacting antibodies were found in both assays. The mean number of mucosal mast cells in sections of the abomasum was significantly higher in the O-O group (73 per 2 mm 2) compared with the other groups (34-39 per 2 mm 2) ( P < 0.005). No differences were found between the groups in the number of globular leucocytes and eosinophils.
4. Discussion No conclusive evidence of an effect of C. oncophora on the course of an O. ostertagi infection in calves could be shown in the present study. Following a challenge with C. oncophora and O. ostertagi of C. oncophora primed calves, we were not able to demonstrate significant reductions in establishment rate, faecal egg counts, worm length or %EL 4. However, there was a trend to lower values of these parasitological parameters compared with naive calves (N-O group). This was also demonstrated by Hilderson et al. (1995) in Cooperia immunised calves following a challenge with Ostertagia. There were also cross-reacting antibodies, both in the Cooper& and in the Ostertagia ELISA. Whether these antibodies have any direct relation to resistance or are merely indicators for the developing immune response still needs to be resolved (Hilderson et al., 1995). Ploeger et al. (1995) noted that antibody responses against O. ostertagi, which are probably T-cell mediated, do occur long before protective immunity against this species becomes evident. The present study also confirmed the very different degrees of immunity conferred to homologous challenge following immunisation with either C. oncophora or O. ostertagi (Armour, 1989; Michel et al., 1973). While a protective immunity was generated in the case of C. oncophora, continuous infection of calves with 420000 L 3 of O. ostertagi during almost five months induced immune reactions which only affected growth and fecundity of the worms but not the establishment rate. Ploeger et al. (1995) demonstrated that the development of immunity to O. ostertagi depends both on magnitude and duration of exposure. They also found a quantitative relationship between the establishment of the challenge with O. ostertagi and the level of infection to which calves had been previously exposed. Protective immunity was only conferred after immunisation with more than 900000 L 3 of O. ostertagi over a 5 month period. Thus, it appears that protective immunity in ostertagiosis develops slowly and is not likely to be affected by a previous immunisation by C.
oncophora. Dineen et al. (1977) suggested that the final rejection mechanism in immunologically mediated interactions is not antigen-specific. On the other hand the 'trigger' for such a
P. Dorny et al. / Veterinary Parasitology 70 (1997) 143-151
149
reaction is antigen specific. The same authors demonstrated that in sheep a non-specific immune reaction is only possible for worm species which share the same or 'downstream' niches in the gut but has no effect on species in 'upstream' localisations. These findings might explain the lack of protective immunity conferred by a C. oncophora immunisation and challenge on the establishment of the abomasal O. ostertagi. However, Hilderson et al. (1995) showed that, following a O. ostertagi challenge, a higher number of globular leucocytes were present in the abomasal mucosa of C. oncophora immunised calves than in uninfected controls. These observations suggested an immunological reaction in the abomasum provoked by an intestinal C. oncophora immunisation. In the present study no differences in the number of globular leucocytes were demonstrated between the Ostertagia challenged groups. This could be due to the setup of the present experiment. Indeed, all animals were slaughtered 24 days following a heavy challenge spread over 12 days, which may be sufficient to elicit an increase in the number of mucosal globular leucocytes, even in previously non-sensitised animals and therefore may have masked histological changes resulting from priming (Claerebout et al., 1996). Also the role of globular leucocytes in the immune mechanism of ruminants against worm infections is not well understood. In sheep and cattle the increase in the numbers of globular leucocytes has been associated with the development of resistance to a worm infection (Jarrett et al., 1967; O'Sullivan and Donald, 1973; Miller, 1984). In sheep, Stear et al. (1995) demonstrated a significant negative relationship between the number of globular leucocytes and the number of Ostertagia circumcincta. However, Huntley et al. (1995) showed that in contrast to sheep, in goats an increase in the proportion of globular leucocytes following nematode infections did not result in an expulsion of worms. Differences in the functional activity of the globular leucocytes between ruminant species are therefore not excluded. Also it was shown in laboratory animals that differences in effector mechanisms exist for different worm species. In rats, mucosal mast cells are involved in the expulsion of Strongyloides ratti, while goblet cells are involved in the case of Nippostrongylus brasiliensis (Nawa et al., 1994). In conclusion no effect of a C. oncophora immunisation on the establishment and fecundity of an experimental infection of O. ostertagi, given concurrently with C. oncophora, could be demonstrated. Therefore, it is unlikely that on pastures contaminated with these two trichostrongyle species the uptake of C. oncophora in the early part of the grazing season will affect the establishment rate of O. ostertagi in the second half of the season.
Acknowledgements The research presented in this paper was supported by an IWONL grant (no. 5399A, section O). The authors would like to thank A. Dereu, P. Meeus, M. Bouch6 and L. Braem for technical assistance, and D.J. Shaw for help in the statistical analysis.
References Anonymous, 1986. Manual of Veterinary Parasitological Laboratory Techniques, 3rd edn., Ref. book 418. Ministry of Agriculture, Fisheries and Food, UK, 160 pp.
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Armour, J., 1989. The influence of host immunity on the epidemiology of trichostrongyle infections in cattle. Vet. Parasitol., 32: 5-19. Berghen, P., Dorny, P. and Vercruysse, J., 1987. Evaluation of a simplified blood pepsinogen assay. Am. J. Vet. Res., 48: 664-669. Canals, A. and Gasbarre, L.C., 1990. Ostertagia ostertagi: isolation and partial characterization of somatic and metabolic antigens. Int. J. Parasitol., 20: 1047-1054. Claerebout, E., Hilderson, H., Meeus, P., de Marez, T., Behnke, J., Huntley, J. and Vercruysse, J., 1996. The effect of truncated infections with Ostertagia ostertagi on the development of acquired resistance in calves. Vet. Parasitol., 66: 225-239. Coop, R.L., Jackson, F., Graham, R.B. and Angus, K.W., 1988. Influence of two levels of concurrent infections with Ostertagia circumcincta and Trichostrongylus Htrinus on the growth performance of lambs. Res. Vet. Sci., 45: 275-280. Dineen, J.K., Gregg, P., Windon, R.G., Donald, A.D. and Kelly, J.D., 1977. The role of immunologically specific and non-specific components of resistance in cross-protection to intestinal nematodes. Int. J. Parasitol., 7: 211-215. Dobson, RJ., Barnes, E.H. and Windon, R.G., 1992. Population dynamics of Trichostrongylus colubriformis and Ostertagia circumcincta in single and concurrent infections. Int. J. Parasitol., 22: 997-1004. Douch, P.G.C., Harrison, G.B.L., Elliott, D.C., Buchanan, L.L. and Greer, K.S., 1986. Relationship of gastrointestinal histology and mucus antiparasite activity with the development of resistance to trichostrongyle infection in sheep. Vet. Parasitol., 20: 315-331. Emery, D.L., Wagland, B.M. and McClure, S.J., 1993. Rejection of heterologous nematodes by sheep immunized with larval or adult Trichostrongylus colubriformis. Int. J. Parasitol., 23: 841-846. Frankena, K., 1987. The interaction between Cooperia spp. and Ostertagia spp. (Nematoda: Trichostrongylidae) in cattle. Ph.D. Thesis, Agricultural University Wageningen, Netherlands, 101 pp. Hilderson, H., Vercruysse, J., Claerebout, E., de Graaf, D.C., Fransen, J. and Berghen, P., 1995. Interactions between Ostertagia ostertagi and Cooperia oncophora in calves. Vet. Parasitol., 56:107-119. Huntley, J.F., Patterson, M., MacKellar, A., Jackson, F., Stevenson, L.M. and Coop, R.L., 1995. A comparison of the mast cell and eosinophil responses of sheep and goats to gastrointestinal nematode infections. Res. Vet. Sci., 58: 5-10. Jarrett, W.F.H., Miller, H.R.P. and Murray, M., 1967. The relationships between mast cells and globule leucocytes in parasitic infections. Vet. Rec., 80: 505-596. Kloosterman, A., Albers, G.A.A. and van der Brink, R., 1984. Negative interactions between Ostertagia ostertagi and Cooperia oncophora in calves. Vet. Parasitol., 15: 155-163. Kloosterman, A., Frankena, K. and Ploeger, H.W., 1989. Increased establishment of lungworms (Dictyocaulus uiviparus) in calves after previous infections with gastrointestinal nematodes (Ostertagia ostertagi and Cooperia oncophora). Vet. Parasitol., 33: 155-163. Michel, J.F., Lancaster, M.B. and Hong, C., 1973. Ostertagia ostertagi: protective immunity in calves, The development in calves of a protective immunity to infection with Ostertagia ostertagi. Exp. Parasitol., 33: 179-186. Miller, H.R.P., 1984.The protective mucosal response against gastrointestinal nematodes in ruminants and laboratory animals. Vet. Immunol. Immunopathol., 6: 167-259. Nawa, Y., Ishikawa, N., Tsuchiya, K., Horii, Y., Abe, T., Khan, A.I., Bing-Shi, Itoh, H., Ide, H. and Uchiyama, F., 1994. Selective effector mechanism for the expulsion of intestinal helminths. Parasite Immunol., 16: 333-338. O'Sullivan, B.M. and Donald, A.D., 1973. Responses to infection with Haemonchus contortus and Trichostrongylus colubriformis in ewes of different reproductive status. Int. J. Parasitol., 3: 521-530. Ploeger, H.W., Kloosterman, A. and Rietveld, F.W., 1995. Acquired immunity against Cooperia spp. in calves: effect of level of exposure and timing of the midsummer increase. Vet. Parasitol., 58: 61-74. Ritchie, J.S.D., Anderson, N., Armour, J., Jarret, W.F.H., Jennings, F.W. and Urquhart, G.M., 1966. Experimental Ostertagia ostertagi infections in calves: parasitology and pathogenesis of a single infection. Am. J. Vet. Res., 27: 659-667. Satrija, F. and Nansen, P., 1993. Experimental concurrent infections with Ostertagia ostertagi and Cooperia oncophora in the calf. Res. Vet. Sci., 55: 92-97. Stear, M.J., Bishop, S.C., Doligalska, M., Duncan, J.L., Holmes, P.H., Irvine, J., McCririe, L., McKellar,
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Q.A., Sinski, E. and Murray, M., 1995. Regulation of egg production, worm burden, worm length and worm fecundity by host responses in sheep infected with Ostertagia circumcincta. Parasite Immunol., 17: 643-652. Thienpont, D., Rochette, F., van Parijs, O.F.J., 1979. Diagnosing helminthiasis by coprological examination. Janssen Research Foundation, Beerse, Belgium, 187 pp. Turner, J.H., Kates, K.C. and Wilson, G.I., 1962. The interaction of concurrent infections of the abomasal nematodes, Haemonchus contortus, Ostertagia circumcincta and Trichostrongylus axei (Trichostrongylidae) in Iambs. Proc. Helminthol. Soc. Wash., 29: 210-216.
Veterinary Parasitology70 (1997) 143-151
The influence of a Cooperia oncophora priming on a concurrent challenge with Ostertagia ostertagi and C. oncophora in calves P. Domy a,*, E. Claerebout a, j. Vercruysse a, H. Hilderson a,l, J.F. Huntley b a Department of Parasitology, Faculty of Veterinary Medicine, Salisburylaan 133, B9820 Merelbeke, Belgium b Moredun Research Institute, 408 Gilmerton Road, Edinburgh EH17 7JH, UK Received 23 August 1996; accepted 14 November 1996
Abstract The development of immunity to Ostertagia ostertagi and Cooperia oncophora and interactions between these species were investigated in experimentally infected calves. Parasitological, serological and histological parameters were used for assessing immune responses. No conclusive evidence of an effect of C. oncophora on the course of an O. ostertagi infection in calves could be shown. Following a challenge with C. oncophora and O. ostertagi of C. oncophora primed calves, no significant reductions in establishment rate, faecal egg counts, worm length or the percentage of early fourth stage larvae could be demonstrated. Results also confirmed earlier work showing the very different degrees of immunity conferred following immunisation with either C. oncophora or O. ostertagi. While a protective immunity was generated in the case of C. oncophora, continuous infection of calves with 420000 L 3 of O. ostertagi during almost 5 months induced immune reactions which affected growth and fecundity of the worms but not the establishment rate.
Keywords: Ostertagia ostertagi; Cooperia oncophora; Cattle-Nematoda;Immunity-Nematoda
1. Introduction Ruminants on pasture are exposed to gastrointestinal nematodes of several species. Interactions, either positive or negative, immunological a n d / o r pathophysiological, have
* Correspondingauthor. i Present address: Hoechst Belgium N.V., Charleroisesteenweg 111-113, B1060 Brussels, Belgium. 0304-4017/97/$17.00 Copyright © 1997 Elsevier ScienceB.V. All rights reserved. PH S0304-4017(96)01142-9
144
P. Dorny et al. / Veterinao' Parasitology 70 (1997) 143-151
been observed between many parasite species (Turner et al., 1962; Dineen et al., 1977; Kloosterman et al., 1984, 1989; Coop et al., 1988; Dobson et al., 1992; Emery et al., 1993). In temperate regions the most common nematodes of cattle are Ostertagia ostertagi and Cooperia oncophora, which live in the abomasum and the small intestine, respectively. They generally occur concurrently but differ in fecundity, pathogenicity and the immunological relationship with their host. Kloosterman et al. (1984) demonstrated a reciprocal negative interaction between these nematode species. It was suggested that this interaction is immune mediated and caused by antigenic substances shared by both species (Frankena, 1987). Satrija and Nansen (1993) and Hilderson et al. (1995) failed to show any protective immunity towards O. ostertagi in calves which had previously been infected with C. oncophora during 17 and 19 weeks, respectively. However, the latter authors noticed that priming with C. oncophora induced the presence of globular leukocytes in the abomasal mucosa. This cell type has been associated with resistance to gastrointestinal nematodes in sheep (Miller, 1984; Douch et al., 1986). The aim of the present experiment was to investigate whether a challenge with C. oncophora in C. oncophora primed calves would elicit immunological reactions which might affect a challenge with O. ostertagi, given concurrently. This was studied using parasitological, serological and histological parameters.
2. Materials and methods
Twenty-four Holstein bull calves, 5-7 months old and reared under worm-free conditions were selected for this experiment. They were individually housed and fed maize silage and soy based concentrate. The calves were allocated to four similar groups of six animals on the basis of age (groups C-C, C-CO, N-O and O-O). During the priming period, calves in groups C-C and C-CO received 20000 third stage larvae ( L 3) week i of C. oncophora, group O-O calves were infected with 20000 L 3 week-1 of O. ostertagi, and group N-O remained uninfected. The weekly infections were spread equally over 3 days (Monday, Wednesday, Friday) and the priming period lasted 21 weeks (Days 1-144). Following the last infection on Day 144, all calves were treated with oxfendazole at 9.0 mg kg -1 for 3 consecutive days (Days 147-149). From Days 154 to 165, calves of all groups were challenged with a total of 156000 L 3 of C. oncophora for group C-C, 156000 L 3 of O. ostertagi for groups N-O and O-O or 156000 L 3 of each for group C-CO. The challenge was administered daily as follows: 4000 L 3 o n Days 154-156, 8000 L 3 o n Days 157-159, 16000 L 3 o n Days 160-162 and 24000 L 3 o n Days 163-165. Faecal and blood samples were taken every week. Twenty-four days following the last challenge infection (Day 189) all calves were necropsied for worm counts. The strains of O. ostertagi and C. oncophora were initially isolated from a commercial dairy farm in 1987 and 1990, respectively, and were maintained in the laboratory by routine passage through nematode-free calves. Third-stage larvae of O. ostertagi and C. oncophora were collected from faecal culture by baermannisation and stored at 10°C until further use. Larvae administered to the calves were less than 10 weeks old.
P. Dorny et al. / Veterinary Parasitology 70 (1997) 143-151
145
Faecal egg counts were estimated using a modified McMaster technique (Thienpont et al., 1979). Necropsy, abomasal and intestinal washings and abomasal digests (HC1pepsin) were done according to standard techniques (Ritchie et al., 1966; Anonymous, 1986). In total, 2% of the worm burden was counted. The lengths of 50 adult Ostertagia from each calf were measured. Serum pepsinogen concentration was determined according to Berghen et al. (1987) and expressed as mU tyrosine ml-1 O. ostertagi and C. oncophora IgG antibodies were determined with an antibody enzyme-linked immunosorbent assay (ELISA) (Canals and Gasbarre, 1990), using crude adult worm soluble extract for O. ostertagi and crude fourth stage soluble extract for C. oncophora. One dilution was used (1/400) for each serum in duplicate. Antibody concentrations are expressed as optical density (OD) at 492 nm. Tissues from the abomasal and small intestinal wall were taken at necropsy before washing and fixed in 4% paraformaldehyde and in Carnoy's fixative. For the quantitative observation of mucosal mast cells a toluidine-blue staining technique (pH 0.5) was used. Eosinophils and globular leucocytes were observed on haematoxylin-eosin stained sections. The number of each cell type was estimated on a total surface of 2 mm 2. Egg counts and worm burdens are expressed as geometric means after transformation to In (count + 1). Significance of differences between groups was calculated on In transformed data using ANOVA. Tukey's HSD multiple range test was used as post hoc test. The Kruskal-Wallis test was used for analysis of cell counts. Probability values of P < 0.05 were considered to indicate significant differences.
3. Results The weekly mean faecal egg counts of the four groups are shown in Fig. I(A). Three weeks post-infection all calves of groups C-C, C-CO and O-O were shedding strongyle eggs in their faeces. The egg count profiles of these groups were similar in the first half of the priming period: the highest faecal egg counts were observed in the three groups
Table 1
Ostertagia ostertagi burdens and measurements of groups C-CO, N-O and O-O Group
O. ostertagi burden a
N-O
92335a (66850-124850) 75436a (61450-103750) 67035a (45300-107900)
Establishment rate
% L4
(%)
C-CO O-O
59.2a
41.4a
48.4a
50.4a
43.0a
73.0b
Length b (mm) Female
Male
7.06a (+0.18) 6.95a (+0.19) 6.45b ( -t-0.23)
5.94b (+0.16) 5.77ab (+0.10) 5.63a ( + 0.22)
a Range in parentheses. b Mean 5: SD. Means in the same column followed by different letters are significantly different at P < 0.05.
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P. Dorny et al. / Veterinary. Parasitology 70 (1997) 143-151 1800
_~--C-C
1600
-.
•
~ - -
-- N-O
C-CO
•
O-O
A
1400 1200
1000 W
8OO 800 4OO 2OO 0
r
4500 4000 3500 3000 2500 2000 1500 1000 5OO 0
-10
I
!
I
~
10
30
50
70
I
f
I
!
I
I
90 110 130 150 170 190 Days Tr
i n f e cDt0-144 i o n sP ~rm i Jary~ l ~ ' ~ a l l e n g e " "
Fig. 1. Geometric mean faecal egg counts (A) and mean serum pepsinogen concentrations (B) in four groups of calves.
on Day 50, after which they decreased in all groups. In the second half, geometric mean egg counts of the Cooperia primed groups (C-C, C-CO) were less than 50 eggs per gram (EPG), whereas the Ostertagia infected group (O-O) fluctuated around 100 EPG. Following anthelmintic treatment all counts became zero. After challenge faecal egg counts rose steadily to reach by Day 189, geometric means o f 1670 (range 6 0 0 - 3 2 5 0 ) EPG and 1168 (550-2350) EPG in groups N-O and C-CO, respectively. In the homologous challenged groups, faecal egg output post-challenge was low, 4 ( 0 - 2 5 ) EPG in the C-C and 60 ( 2 5 - 1 5 0 ) EPG in the O-O groups on Day 189. Statistical analysis of the Ostertagia-challenged groups showed significant differences in egg
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P. Dorny et al. / Veterinary Parasitology 70 (1997) 143-151
counts on Day 189 ( P < 0.004) and of the post-challenge cumulative egg counts ( P < 0.001) between the three groups. However, no significant differences between the C-CO and N-O groups were found. Data on worm burdens at slaughter and worm measurements are given in Table 1. The C. oncophora burdens are not shown because they were very low (less than 500), in groups C-C and C-CO. No significant differences could be shown in the total Ostertagia population and in the establishment rate. However, the percentage of early fourth stage larvae (EL 4) ( P < 0.01) and adult male ( P < 0.05) and female worm length ( P < 0.01) were significantly affected by the priming history, the O-O group showing a significantly higher percentage of L 4 and shortened worms compared with the C-CO and N-O groups.
----o~C-C
- ~ - -
C-CO
--
-N-O
•
-
O-O
1,8
A
1,6
1,4 1,2
1 0,8 0,6 0,4 0,2 I
I
I
I
I
I -----
I
I
I
I
----4
I= 1,4 1,2
O 1 0,8 0,6 0,4 0,2 0
-10
I
I
I
I
I
10
30
50
70
90
Days
I
~
I
110 130 150 170 190 Tr
0-144 Primary infections ~ l ~ a l l e n g e Fig. 2. Mean serum antibody concentrations of four groups of calves to soluble extract of fourth stage C. oncophora (A) and soluble extract of adult O. ostertagi (B) in an ELISA.
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P. Dorny et aL / Veterina~ Parasitology 70 (1997) 143-151
Mean serum pepsinogen concentrations of the four groups are presented in Fig. I(B). Concentrations rose quickly to high values (over 4000 mU tyrosine) during priming with Ostertagia in group O-O and decreased after treatment. In the Cooperia-primed groups (C-C and C-CO) a slight increase of pepsinogen concentration was noted. After challenge all Ostertagia-challenged groups showed a quick increase to 4000-4500 mU tyrosine. Antibody concentrations to Ostertagia and Cooperia are shown in Fig. 2(A)Fig. 2(B), respectively. Specific antibody responses were found in both the Cooperia and Ostertagia primed groups, reaching a plateau approximately 3 months after the first infection. Slight cross-reacting antibodies were found in both assays. The mean number of mucosal mast cells in sections of the abomasum was significantly higher in the O-O group (73 per 2 mm 2) compared with the other groups (34-39 per 2 mm 2) ( P < 0.005). No differences were found between the groups in the number of globular leucocytes and eosinophils.
4. Discussion No conclusive evidence of an effect of C. oncophora on the course of an O. ostertagi infection in calves could be shown in the present study. Following a challenge with C. oncophora and O. ostertagi of C. oncophora primed calves, we were not able to demonstrate significant reductions in establishment rate, faecal egg counts, worm length or %EL 4. However, there was a trend to lower values of these parasitological parameters compared with naive calves (N-O group). This was also demonstrated by Hilderson et al. (1995) in Cooperia immunised calves following a challenge with Ostertagia. There were also cross-reacting antibodies, both in the Cooper& and in the Ostertagia ELISA. Whether these antibodies have any direct relation to resistance or are merely indicators for the developing immune response still needs to be resolved (Hilderson et al., 1995). Ploeger et al. (1995) noted that antibody responses against O. ostertagi, which are probably T-cell mediated, do occur long before protective immunity against this species becomes evident. The present study also confirmed the very different degrees of immunity conferred to homologous challenge following immunisation with either C. oncophora or O. ostertagi (Armour, 1989; Michel et al., 1973). While a protective immunity was generated in the case of C. oncophora, continuous infection of calves with 420000 L 3 of O. ostertagi during almost five months induced immune reactions which only affected growth and fecundity of the worms but not the establishment rate. Ploeger et al. (1995) demonstrated that the development of immunity to O. ostertagi depends both on magnitude and duration of exposure. They also found a quantitative relationship between the establishment of the challenge with O. ostertagi and the level of infection to which calves had been previously exposed. Protective immunity was only conferred after immunisation with more than 900000 L 3 of O. ostertagi over a 5 month period. Thus, it appears that protective immunity in ostertagiosis develops slowly and is not likely to be affected by a previous immunisation by C.
oncophora. Dineen et al. (1977) suggested that the final rejection mechanism in immunologically mediated interactions is not antigen-specific. On the other hand the 'trigger' for such a
P. Dorny et al. / Veterinary Parasitology 70 (1997) 143-151
149
reaction is antigen specific. The same authors demonstrated that in sheep a non-specific immune reaction is only possible for worm species which share the same or 'downstream' niches in the gut but has no effect on species in 'upstream' localisations. These findings might explain the lack of protective immunity conferred by a C. oncophora immunisation and challenge on the establishment of the abomasal O. ostertagi. However, Hilderson et al. (1995) showed that, following a O. ostertagi challenge, a higher number of globular leucocytes were present in the abomasal mucosa of C. oncophora immunised calves than in uninfected controls. These observations suggested an immunological reaction in the abomasum provoked by an intestinal C. oncophora immunisation. In the present study no differences in the number of globular leucocytes were demonstrated between the Ostertagia challenged groups. This could be due to the setup of the present experiment. Indeed, all animals were slaughtered 24 days following a heavy challenge spread over 12 days, which may be sufficient to elicit an increase in the number of mucosal globular leucocytes, even in previously non-sensitised animals and therefore may have masked histological changes resulting from priming (Claerebout et al., 1996). Also the role of globular leucocytes in the immune mechanism of ruminants against worm infections is not well understood. In sheep and cattle the increase in the numbers of globular leucocytes has been associated with the development of resistance to a worm infection (Jarrett et al., 1967; O'Sullivan and Donald, 1973; Miller, 1984). In sheep, Stear et al. (1995) demonstrated a significant negative relationship between the number of globular leucocytes and the number of Ostertagia circumcincta. However, Huntley et al. (1995) showed that in contrast to sheep, in goats an increase in the proportion of globular leucocytes following nematode infections did not result in an expulsion of worms. Differences in the functional activity of the globular leucocytes between ruminant species are therefore not excluded. Also it was shown in laboratory animals that differences in effector mechanisms exist for different worm species. In rats, mucosal mast cells are involved in the expulsion of Strongyloides ratti, while goblet cells are involved in the case of Nippostrongylus brasiliensis (Nawa et al., 1994). In conclusion no effect of a C. oncophora immunisation on the establishment and fecundity of an experimental infection of O. ostertagi, given concurrently with C. oncophora, could be demonstrated. Therefore, it is unlikely that on pastures contaminated with these two trichostrongyle species the uptake of C. oncophora in the early part of the grazing season will affect the establishment rate of O. ostertagi in the second half of the season.
Acknowledgements The research presented in this paper was supported by an IWONL grant (no. 5399A, section O). The authors would like to thank A. Dereu, P. Meeus, M. Bouch6 and L. Braem for technical assistance, and D.J. Shaw for help in the statistical analysis.
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Q.A., Sinski, E. and Murray, M., 1995. Regulation of egg production, worm burden, worm length and worm fecundity by host responses in sheep infected with Ostertagia circumcincta. Parasite Immunol., 17: 643-652. Thienpont, D., Rochette, F., van Parijs, O.F.J., 1979. Diagnosing helminthiasis by coprological examination. Janssen Research Foundation, Beerse, Belgium, 187 pp. Turner, J.H., Kates, K.C. and Wilson, G.I., 1962. The interaction of concurrent infections of the abomasal nematodes, Haemonchus contortus, Ostertagia circumcincta and Trichostrongylus axei (Trichostrongylidae) in Iambs. Proc. Helminthol. Soc. Wash., 29: 210-216.