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Localization of immunoglobulin-containing cells in the abomasum of sheep following infection with Haemonchus contortus
Veterinary Immunology and Immunopathology, 31 (1992) 179-187
179
Elsevier Science Publishers B.V., Amsterdam
Localization of immunoglobulin-containing cells in the abomasum of sheep following infection with Haemonchus contortus H.S. Gill a'~, A.J. Husband b and D.L. Watson c aDepartment of Animal Science, Universityof New England, Armidale, NSW, Australia bFaculty of Medicine, Universityof Newcastle, Newcastle, NSW, Australia cCSIRO Division of Animal Health, Pastoral Research Laboratory, Armidale, NSW, Australia (Accepted 20 February 1991 )
ABSTRACT Gill, H.S., Husband, A.J. and Watson, D.L., 1992. Localization of immunoglobulin-containingcells in the abomasum of sheep following infection with Haemonchus contortus. Vet. lmmunol, lmmunopathol., 31: 179-187. Abomasal cannulation followed by serial collection of biopsies was used to study the kinetics of appearance of IgA-, IgG 1-, IgG2- and IgM-containingcells in the abomasum of sheep following infection with Haemonchus contortus. Very few immunoglobulin-containingcells (ICC) were found in the abomasum of sheep before infection. Following H. contortus infection, there were increased numbers of ICC in the submucosa of the abomasum. Seven days after infection, the numbers of IgA-, IgG 1and IgM-containingcells were six times greater than for uninfected control animals. The numbers of ICC continued to rise as the infection progressed, and the peak response was observed between 21 and 28 days after infection. IgA-containingcells (68-84%) were the most frequent cell types at all the observation times, followed by IgGl and IgM. IgG2-containingcells were minimal throughout the experiment. As there were no significant changes in the numbers of ICC in the abomasum of uninfected controls it is concluded that H. contortus stimulated a local immune response in the abomasum of parasitized sheep. ABBREVIATIONS FITC, fluorescein isothiocyanate; ICC, immunoglobulin-containingcells; PCV, packed cell volume.
INTRODUCTION
Infections with gastro-intestinal nematodes are a major constraint on sheep and goat production in many parts of the world. To date, the control ofgastro1Author to whom correspondence should be addressed.
© 1992 Elsevier Science Publishers B.V. All fights reserved 0165-2427/92/$05.00
180
H.S. GILL ET AL.
intestinal parasitism has depended largely on the use of anthelmintics. However, the development of resistance by nematode parasites to various drugs and the increasing market prejudice against drug residues in meat has stimulated the quest for other methods of parasite control. Recent advances in biotechnology offer exciting new prospects for the development of successful vaccines against parasites. An essential prerequisite for the development of such vaccines is an adequate understanding of the inflammatory and immunological responses that mediate protection. Local i m m u n e responses in the gastrointestinal tract are responsible for immunity to a number of pathogens (Porter et al., 1979; Befus et al., 1986) and it was therefore considered worthwhile to study the development of mucosal immunity in the abomasum in relation to haemonchosis in sheep. The aim of the present investigation was to determine whether abomasal cannulation followed by serial biopsying could be used to study the kinetics of local i m m u n e responses to Haemonchus contortus in sheep. MATERIALS AND METHODS
Animals Five, 1-year-old Merino ewes reared on pasture were used in this study. Three weeks before infection, the sheep were dosed with oxfendazole at double the recommended dose to remove any existing worms and then housed indoors on slatted floors for the period of this study.
Parasite Infective larvae of H. contortus (McMaster strain ) used in this study were obtained from culture of faeces from sheep infected with H. contortus.
Surgical procedure The abomasum of all the sheep were cannulated under general anaesthesia using the technique described by Kondos ( 1967 ). The cannula used in this study were made of rubber (Macam Rubber Sales Pty. Ltd., Northmead, Australia) and had an internal diameter of 2 cm. Each animal was allowed 2 weeks to recover from surgery before biopsy.
Biopsy procedure The sheep were fasted for 48 h and were then sedated by injecting a 2% solution of R o m p u n (Bayer) intramuscularly (0.3 ml 50 k g - i body weight ). When recumbent, the animal was placed onto a surgical table. After removing
LOCAL IMMUNE RESPONSES TO HAEMONCHUS CONTORTUS IN SHEEP
181
the bung from the cannula, the wall of the abomasum was biopsied using biopsy cutting forceps (Karl Storz GmbH, Tuttligen, Germany) attached to a Karl Storz endoscope.
Preparation of antisera Antisera to sheep immunoglobulins were prepared in rabbits and conjugated to fluorescein isothiocyanate (FITC) according to the method described by Scicchitano et al. ( 1984 ).
Processing of tissues Abomasal biopsies were fixed in cold (4 °C) ethanol and processed for fluorescent antibody staining using the method of Sainte-Marie (1962).
Immunofluorescence Sections (4/~m) were prestained with Chromotrope to block autofluorescence and then stained for immunoglobulin-containingcells by incubating with FITC-conjugated rabbit antisera to ovine IgA, IgG 1, IgG2 or IgM. Slides were mounted in buffered-glycerol (one part of PBS to nine parts of glycerol) and examined using a Zeiss microscope.
Enumeration of immunoglobulin-containing cells (ICC) The mean number of ICC m m - 2 of tissue was calculated by counting the number of ICC per field in 30-50 fields (magnification × 500 ) scanning from the top of the villi to the base of the mucosa as previously described (Husband et al., 1979).
Experimental design Two weeks after the surgical procedures the sheep were allocated to two groups, Group A (three ewes) and Group B (two ewes). The animals in group A were infected intraruminally with approximately 20 000 infective larvae of H. contortus. The animals in Group B served as uninfected controls. At 0, 7, 14, 21 and 28 days post infection three tissue biopsies were taken from the abomasum of each animal for quantification of ICC. They were also bled at the same time to measure changes in packed cell volume (PCV). The temperature, appetite and general condition of all the animals were monitored daily to determine the effect of cannulation and biopsy. All the animals were slaughtered for worm counts 28 days after infection.
182
H.S. GILL ET AL.
RESULTS
Clinical findings No overt clinical signs associated with abomasal cannulation and biopsy were seen in any of the sheep. The body temperature, food consumption and water intake of all the animals remained normal throughout the experiment.
Worm counts, PCV and weight gain Worm-burdens and PCVs of H. contortus-infected sheep are presented in Table 1. The uninfected sheep showed no significant changes in PCV during the experiment and no worms were recovered from the abomasum at slaughter. The mean live-weight gain in H. contortus-infected sheep over 28 days was 1.2 kg compared with 3.8 kg for the uninfected controls.
Kinetics of appearance of lg-containing cells The changes in the populations of ICC in the abomasum of uninfected (controls) and H. contortus-infected sheep during the experiment are shown in Fig. 1.
IgA-containing cells A small number of IgA-containing cells ( 12-20 m m -2) were found in the abomasum of uninfected sheep (Fig. 2 ). Following infection, the number of IgA-containing cells rose significantly (Fig. 3 ), reaching peak levels on Day 21 ( 375 cells m m - 2) before declining to half that number by Day 28 (212 cells mm -2). The number of IgA-containing cells in the abomasum of uninfected controls remained unaltered over the period of observation. TABLE 1 The worm burdens and packed cell volumes of H. contortus-infected and control sheep Group
Inaction
Control
Animal No.
10 11 12 20 21
No. of worms recovered
3300 6480 5200 -
Packed cell volume (%) Before infection
4 weeks post infection
32 37 34 25 33
22 32 25 38 46
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Fig. l. Kinetics of appearance of Ig-containing cells in the abomasum of uninfected (control) ( • ) a n d H. contortus-infected sheep ( • ).
IgG l-containing cells Very few IgGl-containing cells were detected in the abomasum of uninfected sheep. There was a significant increase in the number of IgG 1-containing cells as early as 7 days after infection. The IgG 1 response reached a maximum on Day 28 and was 22 times greater than that of uninfected controls (Figs. 1 and 4).
IgG2-containing cells IgG2-containing cells were virtually absent from the abomasum of uninfected sheep. Following infection, a small number of IgG2-containing cells were observed on Days 14, 21 and 28 post infection. However, the response was negligible when compared with IgA- and IgG l-containing cells.
IgM-containing cells Very few IgM-containing cells were found in the abomasum of uninfected sheep. The numbers of IgM-containing cells increased dramatically following
184
H.S. GILL ET AL.
Fig. 2. Micrograph showing a small number of IgA-containing cells in the abomasum of an uninfected sheep. Immunofluorescence× 240.
Fig. 3. Micrograph showing numerous IgA-containingcells in the abomasum of a sheep 14 days after infection with 20 000 H. contortus larvae. Immunofluorescence× 240.
LOCALIMMUNERESPONSESTO HAEMONCHUSCONTORTUSIN SHEEP
18 5
Fig. 4. Micrograph showing IgGl-containing cells in the abomasum of a sheep 28 days after infection with 20 000 H. contortus larvae. Immunofluorescence X 240.
Fig. 5. Micrograph showing IgM-containing cells in the abomasum of a sheep 21 days after infection with 20 000 H. c o n t o r t ~ larvae. Immunofluorescence X 240,
186
H.S. GILL ET AL.
infection (Fig. 5 ). They were recorded in significantly greater numbers in H. contortus-infected sheep at 7, 14, 21 and 28 days compared with those in uninfected controls. Peak numbers of IgM-containing cells were observed on Day 21, when they were 37 times those of control levels. DISCUSSION In the present study, H. contortus became established in each of the three sheep which were infected with larvae. The infection was accompanied by an increased proliferation of IgA-, IgG 1-, IgG2- and IgM-containing cells in the abomasum of infected sheep. As these changes were not detected in the abomasum of uninfected (control) sheep it is inferred that H. contortus stimulated a local immune response in the abomasum of parasitized sheep. The sheep used in this study were raised on pasture and are likely to have experienced light infections with gastro-intestinal nematodes (including H. contortus) commonly found in the Northern Tablelands of New South Wales. Thus, the prompt and vigorous response detected as early as 7 days after infection is suggestive of an anamnestic response to infection. The numbers of ICC continued to rise further as the infection progressed. Twenty-one days after infection, the numbers of IgA-, IgG 1- and IgM-containing cells in the abomasum of infected sheep were between 20 and 40 times greater than those of uninfected controls. The peak ICC response coincided with the finding of H. contortus eggs in the faeces. This might suggest that most of the ICC response was directed against the excretory-secretory products of the adult worms. Contrary to this however, Curtain and Anderson ( 1971 ) found no difference in the numbers of IgA-containing cells in the abomasum of wormfree and parasitized/immune sheep. Whether this was due to the specificity of the reagents used to detect IgA cells or some other reason is not clear. Elevated levels of specific IgA a n d / o r IgG 1 antibodies in the abomasal mucosa (Smith, 1977) and faecal extracts ofH. contortus-infected sheep (Gill et al., 1989 ), and in the intestinal secretions of sheep infected with Trichostrongylus colubriformis (Cripps and Rothwell, 1978 ) has been reported. The appearance of antibodies in the gut secretions could be due to local production or transudation from serum. The predominance of IgA- (68-84% of the total ICC ) and IgG 1-containing cells (8-30%), and a significant increase in their numbers following infection (Fig. 1 ) suggests that most of the parasite-specific IgA and IgG 1 antibodies were locally produced and may play a role in host defense (Miller, 1984). However, nematodes are known to have mitogenic properties (Cross et al., 1986; Gill et al., 1991 ) and it is therefore possible that the observed increase in the numbers of ICC in the abomasum of H. contortus-infected sheep was at least partially non-specific rather than parasite-specific. Further studies are necessary to determine the specificity of the response.
LOCALIMMUNERESPONSESTOHAEMONCHUSCONTORTUSIN SHEEP
187
The results of the present study show that abomasal cannulation and repeated biopsy had no adverse effect on the well-being of the animals. Nor did it have a significant effect on the number of ICC in the abomasum of uninfected controls during the period of observation. This study confirms that gut cannulation and mucosal biopsy is a valuable technique for studying the kinetics of local immune responses to gut pathogens in sheep. ACKNOWLEDGMENTS
This work was supported by a grant from the Wool Research Trust Fund. We thank J. Lea and S. Burgess for their technical assistance and I. Schmidt for typing the manuscript.
REFERENCES Befus, D., Lee, T., Ernst, P., Egwang, T., McElroy, P., Gauldie, J. and Bienenstock, J., 1986. Unique characteristics of local immune responses in host resistance to mucosal parasitic infections. Vet. Parasitol., 20:175-194. Cripps, A.W. and Rothwell, T.L.W., 1978. Immune responses of sheep to parasitic nematode Trichostrongylus colubriformis infections in Thiry Vella loops. Aust. J. Exp. Biol. Med. Res., 56: 99-106. Cross, D.A., Klesius, P.H. and Haynes, T.B., 1986. Lymphocyte blastogenic responses of calves experimentally infected with Ostertagia ostertagi. Vet. Parasitol., 22: 49-55. Curtain, C.C. and Anderson, N., 197 I. Immunocytochemical localization of the ovine immunoglobulins IgA, IgG1, IgGla, and IgG2: effect of gastro-intestinal parasitism in the sheep. Clin. Exp. Immunol., 8:151-162. Gill, H.S., Gray, G.D. and Watson, D.L., 1989. Antibody responses to Haemonchus contortus in genetically resistant and susceptible sheep. Proc. Aust. Soc. Parasitol., p. 77. Gill, H.S., Pomroy, W.E., Charleston, W.E. and Moriarty, K.M., 1991. Caprine haemonchosis: lymphocyte responses to parasite antigen and mitogens. Small Rumin. Res., 4: 101-108. Husband, A.J., Beh, K.J. and Lascelles, A.K., 1979. IgA-containing cells in the ruminant intestine following intraperitoneal and local immunization. Immunology, 37:597-601. Kondos, A.C., 1967. A new method for cannulation of the abomasum of sheep. Aust. Vet. J., 43: 149-151. Miller, H.R.P., 1984. Protective immune responses against gastro-intestinal nematodes in ruminants and laboratory animals. Vet. Immunol. Immunopathol., 6:167-259. Porter, P., Parry, S.H. and Allen, W.D., 1979. Significance of immune mechanisms in relation to enteric infections of the gastrointestinal tract in animals. Ciba Found. Symp., 46: 55-76. Sainte-Marie, G., 1962. A paraffin embedding for studies employing immunofluorescence. J. Histochem. Cytochem., 10: 250-258. Scicchitano, R., Husband, A.J. and Cripps, A.W., 1984. Immunoglobulin-containing cells and the origin of immunoglobulins in the respiratory tract of sheep. Immunology, 52: 529-537. Smith, W.D., 1977. Anti-larval antibodies in the serum and abomasal mucus of sheep hyperinfected with Haemonchus contortus. Res. Vet. Sci., 22: 334-338.
179
Elsevier Science Publishers B.V., Amsterdam
Localization of immunoglobulin-containing cells in the abomasum of sheep following infection with Haemonchus contortus H.S. Gill a'~, A.J. Husband b and D.L. Watson c aDepartment of Animal Science, Universityof New England, Armidale, NSW, Australia bFaculty of Medicine, Universityof Newcastle, Newcastle, NSW, Australia cCSIRO Division of Animal Health, Pastoral Research Laboratory, Armidale, NSW, Australia (Accepted 20 February 1991 )
ABSTRACT Gill, H.S., Husband, A.J. and Watson, D.L., 1992. Localization of immunoglobulin-containingcells in the abomasum of sheep following infection with Haemonchus contortus. Vet. lmmunol, lmmunopathol., 31: 179-187. Abomasal cannulation followed by serial collection of biopsies was used to study the kinetics of appearance of IgA-, IgG 1-, IgG2- and IgM-containingcells in the abomasum of sheep following infection with Haemonchus contortus. Very few immunoglobulin-containingcells (ICC) were found in the abomasum of sheep before infection. Following H. contortus infection, there were increased numbers of ICC in the submucosa of the abomasum. Seven days after infection, the numbers of IgA-, IgG 1and IgM-containingcells were six times greater than for uninfected control animals. The numbers of ICC continued to rise as the infection progressed, and the peak response was observed between 21 and 28 days after infection. IgA-containingcells (68-84%) were the most frequent cell types at all the observation times, followed by IgGl and IgM. IgG2-containingcells were minimal throughout the experiment. As there were no significant changes in the numbers of ICC in the abomasum of uninfected controls it is concluded that H. contortus stimulated a local immune response in the abomasum of parasitized sheep. ABBREVIATIONS FITC, fluorescein isothiocyanate; ICC, immunoglobulin-containingcells; PCV, packed cell volume.
INTRODUCTION
Infections with gastro-intestinal nematodes are a major constraint on sheep and goat production in many parts of the world. To date, the control ofgastro1Author to whom correspondence should be addressed.
© 1992 Elsevier Science Publishers B.V. All fights reserved 0165-2427/92/$05.00
180
H.S. GILL ET AL.
intestinal parasitism has depended largely on the use of anthelmintics. However, the development of resistance by nematode parasites to various drugs and the increasing market prejudice against drug residues in meat has stimulated the quest for other methods of parasite control. Recent advances in biotechnology offer exciting new prospects for the development of successful vaccines against parasites. An essential prerequisite for the development of such vaccines is an adequate understanding of the inflammatory and immunological responses that mediate protection. Local i m m u n e responses in the gastrointestinal tract are responsible for immunity to a number of pathogens (Porter et al., 1979; Befus et al., 1986) and it was therefore considered worthwhile to study the development of mucosal immunity in the abomasum in relation to haemonchosis in sheep. The aim of the present investigation was to determine whether abomasal cannulation followed by serial biopsying could be used to study the kinetics of local i m m u n e responses to Haemonchus contortus in sheep. MATERIALS AND METHODS
Animals Five, 1-year-old Merino ewes reared on pasture were used in this study. Three weeks before infection, the sheep were dosed with oxfendazole at double the recommended dose to remove any existing worms and then housed indoors on slatted floors for the period of this study.
Parasite Infective larvae of H. contortus (McMaster strain ) used in this study were obtained from culture of faeces from sheep infected with H. contortus.
Surgical procedure The abomasum of all the sheep were cannulated under general anaesthesia using the technique described by Kondos ( 1967 ). The cannula used in this study were made of rubber (Macam Rubber Sales Pty. Ltd., Northmead, Australia) and had an internal diameter of 2 cm. Each animal was allowed 2 weeks to recover from surgery before biopsy.
Biopsy procedure The sheep were fasted for 48 h and were then sedated by injecting a 2% solution of R o m p u n (Bayer) intramuscularly (0.3 ml 50 k g - i body weight ). When recumbent, the animal was placed onto a surgical table. After removing
LOCAL IMMUNE RESPONSES TO HAEMONCHUS CONTORTUS IN SHEEP
181
the bung from the cannula, the wall of the abomasum was biopsied using biopsy cutting forceps (Karl Storz GmbH, Tuttligen, Germany) attached to a Karl Storz endoscope.
Preparation of antisera Antisera to sheep immunoglobulins were prepared in rabbits and conjugated to fluorescein isothiocyanate (FITC) according to the method described by Scicchitano et al. ( 1984 ).
Processing of tissues Abomasal biopsies were fixed in cold (4 °C) ethanol and processed for fluorescent antibody staining using the method of Sainte-Marie (1962).
Immunofluorescence Sections (4/~m) were prestained with Chromotrope to block autofluorescence and then stained for immunoglobulin-containingcells by incubating with FITC-conjugated rabbit antisera to ovine IgA, IgG 1, IgG2 or IgM. Slides were mounted in buffered-glycerol (one part of PBS to nine parts of glycerol) and examined using a Zeiss microscope.
Enumeration of immunoglobulin-containing cells (ICC) The mean number of ICC m m - 2 of tissue was calculated by counting the number of ICC per field in 30-50 fields (magnification × 500 ) scanning from the top of the villi to the base of the mucosa as previously described (Husband et al., 1979).
Experimental design Two weeks after the surgical procedures the sheep were allocated to two groups, Group A (three ewes) and Group B (two ewes). The animals in group A were infected intraruminally with approximately 20 000 infective larvae of H. contortus. The animals in Group B served as uninfected controls. At 0, 7, 14, 21 and 28 days post infection three tissue biopsies were taken from the abomasum of each animal for quantification of ICC. They were also bled at the same time to measure changes in packed cell volume (PCV). The temperature, appetite and general condition of all the animals were monitored daily to determine the effect of cannulation and biopsy. All the animals were slaughtered for worm counts 28 days after infection.
182
H.S. GILL ET AL.
RESULTS
Clinical findings No overt clinical signs associated with abomasal cannulation and biopsy were seen in any of the sheep. The body temperature, food consumption and water intake of all the animals remained normal throughout the experiment.
Worm counts, PCV and weight gain Worm-burdens and PCVs of H. contortus-infected sheep are presented in Table 1. The uninfected sheep showed no significant changes in PCV during the experiment and no worms were recovered from the abomasum at slaughter. The mean live-weight gain in H. contortus-infected sheep over 28 days was 1.2 kg compared with 3.8 kg for the uninfected controls.
Kinetics of appearance of lg-containing cells The changes in the populations of ICC in the abomasum of uninfected (controls) and H. contortus-infected sheep during the experiment are shown in Fig. 1.
IgA-containing cells A small number of IgA-containing cells ( 12-20 m m -2) were found in the abomasum of uninfected sheep (Fig. 2 ). Following infection, the number of IgA-containing cells rose significantly (Fig. 3 ), reaching peak levels on Day 21 ( 375 cells m m - 2) before declining to half that number by Day 28 (212 cells mm -2). The number of IgA-containing cells in the abomasum of uninfected controls remained unaltered over the period of observation. TABLE 1 The worm burdens and packed cell volumes of H. contortus-infected and control sheep Group
Inaction
Control
Animal No.
10 11 12 20 21
No. of worms recovered
3300 6480 5200 -
Packed cell volume (%) Before infection
4 weeks post infection
32 37 34 25 33
22 32 25 38 46
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Fig. l. Kinetics of appearance of Ig-containing cells in the abomasum of uninfected (control) ( • ) a n d H. contortus-infected sheep ( • ).
IgG l-containing cells Very few IgGl-containing cells were detected in the abomasum of uninfected sheep. There was a significant increase in the number of IgG 1-containing cells as early as 7 days after infection. The IgG 1 response reached a maximum on Day 28 and was 22 times greater than that of uninfected controls (Figs. 1 and 4).
IgG2-containing cells IgG2-containing cells were virtually absent from the abomasum of uninfected sheep. Following infection, a small number of IgG2-containing cells were observed on Days 14, 21 and 28 post infection. However, the response was negligible when compared with IgA- and IgG l-containing cells.
IgM-containing cells Very few IgM-containing cells were found in the abomasum of uninfected sheep. The numbers of IgM-containing cells increased dramatically following
184
H.S. GILL ET AL.
Fig. 2. Micrograph showing a small number of IgA-containing cells in the abomasum of an uninfected sheep. Immunofluorescence× 240.
Fig. 3. Micrograph showing numerous IgA-containingcells in the abomasum of a sheep 14 days after infection with 20 000 H. contortus larvae. Immunofluorescence× 240.
LOCALIMMUNERESPONSESTO HAEMONCHUSCONTORTUSIN SHEEP
18 5
Fig. 4. Micrograph showing IgGl-containing cells in the abomasum of a sheep 28 days after infection with 20 000 H. contortus larvae. Immunofluorescence X 240.
Fig. 5. Micrograph showing IgM-containing cells in the abomasum of a sheep 21 days after infection with 20 000 H. c o n t o r t ~ larvae. Immunofluorescence X 240,
186
H.S. GILL ET AL.
infection (Fig. 5 ). They were recorded in significantly greater numbers in H. contortus-infected sheep at 7, 14, 21 and 28 days compared with those in uninfected controls. Peak numbers of IgM-containing cells were observed on Day 21, when they were 37 times those of control levels. DISCUSSION In the present study, H. contortus became established in each of the three sheep which were infected with larvae. The infection was accompanied by an increased proliferation of IgA-, IgG 1-, IgG2- and IgM-containing cells in the abomasum of infected sheep. As these changes were not detected in the abomasum of uninfected (control) sheep it is inferred that H. contortus stimulated a local immune response in the abomasum of parasitized sheep. The sheep used in this study were raised on pasture and are likely to have experienced light infections with gastro-intestinal nematodes (including H. contortus) commonly found in the Northern Tablelands of New South Wales. Thus, the prompt and vigorous response detected as early as 7 days after infection is suggestive of an anamnestic response to infection. The numbers of ICC continued to rise further as the infection progressed. Twenty-one days after infection, the numbers of IgA-, IgG 1- and IgM-containing cells in the abomasum of infected sheep were between 20 and 40 times greater than those of uninfected controls. The peak ICC response coincided with the finding of H. contortus eggs in the faeces. This might suggest that most of the ICC response was directed against the excretory-secretory products of the adult worms. Contrary to this however, Curtain and Anderson ( 1971 ) found no difference in the numbers of IgA-containing cells in the abomasum of wormfree and parasitized/immune sheep. Whether this was due to the specificity of the reagents used to detect IgA cells or some other reason is not clear. Elevated levels of specific IgA a n d / o r IgG 1 antibodies in the abomasal mucosa (Smith, 1977) and faecal extracts ofH. contortus-infected sheep (Gill et al., 1989 ), and in the intestinal secretions of sheep infected with Trichostrongylus colubriformis (Cripps and Rothwell, 1978 ) has been reported. The appearance of antibodies in the gut secretions could be due to local production or transudation from serum. The predominance of IgA- (68-84% of the total ICC ) and IgG 1-containing cells (8-30%), and a significant increase in their numbers following infection (Fig. 1 ) suggests that most of the parasite-specific IgA and IgG 1 antibodies were locally produced and may play a role in host defense (Miller, 1984). However, nematodes are known to have mitogenic properties (Cross et al., 1986; Gill et al., 1991 ) and it is therefore possible that the observed increase in the numbers of ICC in the abomasum of H. contortus-infected sheep was at least partially non-specific rather than parasite-specific. Further studies are necessary to determine the specificity of the response.
LOCALIMMUNERESPONSESTOHAEMONCHUSCONTORTUSIN SHEEP
187
The results of the present study show that abomasal cannulation and repeated biopsy had no adverse effect on the well-being of the animals. Nor did it have a significant effect on the number of ICC in the abomasum of uninfected controls during the period of observation. This study confirms that gut cannulation and mucosal biopsy is a valuable technique for studying the kinetics of local immune responses to gut pathogens in sheep. ACKNOWLEDGMENTS
This work was supported by a grant from the Wool Research Trust Fund. We thank J. Lea and S. Burgess for their technical assistance and I. Schmidt for typing the manuscript.
REFERENCES Befus, D., Lee, T., Ernst, P., Egwang, T., McElroy, P., Gauldie, J. and Bienenstock, J., 1986. Unique characteristics of local immune responses in host resistance to mucosal parasitic infections. Vet. Parasitol., 20:175-194. Cripps, A.W. and Rothwell, T.L.W., 1978. Immune responses of sheep to parasitic nematode Trichostrongylus colubriformis infections in Thiry Vella loops. Aust. J. Exp. Biol. Med. Res., 56: 99-106. Cross, D.A., Klesius, P.H. and Haynes, T.B., 1986. Lymphocyte blastogenic responses of calves experimentally infected with Ostertagia ostertagi. Vet. Parasitol., 22: 49-55. Curtain, C.C. and Anderson, N., 197 I. Immunocytochemical localization of the ovine immunoglobulins IgA, IgG1, IgGla, and IgG2: effect of gastro-intestinal parasitism in the sheep. Clin. Exp. Immunol., 8:151-162. Gill, H.S., Gray, G.D. and Watson, D.L., 1989. Antibody responses to Haemonchus contortus in genetically resistant and susceptible sheep. Proc. Aust. Soc. Parasitol., p. 77. Gill, H.S., Pomroy, W.E., Charleston, W.E. and Moriarty, K.M., 1991. Caprine haemonchosis: lymphocyte responses to parasite antigen and mitogens. Small Rumin. Res., 4: 101-108. Husband, A.J., Beh, K.J. and Lascelles, A.K., 1979. IgA-containing cells in the ruminant intestine following intraperitoneal and local immunization. Immunology, 37:597-601. Kondos, A.C., 1967. A new method for cannulation of the abomasum of sheep. Aust. Vet. J., 43: 149-151. Miller, H.R.P., 1984. Protective immune responses against gastro-intestinal nematodes in ruminants and laboratory animals. Vet. Immunol. Immunopathol., 6:167-259. Porter, P., Parry, S.H. and Allen, W.D., 1979. Significance of immune mechanisms in relation to enteric infections of the gastrointestinal tract in animals. Ciba Found. Symp., 46: 55-76. Sainte-Marie, G., 1962. A paraffin embedding for studies employing immunofluorescence. J. Histochem. Cytochem., 10: 250-258. Scicchitano, R., Husband, A.J. and Cripps, A.W., 1984. Immunoglobulin-containing cells and the origin of immunoglobulins in the respiratory tract of sheep. Immunology, 52: 529-537. Smith, W.D., 1977. Anti-larval antibodies in the serum and abomasal mucus of sheep hyperinfected with Haemonchus contortus. Res. Vet. Sci., 22: 334-338.