A. Pfeffer

Sequential cellular and humoral responses in the abomasal mucosa and blood of Romney sheep dosed with Trichostrongylus axei

Abomasal cannulae were surgically placed in 7 2-year-old New Zealand Romney sheep which had been maintained parasite-free from birth. Four of these sheep were randomly selected and dosed orally with 10,000 infective Trichostrongylus axei larvae per week for 8 weeks, while the remaining 3 sheep served as uninfected controls. Abomasal biopsy, blood and faecal samples were obtained from all sheep at regular intervals from 5 days before and until 58 days after the first infection. The sheep were then killed, worm burdens assessed and abomasal and small intestinal samples collected Faecal egg counts of all 4 dosed sheep were low and only one (No. 701) had a substantial worm burden (8400) post mortem. Overall, levels of mucosal mast cells/globule leukocytes, eosinophils, T19+ cells and larval migration inhibitory activity increased significantly in the abomasal mucosa of the dosed sheep compared to the controls. The CD4+:CD8+ cell ratio in the abomasal mucosa of the dosed sheep also increased compared to that of the controls (P = 0.06). In blood, T. axei-specific antibody (total and IgG1) and eosinophil numbers increased significantly in the dosed sheep. Mucosal cells staining for IgE (IgE+), and blood and mucosal eosinophils showed the earliest substantive increases in number followed by increases in specific serum antibody levels, numbers of mucosal cells fluorescing under UV light (UVf) and T19+ cells. The difference in the IgE+ and UVf cell responses indicated that expansion of globule leukocyte numbers lagged behind that of mucosal mast cells. The results supported the concept of CD4+ T cell help in the abomasal mucosa and defined the sequential expression of components of the immunological responses potentially mediating resistance to T. axei. In sheep No. 701, persistence of adult worms was associated with lower mucosal IgE+ cell and eosinophil responses compared with the other dosed sheep.

Studies on the role of mucus and mucosal hypersensitivity reactions during rejection of Trichostrongylus colubriformis from the intestine of immune sheep using an experimental challenge model

Nematode-naive sheep and sheep immunised by truncated infections with Trichostrongylus colubriformis were fitted with intestinal cannulae to allow administration of challenge infection and collection of intestinal fluids. Sheep were slaughtered at various times after challenge and the distribution of larvae along the small intestine was determined. Results showed that immune sheep had significantly fewer larvae in their intestines and that some sheep could expel the challenge infection within 2 h. Mucus samples from immune sheep contained increased parasite-specific antibody, histamine and anti-parasite activity as measured by larval migration inhibition assay. Higher levels of antibody and histamine were seen in intestinal fluids of immune sheep after challenge. Immunisation of sheep by truncated infections stimulated serum IgE and resulted in significantly higher numbers of IgE-positive cells in gut tissue sections before challenge and at 2 h and 24 h after challenge. Immune sheep also had greater numbers of mucosal mast cells and globule leucocytes after challenge, compared with naive sheep. When challenge larvae were mixed with mucus from immune sheep and infused back into naive recipient sheep, there was a distinct displacement of the larval population towards the distal part of the intestine, compared with the profile of larval establishment after infusion with mucus from naive sheep. These results are further evidence for an immediate hypersensitivity reaction in the intestine of immune sheep, where challenge larvae are expelled within 2 h and confirm the direct anti-larval properties of mucus. The cannulated-sheep challenge model described here will be a useful tool to unravel the mechanism of larval rejection from immune sheep and could lead to novel therapies.

Immunization of sheep against parasitic nematodes leads to elevated levels of globule leukocytes in the small intestine lumen

In sheep that had been given three immunizing infections with Trichostrongylus colubriformis and Ostertagia circumcincta infective (L3) larvae, drenched after the last infection and challenged with larvae of the same species, there was a significant increase in numbers of small intestine mucosal tissue globule leukocytes (TGLs) and lumenal globule leukocytes (LuGLs) compared with sheep that had only been drenched and challenged. There was a positive correlation between the numbers of LuGLs and TGLs in the small intestine but the ratio of these two cell types was lower in non-immunized than immunized sheep. In immunized sheep positive correlations were observed between LuGLs and levels of arylsulphatase and peroxidase in the intestinal mucus and between arylsulphatase and larval migration inhibition (LMI) activity in mucus. Lumen eosinophils correlated with blood eosinophils, serum antibody against T. colubriformis correlated with peroxidase in the mucus and blood eosinophils correlated with nematode specific IgM levels in the intestinal mucus. In the abomasum, TGLs were present but not LuGLs. Sheep repeatedly infected with T. axei also had significantly more LuGLs in the small intestine than control animals. Two sheep that had a surgically prepared isolated small intestinal loop, after oral infection with T. colubriformis had TGLs and LuGLs in the intact intestine, but not in the isolated loop. Significantly more LuGLs were produced in sheep by allowing repeated T. colubriformis L3 infections to develop to adult stages compared to sheep treated with the same number of larvae, but where the infections were terminated by drenching at various intervals.

Cross-reacting antibodies to Sarcoptes suis, Chorioptes bovis and Notoedres cati and anti-P. ovis IgE in sera from sheep infested naturally with Psoroptes ovis

For the development of immunodiagnostic tests to detect mange mite infections in man and animals, it is necessary to know about antigen structure and cross-reactivity between different mite species and other arthropods in contact with the host. Sera from sheep infected with Psoroptes ovis (sheep anti-P. ovis sera) showed positive reactions in dot blots to P. ovis antigen and cross-reactivity to crude antigen extracts from Sarcoptes suis, Notoedres cati and Chorioptes bovis. Using sheep Specific Pathogen Free (SPF) sera in dot blots, weak reactions were seen to all but the Ch. bovis antigen. In SDS PAGE-separated P. ovis antigens at least 35 different proteins could be distinguished. In western blots, at least 24 out of these 35 were recognized as antigens by sheep anti-P. ovis immunoglobulins. At least 13 were recognized by sheep anti-P. ovis IgE. One of these, at 19 kDa, was recognized only with sheep anti-P. ovis IgE, the other 12 also with anti-P. ovis immunoglobulins. Cross-reactive antigens were recognized by sheep anti-P. ovis immunoglobulins in SDS PAGE-separated, and nitrocellulose transferred mite extracts in western blots as follows: 13 antigens in S. suis extracts, 9 in N. cati and 8 in Ch. bovis. Sheep SPF sera recognized an antigen at 67 kDa in each of these 4 mite species. Rabbit anti-sheep immunoglobulins in the P. ovis antigen control bound to a protein at 28 kDa which may be sheep IgG light chain taken up by P. ovis feeding on sheep. Despite the antigenic similarities between mange mites, sufficient differences were apparent to make immunodiagnostic tests for mange feasible.

Detection of homocytotropic antibody in lambs infested with the louse, Bovicola ovis, using a basophil histamine release assay

The utility of a basophil histamine-release assay using washed whole blood cells was examined in lambs and was used to determine if homocytotropic antibody with specificity for Bovicola ovis was produced in response to infestation with the louse. Maximal histamine release in the assay in response to Concanavalin A, anti-ovine IgE monoclonal antibody and, in sensitized lambs, to B. ovis antigen ranged from 18 to 48%. Histamine release from blood cells in response to B. ovis antigen was significantly higher in louse-infested lambs than in louse-naive lambs and was significantly correlated with louse and cockle scores. Passive cutaneous anaphylaxis (PCA) tests were negative with sera obtained from the lambs at the same time as blood for the basophil histamine-release assay. Serum histamine levels also were significantly higher in the louse-infested lambs than in louse-naive lambs and were significantly correlated with louse and cockle scores. The present results support a role for B. ovis-specific homocytotropic antibody in the development of cockle and indicate that the basophil histamine-release assay is more sensitive than the PCA test.

The development of cockle, a sheep pelt defect, in relation to size of infestation and time of exposure to Bovicola ovis, the sheep-biting louse

Groups of ten louse-naive lambs were infested with one, ten or 100 female Bovicola ovis and killed 84 days later when an examination of their pelts was made to detect cockle. In a second experiment groups of ten lambs were infested with ten or 100 female B. ovis and groups of lambs were killed every fortnight up to 84 days post-infestation. The pelts were examined in order to detect the earliest time at which cockle could be detected following a louse infestation. Cockle is a nodular condition of the skin arising in response to infestation with B. ovis and is possibly a hypersensitivity on the part of some sheep to antigens of louse origin. In the first experiment cockle did not develop in lambs that had remained louse free or which had been initially infested with one louse. However, five of ten lambs that had been infested with ten lice and all lambs that had been infested with 100 lice developed cockle. In general cockle severity was positively related to the size of the terminal louse population. Group mean louse counts only slightly exceeded the initial infestation in the lambs infested with either ten or 100 lice, and were less than the initial infestation in lambs given only one louse. In the second experiment cockle was first seen 54 days post-infestation, but only in sheep infested initially with 100 lice.

Proliferative responses of lymphocytes in Bovicola ovis-infested lambs.

Proliferative responses of peripheral blood and prescapular lymph node lymphocytes from 8 B. ovis-infested and 8 naive lambs to concanavalin A (Con A) and soluble antigen of B. ovis were examined in vitro. The numbers of lice and the extent of cockle were also assessed on each lamb. Prescapular lymph node lymphocytes from infested lambs showed significantly higher responses to B. ovis antigen than those from naive lambs. Only a marginal difference was observed between infested and naive lambs in the response of lymphocytes from peripheral blood. Proliferative responses to Con A by prescapular lymph node or blood cells were not significantly different between groups. The results indicated a relationship between the intensity of louse infestation and the proliferative response of prescapular lymph node lymphocytes to B. ovis antigen.

Comparison of local and systemic responsiveness of lymphocytes in vitro to Bovicola ovis antigen and Concanavalin A in B. ovis-infested and naive lambs

The in vitro proliferation assay was used to determine lymphocyte responsiveness to soluble antigen of B. ovis and to Concanavalin A (Con A) in peripheral blood, spleen and various lymph nodes from B. ovis-infested and naive lambs. From March to July, an assay of monthly blood samples showed generally higher proliferative responses to antigen and Con A in B. ovis-infested than naive lambs. The proliferative response of cells from the skin-draining prescapular lymph nodes to B. ovis antigen was significantly higher in B. ovis-infested than naive lambs. Responses of cells from the medial iliac, mediastinal and mesenteric lymph nodes (which do not receive lymph from the skin) and spleen showed no significant differences between groups. Within the B. ovis-infested lambs, the response of cells from the prescapular lymph node was significantly higher than that from any other lymphoid organ examined. Responsiveness of the prescapular, medial iliac and mesenteric lymph node and spleen cells to Con A was not significantly different between groups, while mediastinal lymph node cells showed a significantly higher response in B. ovis-infested lambs. The data indicate that the antigen-specific cellular immune response is operating mainly locally, at the level of the skin and draining lymph nodes. Responses to the T cell mitogen Con A did not support non-specific immunodepression as reported in other ectoparasite/host systems.

Experimental respiratory infection of goats with caprine herpesvirus and Pasteurella haemolytica.

Groups of six male goats were inoculated intratracheally and intranasally with either caprine herpesvirus followed 6 days later by Pasteurella haemolytica, canine herpesvirus alone or P. haemolytica alone. Pneumonic lesions were observed in five of the six goats inoculated with caprine herpesvirus followed by P. haemolytica and in three of the six goats inoculated with P. haemolytica alone, but were not observed in goats inoculated with caprine herpesvirus alone or in non-infected controls. Pasteurella haemolytica was isolated from seven of eight lungs with pneumonia, but only from one of sixteen lungs without pneumonia. The lesions ranged from fatal acute exudative necrotising pneumonia to predominantly proliferative pneumonia. Half of the caprine herpesvirus-inoculated goats developed a clinical catarrhal rhinitis five days post-inoculation and the only virus-specific histopathological lesion was a mild tracheitis. Canine herpesvirus was recovered from the nasal swabs of all caprine herpesvirus- inoculated goats developed a clinical catarrhal rhinitis five days post-inoculation and the only virus-specific histopathological lesion was a mild tracheitis. Canine herpesvirus was recovered from the nasal swabs of all canine herpesvirus-inoculated goats and from the lungs of three goats inoculated with caprine herpesvirus alone. The experimental inoculations demonstrated that P. haemolytica alone can produce pneumonia in goats. In addition, the study showed that caprine herpesvirus readily proliferates in the upper respiratory tract and lungs of goats but the role of caprine herpesvirus in the aetiology of pneumonia remains uncertain.

A caprine pneumonia outbreak associated with caprine herpesvirus and Pasteurella haemolytica respiratory infections.

In a field disease outbreak, 60 female goats died over a 2-3 week period. Necropsies of seven of these does revealed that six had an acute exudative necrotising broncho-pneumonia, and moderate to high numbers of Pasteurella haemolytica were isolated from their lungs. Caprine herpesvirus, identified as Bovid herpesvirus type 6, was isolated from the lungs of two of these does, including one with pneumonia, and from nasal swabs of in-contact goats. Clinical disease was only observed in does, and deaths began 3 weeks after the introduction of a mob of goat hoggets from another farm.