The binding of soybean agglutinin (SBA) to the intestinal epithelium of Atlantic salmon, Salmo salar and rainbow trout, Oncorhynchus mykiss, fed high levels of soybean meal.

A lectin present in soya, soybean agglutinin (SBA), was identified in electrophoretic profiles and immunoblots of dehulled solvent-extracted soybean meal (DSSM), full-fat soybean meal (FFSM) and of aqueous extracts of feeds incorporating them in their formulation. A quantitative estimation was made of the proportion of SBA comprising the total protein in FFSM and a trial diet was prepared containing an amount of pure SBA similar to that in diets incorporating high levels of the whole soya product. Fish fed with this diet exhibited similar pathological disruption of the intestinal tract to that observed in fish given a diet with a high level of DSSM (60% of the diet). Furthermore, immuno-histochemistry revealed the binding of the SBA to the enterocytes lining the intestinal villi both of fish fed a diet incorporating pure SBA and those fed a diet containing a high-level of soya (60%). Our results suggest that SBA binds in vivo to the intestinal epithelium of fish and has a contributory role in pathological changes associated with fish feeds containing high levels of soybean proteins.

Interferon-gamma and interleukin-2 release by lymphocytes derived from the blood, mesenteric lymph nodes and intestines of normal sheep and those affected with paratuberculosis (Johne's disease).

This study sought to determine if T-cell cytokine responses to mycobacterial infections in sheep were similar to those in other species and if such responses correlated with prevailing gut pathology. Lymphocytes were isolated from the blood (PBL), mesenteric lymph nodes (MLN) and ileal lamina propria (LPL) of control sheep and of sheep with clinical Johne's disease due to infection with Mycobacterium avium ssp. paratuberculosis (M.a. paratuberculosis). These animals had previously been categorised into two groups exhibiting either the 'tuberculoid' (paucibacillary) form of lesion or the 'lepromatous' (multibacillary) form. Lymphocytes were examined for their capacity, following stimulation with johnin-PPD, to release interferon-gamma (IFN-gamma) and interleukin 2 (IL-2) characteristic of the Th1 subset of MHC Class II-restricted CD4+ (helper) T-cells in other species. The expression of the two cytokines appeared related to the type of histological lesion observed. Antigen-stimulated lymphocytes from the tuberculoid group exhibited greater release of IFN-gamma and IL-2 than lymphocytes from the lepromatous group suggesting a Th1-type of response in the former in which expression of IFN-gamma by PBL showed a significant positive correlation with that expressed by MLN and LPL. Lymphocytes from animals with lepromatous lesions released lesser mycobacterium-induced IFN-gamma and IL-2 indicating a diminished role for a Th1 subset in this group of sheep. Differences in cytokine expression were much more apparent with lymphocytes which were derived from MLN.

Immunological, physiological and pathological responses of rainbow trout (Oncorhynchus mykiss) to increasing dietary concentrations of soybean proteins.

High concentrations of dietary soya were shown to suppress salmonid growth rates and non-specific immune capacity. The immunosuppression became evident at dietary inclusion rates of 60-70% and was coincident with a reduction in weight gains and the appearance of demonstrable pathological changes in the distal intestine. Further increases in soya concentrations to 80-89% caused a progressive decline in specific growth rates and exacerbation of the intestinal pathology. There was no evidence of circulating antibody responses to dietary soybean proteins at any of the rates of inclusion. These observations confirm the findings of other authors that, at concentrations of up to 20-30% inclusion in diets, soybean proteins can provide a partial replacement for fish meal, but at higher concentrations detrimental effects become apparent, not only through reduced weight gains, but also through other physiological and immunological changes.

A study of immunological responses of sheep clinically-affected with paratuberculosis (Johne's disease). The relationship of blood, mesenteric lymph node and intestinal lymphocyte responses to gross and microscopic pathology.

Nineteen adult sheep diagnosed as having clinical paratuberculosis (Johne's disease) and 16 unaffected controls were examined in this study. Animals were tested for the presence of circulating antibodies of Mycobacterium avium ssp. paratuberculosis (M. a. paratuberculosis) and lymphocytes derived from the blood, mesenteric lymph nodes and intestines were examined for cell-mediated immune (CMI) responses to Johnin pure protein derivative (Johnin-PPD: J-PPD). Bacteriological examinations were carried out on faeces and tissues and any mycobacterial isolates identified as M. a. paratuberculosis (IS900+) or M. avium ssp. silvaticum (M. a. silvaticum) (IS901+) by polymerase chain reaction (PCR). Full necropsy and histopathological studies were performed and diseased animals were categorised on the basis of having a lepromatous or tuberculoid form of intestinal pathology. Unaffected control sheep were generally antibody-negative and demonstrated varying CMI responses to J-PPD. Clinically-affected animals were almost always antibody-positive with variable CMI responses. A correlation was observed between the histological lesion type in the intestine and the cellular immune response. Tuberculoid-type lesions were associated with strong CMI responses in lymphocytes derived from the peripheral blood, mesenteric lymph node and intestine, whereas lepromatous-type lesions were associated with weak CMI responses in all tissues examined.

Detection of specific T cell reactivity in sheep infected with Mycobacterium avium subspecies silvaticum and paratuberculosis using two defined mycobacterial antigens.

A 30 kDa antigen (P30) from Mycobacterium avium ssp. paratuberculosis (M. a. paratuberculosis) and a 40 kDa (P40) antigen from Mycobacterium avium ssp. silvaticum (M. a. silvaticum) were employed in two different assays to measure the cell-mediated immune reactivity of ovine peripheral blood lymphocytes. In lymphocyte stimulation assays, proliferative responses to the P30 were observed only with lymphocytes from sheep inoculated with live M. a. paratuberculosis or M. a. silvaticum. Although this antigen was not subspecies-specific it differentiated between animals given live organisms and those inoculated with an inactive lysate. The P40 protein from M. a. silvaticum showed subspecies specificity by eliciting in vitro responses only with lymphocytes derived from sheep inoculated with live M. a. silvaticum. Similar results were obtained using an interferon-gamma release assay which proved to be a more rapid and sensitive system.

Phenotypic analysis of lymphocytes obtained by bronchoalveolar lavage of normal sheep.

Lungs were excised from apparently healthy sheep at slaughter and lymphocytes obtained by bronchoalveolar lavage were separated by buoyant density and characterised phenotypically. In most animals T-lymphocytes (CD5+ cells) accounted for almost the entire population, with B-lymphocytes (sIg+ cells) generally accounting for less than 10%. In the main, CD4+ T cells exceeded CD8+ T cells by at least a factor of three and CD4-/CD8- (gamma delta) T cells represented around 6% of the population. Proportions of subtypes of lymphocytes obtained by bronchoalveolar lavage differed from those in peripheral blood. The normal range of lymphocyte phenotypes in the ovine lung established in this study will be of value in subsequent work on animals with defined respiratory disease.

Phenotypic analysis of lymphoblastoid cell lines derived from cattle and deer affected with "sheep-associated" malignant catarrhal fever.

Established lymphoblastoid cell lines with natural killer cell-like activity have been derived from cattle and deer affected with malignant catarrhal fever. They were examined phenotypically using monoclonal antibodies chosen for their cross-reactivity with peripheral blood lymphocytes from these species. Cell lines established from three of four cattle were identified as cytotoxic/suppressor lymphocytes (CD4-/CD8+/T19-) whilst the other was shown to be of the helper cell phenotype (CD4+/CD8-/T19-). Two other cell lines, one derived from a red deer and the other from a Père David's deer, were both CD4-/CD8-/T19. All of the lines examined expressed a T cell receptor (CD2+).

Immune responses of the ovine lymph node to Chlamydia psittaci. A cellular study of popliteal efferent lymph.

The popliteal efferent lymphatics were cannulated in sheep of two categories, seronegative or immune to Chlamydia psittaci. Following subcutaneous injection of live C. psittaci or control material into the draining area of the popliteal node, sequential samples of efferent lymph were collected and analysed. Both categories of sheep responded to C. psittaci with increased outputs of lymphocytes and blast cells. Numbers of blast cells rose both absolutely and as a proportion of the total. Plasmablasts increased in number only in seronegative sheep. Outputs of total T cells (CD5+), helper T cells (CD4+), cytotoxic/suppressor T cells (CD8+) and non-helper, non-suppressor T cells (T19) were maximal 4 and 7 days after challenge in immune and seronegative sheep, respectively. Proportionally, CD4+ T cells declined, CD8+ T cells increased and T19 cells were unaltered with time after infection. Chlamydial antigens could not be demonstrated in the cells of efferent lymph by an immunoperoxidase method. The results of this preliminary study show that both T and B cell responses are involved in immunity to C. psittaci.

Lymphocyte subpopulations in the blood of sheep persistently infected with border disease virus.

The surface phenotypes of peripheral blood lymphocytes in groups of lambs and adult sheep persistently infected with Border disease virus (P-I BD) were compared with those of healthy controls. The proportion and number of lymphocytes bearing surface immunoglobulin (sIg+) and expressing class II MHC antigen (B cells) were significantly increased. A significant increase in CD1+ lymphocytes was also evident. Conversely, the proportion of T lymphocytes in P-I BD lambs was reduced. A marked reduction in the proportion of circulating lymphocytes expressing class I MHC antigen was also observed. These findings were not affected by differences in the strain of the virus responsible for the persistent infection.

Bronchoalveolar lavage of the live anaesthetised sheep.

Segmental lavage was performed on anaesthetised adult sheep using either endoscopic catheterisation or fibreoptic bronchoscopy. Cellular and humoral data were compared with results obtained from gross lavage of whole excised lungs. Both techniques proved rapid, convenient and reproducible, providing sufficient material for qualitative and quantitative assays of respiratory cells and fluid. Although total cells per unit volume of fluid obtained by catheterisation or by bronchoscopy were decreased compared with whole lung lavage, differential cell populations, fluid recovery and immunoglobulin and complement (C3) concentrations were comparable.

Immunity of specific pathogen-free lambs to challenge with an aerosol of Pasteurella haemolytica biotype A serotype 2. Pulmonary antibody and cell responses to primary and secondary infections.

Specific pathogen-free (SPF) lambs previously exposed to an aerosol of P. haemolytica biotype A serotype 2 (A2) were immune to subsequent challenge with an aerosol of P. haemolytica A2. Untreated control lambs were not immune to this challenge. The local immune responses of the lung to these challenges were examined. High IgG and IgA titres to P. haemolytica and high levels of opsonizing antibody against P. haemolytica were present in the lung washings from previously infected immune lambs at autopsy, seven days after the second infection. Lung washings from control lambs, 7 days after challenge with P13 virus and P. haemolytica A2, had no IgG titres, very little opsonizing activity but did have IgA titres which were significantly higher than in unchallenged control lambs. The cellular response of animals challenged with P13 virus and P. haemolytica was significantly greater than that of unchallenged controls or of lambs exposed only to P. haemolytica. However, this finding was complicated by the response to P13 virus. Lymphocytes from lung washings of all lambs failed to respond in a lymphocyte stimulation test to phytohaemagglutinin while blood lymphocytes did respond. There was little specific response to P. haemolytica antigen in the test.

Cellular and humoral elements of the lower respiratory tract of sheep. Immunological examination of cells and fluid obtained by bronchoalveolar lavage of normal lungs.

Bronchoalveolar washings were harvested from the excised lungs of 68 normal sheep of 3 different age groups (A: birth to 8 weeks; B: 6 months to 2 years; C: older than 2 years). Cells and fluid obtained were examined quantitatively and functionally. Fewer cells were present in the lavage fluids of Group A sheep compared with those of Groups B and C. Macrophages were the predominant cell type (70-80%) in all sheep, with lymphocytes being second in number. The lower proportion of lymphocytes in young sheep (Group A) was attributable to lack of B-lymphocytes. As a proportion of the lymphocyte population T-cells were in the majority (60-80%) in all sheep. The proportion of null cells was higher in young sheep than in adults. Pulmonary lymphocytes from sheep of all ages responded poorly to stimulation by the non-specific mitogens phytohaemagglutinin, concanavalin A, pokeweed mitogen and lipopolysaccharide of Escherichia coli. The proportion of neutrophils was higher in sheep over 2 years old compared with younger animals. Eosinophils were present in all age groups but their proportion was greatly increased in animals over 2 years of age. Basophils were absent in young sheep and were present in only low numbers in the lungs of adult animals. In young sheep (Group A) IgG was the predominant immunoglobulin. With age, the percentage of IgG in lung fluid decreased while that of IgA increased so that IgA became the predominant immunoglobulin in older animals (Group C). In sheep of all ages IgM was present in negligible amounts. The highest value of complement (C3) occurred in adult sheep (Group B). The total white blood cell counts and differential blood counts of all the sheep were within accepted normal ranges. As in the lungs, there was an age-associated reduction in the proportions of blood T-lymphocytes and an increase in B-lymphocytes. In contrast to lung lymphocytes, those from the blood of sheep of all ages exhibited a wide range of responses to mitogens. The lowest stimulation indices were observed in the oldest sheep (Group C). The results provide background data against which assessment can be made of changes consequent upon infection with respiratory pathogens.

Antigenic relationships between the serotypes of Pasteurella haemolytica demonstrable by enzyme-linked immunosorbent assay (ELISA).

Mice and rabbits were immunised with sodium salicylate extracts (SSE) prepared from each of 12 serotypes of Pasteurella haemolytica, and the antisera to each were used in cross-indirect haemagglutination (IHA) tests and cross-enzyme-linked immunosorbent assays (ELISA) to study antigenic relationships between the serotypes. An indirect micro-ELISA demonstrated common antigenic relationships which were not apparent by IHA. Antisera from both species revealed considerable shared antigenicity between all the serotypes. Rabbit antisera presented clearer differences between the A biotypes on one hand and the T biotypes on the other, the T biotypes exhibiting much less cross-relatedness than that shown between the A serotypes.

Specificity of the enzyme-linked immunosorbent assay (ELISA) for antibodies in the sera of specific pathogen-free lambs vaccinated with Pasteurella haemolytica antigens.

Pooled serum from specific pathogen-free (SPF) lambs vaccinated with sodium salicylate extracted (SSE) antigens of Pasteurella haemolytica serotype A1 was shown to contain antibody to other A serotype SSE antigens when tested by the enzyme-linked immunosorbent assay (ELISA). Specific antibody to serotype A1 SSE antigens was demonstrated by absorption of the serum pool with heterologous serotype SSE antigens. The type-specific antigens of serotypes A1 and A9 were prepared by phenol--water extraction (PWE) of their respective SSE antigens. The PWE antigens were examined in a sandwich ELISA where rabbit IgG anti-P. haemolytica A1 cells or A9 cells was used as a coating layer to bind PWE antigens. The specificity of these antigens was demonstrated by marked reduction of reactivity between serum from SPF lambs vaccinated with SSE of serotypes A1 or A9.

IgG1 antibody in milk protects lambs against rotavirus diarrhoea.

Newborn gnotobiotic lambs were fed a diet of diluted evaporated milk supplemented either with normal ewes' milk or with milk obtained from ewes injected parenterally during gestation with rotavirus. Lambs fed 150 ml per day of milk collected 5 days after lambing from normal ewes were susceptible to rotavirus infection and diarrhoea, while lambs fed milk from vaccinated ewes collected either 5 or 12 days after lambing were protected. Analysis of the milk by column chromatography showed the anti-rotavirus activity to be in the fractions containing IgG1.

The effect of pre-natal thymectomy on lymphocyte sub-populations in the sheep.

Ten sheep foetuses were thymectomized between 55 and 77 days gestation. The subsequent growth of the lambs was not affected for periods up to 500 days after birth. Prior to 240 days of age the thymectomized lambs were markedly lymphopenic and the response of their blood lymphocytes to phytohaemagglutinin (PHA) was significantly reduced. Peanut agglutinin-binding cells were found to be depleted in the blood of thymectomized lambs, while an unmarked 'null' cell population was virtually absent. The absolute numbers of E-rosette forming cells and sIg+ cells were similar for both groups. These findings indicated that 'null' cells in sheep may be immature thymus-dependent lymphocytes. The effect of thymectomy on blood counts, PHA responsiveness and the numbers of 'null' cells were less evident in thymectomized sheep that survived beyond 240 days. Possible differentiation pathways for sheep T-cells are discussed, together with the role played by the thymus in the maturation of T-cells.

Serum antibody response of ewes and their lambs to Pasteurella haemolytica.

Vaccination of pregnant ewes with a Pasteurella haemolytica adjuvanted vaccine which contained serotypes A1, A2 and A6 antigens caused significant increases in serum antibody titres to A1 and A6 measured by the indirect haemagglutination test (IHA). The response to vaccination with the serotype A2 antigen contained in this vaccine cannot be measured by the IHA test. There were also increased antibody titres in the colostrum from the vaccinated ewes and in the serum of their lambs after sucking compared with the corresponding titres in unvaccinated ewes and their lambs. The inoculation of either 10 +/- 2- or 18 +/- 2-day-old lambs from both vaccinated or unvaccinated ewes with the same vaccine induced the active production of antibodies to serotypes A1 and A6 despite the presence of passively acquired antibodies. By four weeks after vaccination the group geometric mean serum antibody titres of lambs from both vaccinated and unvaccinated were similar whether the lambs were vaccinated at 10 or 18 days of age. Successful vaccination of young lambs with this type of vaccine is therefore possible. Optimum protection would be obtained by vaccinating ewes in late pregnancy and their lambs within the first two weeks of life.