Suzan H.M. Jeurissen

Immunomodulation by probiotic lactobacilli in layer- and meat-type chickens

1. The aim of the experiments was to evaluate whether selected probiotic lactobacillus strains have different immunomodulating effects in layer- and meat-type strain chickens. 2. Humoral and cellular specific and non-specific immune responses were studied by experiments on cellular proliferation, entry and survival of Salmonella bacteria in gut and spleen leukocytes, immunoglobulin isotypes and specific immunoglobulin titres. 3. The effects of two different feeding regimes (short and continuous feeding) and doses for administration of lactobacilli were studied. 4. The lactobacillus strains that were evaluated showed modulating effects on the immune system of layer- and meat-type chickens. 5. In meat-type strain chickens the lactobacilli had a stimulating effect when the chickens were young (up to 3 weeks) and the dose was relatively high, whereas in layer-type chickens a lower effective dose and discontinuous administration was also effective. 6. Immunoprobiotic lactobacilli can have a positive effect on humoral and cellular immune responses in layer- and meat-type strain chickens, but the lactobacillus strain to be used, the age of the animals and effective dose of lactobacilli to be administered need to be optimised.

Postnatal development of mucosa-associated lymphoid tissues in chickens

SummaryThe postnatal development of chicken mucosa-associated lymphoid tissues of the eyes, lungs, and intestines were investigated with monoclonal antibodies specific for either all leucocytes, B lymphocytes, mononuclear phagocytes, IgM, IgG, or IgA. Attention has been paid to the relation of lymphoid infiltrates with their surrounding mucosae, the segregation into B-cell and T-cell areas, development of germinal centers, and secretory immunoglobulins. Abudant secretory IgM and IgA was detected in the epithelium of the Harderian glands in the orbits, even though they lacked large leucocyte infiltrates with germinal centers. Lymphoid tissues in the mucosae of lungs and intestines developed separate B-cell and T-cell areas. The proventriculus, Meckels diverticulum, and Peyers patches generally contained germinal centers from 12 weeks of age on. Because chickens as young as 2 weeks old had germinal centers in bronchus-associated lymphoid tissue and cecal tonsils, these areas were probably highly stimulated by antigens. Isotype-specific monoclonal antibodies were used to detect IgM-, IgG-, and IgA-bearing follicular cells in the same germinal center.

Immunological differences between layer- and broiler-type chickens

In commercial poultry husbandry, alternatives for the use of antibiotics and vaccines are under investigation, which preferably have to be applicable for both layer- and broiler-type chickens. There are indications that the defense mechanisms vary between layer- and broiler-type chickens. Therefore, the difference in immune response between layer- and broiler-type chickens of the same age was investigated, using TNP-KLH (trinitrophenyl-conjugated keyhole limpet hemocyanin) as antigen without adjuvant. First different routes of immunization (intravenously, intramuscular, subcutaneous and ocular) were examined to find out which immunization route gives the highest antibody titers. The intravenous immunization route resulted in higher TNP-specific antibody responses than the other immunization routes tested and therefore this immunization route was used in both following experiments. In order to investigate the optimal dose of antigen needed for immunization, a dose-response curve in broiler- and layer-type chickens was completed. The humoral immune response was measured in serum by a TNP-specific ELISA and the in vitro cellular immune response by an antigen-specific lymphocyte proliferation assay. The antibody response of layer- and broiler-type chickens appeared to differ, not only in optimal dose and response, but also in kinetics of the response itself. Broiler chickens generated higher IgM anti-TNP titers whereas layer-type chickens generated higher IgG anti-TNP titers. This specific antibody response in broiler-type chickens did not last as long as in layer-type chickens. The TNP-specific cellular immune response was detectable in layer-type chickens, but not in broilers. Both types generate a non-specific cellular immune response, although this response in broilers is lower than in layer-type chickens. From these results, we conclude that broilers primarily respond to TNP-KLH with a high IgM antibody response whereas layer-type chickens respond with a high IgG response. In addition, the cellular response of layer-type chickens is much higher than the response of broilers. The results suggest that broilers are specialized in the production of a strong short-term humoral response and layer-type chickens in a long-term humoral response in combination with a strong cellular response, which is in conformity with their life expectancy.

Monoclonal antibodies as probes for defining cellular subsets in the bone marrow, thymus, bursa of Fabricius, and spleen of the chicken

Using immunohistochemistry, the distribution and characteristics of cells detected by the newly developed monoclonals HIS-CI (B lymphocytes), HIS-C7 (leucocytes), HIS-C12 (IgM), CVI-ChIgM-59.7 (IgM), CVI-ChIgG-47.3 (IgG), and CVI-ChIgA-46.5 (IgA) are described in bone marrow, thymus, bursa of Fabricius, and spleen of chickens of different ages. Furthermore, quantification of cells positive with the described monoclonal antibodies was performed on cytocentrifuge preparations. The specificities of the monoclonal antibodies are discussed.

The monoclonal antibody CVI-CHNL-68.1 recognizes cells of the monocyte-macrophage lineage in chickens

The characteristics of monoclonal antibody CVI-ChNL-68.1, which specifically reacts with a group of chicken non-lymphoid cells, are described. Both tissue distribution shown on cryostat sections using immuno-enzyme histochemistry, and quantitative data obtained on cell suspensions are presented. Functional characteristics of CVI-ChNL-68.1-positive cells, such as antigen uptake and glass adherence, are determined. Results show that CVI-ChNL-68.1 reacts with monocytes, macrophages, and interdigitating cells. Possible relationships between the various non-lymphoid cells are discussed.

Ontogeny and Function of Two Non-Lymphoid Cell Populations in the Chicken Embryo

The purpose of the study was to determine what type of non-lymphoid cells develop in chicken embryos during ontogeny, and whether these cells are functional. To detect these cells, we used monoclonal antibodies specific to two groups of non-lymphoid cells: CVI-ChNL-68.1, specific for mononuclear phagocytes, and CVI-ChNL-68.2, specific for a subpopulation of reticulum cells in spleen, liver and bursa. Monoclonal antibodies HIS-C7, HIS-C1, and HIS-C12, which are specific to leukocytes, B lymphocytes, and IgM respectively, were used to correlate the ontogeny of non-lymphoid cells and lymphoid cells. Mononuclear phagocytes and reticulum cells were detected in the liver, spleen, yolk sac, bursa, gut, and thymus at about the same time as leukocytes, but earlier than B lymphocytes. To determine whether mononuclear phagocytes and reticulum cells in spleen and liver absorb antigen, we injected embryos intravenously with colloidal carbon and the antigen FITC-Ficoll. In addition, acid phosphatase was used as a marker for phagocytic activity. Reticulum cells in the liver and spleen were functional from the first point of detection, whereas mononuclear phagocytes in the liver and spleen started to absorb antigen a few days after their development.

Distribution and function of non-lymphoid cells positive for monoclonal antibody CVI-ChNL-68.2 in healthy chickens and those infected with Marek's disease virus

Immuno-enzyme histochemistry was used to study the staining pattern and tissue distribution of monoclonal antibody CVI-ChNL-68.2 that specifically reacts with a subset of non-lymphoid cells in healthy chickens and those infected with Mareks disease virus (MDV). Functional characteristics of CVI-ChNL-68.2-positive cells, e.g. antigen uptake, are determined. In the liver CVI-ChNL-68.2 recognizes reticulum cells, whereas in the bursa of Fabricius it detects single cells in the interfollicular connective tissue. In the spleen CVI-ChNL-68.2 reacts selectively with the reticulum cells of the ellipsoid. In some MDV-infected chickens the splenic reticulum cells show a different staining and distribution pattern. Furthermore, the proliferative lesions associated with Mareks disease contain many CVI-ChNL-68.2-positive cells. The possible role of CVI-ChNL-68.2-positive cells in disseminating Mareks disease virus is discussed.

Characterization of the innate and adaptive immunity to Salmonella enteritidis PT1 infection in four broiler lines

Four broiler lines were inoculated orally with Salmonella enteritidis phage type 1 at the age of 7 days (experiment A: lines 1 and 2) and at the age of 1 day (experiment B: lines 3 and 4). At various days post-infection chickens were sacrificed and the number of Salmonella in the caeca, liver, and spleen were determined. Furthermore, phagocytic activity, cellular immune responses, and humoral responses were determined using, respectively, single-cell suspensions of spleen or intestine and serum. In both experiments, similar trends were seen. Increased numbers of S. enteritidis were found in the caeca of lines 1 and 3, whereas at the same time a decreased colonization was found in the spleen and in the liver, as compared to lines 2 and 4. In the latter two lines, the phagocytic activity of the phagocytes was higher and the humoral responses were lower. Observations from this study suggest that lower activity of phagocytes and higher humoral activity prevent systemic S. enteritidis infection.

Defence mechanisms against viral infection in poultry: a review

Summary Defence against viral infections in poultry consists of innate and adaptive mechanisms. The innate defence is mainly formed by natural killer cells, granulocytes, and macrophages and their secreted products, such as nitric oxide and various cytokines. The innate defence is of crucial importance early in viral infections. Natural killer cell activity can be routinely determined in chickens of 4 weeks and older using the RP9 tumour cell line. In vitro assays to determine the phagocytosis and killing activity of granulocytes and macrophages towards bacteria have been developed for chickens, but they have not been used with respect to virally infected animals. Cytokines, such as interleukin (IL)‐1, IL‐6 and tumour necrosis factor (TNF)‐α, are indicators of macrophage activity during viral infections, and assays to measure IL‐1 and IL‐6 have been applied to chicken‐derived materials. The adaptive defence can be divided into humoral and cellular immunity and both take time to develop and thus are more important later on during viral infections. Various enzyme‐linked immunosorbent assays (ELISAs) to measure humoral immunity specific for the viruses that most commonly infect poultry in the field are now commercially available. These ELISAs are based on a coating of a certain virus on the plate. After incubation with chicken sera, the bound virus‐specific antibodies are recognized by conjugates specific for chicken IgM and IgG. Cytotoxic T lymphocyte activity can be measured using a recently developed in vitro assay based on reticuloendotheliosis virus‐transformed target cells that are loaded with viral antigens, e.g. Newcastle disease virus. This assay is still in an experimental stage, but will offer great opportunities in the near future for research into the cellular defence mechanisms during viral infections.

Comparison of natural resistance in seven genetic groups of meat-type chicken

1. Several studies have shown that genetic variation exists in response to various Salmonella strains in mammals and poultry. In the current study immunocompetence traits related to natural resistance to Salmonella were measured in 7 genetic groups of meat-type chickens (in total 296 chickens involved). 2. Variables were measured of both innate (phagocytic activity) and adaptive immune responses that are important after a natural or experimental Salmonella enteritidis infection. Two traditional Old Dutch Breeds (groups 1 and 2), four commercial broiler groups (groups 3 to 6), and one experimental broiler group (group 7) were used. In two periods, birds of each group were killed for examination at ages between 14 and 35 d post hatch. 3. Significant differences between groups were found for most immune variables measured, with significant correlations between several of them. All groups produced an adequate immune response, of either the innate or the adaptive type. 4. In the current study, group 2 showed the highest overall natural resistance, though none of the groups was uniformly superior with respect to all traits measured. 5. In conclusion, for reliable measurements of general immunocompetence or resistance to Salmonella, for example, it is important to measure several aspects of the immune system.