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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.

The effect of an in ovo infection with a Dutch isolate of avian leukosis virus subgroup J (ALV-J) on the growth of specific pathogen free (SPF) broiler chickens was analysed. During this study, possible immune suppressive effects of ALV-J were assessed by measuring delayed-type hypersensitivity with keyhole limpet haemocyanin (KLH), natural killer (NK) cell activity, the production of radicals of nitric oxide (NO) by macrophages, humoral immune response against Newcastle and infectious bursal disease vaccine viruses, and automated total and differential leukocyte counts. In an attempt to elucidate the underlying causal mechanisms of the induced growth retardation, 3,3',5-triiodothyronine (T3) concentrations in serum were measured. Four experiments were conducted. In experiment 1, ALV-J-injected birds were compared with ALV subgroup A (ALV-A)-injected and negative control chickens. In experiment 2, ALV-J-injected birds were only compared with negative controls. Finally, in experiments 3a and 3b, ALV-J-injected chickens were compared with negative controls and a group of chickens in which only 10% of birds had been injected with ALV-J. Birds were injected in ovo at day 7 of incubation with 10(4) median tissue culture infectious dose (TCID(50)) ALV-J or ALV-A, except in experiment 3a where 10(2) TCID(50) ALV-J was injected. Significant growth suppression was found in all 100% of ALV-J-infected groups. The average growth retardation of ALV-J-infected birds compared with negative controls at 6 weeks of age was approximately 8, 11, 2.5 and 6% for the four successive experiments performed. The delayed-type hypersensitivity test against KLH of ALV-J-infected birds showed a tendency towards lower wattle thickness; however, the difference with controls was not significant (P > 0.05). The same was true for NK cell activity and NO production by macrophages, although the difference was not significant. The total and differential leukocyte counts performed on blood samples from birds at 3, 4 and 6 weeks of age as well as the humoral immune response against Newcastle and infectious bursal disease vaccine viruses did not show significant differences between treatment groups either. Only the number of basophils were significantly higher (P = 0.02) in ALV-J-infected birds at 3 weeks of age. No significant lower T(3) levels were found in ALV-J-infected birds in weeks 2 and 3 (experiment 2) and weeks 3 and 5 (experiment 3b); however, at 4 weeks (experiment 2) and 6 weeks (experiment 3b) of age, T(3) levels were significantly lower suggesting mild hypothyroidism in these broilers. In conclusion, the present experiments show the occurrence of significant growth retardation in SPF broilers after an ALV-J in ovo infection. The various studies performed to assess the immune competence of ALV-J-infected chickens did not show significant differences in immune responsiveness. The assays on cellular immunity showed a tendency to a lower response in ALV-J-infected birds, but these differences were not statistically significant.

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 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)-alpha, 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.

For poultry as well as for mammalian species used for scientific research, many immunocytochemical techniques have been developed to investigate in detail the interaction between infectious micro-organisms and the nonspecific and specific immune systems of the host. In this review three techniques have been described with all technical details necessary to perform them correctly: (1) single immunocytochemical staining to detect the infectious micro-organisms in situ at their site of infection, (2) double immunocytochemical staining to visualize simultaneously the infectious micro-organism and the host cellular response to investigate their interactions, and (3) detection of plasma cells producing antibodies specific to the micro-organism. Of the three techniques the results are described when applied on chicken tissues infected with various micro-organisms, such as Marek's disease virus, chicken anemia virus, infectious bursal disease virus and Eimeria tenella.

The developmental sequence of markers for hematopoietic, hormone-producing, and folliculo-stellate cells in the chicken adenohypophysis is described using immunohistochemical staining techniques. Hematopoietic cells are detected in cryosections of the adenohypophysis starting from 10- or 12-day embryos, using chicken-specific monoclonal antibodies against the leukocyte common antigen (CD45) and the macrophage antigen CVI-ChNL-68.1, respectively. During the second half of embryonic development, CVI-ChNL-68.1-positive macrophages, which are also found in several lymphoid and nonlymphoid tissues of the embryo, progressively populate the adenohypophysis, simultaneously with the maturation of the different hormone-producing cell types in their characteristic topographical location. In cryosections of embryonic chicken adenohypophyses, from day 10, distinct cell populations gradually become immunoreactive to chicken-specific monoclonal antibodies against proopiomelanocortin, the beta-subunit of luteinizing hormone, growth hormone, and prolactin. At hatching, these pituitary hormones are immunohistochemically detectable in a topographical pattern corresponding to the known distribution of hormone-producing cells in the adult chicken adenohypophysis. However, in the hatchling, there is no immunoreactivity to the S100 protein, a marker characteristic for the non-hormone-producing folliculo-stellate (FS) cells in the adult adenohypophysis, although FS cells in the 4-week-old chicken show a strong immunoreactivity to a polyclonal antiserum against bovine S100. Immunoreactivity to the major histocompatibility complex class II (MHC-class II of the chicken) is also absent in the embryonic adenohypophysis, thereby corroborating the absence of these characteristic markers of dendritic cells (MHC class II) and FS cells (S100) in the perinatal adenohypophysis, as in the rat. It is concluded that, whereas early macrophages populate the adenohypophysis simultaneously with the maturation of hormone-producing cells (i.e., during the second half of embryonic development), the FS cell-specific expression of S100 protein does not take place before hatching, and neither does the expression of MHC-class II antigens in the embryonic chicken adenohypophysis., (Copyright 1999 Academic Press.)