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.

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.

Entry and survival of Salmonella enterica serotype Enteritidis PT4 in chicken macrophage and lymphocyte cell lines.

Various leukocytes are involved in the reaction to counter Salmonella infection in chicken. The various leukocyte types react differently after an infection, since some clear the infection while others may cause dissemination of Salmonella throughout the chicken. Therefore, we investigated in vitro the entry and survival of Salmonella enterica serotype Enteritidis in chicken cell lines of various cell types, including two macrophage cell lines, HD11 and MQ-NCSU (NCSU), two B-cell lines LSCC-1104-X5 (1104) and LSCC-RP9 (RP9), and a T-cell line MDCC-MSB-1 (MSB-1). The macrophages were able to internalize high numbers of S. Enteritidis. In contrast and as expected, cells of the T-cell line MSB-1 and the B-cell line RP9 internalized bacteria at a much lower level. After S. Enteritidis entered the macrophages, the number of intracellular S. Enteritidis decreased over time, so that after 48h no more than 20% of the bacteria, which had entered, survived intracellularly. In contrast to macrophages, the number of S. Enteritidis in cells of the T-cell line MSB-1 and the B-cell line RP9 increased rapidly within 12h post-inoculation. Thereafter the number of intracellular S. Enteritidis decreased only slowly. In conclusion, all three different cell types were able to control and to start clearing S. Enteritidis, although macrophages were far more effective compared to T- and B-cells. However, none of the cell lines were able to clear S. Enteritidis fully within 48h. These results suggest that the three cell types play an important but different role in the dissemination and elimination of S. Enteritidis throughout the animal.

The localization and uptake of in ovo injected soluble and particulate substances in the chicken.

The localization of in ovo injected substances in the chicken was investigated. We determined that localization is dependent on the nature of substances and the time of in ovo injection. In ovo injections with soluble bromodeoxyuridin (BrdU), particulate colloidal carbon, 40 nm fluorescent microspheres, and live Infectious Bursal Disease Virus (IBDV) were performed with a 25-mm (1-in) needle at Days 16 and 18 of incubation (DI-16 and DI-18, respectively). Localization of injected substances was determined in several organs using immunocytochemical methods. At DI-16, approximately 50% of the substances were detected in the organs; therefore, the localization of substances was not consistent. At DI-18, the substances were injected into the amnion. The substances entered the embryo by the mouth and were ingested into the intestinal and respiratory tract. All substances reached the lungs of the embryo via the trachea and the bronchi and were absorbed by the gas exchange tissue. In addition, the substances were absorbed by the bursa. Particulate colloidal carbon and microspheres remained in the organs where they were taken up initially for the rest of time of the experiment. Live IBDV, however, was distributed to other organs of the embryo. Soluble BrdU was found in all investigated organs of the embryo in high amounts. These results demonstrate that in ovo injection at DI-18 is an effective route to introduce substances into the chicken embryo, whereby the characteristics of the substance determine its final localization.

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.

Alveolar macrophages, surfactant lipids, and surfactant protein B regulate the induction of immune responses via the airways.

The influences of alveolar macrophages (AM) and pulmonary surfactant on the induction of immune responses via the airways were assessed. Mice were depleted of their AM by intratracheal instillation of multilamellar vesicles containing dichloromethylene-diphosphonate followed by intratracheal instillation of a T cell--dependent antigen, trinitrophenyl--keyhole limpet hemocyanin, in vesicles of various compositions. The primary immune response was determined in the spleen of these animals using an ELI-Spot assay. The secondary immune responses in the sera of the mice were assessed using enzyme-linked immunosorbent assays. An immune response was detected in animals depleted of their AM and intratracheally instilled with antigen in small unilamellar vesicles consisting of either phosphatidylcholine cholesterol or surfactant lipids. Incorporation of surfactant protein (SP)-B in the antigen vesicles enhanced the immune response, whereas SP-A or SP-C in the antigen vesicle did not have an effect. Strikingly, intratracheal instillation of SP-B containing antigen vesicles can induce an immunoglobulin M immune response in mice without depletion of AM. These results indicate that SP-B containing vesicles can enhance the induction of immune responses via the airways and further illustrate the important roles of both AM and pulmonary surfactant in the pulmonary immune system.

Defence mechanisms against viral infection in poultry: a review.

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.

Immunocytochemical techniques to investigate the pathogenesis of infectious micro-organisms and the concurrent immune response of the host.

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.

Further characterization of M cells in gut-associated lymphoid tissues of the chicken.

M cells are considered to be the most effective cells for the transport of antigens from the intestinal lumen into the gut-associated lymphoid tissue. M cells are characterized by their ultrastructural appearance, the selective uptake of antigens, the binding of lectins, and the presence of underlying lymphocytes. Little attention has been paid to the interaction of intra-epithelial leucocytes and M cells in chickens; therefore, we have investigated both cell types separately and using double immunocytochemical staining in cecal tonsils and Meckel's diverticulum. In the follicle-associated epithelium (FAE), cells were present that differ from their neighbors by short, irregular microvilli. Ferritin was absorbed by these putative M cells, but also by other epithelial cells. The lectins of Triticum vulgaris (WGA) and Glycine max (SBA) showed a patchy staining of the FAE. The numbers of intra-epithelial leucocytes (IEL) increased rapidly after hatch, reaching innumerable at 6 wk of age. Most IEL were T lymphocytes expressing CD8 and only about 30% of them were B lymphocytes. Nevertheless, double staining of M cells (WGA/SBA) and IEL showed that M cells were much fewer than IEL. These results indicate that M cells are not solely induced by the intra-epithelial localization of leucocytes. Because the phenotype of IEL reflected the content of the adjacent underlying lamina propria, IEL immigrate the FAE locally and do not migrate along with the epithelial cells from the crypts. In conclusion, M cells exist in the chicken, but their phenotype and function are less well demarcated from neighbor epithelial cells than is seen in mammals.

Prenatal development of hematopoietic and hormone-producing cells in the chicken adenohypophysis.

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.

The use of chicken-specific antibodies in veterinary research involving three other avian species.

In this study we investigated a panel of 20 polyclonal and monoclonal antibodies specific for chicken cells and tissues for cross-reactivity with other avian species, i.e. turkey, duck and quail, using immunoperoxidase staining on cryostat sections. The number of cross-reacting antibodies was highest for turkey (12/20) and quail (10/20) and lowest for duck (5/20), reflecting the phylogenetic distance between these birds. In ducks only antibodies specific for mononuclear phagocytes and for stromal molecules in mesenchym, muscle cells, endothelial, and epithelial cells cross-reacted. In turkeys and quails cross-reaction was seen with antibodies against T-helper lymphocytes, IgM- and IgG-positive B cells and plasma cells, and mononuclear phagocytes. In addition, most antibodies specific for stromal molecules reacted against turkey or quail molecules. The panel of reacting antibodies can be used for research into the natural and specific defence mechanisms of turkeys and quails.

The working mechanism of an immune complex vaccine that protects chickens against infectious bursal disease.

The role of immune complexes (Icx) in B-cell memory formation and affinity maturation allow for their potential use as vaccines. Recently, a new immune complex vaccine has been developed that is currently under field trials conducted in commercial poultry. This immune complex vaccine is developed by mixing live intermediate plus infectious bursal disease virus (IBDV) with hyperimmune IBDV chicken serum (IBDV-Icx vaccine). Here we have investigated the infectivity of this vaccine as well as the native IBDV (uncomplexed) vaccine in terms of differences in target organs, in target cells and speed of virus replication. At various days after inoculation on day 18 of incubation (in ovo) with either one dose of virus alone or the IBDV-Icx vaccine, the replication of IBDV and the frequency of B cells and other leucocyte populations were examined in the bursa of Fabricius, spleen, and thymus using immunocytochemistry. With both vaccines, IBDV was detected associated with B cells, macrophages and follicular dendritic cells (FDC) in bursa and spleen, although complexing IBDV with specific antibodies caused a delay in virus detection of about 5 days. Most remarkable was the low level of depletion of bursal and splenic B cells in IBDV-Icx vaccinated chickens. Furthermore, in ovo inoculation with the IBDV-Icx vaccine induced more germinal centres in the spleen and larger amounts of IBDV were localized on both splenic and bursal FDC. From these results we hypothesize that the working mechanism of the IBDV-Icx vaccine is related to its specific cellular interaction with FDC in spleen and bursa.

Induction of a local and systemic immune response using cholera toxin as vehicle to deliver antigen in the lamina propria of the chicken intestine.

In this study, the humoral mucosal immune response to a recombinant Eimeria antigen (Ea1A) was enhanced using cholera toxin (CT). Chickens were primed intra-intestinally with Ea1A either conjugated or not to CT. The local and systemic antibody responses to both Ea1A and CT were determined to find out whether the chickens could respond to CT and whether both antigens had reached the lamina propria. In addition the effects of CT on lamina propria leukocytes were examined. The results showed that chickens had receptors on the caecal epithelium that could bind CT. At day 7 after administration, the number of CD4+ and CD8+ T lymphocytes in the lamina propria of the caecum had increased, indicating that CT had a specific immunological effect. At this timepoint, anti-CT antibody containing cells were detected locally in the lamina propria of the caecum. In serum all antigen preparations containing CT induced IgM and IgG antibody titres specific for CT within 10 days after priming. In addition, the recombinant Ea1A antigen also induced serum responses when administered together with CT or conjugated to CT, thus both CT and the antigen had reached the lamina propria. Nevertheless, the Ea1A specific response was much higher in the primary response and after booster immunization when the antigen was conjugated to CT than when only mixed with CT. Therefore, we conclude that CT is a suitable adjuvant for intra-intestinal application in chickens, especially when the antigen is conjugated to it.

Inadequate anti-polysaccharide antibody responses in the chicken.

Chickens are notorious for the fact that they carry bacteria such as Salmonellae and Campylobacter, which can cause zoonoses by contamination of the end product, without hampering growth and development of the chicken itself. This carrier status can only been explained by the inability of the chickens immune system to eliminate the pathogen, this in turn being due to insufficient humoral responses towards the polysaccharides of the bacterial capsule. In a previous study, we demonstrated that in chickens a model thymus-independent type 2 (TI-2) polysaccharide antigen, trinitrophenylated Ficoll (TNP-Ficoll), hardly evokes a humoral immune response. Furthermore this TI-2 antigen was shown to exhibit a very specific initial localization pattern after intravenous injection, i.e. in the periellipsoidal lymphocyte sheaths (PELS) and the surrounding ring of macrophages. The functional equivalent of these macrophages in mammals, the marginal zone macrophages, were shown to suppress the humoral responses against TI-2 antigens. Therefore we investigated whether other standard TI-2 antigen models also induce low antibody responses, whether this low response is dose-dependent, and whether macrophages are responsible for this low response. It was found that other TI-2 antigens, such as hydroxyethyl starch and detoxified lipopolysaccharides, also induced very low IgM and IgG responses, indicating a general phenomenon that could not be overcome by using a higher dose of antigen. In addition, selective depletion of splenic macrophages with liposomes containing dichloromethylene diphosphonate prior to immunization increased the specific humoral response to TD and TI-1 antigens, but failed to do so for TI-2 antigen. This result indicates that the low humoral responses are not (only) due to a macrophage suppressive activity but also to other yet unknown mechanisms, for example the lack of responsive B cells in the splenic PELS.

Eimeria tenella infections in chickens: aspects of host-parasite: interaction.

Intestinal coccidiosis, caused by various species of Eimeria, has become an economically important disease of poultry and livestock throughout the world. Infection of chickens starts after ingestion of oocysts when sporozoites penetrate the epithelium of the villi. After passage through the lamina propria, they enter crypt epithelial cells where they undergo several rounds of asexual and sexual proliferation, thus forming merozoites and later, gametocytes. When macrogametes are fertilized by microgametes, oocysts are formed that are shed in the faeces. Nowadays, coccidiosis is prevented by anticoccidial drugs that are added to food, but the prolonged use of these drugs leads inevitably to the emergence of resistant Eimeria strains. During infection, there are three stages when the chicken immune system can inhibit parasitic development. The first is when the sporozoite searches for a site of penetration and binds to the epithelium. The second is when the sporozoite is in the villus epithelium amongst intra-epithelial leucocytes. The third is during its passage through the lamina propria to the crypt epithelium. To investigate this, the decisive factors in the induction and effector phase of immunity against coccidiosis have been investigated in situ. Our studies have revealed that three phenomena are responsible for immunity against Eimeria infections. First, the actual passage and presence of parasites in the lamina propria to induce immunity. Second, the sporozoite seems to be the most important parasite stage for immunity, and third, cytotoxic T cells are necessary to inhibit parasites.

The B cell compartment of two chicken lines divergently selected for antibody production: differences in structure and function.

In the present study differences in the B cell compartment of two chicken lines selected for either high (H) or low (L) antibody response to sheep red blood cells (SRBC) are investigated. In non-immunized chicks, flow cytometry revealed generally more circulating Ig+ leukocytes in the H line, while in the L line slightly more CD4+ and in week 5, more CD8+ cells were found. In the L line spleen more CD8+ were found and in the H line spleen more CD4+ cells. In week 6, half of the chicks were immunized. Both lines were similarly affected by immunization. Immunization reduced the percentages of the circulating T cell subpopulations, while Ig+ cells were enhanced, compared with non-immunized chicks. Histological determinations with specific mAbs on spleens of young, non-immunized chicks, showed large dense T cell areas in the L line, while in the H line more and larger germinal centres were found. In the H line, also, more B cells were found in the peri-ellipsoid lymphoid sheaths (PELS). No line differences in mononuclear phagocytes were found other than associated with line differences in numbers of PELS and germinal centres. After immunization with TNP-BSA, both higher numbers of TNP-specific antibody producing cells and higher levels of circulating antibody were found in the H line. Moreover, more TNP-specific plasma cells were found in non TNP-immunized H line chicks, than in the L line chicks. The H line had also higher ELISA-titers to KLH 5 days after immunization with KLH. Therefore it was concluded that selection for antibody response has affected the B cell compartment. The H line has relatively more B cells and the splenic structure of the H line differs from the L line, in the H line probably resulting in a more optimal organization for antibody response to T cell dependent antigens.

In situ characterization of leucocyte subpopulations after infection with Eimeria tenella in chickens.

We characterized the leucocyte subpopulations after infection with Eimeria tenella in both naive and immune chickens. Immunocytochemical staining was used to characterize the cells in situ, so that the interaction between host and parasite could be studied. More leucocytes were detected in the lamina propria of immune chickens, and leucocytes infiltrated the ceca more rapidly than in naive chickens, but the infiltration was less pronounced than in naive chickens. In naive chickens, most infiltrated leucocytes were macrophages and T cells. Two days after inoculation the number of CD4+ cells had increased greatly. In immune chickens, mainly T cells (CD4+ and CD8+) infiltrated the lamina propria, and in contrast to naive chickens, the number of CD8+ cells exceeded the number of CD4+ cells. Furthermore, we characterized which cells contained a parasite and which cells were detected next to the parasites, because these cells are probably involved in the arrested development of the parasites. In naive chickens, sporozoites were significantly more often located within or next to macrophages than in immune chickens. In immune chickens, sporozoites were significantly more often located within or next to CD3+, CD8+, and TCR2+ cells. In conclusion, the marked increase of CD4+ cells after primary infection suggests that these cells are involved in the induction of the immune response, whereas the increase of CD8+ cells after challenge infection suggests that these cells act as effector cells.

Differences in distribution of lymphocyte antigens in chicken lines divergently selected for antibody responses to sheep red blood cells.

The proportion of cells showing differentiation antigens specific for T cells, B cells and leukocytes was studied at various ages in peripheral blood, and at 14 weeks of age in the bursa of Fabricius, spleen and thymus of two lines of chicken that had been selected over 13 generations for either high (H) or low (L) antibody responses to sheep red blood cells (SRBC), and also in a randombred control (C) line. Flow cytometry showed no consistently significant differences between the three lines in numbers of circulating lymphocytes and other leukocytes after hatching. However, higher percentages of CD4+ cells and B cells were present in the spleen and thymus from the H line compared with the L line. However, the L line was characterized by a higher proportion of splenic CD8+ cells and spleen cells expressing gamma-delta T-cell receptors. Immunization with sheep red blood cells had no effect on the distribution of CD4+ or CD8+ cells in the various tissues at 2 and 7 days after immunization. These results suggest that previously reported differences in in vivo immune responses between these chicken lines may be related to the differences in resident T-lymphocyte subpopulations in the lymphoid tissues. The involvement of T-cell subsets and non-antigen-specific mechanisms in divergent selection on humoral immune responses in chickens is discussed.

Eimeria tenella: sporozoites rarely enter leukocytes in the cecal epithelium of the chicken (Gallus domesticus).

In this study, the intraepithelial leukocytes supposedly involved in the transportation of Eimeria tenella sporozoites through the cecal lamina propria were phenotypically characterized. The ceca of naive and immune chickens were examined at various times after inoculation by light microscopy and immunocytochemical techniques. The distribution of sporozoites within the villus differed markedly between both groups. From 16 hr postinoculation, significantly fewer sporozoites had reached the crypts in immune chickens, and schizont formation was inhibited. In the villus epithelium of both naive and immune chickens, few sporozoites were found within a leukocyte. Using anti-sporozoite and anti-CD45 monoclonals, we showed that even when intraepithelial leukocytes are abundantly present, only a few sporozoites were inside them. In the lamina propria of immune chickens significantly more sporozoites were found within leukocytes than in the lamina propria of naive chickens. The phenotype of the few leukocytes that harbored sporozoites was similar in naive and immune chickens. A few sporozoites were detected in B cells, 10% in macrophages, and 50% in T cells, especially CD8+ cells. These results show that E. tenella sporozoites rarely enter intraepithelial leukocytes and therefore their putative role in transporting sporozoites through the lamina propria is doubtful.