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.

Circular double-stranded replication intermediates were identified in low-molecular-weight DNA of cells of the avian leukemia virus-induced lymphoblastoid cell line 1104-X-5 infected with chicken anemia virus (CAV). To characterize the genome of CAV, we cloned linearized CAV DNA into the vector pIC20H. Transfection of the circularized cloned insert into chicken cell lines caused a cytopathogenic effect, which was arrested when a chicken serum with neutralizing antibodies directed against CAV was added. Chickens inoculated at 1 day of age with CAV collected from cell lines transfected with cloned CAV DNA developed clinical signs of CAV. The 2,319-bp cloned CAV DNA contained all the genetic information needed for the complete replication cycle of CAV. The CAV DNA sequence has three partially overlapping major reading frames coding for putative peptides of 51.6, 24.0, and 13.6 kDa. The CAV genome probably contains only one promoter region and only one poly(A) addition signal. Southern blot analysis using oligomers derived from the CAV DNA sequence showed that infected cells contained doubleand single-stranded CAV DNAs, whereas purified virus contained only the minus strand. It is the first time that the genome of one of the three known single-stranded circular DNA viruses has been completely analyzed.

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After infection of 1-day-old chickens, chicken anemia virus (CAV) causes a complete depletion of the thymic cortex by day 14. Since cell death can be caused either by necrosis or by apoptosis, we investigated which type of cell death occurs after in vivo and in vitro infections with CAV. Using electron microscopy and biochemical methods, we demonstrated that CAV induces apoptosis of cortical thymocytes after in vivo infection and of lymphoblastoid cell lines after in vitro infection. At day 13 after in vivo infection, virus-like particles were detected in apoptotic bodies that were absorbed by epithelial cells. These results show that apoptosis, a phenomenon that has been observed for a few other viruses, is also an important phenomenon during the pathogenesis of CAV.

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SUMMARY Using commercially available reagents we developed an enzyme-linked immunosorbent assay to measure lactoferrin in normal human tears. Tears obtained from 38 normal human subjects contained a mean lactoferrin content of 2-2 mg/ml (g/l). No chloride and 0-1% Tween 20 (incubation buffer). Tear samples were also collected with calibrated Schirmer tear strips. The wet strip was cut off so as to contain 10 IlI of tears and immediately placed in 0 49 ml incubation buffer. Control studies whereby known lactoferrin concentrations were applied on Schirmer tear strips showed that over 90% of the lactoferrin was eluted from the strips by this procedure.Lactoferrin in the diluted tear samples was determined by an enzyme-linked immunosorbent assay (ELISA). The method used is based on a competition between solid-phase (constant) and fluid-phase (experimental) lactoferrin for a rabbit anti-human lactoferrin antibody. The solid phase bound rabbit antibody is subsequently measured with a peroxidase-coupled goat anti-rabbit immunoglobulin antibody. The details of the assay are as follows: plastic microcuvettes were coated with lactoferrin by incubating them for one hour at room temperature with a 300 ,lI solution of a commercial preparation of human milk lactoferrin (5 pug/ml) in coating buffer (IP6 g Na2co3, 2-9 g NaHCO3, 0-2 g NaN3 in 11 H20, pH 9-6).After coating the cuvettes they were rinsed 3 times with phosphate-buffered saline (PBS) containing 0 1% Tween 20 (PBS-Tween). To the empty cuvettes we subsequently added 200 ,ul of a diluted (1/500) tear sample or tear lactoferrin standards in incubation buffer (0-20 ,ug/ml), which was followed by 200 ,lI of a 1/1000 dilution of a rabbit anti-human lactoferrin antibody preparation (in PBS-Tween). The cuvettes were incubated in a 37°C shaking waterbath for one hour and rinsed 3 times in PBS-Tween. Then 300 gl of

The 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. Abundant 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, Meckel's diverticulum, and Peyer's 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.

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.