Immunomodulating effects of levamisole in chicks immunocompromised by infectious bursal disease virus.

Immunomodulating effects of levamisole in experimentally IBD induced immunosuppressed 7-days old White Leghorn chicks have been observed. For this, infectious bursal disease (IBD) virus (Poona strain) was used. All the chicks were immunised with sheep red blood cells to monitor antibody responses. A group of chicks each from infected (PS-L) and uninfected (PBS-L) groups were given 4 injections of levamisole hydrochloride at the daily dose of 1.5 mg per 100 g body weight starting from the second day post inoculation with IBD virus. Serum samples were collected and the haemagglutination titre against sheep red blood cells was determined. IBDV infected chicks showed a significant decrease in HA titre compared with uninfected control chicks. In levamisole treated IBDV infected birds the HA titres were comparable to those of uninfected controls. However, uninfected chicks treated with levamisole showed no significant increase in HA titre to SRBC compared with uninfected untreated control chicks.

Prevalence of subclinical infectious bursal disease and its significance in India.

From a total of 32 poultry flocks, 1,176 serum samples were screened by the agar gel precipitation test and 314 (26.7%) were positive for antibodies to infectious bursal disease virus (IBDV). Prevalence of infection on the farms ranged from 8.88 to 53.84 per cent. The prevalence was highest (61.82%) in chickens between 7 and 11 weeks old and lowest (3.92%) in those above 22 weeks of age. In commercial broilers and layers 51.61 and 17.78% respectively were seropositive reactors. The high prevalence of subclinical IBD and its economic significance are discussed.

Detection of anti-equine neutrophil antibody by use of flow cytometry.

Flow cytometric and conventional fluorescence microscopic methods were compared to detect heterologous (rabbit) neutrophil antibody bound to equine neutrophils. Unfixed and paraformaldehyde-fixed neutrophils were treated with normal rabbit serum or various dilutions of an antineutrophil serum. The cells were then reacted with fluorescein conjugates of goat anti-rabbit IgG, staphylococcal protein A, and streptococcal protein G. Antibody binding was evaluated by use of fluorescence microscopy and flow cytometry. Unfixed neutrophils treated with normal rabbit serum did not fluoresce, whereas many of the fixed neutrophils had distinct cytoplasmic and some membranous (nonspecific) fluorescence. Unfixed cells treated with the antiserum had localized areas (capping) of intense membrane fluorescence, whereas fixed cells had bright uniform membranous fluorescence. The intensity of specific fluorescence varied with the antiserum dilution and the conjugate. On flow cytometry, over 80% of unfixed cells treated with antiserum dilutions up to 1:1,024, 1:2,048, and 1:256 fluoresced, respectively, with anti-IgG, protein-G, and protein-A conjugates. Fixed cells generally had similar percentages of fluorescent cells, but at a higher (1-step) antiserum dilution. It was concluded that flow cytometry is more sensitive than conventional fluorescence microscopy to detect antibodies associated with equine neutrophils.

Detection of equine antiplatelet and antineutrophil antibodies by enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay (ELISA) was standardised and applied for the detection of antiplatelet and antineutrophil antibodies using a heterologous system consisting of equine platelets or neutrophils and antisera raised in rabbits. The standardised technique consisted of using Immulon type 3 plate, 1 per cent gelatine as a blocking solution, poly-L-lysine buffer as a coating solution, unfixed antigen, 90 microliters test serum, horseradish peroxidase conjugated antibody and o-phenylenediamine dihydrochloride as a substrate. The number of unfixed platelets or neutrophils required for optimum detection of antibodies was 250,000 per well. Unfixed cellular antigens were as good as their extracts and superior to paraformaldehyde-fixed antigens in detecting specific antibodies. Microtitre plates coated with platelet or neutrophil antigens could be stored at 4 degrees and -70 degrees C for four to five weeks without significant loss of antigenicity. The ELISA was very sensitive in that antiplatelet antibody was detected up to a titre of 1:204,800 and antineutrophil antibody to a titre of 1:51,200. Some cross-reactivity (1:1600) was detected in antiplatelet and antineutrophil sera for neutrophil and platelet antigens, respectively. Platelet-associated antibody was also detected in extracts from platelets pretreated with 1:2 and 1:8 dilutions of antiplatelet serum. Standardised ELISA detected antiplatelet antibodies in nine and antineutrophil antibodies in three of 100 isologous equine blood typing sera.

Ultrastructural and biochemical observations on antineutrophil antibody- and complement-induced immuno-injury to equine neutrophils.

Antibody-induced damage to neutrophils was studied to elucidate processes associated with destruction of neutrophils in immune-mediated neutropenias. Cytomorphological changes and release of certain cellular constituents were determined for neutrophils treated with an antineutrophil serum in the presence or absence of rabbit complement. Neutrophils exposed to the antineutrophil serum alone showed endocytotic vacuoles and degranulation. In contrast, neutrophils exposed to the antineutrophil serum and complement showed marked morphologic changes. The plasma membrane developed numerous vesicles, villous processes and minute areas of bilayer discontinuity. Highly damaged cells exhibited cellular and nuclear swellings, disruption of cytoplasmic integrity and disordered distribution of lysosomal granules. Cytoplasmic constituents (K+ and lactate dehydrogenase) were released extracellularly from neutrophils exposed to the antineutrophil serum with or without complement. Cytological changes induced by the antineutrophil serum and complement were analogous to those reported for leucocytes exposed to the activated complement components C5b-9 (the membrane attack complex) and bacterial toxins. It was concluded that the cytological abnormalities observed were most probably associated with immune-mediated damage to the cell membrane, leading to leakage of cytoplasmic constituents like K+, colloidal osmotic swelling, and disruption of the cytoskeletal system.

Pathology of reversed passive Arthus reaction in the chicken.

Sequential study of gross and microscopic changes in the chicken skin revealed that it was possible to induce a reversed passive Arthus reaction in 14- to 20-week-old chickens, using bovine serum albumin and anti-bovine serum albumin. However, high doses of immune reactants were required to elicit lesions of optimal intensity. The lesions were characterised by erythema, oedema, and the formation of thrombi in the vessels of the superficial dermis. Thrombosis, caused by the adherence of thrombocytes to vascular endothelium, induced widespread necrosis and haemorrhage. The inflammatory changes, which were confined to the deep dermis, included a necrotising vasculitis with infiltration of heterophils, monocytes and basophils. Phagocytosis of carbon particles by heterophils and basophils appeared to be sensitisation-dependent. The reaction was also characterised by the development of perivascular lymphoid foci. The findings indicate that in chickens the thrombocyte appears to be the principal cell involved in the induction of tissue damage.

Pathology of the active Arthus reaction in the chicken.

It was possible to produce an active Arthus reaction in chicken skin which resulted in gross and microscopic lesions. Histologically, the reaction was predominantly thrombotic in nature and restricted to the upper dermis. The thrombi appeared to develop as a consequence of immune complex deposition with adherence and aggregation of thrombocytes at the vascular endothelium. Thrombosis induced widespread necrosis and haemorrhage and vasculitis occurred in the lower dermis. Up to four hours after inoculation, the cell population comprised an infiltration of heterophils, monocytes and basophils, suggesting an immediate hypersensitivity reaction. This was followed by an Arthus type reaction for four to 12 hours involving both heterophils and monocytes. A characteristic feature was the development of early perivascular lymphoid foci. After 12 hours the reaction resembled a delayed hypersensitivity. The use of colloidal carbon suggested that whereas phagocytic activity of the heterophils and basophils appeared sensitisation dependent, that of thrombocytes and monocytes was independent of it. The findings indicate that in the Arthus reaction in the chicken the thrombocyte appears to be the principal cell producing tissue damage by thrombosis. A comparison was made with the active Arthus reaction in the rabbit.