Traumatic ventriculitis in chickens.

Nine cases of traumatic ventriculitis in chickens, with associated complications, are reported. The findings suggest that chickens with traumatic ventriculitis may die either from extreme emaciation or from toxemia.

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.

Observations on the pathology of experimental avian spirochaetosis.

In experimental infection of chickens with a virulent strain of Borrelia anserina, peak spirochaetaemia was recorded from 72 to 96 hours. Progressive enlargement of the spleen with mottling was the predominant gross finding. Spleen, liver and small intestine showed extensive erythrophagocytosis, which continued even after the disappearance of spirochaetes from blood and tissues. While haemosiderosis was mild in the lungs, it was absent from the heart, kidney and brain. Spirochaetes were demonstrable in the spleen, liver, intestine, kidney and to a lesser extent in the lungs, but absent from the heart and brain. Widespread erythrophagocytosis and extravascular haemolysis suggest involvement of an immune mechanism in the pathogenesis of splenomegaly.

Enteritis and green diarrhoea in experimental avian spirochaetosis.

Chickens infected with a virulent strain of Borrelia anserina showed acute enteritis, haemosiderosis in intestinal villi and green diarrhoea. Although spirochaetes in the intestine had begun to disintegrate 96 hours after infection, enteritis and green diarrhoea continued. It is suggested that the greenish discoloration may be a consequence of enteritis and haemosiderosis.

Demonstration of the phagocytic activity of chicken thrombocytes using colloidal carbon.

Chicken thrombocytes showed marked phagocytosis of carbon particles in both in vivo and in vitro systems. Initially, in both systems, thrombocytes containing carbon particles tended to form clumps. In the later stages, however, clumping was less obvious in the in vivo system. In addition to the thrombocytes, monocytes also contained carbon particles but heterophils did not. The present findings suggest that thrombocytes play a useful role in helping to clear the blood of foreign materials.

Studies on acute inflammation in the chicken using mesentery as a test system.

An acute inflammatory response was produced in the chicken by the intraperitoneal injection of turpentine and vascular permeability was assessed by the technique of carbon labelling. This revealed a biphasic pattern of increased permeability which comprised an immediate transient reaction and a delayed more prolonged response. In both phases, the increased permeability remained confined to venules only. The vascular permeability was dissociated in time from leucocyte emigration. The pattern of leucocyte emigration revealed that heterophils and monocytes emigrated concurrently. Participation of basophils in significant numbers in the early stages indicated their possible role in avian inflammation. Examination of mast cells revealed morphological changes of degranulation and disruption. There was a coincident reduction in the population of these cells. It was thought likely that vasoactive amines released from the degranulated mast cells and the basophils might initiate the early vascular changes by acting on the venules.

A topographical study of increased vascular permeability in acute inflammatory reaction in the chicken skin.

An inflammatory reaction was produced in the skin of the chicken by various stimuli. Then the colloidal carbon technique was used to identify vessels showing an increased permeability. The type of vessel affected, the extent and duration of reaction after each procedure were compared. Intradermal histamine, 5-hydroxytryptamine and bradykinin caused extensive but transient carbon labelling which was confined to venules. Topical application of xylol produced a more persistent delineation of venules. Intradermal turpentine and carrageenin affected extensive and restricted portions, respectively, of venules, and to a lesser extent capillaries. Following thermal injury of 65 degrees C for 30 sec, the superficial capillary network was initially involved but the venular response persisted for up to 18 h. These findings are compared and contrasted with those reported in laboratory animals. The possibility of distinction between direct vascular injury and chemical mediation is discussed.

Microscopic study of increased vascular permeability and leucocyte emigration in the chicken wing web.

The components of an acute inflammatory response in the chicken were investigated using a combination of histological and colloidal carbon techniques. The experimental model was the inflammatory reaction induced by the subcutaneous injection of turpentine or carrageenin into the wing web. Increased vascular permeability was shown to be mainly venular in origin. There did not appear to be an ordered migration of heterophils and monocytes and the process was unrelated to altered vascular permeability. Special features distinct from acute mammalian inflammation included a basophilic response, giant cell formation and the development of perivascular lymphoid foci.

Demonstration of the phagocytic activity of chicken basophils in the reversed Arthus reaction using colloidal carbon.

Chicken basophils showed phagocytosis of carbon particles in reversed passive Arthus reaction, induced in the skin. Heterophils, monocytes and thrombocytes also contained carbon particles, but eosinophils revealed them only occasionally. The findings suggest that basophils may share phagocytic properties with other cells under immunological stimulus.

Pathology of concanavalin A-induced cutaneous reaction in the chicken.

Sequential study of permeability and cellular responses following intradermal concanavalin A in the chicken skin, using the colloidal carbon technique, revealed an increase in vascular permeability which was mostly confined to venules. A noteworthy feature of the reaction was marked accumulation of basophils, even in the later stages, and the early appearance of perivascular lymphoid aggregations. The occurrence of well formed giant cells, hypertrophy and hyperplasia of vascular endothelium and marked acanthosis of the epidermis were the other prominent changes. The findings suggest that Con A, in the chicken, appears to have a more general effect on the different types of cells and that it may act as a mitogen not only for T lymphocytes but also for endothelial and epithelial cells.

Attempted induction of local Shwartzman reaction in the chicken.

Chickens and rabbits were injected intradermally with an endotoxin, namely Escherichia coli lipopolysaccharide (LPS). Twenty-four hours later, LPS was again administered intravenously to induce a local Shwartzman reaction. A typical cutaneous inflammatory reaction developed in rabbits, but not in chickens. Even very high doses of LPS, that made the birds visibly sick, failed to elicit the reaction. The results suggest that chickens are refractory to the Shwartzman reaction. A noteworthy feature of the chickens' response to intradermal endotoxin was the formation of prominent perivascular lymphoid aggregates.

Increased vascular permeability and leucocyte emigration in Escherichia coli endotoxin injury in the chicken skin.

The permeability response was examined in chickens following intradermal injection of Escherichia coli endotoxin. The 'dye' and 'colloidal carbon' techniques were employed. The endotoxin evoked a monophasic response of immediate-prolonged type. The increase in vascular permeability was confined to venules and small veins only, indicating its mediation by endogenous permeability factors. The carbon labelling exhibited arboreal, disjointed and rectangular to hexagonal patterns. Histologically, a striking feature of the reaction was an accumulation of basophils in unusually large numbers. No other type of stimulus appears to induce basophilic response of a similar magnitude in the chicken. The results suggest that endotoxin, being a bacterial product, may exert a chemotactic effect on basophils. Hyperaemia, oedema, necrosis and formation of perivascular lymphoid aggregates were also recognised.

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.

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.

Haematological studies on normal lactating Indian water buffaloes.

Haematological studies were conducted on 50 clinically normal lactating Murrah buffaloes in India. The range and mean (with one standard deviation), respectively, for the parameters examined were: red blood cells, 5.07 to 8.27, 6.54 +/- 0.77 million per microliter; haemoglobin, 9 to 13.5, 11.1 +/- 0.96 g/dl; packed cell volume, 0.26 to 0.34, 0.31 +/- 0.02; mean corpuscular volume, 40.6 to 55.2, 48.2 +/- 4.6 fl; mean corpuscular haemoglobin concentration, 30.9 to 38.5, 35.2 +/- 2.34 g/dl; mean corpuscular haemoglobin, 13.5 to 20.5, 17.10 +/- 1.85 pg; icterus index, 2 to 5, 2 +/- 1.25 units; erythrocyte sedimentation rate, 17 to 69, 53 +/- 12.3 mm at one hour; plasma protein, 6 to 9, 7.8 +/- 0.7 g/dl; fibrinogen, 0.2 to 0.8, 0.37 +/- 0.2 g/dl; reticulocytes, 0 per cent; white blood cells, 6250 to 13,050, 9676 +/- 1789 microliters; band cells, 0 to 1, 0.2 +/- 0.34 or 0 to 106, 18 +/- 40/microliters; neutrophils, 13 to 54, 32.9 +/- 8.74 per cent or 1285 to 6893, 3257 +/- 1262/microliters; lymphocytes, 26 to 75, 52.7 +/- 12.0 per cent or 2554 to 9637, 5065 +/- 1595/microliters; monocytes 1 to 11.5, 5.9 +/- 2.63 per cent or 63 to 1349, 584 +/- 301/microliters; eosinophils, 2 to 14.0, 6.9 +/- 4.64 per cent or 170 to 1471, 592 +/- 452/microliters and basophils, 0 to .5, 1.4 +/- 1.02 per cent or 0 to 326, 131 +/- 98 microliters. Normal species characteristics evident from this investigation included average size of the erythrocytes similar to that in cattle, low icterus index, conspicuous erythrocyte sedimentation rate, absence of reticulocytes and predominance of lymphocytes over neutrophils.

Haematological changes in buffalo calves inoculated with Escherichia coli endotoxin and corticosteroids.

Haematological studies were conducted on 10 clinically normal water buffalo calves to determine leucocytic responses to Escherichia coli endotoxin, prednisolone and dexamethasone. Intravenous injection of 10 micrograms endotoxin induced minimal decreases in leucocyte numbers, whereas 20, 50 and 100 micrograms produced a marked leucopenia within one hour. Moderate to marked leucopenia, neutropenia and lymphopenia persisted for three to 14 hours. Significant rebound neutrophilia was evident at six to eight hours after inoculation in calves given only 10 and 20 micrograms. Intramuscular injection of prednisolone (100 mg) and dexamethasone (5 mg) produced increases in total leucocyte counts and neutrophil numbers within two hours. Moderate to marked leucocytosis and neutrophilia persisted for eight to 24 hours. Lymphocyte response was unlike that in other species in that lymphopenia was not a consistent feature of the corticosteroid response. A transient monocytosis was seen following administration of prednisolone but not of dexamethasone, while eosinopenia and basopenia developed in both cases. In conclusion, endotoxin and corticosteroid induced changes in total and differential leucocyte counts in water buffalo were largely similar to those seen in cattle.

Methods for detection of immune-mediated neutropenia in horses, using antineutrophil serum of rabbit origin.

Equine neutrophil antibody was raised in rabbits inoculated with equine neutrophils isolated to purity greater than 99.0%, using Percoll density-gradient sedimentation. Neutrophil antibody was detected by use of agar gel diffusion, leukoagglutination, indirect immunofluorescence, staphylococcal protein A and streptococcal protein G binding, and phagocytic inhibition techniques. Precipitin lines and leukoagglutination were seen in antiserum dilutions of 1:4 and 1:64, respectively. The specific nature of leukoagglutination was characterized by the formation of rosette-like clumps of neutrophils. Specific bright membranous fluorescence was seen in neutrophils treated with the antiserum and exposed to fluorescein-conjugated goat anti-rabbit immunoglobulin, and staphylococcal protein A and streptococcal protein G. Whereas the indirect immunofluorescence and protein G-binding tests were equally sensitive and resulted in titer of 1:256, the protein A-binding test was less sensitive and resulted in titer of only 1:32. Nonspecific binding of protein A and protein G was noticed as uniform or patchy cellular fluorescence in a small number of neutrophils. Treatment of neutrophils with antiserum up to dilution of 1:8 resulted in a significant (P less than 0.05) suppression of phagocytosis of opsonized zymosan particles. Thus, protein G-binding and indirect immunofluorescence tests are highly sensitive to detect neutrophil antibody and may be used to diagnose immune-mediated neutropenias in horses and, possibly, in other animal species.

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.

Studies on acute inflammation in the chicken using turpentine-induced pleuroperitonitis as a test system.

An acute inflammatory reaction was induced in chickens by intraperitoneal injection of turpentine. The birds were then killed at various intervals and the peritoneal exudate sampled. The antihistamine mepyramine maleate was administered to another group of birds and the procedure repeated. Following turpentine injections there was a biphasic increase in vascular permeability, the early phase apparently mediated by histamine.

Dissecting aneurysm and cardiac tamponade.

A Gir cow which died suddenly had a dissecting aortic aneurysm. Death was caused by cardiac tamponade which followed rupture of the intrapericardial aorta after it had been damaged by onchocercosis.