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Biomedical subjects

S K Maheswaran

Publications and source records attributed to S K Maheswaran.

At least 73 records · Page 4Linked to original sources
PubMed1982

Model infection of the chicken embryo with Haemophilus somnus.

Infectious thromboembolic meningoencephalitis of cattle which is caused by Haemophilus somnus has been difficult to study due, in part, to lack of model infection in a laboratory animal. Model infection was induced in 12-day-old chicken embryos after IV injection of H somnus. Death with typical lesions (hemorrhage, degeneration of brain, congestion of venules with leukocytes and meningitis) was seen in the chicken embryo between 2 and 6 days after injection of organisms. Bacteria could be demonstrated in lesions, using Warthin-Starry silver strain. Three stable colonial variants of H somnus were cultured from dead embryos. They appeared as small translucent (ST), small opaque (SO), and large opaque (LO). Lethal dose50 values of ST was 4.4 x 10(4); for SO, 3.6 x 10(4); and for LO, 8 x 10(6). Statistical analysis indicated that ST and SO were significantly more lethal (P less than 0.001) than LO.

Animals↗
PubMed1981

Radiolabeled substrate assay to measure inhibition of growth of Haemophilus somnus by normal bovine serum.

An assay was developed to measure the inhibition of cell division of Haemophilus somnus by normal bovine serum. Dilution plate counts, turbidometric measurements, and release of radiolabeled cell constituents were found to be time-consuming or difficult to perform and reproduce. This assay determined the inhibitory effect of normal serum on population growth, by measuring the relative rate of macromolecular (DNA) synthesis in exponential cultures. This method was simple to perform and gave reliable results in 1 day. When this assay was used, a significant difference (P less than 0.025) in serum susceptibility was demonstrated between a H somnus isolate recovered from the trachea of a healthy calf and an isolate recovered at necropsy from a brain lesion of a cow. The implication of this finding, in relation to the pathogenicity of the organism, it briefly discussed.

Animals↗
PubMed1981

Interaction of Pasteurella haemolytica with bovine neutrophils: identification and partial characterization of a cytotoxin.

Timed cultures of Pasteurella haemolytica 12296 strain in RPMI 1640 medium (with L-glutamine, pH 7.4) were used to determine the correlation between cytotoxin production and the age of the culture. Cytotoxic activity was measured by a 51Cr-release assay and trypan blue exclusion test with bovine neutrophils as target cells. Results demonstrated that optimal cytotoxin production occurred during the logarithmic phase (peaked at 6 hours) and decreased during the stationary phase of bacterial growth. The cytotoxin was concentrated by sequential ultrafiltration on Diaflo XM 50, XM 100, and XM 300 membranes. The cytotoxin was retained on an XM 300 membrane. These studies indicated that the molecular weight of cytotoxic substance was 300,000 or more. The cytotoxin was heat labile, oxygen stable, and susceptible to extremes of pH and killed bovine neutrophils and mononuclear leukocytes. It was not hemolytic to bovine or ovine RBC. The cytotoxic activity was inactivated by trypsin and did not contain any detectable endotoxin. Bovine fetal serum and serum collected before immunization from neonatal calves did not neutralize the cytotoxic effects of toxin on neutrophils. However, adult bovine serum from 6 cows and an antiserum (against the cytotoxin) neutralized the cytotoxin, as revealed by both the 51Cr-release assay and the trypan blue exclusion test. This was confirmed by transmission electron microscopy. These results indicated that the cytotoxin may be antigenic in cattle. The significance and implications of these findings to bovine pasteurellosis are discussed.

Animals↗
PubMed1981

Cytotoxic effects of Pasteurella haemolytica on bovine neutrophils.

The interaction of logarithmic- and stationary-phase organisms of Pasteurella haemolytica with bovine neutrophils was evaluated by an opsonocytophagic assay. Only 5% to 8% of the logarithmic-phase P haemolytica 12296 organisms opsonized with normal bovine serum or antiserum were phagocytized. Results from cytotoxicity assays, using the 51Cr-release technique and the trypan blue exclusion test, indicated that the logarithmic-phase organisms liberated a soluble material that was cytotoxic to neutrophils and destroyed their phagocytic capabilities. This hypothesis was verified by transmission electron microscopy studies. Opsonized stationary-phase organisms were completely phagocytized and degraded when exposed to neutrophils at a bacteria/neutrophil ratio of 10:1. However, at a high bacteria/neutrophil ratio of 100:1, only 31% of the bacteria were phagocytized. Prolonged incubation of this mixture resulted in cytotoxic changes in the neutrophils. Seemingly, excess unphagocytized bacteria liberated a soluble substance that was toxic to neutrophils. These findings were confirmed by cytotoxicity assays and transmission electron microscopy studies.

Animals↗
PubMed1980

Kinetics of interaction and fate of Pasteurella hemolytica in bovine alveolar macrophages.

To study the role of pulmonary alveolar macrophages (PAMs) in phagocytizing Pasteurella hemolytica, we developed an in vitro cultivation method for preparing them. This procedure provided an adherent monolayer of PAMs which were nonspecific esterase-positive and phagocytized latex beads. The phagocytosis and fate of P. hemolytica (biotype A, serotype 1) by PAMs in suspension were studied. The kinetics of phagocytosis were determined by quantitatively measuring the uptake of 24-h [(3)H]thymidine-labeled bacteria by the PAMs in the presence of opsonins. Results showed that the uptake of P. hemolytica was enhanced in the presence of normal serum or antiserum. A total of 90% of the bacteria were phagocytized in the presence of normal adult bovine serum, and up to 95% were phagocytized in the presence of an antiserum. These studies also showed that normal serum, but not fetal calf serum, contained heat-stable natural antibodies which readily initiated the opsonization of P. hemolytica. The heat-labile complement system was also involved in the opsonization. The fate of P. hemolytica inside the PAMs was investigated by transmission electron microscopy and by the viable plate count method. Approximately 90% of the normal serum- or antiserum-opsonized P. hemolytica were phagocytized by PAMs at a bacteria/PAM ratio of 20:1 and were completely degraded after 60 min of exposure. Prolonged incubation of this mixture of bacteria and PAMs resulted in cytotoxic changes and destruction of PAMs. At a low bacteria/PAM ratio (10:1 or less), there was phagocytosis and killing of bacteria but no cytotoxic changes on the PAMs. The exact mechanism which initiated this phenomenon was not demonstrated. Perhaps toxic substance(s) released by the excess unphagocytized bacteria caused the cytotoxic changes to the PAMs.

Animals↗
PubMed1979

Influence of encapsulation on phagocytosis of Pasteurella multocida by bovine neutrophils.

The effect of encapsulation on phagocytosis of Pasteurella multocida by bovine neutrophils was examined by using two encapsulated strains, NA77 (capsular type A) and C42 (capsular type B), and comparing them with an unencapsulated counterpart strain, 1173. The uptake of [(3)H]thymidine-labeled bacteria by neutrophils was quantitatively measured after incubation of the bacteria in normal bovine serum, heat-inactivated serum, and hyperimmune sera (anti-NA77 and anti-C42). Results showed that all three strains of P. multocida were ineffectively opsonized by the heat-labile serum complement system. The unencapsulated strain was completely phagocytized in the presence of heat-stable opsonins found in normal serum. Although encapsulation of strain C42 was found to interfere with opsonization by normal serum, this strain was completely phagocytized when hyperimmune serum (anti-C42) was used as the opsonin source. These results suggest that specific anticapsular antibodies found in the hyperimmune serum readily opsonized the encapsulated strain C42 and enhanced phagocytosis. The presence of a thick capsule on strain NA77 interfered with phagocytosis in the presence of normal or hyperimmune serum (anti-NA77). This interference was due to the presence of hyaluronic acid which was a major component of the capsule. Treatment of this encapsulated strain with hyaluronidase decapsulated the bacteria. Bacteria treated in this way were almost completely phagocytized (90%) in the presence of heat-stable opsonins. The exact mechanism by which the capsule of P. multocida NA77 interfered with phagocytosis was not demonstrated; perhaps the slimy nature of the hyaluronic acid makes the phagocytic act difficult by changing the physiochemical surface properties, or it may prevent opsonization.

Animals↗
PubMed1979

Pasteurella multocida antigen-induced in vitro lymphocyte immunostimulation, using whole blood from cattle and turkeys.

A whole blood lymphocyte stimulation assay to study cell-mediated immune responses in bovine pasteurellosis was developed. Peripheral blood lymphocytes from cattle artificially immunised with three Asiatic haemorrhagic septicaemia strains of Pasteurella multocida exhibited higher stimulation indices when incubated with antigen preparations from homologous strains than with the heterologous shipping fever strain. Lymphocytes from cattle immunised with the shipping fever strain of P multocida exhibited a higher stimulation index when incubated with an antigen preparation from the homolgous strain than with antigen preparations from heterologous haemorrhagic septicaemia strains. These results suggest that immunogenic differences exist between the haemorrhagic septicaemia strains and the shipping fever strain of P multocida. An assay using turkey whole blood lymphocytes was also developed. The use of small amounts of whole blood, microtitre plates, either 125I iododeoxyuridine or 3H-thymidine as the labelling agent, and a multiple cell-culture harvester makes the method simple, rapid and suitable for the study of immune competence and cell-mediated immune responses in turkeys on a flock basis.

Animals↗
PubMed1978

Acquired resistance in turkey poults to Pasteurella multocida (P-1059 strain) during aflatoxin consumption.

The potential for impairment by aflatoxin of the development of acquired immunity to fowl cholera in 8 week old turkey poults from a commercial flock was investigated by challenge of vaccinated poults at 10, 12, and 16 weeks of age. Sixty poults were allotted to 4 groups: A) aflatoxin fed, nonvaccinated; B) aflatoxin fed, vaccinated; C) control fed, nonvaccinated; and D) control fed, vaccinated. Aflatoxin B-1 (.5 ppm) was added to the control ration of groups A and B from 49 to 70 days of age. The avirulent CU (Clemson University) strain of Pasteurella multocida drinking water vaccine was given for one day to groups B and C. Challenge (5 poults from each group) was by administration of the virulent (P-1059) strain of P. multocida in the drinking water for 4 consecutive days. The presence of aflatoxin B-1 (.5 ppm) in the feed during oral vaccination with the avirulent strain of P. multocida did not impair the development of acquired resistance to later challenge with the virulent strain. Results indicate that age of poults and method of vaccination may be important factors in the development of acquired resistance to fowl cholera when aflatoxin is present in the diet.

Administration, Oral↗
PubMed1978

Immunocompetent cells of the turkey: age and organ distribution patterns of T and B lymphoid cells.

The percentage of lymphoid cells from the bursa of Fabricius, thymus, spleen, peripheral blood, and cecal tonsils reactin with chicken antisera to turkey bursa and thymus were evaluated, using 1-day-old to 5-week-old turkeys. For this, rabbit anti-chicken globulin fluorescein isothiocyanate conjugate was used. The percentage of lymphoid cells showing immunoglobulin surface determinants from these organs also was examined, using a direct immunofluorescence test with a rabbit anti-turkey globulin fluorescein isothiocyanate conjugate. This study suggests that the bursa-specific antigen and immunoglobulin surface determinants could be used as markers for bursa-derived cells in the turkey. It also was found that thymus-specific antigen could be used as a marker for thymus-derived cells.

Age Factors↗
PubMed1976

Immunocompetent cells of the turkey: antigenic surface determinants of turkey lymphoid cells.

The serologic properties of chicken antiserums to turkey bursa and thymus were assayed by the cytotoxicity tests and indirect immunofluorescence. The following antigenic surface determinants were detected, using proper absorptions on thymic and bursal lymphoic cells: (a) common lymphocyte antigens present on both kinds of cells, (b) thymus-specific antigens, (c) bursa-specific antigens, and (d) immunoglobulin surface determinants in bursa cells, as revealed by direct immunofluorescence.

Animals↗
PubMed1975

Development of a microculture system for stimulation of chicken peripheral blood lymphocytes with concanavalin A.

A microculture system in conjunction with a semiautomatic multiple sample harvester (SAMSH) was used to study the in vitro properties of chicken peripheral lymphocytes. This new procedure enabled doing rapid multiple tests, using relatively few cells, and was highly reproducible. Data were presented to show many variables that are involved in studying the concanavalin A (Con A) response of chicken lymphocytes in a microculture system. Analysis indicated that the conditions for optimal Con A stimulation as measured by incorporation of 3H-TdR include: (a) use of 2 x 10(6) cells per culture in RPMI 1640 culture medium in the absence of any serum, (b) use of 0.4 mug of Con A per culture, (c) incubation at 37 degrees C for 72 hours, and (d) addition of 1 muCi of 3H-TdR to each culture 12 to 24 hours prior to termination. This technique could be used to monitor immunocompetence of the chicken.

Animals↗
PubMed1975

A micromethod for evaluating turkey lymphocyte response to phytomitogens.

A microculture system used in conjunction with a semiautomatic sample harvester is described to determine the in vitro properties of turkey peripheral blood lymphocytes. By this new procedure, multiple tests were done rapidly, relatively few cells were used, and results were highly reproducible. Analysis indicated that the conditions for optimal concanavalin A (con A) stimulation, as measured by incorporation of 3H-thymidine (3H-TdR) included (a) 2 times 10(6) cells per culture in RPMI 1640 medium in the absence of any serum; (b) 0.4 mug of con A per culture, incubated in flat-bottom microtitration wells for 72 hours at 37 C; and (c) 1 muCi of 3H-TdR per culture added 12 to 24 hours before termination. Conditions for optimal stimulation with pokeweed mitogen were similar to those used for con A. The exceptions were that 1 times 10(6) cells per culture were incubated in round-bottom microtitration wells. The pokeweed mitogen gave the highest degree of stimulation when used at a concentration of 80 mug per culture.

Animals↗
PubMed1967

Platelet damaging factor, a fifth activity of staphylococcal alpha-toxin.

Crude and purified staphylococcal alpha-toxin were used to demonstrate that the platelet-damaging effect of crude alpha-toxin represents a fifth activity of the alpha-toxin molecule. The homogeneity of the purified toxin employed was demonstrated by ultracentrifugation, Ouchterlony, and immunoelectrophoretic methods. Continuous-flow electrophoretic migration studies demonstrated under a variety of conditions that the platelet-damaging and the alpha-hemolytic activities migrated as a unit. Fractionation studies with the use of Sephadex G-100, carboxymethyl cellulose, and diethylaminoethyl cellulose failed to separate these two activities. Further, when alpha-toxin of demonstrated purity and crude toxin were adjusted to the same hemolytic activity, they possessed the same platelet-damaging activity. In addition, heat-reactivation studies with crude alpha-toxin revealed that the platelet-damaging effect was inactivated and reactivated in parallel with alpha-hemolytic activity. Comparable studies with purified alpha-toxin showed parallel inactivation of both activities at 60 C. Additional heating at 100 C failed to reactivate either activity. Electron micrographs revealed that purified alpha-toxin produced distinct degenerative changes in rabbit platelets. These studies also provided definite evidence that purified alpha-toxin has a damaging effect on human platelets. Monovalent alpha-antisera prevented platelet damage.

Animals↗
PubMed1967

Saphylococcal beta-hemolysin. I. Purification of beta-hemolysin.

The purification of staphylococcal beta-hemolysin was accomplished by the successive use of three protein fractionation methods. The first method employed was a double precipitation with the use of ammonium sulfate at 65% saturation. The second phase of purification used Sephadex G-100 column fractionation. The third phase utilized either carboxymethyl cellulose or diethylaminoethyl cellulose fractionation. The last two fractionation methods both resulted in the separation of a relatively high concentration of cationic hot-cold lysin and a low concentration of anionic hot-cold lysin. Because of the low concentration of the anionic component, its purity could not be assessed. However, the purity of the cationic component was demonstrated by immunodiffusion, microimmunoelectrophoresis, and by disc polyacrylamide gel electrophoresis. In addition, antisera against purified cationic beta-hemolysin yielded one line of precipitate when tested against the original crude beta-hemolysin. The purified cationic beta-hemolysin was stable in the lyophilized state. Crude beta-hemolysin was dermonecrotic, whereas purified cationic beta-hemolysin was not dermonecrotic even after Mg(++) activation.

Animals↗
PubMed1967

Staphylococcal beta-hemolysin. II. Phospholipase C activity of purified beta-hemolysin.

Sheep erythrocyte ghosts released water-soluble organic phosphorus when treated with purified beta-hemolysin. Phospholipid analysis demonstrated that sphingomyelin accounted for 53% of the phospholipids present in sheep erythrocytes. Purified beta-hemolysin showed phospholipase C activity when purified ox brain or sheep erythrocyte sphingomyelin was used as substrate. Such studies have also revealed that the disappearance of sphingomyelin from the reaction mixture was accompanied by a comparable increase in the concentration of phosphoryl choline. Thin-layer chromatography of phospholipids, extracted from sheep erythrocytes which had been exposed to beta-hemolysin, demonstrated that sphingomyelin was rapidly degraded. Activators of beta-hemolysin, such as Mg(++), enhanced the release of organic phosphorus from erythrocyte ghosts and from sphingomyelin. Inhibitors of beta-hemolysin, such as ethylenediaminetetraacetic acid, p-chloromercuribenzoate, and iodoacetamide, also inhibited the release of organic phosphorus from erythrocyte ghosts and from sphingomyelin. These studies strongly suggested that beta-hemolysin enzymatically degraded the sphingomyelin of the erythrocyte membrane. Such degradation probably resulted in the eventual lysis of the erythrocyte.

Animals↗
PubMedDate not supplied

The role of induced virulence factors produced by Pasteurella haemolytica in the pathogenesis of bovine pneumonic pasteurellosis: review and hypotheses.

In the pathogenesis of pneumonic pasteurellosis, there is an abrupt commensal to pathogen shift from a predominance of P. haemolytica serotype 2 (ST2) to serotype 1 (ST1) in the bovine upper respiratory tract (URT) microfloral population. This occurs following periods of stress associated with development of this disease. Data are reviewed from recent publications supporting the contention that surface-expressed ST1-specific factor(s) could be critical in mediating URT adhesion and colonization. Such factors may promote an increase in the number of ST1 organisms deposited through infective droplets into the lungs, beyond that efficiently cleared by normal lung defences. The seeding of these organisms into the lungs may provide numerous foci of infection that eventually progress into characteristic pneumonic lesions seen in the disease.

Animals↗