Biomedical subjects

M M Chengappa

Publications and source records attributed to M M Chengappa.

At least 55 records · Page 3Linked to original sources

PubMed1995

Influence of recombinant bovine interleukin-1 beta and interleukin-2 in pigs vaccinated and challenged with Streptococcus suis.

An experiment was conducted to determine the adjuvanticity of recombinant bovine IL-1 beta (rBoIL-1 beta) and recombinant bovine IL-2 (rBoIL-2) administered in conjunction with a single Streptococcus suis vaccination in pigs. Sixty 4-week-old pigs were allotted to eight groups: nonvaccinated controls; vaccinated controls; rBoIL-beta at 0.1, 1, and 10 micrograms kg-1; rBoIL-2 at 2.5, 25, and 250 micrograms kg-1. All pigs (except nonvaccinated controls) were vaccinated on Day 0 with a commercial Streptococcus suis vaccine (serotypes 1 and 2). At vaccination, pigs were injected intramuscularly with their respective cytokine treatments. Pigs received additional cytokine injections on 2 consecutive days. On Day 21, all pigs were challenged intravenously with 3.2 x 10(9) colony forming units of a log phase culture of S. suis (serotype 2). The highest dose of rBoIL-1 beta exceeded the maximum tolerable dose for the cytokine; however, this dose of rBoIL-1 beta protected pigs from the S. suis challenge. Pigs administered rBoIL-1 beta at 10 micrograms kg-1 had higher antibody responses to S. suis, less severe clinical signs of the disease after challenge, better growth performance during the infection, and less severe gross pathological lesions caused by the bacteria. No pigs in this treatment group died from the bacterial challenge. These data suggest that rBoIL-1 beta (10 micrograms kg-1), administered intramuscularly for 3 consecutive days at vaccination, is more effective than a single S. suis vaccination alone in protecting pigs against a S. suis challenge.

Adjuvants, Immunologic↗

PubMed1995

Partial characterization of the leukotoxin of Pasteurella haemolytica-like bacteria isolated from swine enteritis.

Pasteurella haemolytica-like (PHL) strains isolated from diarrheic pigs are known to produce a leukotoxin that is lethal to ruminant leukocytes. In the present study, 12 PHL strains were screened for leukotoxin production using a tetrazolium dye-reduction assay. Sterile culture supernatant from strain 6213A, the maximum leukotoxin producer, was used as the crude leukotoxin for characterization studies. The leukotoxin was inactivated by heat at 60 degrees C and by trypsin, protease, and amylase. Toxicity was retained over a pH range of 3.0-11.0. The leukotoxin was lethal to polymorphoneutrophils (PMNs) of cattle, sheep, goat, and swine. Chromosomal DNA of all 12 PHL strains hybridized with a 3.9 kb Pasteurella haemolytica A1 leukotoxin probe, indicating similarities between the leukotoxin genes of P. haemolytica and PHL strains.

Animals↗

PubMed1995

Combined PCR-oligonucleotide ligation assay for rapid detection of Salmonella serovars.

We have developed a rapid and sensitive assay for the detection of Salmonella serovars in veterinary clinical specimens. This method utilizes a short cultivation period followed by PCR. For detection of the amplified product, an enzyme-linked immunosorbent assay (ELISA)-based oligonucleotide ligation assay (OLA) was used. In this study, the PCR-OLA technique was compared with conventional culture and membrane hybridization for the detection of Salmonella bacteria. In evaluating the PCR-OLA with Salmonella serovars and non-Salmonella strains of bacteria, A490 readings for 51 Salmonella strains, representing 28 serovars, were significantly higher (P < 0.05) than those for 25 non-Salmonella bacteria. With serial 10-fold dilutions of Salmonella CFU or with known concentrations of purified chromosomal DNA from Salmonella typhimurium ATCC 29946, the PCR-OLA was able to detect > or = 20 CFU per assay or > or = 80 fg of chromosomal DNA (corresponding to 160 molecules of DNA). Of 102 suspect clinical specimens screened, 15 were positive for Salmonella bacteria by both culture and the PCR-OLA procedure (100% sensitivity), and 3 samples were positive only by PCR-OLA (96.6% specificity), indicating a positive predictive value of 83.3% and a negative predictive value of 100%. In all experiments, the PCR-OLA was as sensitive as membrane hybridization. These results indicate that a limited enrichment cultivation and PCR-OLA could be used as a presumptive screening test for the detection of Salmonella serovars from any sample that currently requires extensive cultivation and that this assay would be adaptable to automation.

Animals↗

PubMed1995

Detection of genomic heterogeneity in Streptococcus suis isolates by DNA restriction fragment length polymorphisms of rRNA genes (ribotyping).

Whole-cell chromosomal digests of 54 isolates of Streptococcus suis encompassing all known serotypes from a geographically varied collection were examined by PstI restriction fragment length polymorphisms and then hybridized with a digoxigenin-11-dUTP-labeled cDNA probe transcribed from a mixture of 16S and 23S rRNAs from Escherichia coli MRE600. The hybridization patterns showed genetic heterogeneity within and between S. suis serotypes. Most isolates (87%) representing 28 serotypes contained a common band at approximately 1.8 kb. However, 13% of the isolates representing seven serotypes lacked the 1.8-kb band, indicating that the species as currently defined is diverse. Nonetheless, the 1.8-kb band may be a useful genotypic marker for identification of most S. suis isolates. We tested the ability of this technique to discriminate between virulent and avirulent S. suis type 2 isolates. A virulent strain of S. suis type 2 could be distinguished from avirulent strains by the presence of specific bands. No correlation was obvious between band pattern and hemolysin production.

Animals↗

PubMed1995

Detection of Salmonella typhimurium from rectal swabs of experimentally infected beagles by short cultivation and PCR-hybridization.

A rapid and sensitive cultivation and PCR-hybridization procedure for the detection and identification of Salmonella typhimurium was evaluated over a 42-day period with eight experimentally infected beagles. Rectal swabs were taken at several times postinfection, inoculated into selenite-cystine broth, and plated onto Hektoen-Enteric Enteric agar immediately after incubation for 4 and 24 h. PCRs and hybridizations were also conducted with each sample, and the results were compared with those of standard culture techniques to evaluate the efficiency of the PCR-hybridization procedure. The PCR-hybridization procedure was more sensitive than standard culture techniques at each enrichment incubation (P < 0.05). In addition, the PCR-hybridization procedure was significantly better than culture up through 3 days postinfection (P < 0.05). A nonspecific amplified product, relatively close in size to the 457-bp specifically amplified product, did not hybridize to an internal oligonucleotide probe or to a random-primed labeled probe. Subsequent sequence information revealed that the product had very little similarity to the 457-bp product but had significant similarity to an Escherichia coli aldehyde dehydrogenase gene. This study indicated that a cultivation and PCR-hybridization procedure is significantly better than culture for the identification of S. typhimurium. Additionally, the results confirm the importance of determining specificities of PCR products beyond the gel electrophoresis level by hybridization with a specific probe.

Animals↗

PubMed1995

Influence of dietary beta-glucan on growth performance, nonspecific immunity, and resistance to Streptococcus suis infection in weanling pigs.

Three experiments, using 344 pigs, were conducted to evaluate the influence of beta-glucan on growth performance, neutrophil and macrophage function, haptoglobin production, and resistance to Streptococcus suis challenge in weanling pigs. In Exp. 1, 144 pigs were used to evaluate the influence of .1% dietary beta-glucan in a soybean meal- or milk protein-based diet on growth performance and neutrophil function. Pigs fed beta-glucan from d 7 to 14 after weaning had lower ADFI (P < .01) and, although not significant, ADG was lower for pigs fed beta-glucan than for pigs fed control diets. However, no differences were observed in growth performance or neutrophil function for pigs fed control or diets containing beta-glucan from d 7 to 35 after weaning. Experiment 2 was a 28-d growth assay in which pigs were fed a diet with or without .1% beta-glucan, containing 7.5% spray-dried plasma protein and 25% dried whey from d 0 to 14 after weaning. Pigs then were fed corn-soybean mealbased diets containing 2.5% spray-dried blood meal and 10% dried whey. No differences in growth performance were observed. Experiment 3 was a 35-d assay to evaluate growth performance, neutrophil and macrophage function, and plasma haptoglobin concentration. Pigs were challenged on d 28 postweaning with intravenous S. suis. In Exp. 3, pigs were fed diets without or with .025 or .05% beta-glucan. Dietary beta-glucan did not influence neutrophil or macrophage function. However, pigs fed diets containing .025% beta-glucan had increased (P < .05) ADG and ADFI and were heavier (P < .05) on d 28 after weaning than pigs fed the control diet. No differences in feed efficiency (G/F) were detected between treatments. Pigs fed beta-glucan had decreased (P < .10) plasma haptoglobin on d 14, 21, and 28 after weaning. However, Fisher's Exact test revealed that more (P < .04) pigs fed a diet containing .025% beta-glucan died by d 12 after challenge with S. suis. In conclusion, these data suggest the existence of a complex interaction involving growth performance and resistance to S. suis in pigs fed .025% beta-glucan.

Aging↗

PubMed1995

Sequence analysis of leukotoxin secretion determinants from a Pasteurella haemolytica-like organism.

The pHLBD genes encoding the secretion functions for the 105 kDa RTX leukotoxin of Pasteurella haemolytica-like (PHL) organism has been cloned and sequenced. Like analogous genes from other RTX determinants, the pHLBD genes lie immediately downstream from the leukotoxin structural gene, pHLA. Although isolated from a diverse group of gram-negative organisms, the pHLBD genes and the characterized RTX BD genes from other organisms exhibit a high degree of homology at both the DNA and predicted amino acid sequence levels. We have previously reported the cloning of the leukotoxin gene (pHLCA) (Chang et al., Infect. Immun. 61:2089-2095), which encodes a 105-kda polypeptide with cytotoxic activity. DNA sequence analysis of the pHLBD genes shows 83.93% and 86.05% homologous to that of P. haemolytica IktBD genes, respectively.

ATP-Binding Cassette Transporters↗

PubMed1994

Growth-condition dependent expression of Pasteurella haemolytica A1 outer membrane proteins, capsule, and leukotoxin.

Pasteurella haemolytica, strain P1148 (biotype A, serotype 1) was grown under iron-rich and iron-restricted conditions both with and without serum, and the outer membrane protein (OMP), capsule, and leukotoxin production studied. OMPs were evaluated by SDS-PAGE and examined by immunoblot to identify antigens recognized by sera from P. haemolytica A1 convalescent and vaccinated cattle. Capsule production was evaluated using fluorescent antibody staining and rapid plate agglutination reaction. Leukotoxin production was measured by neutrophil 51Cr-release assay. Expression of specific OMPs, amount and antigenic character of capsule, and quantity of leukotoxin produced by P. haemolytica A1 varied in response to alterations in the growth media. Immunoblots indicated the immune response of convalescent calves differs from vaccinated calves, and convalescent calves produce antibodies to novel OMPs induced by growth in iron-restricted conditions.

Animals↗

PubMed1994

Biochemical and biological characterization of ruminal Fusobacterium necrophorum.

Biochemical characteristics, biological activities, and antimicrobial susceptibilities of ruminal Fusobacterium necrophorum (eight subsp. necrophorum and eight subsp. funduliforme) and of isolates (three of each subsp.) obtained from bovine hepatic abscesses were determined. F. necrophorum subsp. necrophorum strains had higher phosphatase and DNase activities, produced more leukotoxin, and were more pathogenic to mice than subsp. funduliforme strains. The leukotoxin titer for culture supernatants of ruminal subsp. necrophorum strains was approximately 15 times lower than that of hepatic subsp. necrophorum strains. Hemagglutination activity was present in all hepatic, but only in some ruminal, strains of subsp. necrophorum. The antimicrobial sensitivity profile of the ruminal isolates was similar to that of hepatic isolates.

Animals↗

PubMed1994

Influence of interleukin-1 on neutrophil function and resistance to Streptococcus suis in neonatal pigs.

Nonspecific immunity is usually lower in neonates than adults. Consequently, enhancing the neonate's nonspecific immune capability may be beneficial for the health and growth performance of young animals. We conducted two experiments in which neonatal pigs were injected with recombinant bovine interleukin-1 beta (rBoIL-1 beta) at 9 to 11 days of age. Three consecutive daily injections of rBoIL-1 beta increased neutrophil and monocyte numbers, which remained elevated until the animals were challenged with Streptococcus suis at 19 days of age. Neutrophil bactericidal activity was greater in interleukin-1-treated pigs than in saline-injected controls. At lower ratios of effector to target cells, neutrophil-mediated, antibody-dependent cellular cytotoxicity was increased in neonates treated with IL-1. However, natural killer cell activity and neutrophil production of superoxide anion were not affected by treatment with IL-1. Expression of CD18 was increased transiently on neutrophils from IL-1-treated pigs at 15 days of age. Severity of the streptococcal infection was less in pigs that were treated with IL-1 at 9 to 11 days of age. These data suggest that IL-1 treatment in neonates may augment nonspecific immune function and disease resistance.

Animals↗

PubMed1994

Identification of a proline-arginine-rich antibacterial peptide from neutrophils that is analogous to PR-39, an antibacterial peptide from the small intestine.

Neutrophil nonoxidative defense mechanisms include several low molecular weight antimicrobial peptides. We have isolated a proline-arginine-rich antibacterial peptide from porcine neutrophils. This cationic, low molecular weight peptide has a very high degree of identity (97%) to a bactericidal peptide, PR-39, that has been found in the porcine small intestine. Isolation of the same antimicrobial peptide from both neutrophils and cells of the small intestine in the same species is unique and suggests an important role for this protein in innate immune defenses.

Amino Acid Sequence↗

PubMed1994

Isolation, characterization and protection studies in mice of a streptomycin-dependent mutant of Streptococcus suis type 1/2.

An avirulent, streptomycin-dependent (Str-D) mutant of Streptococcus suis type 1/2 was produced and characterized by its antimicrobial susceptibility, growth kinetics, biochemical reactions and reversion rate. Homologous and heterologous vaccine trials in mice resulted in complete protection against challenge with S. suis types 1 and 1/2 and partial protection against challenge with S. suis type 2.

Animals↗

PubMed1994

Purification and quantification of Fusobacterium necrophorum leukotoxin by using monoclonal antibodies.

Monoclonal antibodies (Mabs) were produced to the leukotoxin of Fusobacterium necrophorum. Two mAbs (F7B10 and E12E9) partially neutralized leukotoxin activity, as determined by a tetrazolium (MTT)-dye reduction assay with bovine polymorphonuclear neutrophils as target cells. Immunoblot analysis showed that both clones reacted with antigens of 110 and 131 kilodaltons. Epitope analysis showed that the two mAbs recognized the same epitope. An affinity column containing immobilized mAb F7B10 was used to purify leukotoxin from crude toxin. Affinity chromatography of 1 ml of culture supernatant resulted in 0.67 microgram or 1350 units of leukotoxin. Leukotoxin was quantitated by a sandwich enzyme-linked immunosorbent assay using mAb F7B10 as the capture antibody and as the biotinylated indicator. The minimal detectable level was approximately 1 ng, corresponding to 2 leukotoxin units in the sample.

Animals↗

PubMed1994

Selective enumeration of Fusobacterium necrophorum from the bovine rumen.

A culture medium containing lactate as the sole energy source and antibiotics (bacitracin, gentamicin, and streptomycin) was used for selection and enumeration of Fusobacterium necrophorum from bovine ruminal contents. F. necrophorum growth was determined by indole production, and enumeration was performed by the most-probable-number technique. The number of F. necrophorum cells in cattle fed a 100% forage diet was 7 x 10(5)/g of ruminal contents. The number increased (P < 0.05) 10-fold after the diet was changed to 85% corn grain.

Animal Feed↗

PubMed1994

Partial characterization of Streptococcus suis type 2 hemolysin.

Streptococcus suis type 2 was evaluated for hemolysin production. Supernatants of S. suis type 2 grown in Todd-Hewitt broth were assayed for hemolytic activity by a photometric assay. Twenty-two additional serotypes of S. suis (1,3 to 22, and 1/2) were evaluated for hemolysin production; nine of them (1/2, 1, 4, 5, 14, 15, 17, 19, and 20) were positive. The effects of temperature, atmosphere, centrifugation, sonication, chemicals, bovine serum albumin, fetal calf serum, and enzymes on S. suis type 2 hemolysin activity were studied. Maximum hemolysis occurred after incubation in RPMI 1640 medium at 40 degrees C in 6% CO2 and after growth in Todd-Hewitt broth at 37 degrees C under anaerobic conditions. Hemolytic activity was absent after the addition of fetal calf serum and decreased after the addition of trypsin or amylase. However, treatment of erythrocytes with amylase or trypsin prior to incubation with supernatant also resulted in a decrease in hemolytic activity. The addition of bovine serum albumin caused increased hemolytic activity. Dipyridyl and EDTA had negligible effects on hemolysis. Hemolytic S. suis type 2 culture supernatant injected intraperitoneally failed to cause death in BALB/c mice. Data from our study indicate that S. suis type 2 hemolysin is a secreted or loosely cell bound, thermolabile molecule whose activity is growth condition dependent.

Animals↗

PubMed1994

Detection of Salmonella serovars from clinical samples by enrichment broth cultivation-PCR procedure.

To overcome problems associated with application of PCR to clinical samples, we have combined a short cultivation procedure with a Salmonella-specific PCR-hybridization assay to specifically identify Salmonella serovars from clinical samples of various animal species. The technique was investigated by using fecal samples seeded with known numbers of Salmonella organisms and cultivated for different lengths of time in assorted selective and nonselective enrichment media. The ability of PCR to amplify a Salmonella-specific DNA product (457-bp sequence covering the Salmonella invE and invA genes) was examined in Southern hybridizations with an internal oligonucleotide probe. Forty-seven Salmonella isolates representing 32 serovars were evaluated, and all Salmonella isolates resulted in a 457-bp product that hybridized with the oligonucleotide probe, whereas no hybridizations were evident with 53 non-Salmonella organisms. The assay detected as few as 9 CFU of Salmonella organisms in pure culture and as little as 300 fg of purified chromosomal DNA. Rappaport-Vassiliadis and tetrathionate broths were inhibitory to PCR, whereas brain heart infusion and selenite-cystine broths were not. The PCR-hybridization assay coupled with a brain heart infusion enrichment culture incubated for 2 h detected as few as 80 CFU of Salmonella organisms in seeded feces. We have successfully identified Salmonella serovars in clinical samples from swine, horses, and cattle more rapidly than with conventional culture techniques. The sensitivity and specificity of this assay were both 100% compared with culture results. These results indicate that a combined cultivation-PCR-hybridization assay could be applicable and advantageous in the rapid identification of Salmonella serovars in routine diagnostic situations.

Animals↗

PubMed1994

Serum neutralizing antibodies against Fusobacterium necrophorum leukotoxin in cattle with experimentally induced or naturally developed hepatic abscesses.

The relationship between serum-neutralizing antibody against Fusobacterium necrophorum leukotoxin and hepatic abscesses was investigated in cattle fed diets supplemented with or without tylosin. Sixteen cattle (eight each in tylosin and in control groups) were inoculated intraportally with F. necrophorum. Ultrasonographic scanning showed that all control animals developed hepatic abscesses after inoculation. In the tylosin group, two animals were free of abscess by d 7 and one was free by d 14. Leukotoxin-neutralizing antibody titers were low on d 0, but increased (P < .05) markedly after intraportal inoculation in both groups. In a second study, blood was collected at the time of slaughter from 141 feedlot cattle (36 fed diets with tylosin and 105 fed diets without tylosin), and livers were examined for presence or severity of hepatic abscesses at slaughter. The incidences of hepatic abscesses were 32% in the control group and 6% in the tylosin group. Antibody was detected in all animals; however, antibody titers were greater (P < .05) in cattle with abscessed liver than those without, and greater (P < .01) in the nontylosin than in the tylosin group. Abscess score and antibody titer were correlated (r = .34; P < .0001). We conclude that F. necrophorum leukotoxin is highly antigenic and that anti-leukotoxin antibody titer is related to the severity of hepatic abscesses.

Animals↗

PubMed1994

Conjunctival microbial flora of clinically normal pigs.

Conjunctival swab specimens from healthy pigs were cultured to determine normal microbial population. Four commercial swine operations were selected for study. Pigs of 4 age groups were tested: nursing pigs, nursery pigs, feeder pigs, and sows. Swab specimens were taken from the conjunctival sac of each pig. Bacterial, fungal, and mycoplasmal growth was determined separately. Chlamydia sp was detected by use of an ELISA. Bacteria were recovered from 98% of specimens evaluated. alpha-Streptococcus sp (89%) was the most commonly recovered organism, followed by Staphylococcus epidermidis (39%) and Staphylococcus sp (39%). Mycoplasma sp was not detected in any of the specimens. Chlamydia sp was identified in 28% of all specimens evaluated. These results are similar to reports of normal conjunctival flora in other domestic animals.

Aging↗