Comparison of Pasteurella spp. simultaneously isolated from nasal and transtracheal swabs from cattle with clinical signs of bovine respiratory disease.

Twenty-four matched pairs of isolates of Pasteurella haemolytica and three matched pairs of isolates of Pasteurella multocida were isolated by using a nasal swab and a transtracheal swab from individual calves with clinical signs of bovine respiratory disease. The identity of each matched pair was confirmed biochemically and serologically. The similarity of the isolates obtained from a nasal swab and from a transtracheal swab was compared by using ribotyping and antibiotic susceptibility analyses. Although the calves were sampled only once with a nasal and a transtracheal swab, when both samples were bacteriologically positive the nasal swab identified the same bacterial species as the transtracheal swab 96% of the time. The nasal swab isolate was genetically identical to the transtracheal isolate in 70% of the matched pairs. Six different ribotypes were observed for the P. haemolytica isolates, while only one ribotype was observed for the limited number of P. multocida isolates. Of the six P. haemolytica ribotypes, two ribotypes predominated. All the paired isolates displayed similar susceptibility to ceftiofur, erythromycin, tilmicosin, trimethoprim-sulfamethoxazole, and florfenicol, with some minor variations for ampicillin and spectinomycin. These results suggest that a nasal swab culture can be predictive of the bacterial pathogen within the lung when the isolates are from an acutely ill animal and can be used to determine antibiotic susceptibility.

Presence of the Streptococcus suis suilysin gene and expression of MRP and EF correlates with high virulence in Streptococcus suis type 2 isolates.

Nineteen Streptococccus suis type 2 isolates that had been analyzed previously for hemolysin production, ribotype, and virulence in pigs were examined for presence of the gene coding for suilysin by PCR amplification, and southern blot and hybridization techniques. Based on southern blot and hybridization analysis, all isolates tested contained at least a portion of the suilysin gene. PCR amplification of the entire gene resulted in gene fragments from five of the seven highly virulent isolates and none of the moderately virulent or avirulent isolates. Additional PCR analysis showed that mutation or deletions at the 5' end of the suilysin gene in the less virulent isolates prevented amplification of the sly gene fragment from those isolates. The MRP+ (muramidase-released protein) EF+ (extracellular protein) phenotype was also expressed by the same five highly virulent/sly+ isolates.

Biochemical and biological characterizations and ribotyping of Actinomyces pyogenes and Actinomyces pyogenes-like organisms from liver abscesses in cattle.

Actinomyces pyogenes is the second most frequently encountered pathogen, next only to Fusobacterium necrophorum, in liver abscesses of feedlot cattle. Ninety-one isolates, presumptively identified as A. pyogenes, isolated from liver abscesses of cattle were studied. Biochemical characteristics determined by the API 20 Strep kit were similar to those reported previously for A. pyogenes isolated from other infections, except that 18% of isolates hydrolyzed esculin. Nine isolates that resembled A. pyogenes in morphology and in certain biochemical characteristics, but fermented mannitol and/or raffinose, were called A. pyogenes-like (APL) organisms. The five antimicrobial agents, bacitracin, chlortetracycline, oxytetracycline, tylosin, and virginiamycin were inhibitory to all strains of A. pyogenes and APLs. Generally, APL organisms had higher mean hemolytic and leukotoxic activities than A. pyogenes. All isolates of A. pyogenes and APLs produced proteases and neuraminidases. Ribotyping with endonucleases, including BstEII, ClaI, EcoRI, EcoRV, HaeIII, MboI, PvuII, SalI, and SmaI alone or in combinations, showed considerable genetic heterogeneity in both A. pyogenes and APLs. No specific ribopattern characteristic of each group was observed with any of the endonuclease used. The origin of A. pyogenes and APLs and the relative importance of APLs in causing liver abscesses in feedlot cattle are not known.

Biochemical and ribotypic comparison of Actinomyces pyogenes and A pyogenes-like organisms from liver abscesses, ruminal wall, and ruminal contents of cattle.

OBJECTIVE: To isolate Actinomyces pyogenes and A pyogenes-like (APL) organisms from the ruminal wall and ruminal contents of cattle and compare them with isolates from liver abscesses from the same animals, using ribosomal DNA restriction fragment length polymorphism analysis or ribotyping. PROCEDURE: Specimens of liver abscesses, ruminal walls, and ruminal contents were collected from 59 cattle at slaughter. All beta-hemolytic, pinpoint colonies that were gram positive, pleomorphic rod-shaped, and catalase negative, and that hydrolyzed casein and gelatin were presumptively identified as A pyogenes and were characterized biochemically, using an identification kit. The isolates that resembled A pyogenes but fermented mannitol or raffinose, or both, were called APL organisms. Isolates from the ruminal wall and ruminal contents were compared with liver abscess isolates from the same animal by use of ribotyping. RESULTS: Actinomyces pyogenes and APL organisms were isolated more frequently from the ruminal wall than from ruminal contents. Ruminal isolates of A pyogenes and APL had biochemical characteristics similar to those of the isolates from liver abscesses. Among 6 sets of isolates (4 A pyogenes and 2 APL), 2 isolates from liver abscesses had ribopatterns identical to the corresponding ruminal wall isolates. Also, the APL organisms isolated from the ruminal content matched with the corresponding liver abscess isolates for both sets of specimens tested. CONCLUSIONS: The ruminal wall may be the niche for A pyogenes and APL organisms in the rumen. The genetic similarity, on the basis of ribotyping among isolates from liver abscesses, the ruminal wall, and ruminal contents of the same animal suggests that A pyogenes and APL organisms that cause liver abscesses originate from the rumen.

Use of ribotyping and hemolysin activity to identify highly virulent Streptococcus suis type 2 isolates.

Nineteen Streptococcus suis type 2 isolates were evaluated for their virulence in pigs and mice. Of these, seven were determined to be highly virulent in pigs on the basis of clinical sign scores and gross pathology and histopathology results. Clinical sign scores correlated with gross pathology and histopathology scores at P equal to 0.004 and P equal to 0.009, respectively. The virulence of highly virulent isolates in pigs compared somewhat with virulence in mice, but the correlation was not significant. No correlation of virulence was noted among the moderately virulent and avirulent isolates in pigs and mice. Chromosomal DNAs from all S. suis isolates were evaluated by PstI, PvuII, EcoRI, and HaeIII restriction enzyme digestion followed by hybridization with a digoxigenin-11-dUTP-labeled cDNA probe transcribed from 16S and 23S rRNAs from Escherichia coli. The hybridization patterns (ribotypes) varied depending upon the enzyme used, but a significant number of isolates determined to be highly virulent in pigs had unique hybridization patterns compared with those of the moderately virulent and avirulent isolates (P = 0.002). In addition, hemolysin activity showed a high correlation to virulence (P = 0.00008) and ribotype (P = 0.002).

Ribotyping to compare Fusobacterium necrophorum isolates from bovine liver abscesses, ruminal walls, and ruminal contents.

Restriction fragment length polymorphism analysis of rRNA genes was employed to genetically compare Fusobacterium necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme isolates from multiple abscesses of the same liver and isolates from liver abscesses, the ruminal wall, and ruminal contents from the same animal. Four livers with multiple abscesses and samples of ruminal contents, ruminal walls, and liver abscesses were collected from 11 cattle at slaughter. F. necrophorum was isolated from all liver abscesses, nine ruminal walls, and six ruminal content samples. Chromosomal DNA of the isolates was extracted and single or double digested with restriction endonucleases (EcoRI, EcoRV, SalI, and HaeIII); then restriction fragments were hybridized with a digoxigenin-labeled cDNA probe transcribed from a mixture of 16S and 23S rRNAs from Escherichia coli. EcoRI alone or in combination with EcoRV yielded the most discriminating ribopatterns for comparison. Within the subspecies multiple isolates from the same liver were indistinguishable based on the ribopattern obtained with EcoRI. The hybridization patterns of liver abscess isolates were concordant with those of the corresponding isolates from ruminal walls in eight of nine sets of samples. None of the six ruminal content isolates matched either the liver abscess isolates or the ruminal wall isolates. The genetic similarity between the isolates from liver abscesses and ruminal walls supports the hypothesis that F. necrophorum isolates of liver abscesses originate from the rumen.

Streptococcus suis: past and present.

Steptococcus suis is a Gram-positive, facultatively anaerobic coccus that has been implicated as the cause of a wide range of clinical disease syndromes in swine and other domestic animals. In swine, the disease has spread worldwide but is more prevalent in countries with intensive swine management practices. The disease syndromes caused by S. suis in swine include arthritis, meningitis, pneumonia, septicaemia, endocarditis, polyserositis, abortions and abscesses. S. suis has also been implicated in disease in humans, especially among abattoir workers and swine and pork handlers. In humans, S. suis type 2 can cause meningitis, which may result in permanent hearing loss, septicaemia, endocarditis and death. The pathogenic mechanism of S. suis is not well defined. Several virulence factors have been identified, but their roles in pathogenesis and disease have not been well elucidated. Much work is in progress on characterization of virulence factors and mechanisms, with emphasis on the control of the disease. Because of the non-availability of suitable immunoprophylaxis, control of S. suis infection has depended mainly on the use of antimicrobials.

Identification and partial characterization of an Actinomyces pyogenes hemolysin.

Twenty-two Actinomyces pyogenes isolates were recovered from hepatic abscesses in cattle and evaluated for hemolysin production. Hemolysin was collected from supernatant of cultures grown in 6% CO2 in brain heart infusion (BHI) broth. The effect of oxidizing and reducing agents, enzymes, temperatures and pH on hemolytic activity were studied using sheep erythrocytes as the target cells. Our study showed that A. pyogenes hemolysin is oxygen stable; sensitive to treatment by protease, trypsin, and amylase; and destroyed by treatment at extreme temperatures (56 and 100 degrees C) and pH (pH 3 and 11). Production of hemolysin was studied in BHI, RPMI-1640, and a defined serum-free A. pyogenes medium under aerobic and anaerobic conditions. Maximum hemolysin was produced in BHI incubated aerobically in 6% CO2 and to a lesser degree anaerobically in RPMI-1640. No hemolysin was produced in the defined A. pyogenes medium. Differential filtration, isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis identified two hemolysin proteins with pI values of 3.40 and 9.45 and estimated molecular masses of 62 and 58 kDa, respectively. Cell-free supernatant samples positive for hemolysin activity also were screened for leukotoxin activity. Significant levels of leukotoxin were detected in all samples screened.

Growth and microbial flora of nonmedicated, segregated, early weaned pigs from a commercial swine operation.

OBJECTIVE: To determine whether segregated, early weaned pigs have better growth performance and different microbial flora than those pigs raised on-site. DESIGN: Prospective, observational study. ANIMALS: Pigs from a commercial operation that were known to be infected with several common swine pathogens. PROCEDURE: Pigs (7 to 10 days old) were weaned and segregated from the farm of origin and compared with littermate control pigs (14 to 17 days old) that were weaned and raised on-site. Pig weight was measured and microbial flora were isolated at 14-day intervals for 84 days, beginning when the pigs were 7 to 10 days old. RESULTS: At 50 days of age, the segregated, early weaned pigs had a mean weight of 23.7 kg, compared with a mean weight of 12.5 kg for control pigs. Pasteurella multocida was isolated from fewer segregated, early weaned pigs than from controls. Signs of Mycoplasma hyopneumoniae infection were detected in control pigs but not in segregated early weaned pigs. Clinical, serologic, or bacteriologic signs of early postnatal vertical transmission of Actinobacillus pleuropneumoniae were not detected in either group. CLINICAL IMPLICATION: Vertical transmission of M hyopneumoniae was prevented by weaning pigs at 7 to 10 days of age and segregating them off-site, without the use of medication. Although medicated controls were not compared, results from this herd revealed that use of antibiotics is not the most important factor for disease control in segregated, early weaning programs. Minimizing antibiotic use in disease-control protocols reduces costs as well as removes the need for extra-label drugs.

Ribotyping to differentiate Fusobacterium necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme isolated from bovine ruminal contents and liver abscesses.

Differences in biological activities (hemagglutination, hemolytic, leukotoxic, and virulence) and ribotypes between the two subspecies of Fusobacterium necrophorum of bovine ruminal and liver abscess origins were investigated. Hemagglutination activity was present in all hepatic, but only some ruminal, strains of Fusobacterium necrophorum subsp. necrophorum. Ruminal F. necrophorum subsp. necrophorum had low leukotoxin titers yet was virulent in mice. Fusobacterium necrophorum subsp. funduliforme of hepatic or ruminal origin had no hemagglutination activity, had low hemolytic and leukotoxic activities, and was less virulent to mice. For ribotyping, chromosomal DNAs of 10 F. necrophorum subsp. necrophorum and 11 F. necrophorum subsp. funduliforme isolates were digested with restriction endonucleases (EcoRI, EcoRV, SalI, PstI, and HaeIII) and examined by restriction fragment length polymorphisms after hybridizing with a digoxigenin-labeled cDNA probe transcribed from a mixture of 16 and 23S rRNAs from Escherichia coli. The most discriminating restriction endonuclease enzyme for ribotyping was EcoRI. The presence or absence of two distinct bands of 2.6 and 4.3 kb differentiated the two subspecies. Regardless of the origin, only F. necrophorum subsp. necrophorum, a virulent subspecies, had a ca. 2.6-kb band, whereas F. necrophorum subsp. funduliforme, a less virulent subspecies, had a ca. 4.3-kb band. Ribotyping appears to be a useful technique to genetically differentiate the two subspecies of F. necrophorum.

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.

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.

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.

The effect of chemical dependency on pain perception in persons with AIDS.

A sample of persons with AIDS (N = 71) was drawn from two tertiary care centers and one group residence. Chemical dependence was measured by the Michigan Alcoholism Screening Test (MAST) and the Drug and Alcohol Screening Test (DAST), and the perception of pain was then measured with the Wisconsin Brief Pain Inventory. The hypothesis that the groups would demonstrate a significantly different perception of pain was not supported. Self-report of drug and alcohol use did not correlate well with scores on the MAST and DAST, indicating that these instruments may not measure chemical dependence in persons with AIDS. On scales of zero to 10, mean scores reflecting pain intensity averaged 5.1 and scores reflecting pain's interference with seven aspects of daily life averaged 5.98.

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.

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.

Prevalence of Salmonella in raw meat used in diets of racing greyhounds.

One hundred twelve samples of commercial raw meat used in greyhound diets were collected and cultured for Salmonella using standard procedures. Fifty (44.64%) of these samples were positive for Salmonella. Salmonella typhimurium was the most frequently isolated serovar (48%), followed by S. newport (12.76%), S. agona (8.51%), and S. muenster (6.38%). The remaining 10 serovars recovered in this study represented 27.59% of the total Salmonella isolates. In addition, the meat samples were screened for Salmonella using a commercial DNA probe. Of the 106 samples tested, 70 (66.03%) were positive for Salmonella, which indicated that the DNA probe assay was more sensitive than the culture method for screening of Salmonella in raw meat. Antimicrobial susceptibility testing revealed that most of the Salmonella isolates were sensitive to a variety of antimicrobials, particularly amikacin and apramycin, and resistant to some others, such as clindamycin, erythromycin, penicillin, and sulfadimethoxine. The cumulative percentages of susceptibility (MIC50 and MIC90) of the Salmonella isolates were also determined. Most isolates were susceptible (MIC90) to low concentrations of gentamicin (2.0 micrograms/ml), imipenem (< or = 0.25 microgram/ml), and ciprofloxacin (< or = 0.5 microgram/ml). Marked resistance was found with the other antimicrobial agents. However, the high MIC values found for these isolates would not be achievable in vivo with the normal recommended doses of antimicrobial agents, so their use would not be beneficial. Numerous plasmid patterns were found in 17 randomly selected Salmonella isolates. Eight of the 17 isolates had 2-7 plasmids ranging from 2.4 to 15 kilobases in size. Eight isolates also exhibited large plasmids in the range of 50-60 and 95-105 kilobases.(ABSTRACT TRUNCATED AT 250 WORDS)

Application of polymerase chain reaction for the correlation of Salmonella serovars recovered from greyhound feces with their diet.

The polymerase chain reaction was employed to correlate Salmonella serovars isolated from fecal material of greyhounds suffering from gastroenteritis with those isolated from the diet fed to the greyhounds prior to onset of diarrhea. Kennels around the Abilene, Kansas, area were contacted and supplied with materials needed to collect a portion of the diet each day. With the onset of diarrhea, the kennels were instructed to ship the fecal material and diet from the previous 10 days to the laboratory for testing. Forty-one fecal samples and corresponding diets were screened for Salmonella, Clostridium perfringens, Campylobacter jejuni, Staphylococcus aureus, Staphylococcus intermedius, and pathogenic (piliated) Escherichia coli by direct culture using standard procedures. The fecal material was also screened for coronavirus and parvovirus using electron microscopy. Thirty-five "normal" fecal samples were screened for all of the above mentioned microorganisms as a control. In addition, the fecal material was screened for E. coli verotoxins I and II and clostridial enterotoxins. A total of 61 Salmonella isolates were recovered from the 41 samples of feces and diet submitted for testing; 31 were recovered from the feces and 30 from the diet. Four Salmonella isolates were recovered from the normal fecal samples. Results obtained by PCR, plasmid profiles, antigenic analysis, and antibiogram profiles indicated that 16 of the 31 isolates recovered from the fecal material were the same strain as that recovered from the diet.

Further characterization of Pasteurella haemolytica-like bacteria isolated from swine enteritis.

DNA-DNA hybridization studies were conducted on six Pasteurella haemolytica-like (PHL) organisms recovered from cases of swine enteritis. Chromosomal-enriched fractions of PHL organisms served as the source of DNA for Southern blots or as whole-chromosomal DNA probes. Under stringent hybridization conditions, chromosomal DNA probes of a prototype PHL (strain 6213A) organism distinguished other PHL organisms from Pasteurella haemolytica types A1 and T3, Pasteurella multiocida types A:1 and A:3, Escherichia coli, Pseudomonas aeruginosa, Actinobacillus pleuropneumoniae type 1, and Salmonella cholerasuis. The guanine-cytosine content of the DNA of three PHL strains was 41.2 to 42.8 mol % as calculated from the thermal denaturation midpoint temperatures. The PHL strains are Gram-negative, nonmotile, beta-hemolytic, pleomorphic, oxidase-positive, urease- and indole-negative, fermentative rods with the key characteristics of the species Pasteurella haemolytica. None of the PHL strains reacted with the type-specific antisera of P. haemolytica types 1 through 12 as tested by an agglutination procedure. These swine strains differed in their biochemical differentiation from P. haemolytica types A1 and T3 in that all produced acid from M-inositol and failed to grow on MacConkey agar. Acid production from trehalose and L-arabinose was variable with PHL strains. Leukotoxicity of PHL strains was evaluated by a colorimetric micro-titration assay. Sterile culture supernatants of three of five PHL strains were toxic to bovine neutrophils. Results of these studies suggest that the PHL organisms may belong to a new group of organisms under the genus Pasteurella.