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Several methods have been previously suggested for the presumptive identification of Gardnerella vaginalis in clinical laboratories, but none is entirely satisfactory. We previously found that sodium polyanetholesulfonate (SPS) inhibits G. vaginalis in blood culture media. We compared susceptibility to an SPS-containing paper disk with beta-hemolysis on human blood agar, hippurate hydrolysis, and inhibition by alpha-hemolytic streptococci for identification of 62 previously confirmed G. vaginalis strains. All strains were positive by SPS disk and alpha-hemolytic streptococcus inhibition, 78% were positive by beta-hemolysis, and 81% were positive by hippurate hydrolysis. Although positive reactions occurred with SPS disk and alpha-hemolytic streptococcus tests for 5 and 9 of 84 other bacteria tested, respectively, none of these bacteria were positive for both tests. We conclude that a combination of SPS disk susceptibility and alpha-hemolytic streptococcus inhibition provides excellent identification of G. vaginalis when performed by the methods suggested.

Of 30 sows from a herd believed to be free of Haemophilus pleuropneumoniae infection, 2 had complement-fixing antibodies to H. pleuropneumoniae serotype 5. Necropsy and microbiological examination of the two sows revealed no evidence of H. pleuropneumoniae infection; however, Haemophilus taxon "minor group" and a urease-negative, indole-positive Haemophilus sp. were isolated from numerous respiratory tract sites in both sows. Isolation of these Haemophilus spp. was facilitated by serially diluting specimens in two broth media. Pigs from a closed, respiratory disease-free herd were inoculated with four strains of Haemophilus taxon "minor group" to determine whether the organism induces antibodies which cross-react with H. pleuropneumoniae in the complement fixation test. Antigenic heterogeneity among the taxon "minor group" strains was apparent; however, antibodies cross-reacting between these strains and H. pleuropneumoniae serotypes 1 through 5 and 7 were not detected.

Eighteen field isolates of Haemophilus pleuropneumoniae were studied biochemically and serotyped using the complement fixation test (CFT), agglutination test and the immunodiffusion test. Three biochemical tests (V-dependency, CAMP-reaction and urease activity) were found to be very useful for the biochemical characterization of the H. pleuropneumoniae. Haemolysis on blood agar plates, although present, was not sufficiently pronounced in all cases to warrant absolute dependence on this characteristic. Serological typing revealed the isolates belong to Serotypes 1 and 5. The immunodiffusion test proved to be the most serotype specific, while a marked cross-reaction was observed with the CFT.

When grown on meat-peptone agar with heated blood, different Haemophilus species (H. influenzae, H. parahaemolyticus, H. parasuis, H. pleuropneumoniae), including different H. influenzae serovars (a, b, c, d, e, f), and Pasteurella multocida have identical fatty acid composition, characterized by the prevalence of fatty acids with 16 carbon atoms, constituting about 70% and more of the total number of fatty acids, and a low level of fatty acids with 18 carbon atoms. P. multocida strains cultivated on meat-peptone agar with unheated blood have a greatly increased content of fatty acids with 18 carbon atoms, while the content of fatty acids with 16 carbon atoms is much lower. The identity of fatty acid composition under similar cultivation conditions, together with their similarity in other phenetic signs, is indicative of close phylogenic relationship between bacteria belonging to the genus Haemophilus and P. multocida.

Two hundred and nine strains of Haemophilus influenzae and Haemophilus aegyptius were screened for trooleandomycin susceptibility. Four strains were shown to be sensitive to the drug. Of these four, two were Haemophilus aegyptius (ATCC 11116, NCTC 8134), and the other two were Haemophilus influenzae biotype I (1-605) and IV (80-212. One strain of Haemophilus aegyptius (NCTC 8135) was resistant to trooleandomycin. Restriction enzyme assays and DNA homology were carried out to establish relationships between the strains. It is concluded that trooleandomycin susceptibility has no taxonomic value to differentiate between Haemophilus aegyptius and biotype III Haemophilus influenzae.

Fifty-five strains received as Haemophilus vaginalis or as catalase-negative coryneform bacteria from the vagina together with 61 marker cultures were subjected to numerical phenetic analyses using 149 unit characters. The data were examined using the simple matching (SSM), Jaccard (SJ) and pattern (DP) coefficients and clustering was achieved using the average linkage algorithm. Cluster composition was not markedly affected by the coefficient used or by test error, estimated at 6 . 5%. The H. vaginalis strains formed a tight cluster which was only distantly related to representatives of the genera arthrobacter, Cellulomonas, Corynebacterium sensu stricto, Erysipelothrix, Haemophilus, Kurthia, Lactobacillus, Listeria and Propionibacterium but shared a high overall affinity to unclassified catalase-negative coryneforms which formed a discrete taxon, cluster 9. The H. vaginalis strains could be distinguished from the related strains in cluster 9 by several unrelated phenotypic characters. Using the S1 endonuclease assay, DNA-DNA hybridizations were performed with representative strains from the numerical as well as with reference strains of Bifidobacterium and Actinomyces. Haemophilus vaginalis was found to be a genotypically legitimate group and its DNA showed little homology with DNA from the marker strains tested. The DNA base composition of H. vaginalis was 42 to 44 mol % guanine plus cytosine. A new genus should be created to incorporate strains known as H. vaginalis or Corynebacterium vaginale. The name Gardnerella vaginalis proposed by Greenwood & Pickett (1979) is supported.

Actinobacillus (Haemophilus) actinomycetemcomitans is a facultatively anaerobic, gram-negative coccobacillus which is a possible etiological agent in juvenile periodontitis (JP). In this study, bacterial flora, especially the occurrence of A. actinomycetemcomitans, in the periodontal pockets of one juvenile with gingivitis (G), one JP patients, five rapidly progressive periodontitis (RP) patients and one adult periodontitis(AP) patient, and one adult with healthy periodontium was investigated using a blood agar medium and a selective medium for A. actinomycetemcomitans. Eight hundred and sixty-five bacteria were isolated from the periodontal pockets, examined for their gram-stain, cell morphologies, relations to O2 and CO2 and catalase reaction, and divided into 21 groups on the basis of these characters. Among the isolates, 604 isolates were further characterized biochemically and identified. A. actinomycetemcomitans was found as 0.2% of the flora of a site in the JP patient, as 9% of the flora of a site in the G patient, and as 19% and 1%, respectively, of the flora of a site in the two RP patients. However, the organism was not detected in another lesion site of the JP patient. In our JP and RP patients, Fusobacterium, Wolinella, Streptococcus, and obligately anaerobic, gram-positive cocci were frequently found at high levels. The bacterial flora of the G and AP patients were more heterogeneous and included Bacteroides at relatively high proportions. These results indicate that A. actinomycetemcomitans is not always associated with JP but occurred in some patients with RP and G.

A coagglutination test for the identification and serotyping of Haemophilus pleuropneumoniae is described. A total of 360 H. pleuropneumoniae strains were isolated from pulmonary tissues of feeder pigs which died of acute pleuropneumonia. All of the isolates were serotyped by coagglutination, and the results were confirmed by the ring precipitation test. The most common serotype isolated in Quebec was serotype 1, followed by serotypes 5, 2, and 7. None of the isolates belonged to serotypes 3, 4, or 6. Mixed infections due to H. pleuropneumoniae of more than one serotype in the same animal were encountered. Serotype 1 was the only common isolate among the mixed-serotype infections. The coagglutination test was more sensitive than was the ring precipitation test. Serotyping by the coagglutination test is inexpensive, rapid, reliable, and easy to perform.

The virulence of strains of Haemophilus pleuropneumoniae serotype 1, 2, 3, 7 and strains of the "minor-group" and Haemophilus parasuis were compared by inoculating specific pathogen-free pigs into the lower airways with specified doses of bacteria. Haemophilus pleuropneumoniae, strain W, serotype 1, given in 1 X 10(8) colony-forming units, produced a lethal acute pleuropneumonia in four pigs. Nonlethal localized pulmonary necrosis was induced in four groups of two pigs given 1 X 10(7), 1 X 10(6), 1 X 10(5) and 1 X 10(4) respectively of the same strain. Two groups of four pigs developed chronic lesions when inoculated with 1 X 10(7) colony-forming units of H. pleuropneumoniae, strain Shope 4074, serotype 1 and 1 X 10(7) colony-forming units of H. pleuropneumoniae, strain WF83, serotype 7, respectively. Of 20 pigs given 1 X 10(8) colony-forming units of strain 1536, serotype 2, two died of acute pleuropneumonia and 18 had lesions of pulmonary necrosis or abscessation and pleuritis. A dose of 4 X 10(9) colony-forming units of strain BC181, serotype 3, induced pulmonary necrosis similar to the lesions in pigs given 10(7) colony-forming units or less of strain W, serotype 1, suggesting that the serotype 3 strain is less virulent. No clinical signs, but focal areas of pulmonary fibrosis and pleural adhesions were induced in four pigs inoculated with 4 X 10(9) colony-forming units of the "minor-group" strain 7ATS. Similarly, four pigs inoculated with "minor-group" strain 33PN did not show clinical signs, but had focal necrotic and fibrotic pulmonary lesions and pleural adhesions.(ABSTRACT TRUNCATED AT 250 WORDS)

The fatty acid composition of 35 Haemophilus influenzae strains was found to be grossly similar and characterized by relatively large amounts of 14:0, 3-OH-14:0, 16:1 and 16:0. The three C18 fatty acids 18:2, 18:1 and 18:0 were also present, but in much lower concentrations. This general pattern was also found for most of the other species of Haemophilus examined (H. aegyptius, H. aphrophilus, H. canis, H. gallinarum, H. haemolyticus, and H. parainfluenzae). Small but distinct quantitative discrepancies were detected for H. ducreyi and the haemin-independent species H. paraphrohaemolyticus, H. paraphrophilus and H. suis. Actinobacillus actinomycetemcomitans was found to be indistinguishable from H. influenzae. Pasteurella multocida also exhibited a fatty acid pattern closely related to that of Haemophilus, but could be distinguished by its higher concentration levels of the C18 fatty acids. The fatty acid pattern of H. vaginalis was considerably different from those of the other species examined. This species lacked 3-OH-14:0 and 18:2 and contained small amounts of 14:0 and 16:0, whereas 18:1 and 18:0 were the major constituents.

One hundred and forty-one isolates of Haemophilus pleuropneumoniae from Iowa and Illinois swine were characterized morphologically and biochemically and serotyped by rapid slide agglutination (RSA) and indirect fluorescent antibody (IFA) tests. Hyperimmune antisera were produced in rabbits using inactivated whole-cell suspensions of the reference strains for H pleuropneumoniae serotypes 1 to 7 and strain 202, representing the taxon "minor group." Cross testing of the reference strains and reference antisera indicated the antisera to be essentially serotype-specific, although reactivity of some antisera with heterologous strains was observed. Cultures of the 141 isolates formed adherent or smooth colonies or mixtures of these colony forms. Adherent and smooth colony types were found in all serotypes identified. Microscopic and biochemical characteristics of all isolates were typical of those previously described for H pleuropneumoniae. The overall incidence of H pleuropneumoniae serotypes was serotype 5, 55.3%; serotype 1, 34.0%; serotype 7, 7.8%; and nontypeable, 2.8%. Comparing the 2 test procedures, 87.2% of the isolates could be typed by RSA, and 66.0% could be typed by IFA. Cross-reactions between serotype 4 antisera and serotype 5 and 7 isolates were common with the IFA test. The reactions with serotype 7, but not serotype 5, were eliminated by cross adsorption of serotype 4 antisera. There was good correlation between the 2 test procedures, but RSA was judged to be more specific and sensitive than IFA.

Haemophilus aegyptius and Haemophilus influenzae biotype III are morphologically and biochemically similar; however, their outer membrane protein (Sarkosyl insoluble) profiles are distinct. Of 18 strains of H. aegyptius examined, 15 had a type 1 protein profile, and 3 had a type 2 profile, whereas the 5 strains of H. influenzae biotype III examined had three other protein profile types. All Haemophilus strains examined had 31- and 76-kilodalton (kDa) proteins and minor proteins with molecular masses between 20 and 100 kDa. H. aegyptius, with a type 1 protein profile, had major outer membrane proteins with apparent molecular masses of 27, 35.5, and 41.5 kDa, and H. aegyptius, with a type 2 protein profile, had 26-, 29-, 39.5-, and 41-kDa proteins. The type strain of H. influenzae biotype III had three major outer membrane proteins with apparent molecular masses of 29, 38.5 and 40 kDa. Four other strains designated as H. influenzae biotype III had major outer membrane proteins between 27 and 41.5 kDa representing two additional protein profiles.

Thirty-nine Australian isolates of Haemophilus paragallinarum were compared serologically with 3 reference serotype strains of H. paragallinarum using a plate agglutination test. Twenty-eight of the isolates were serotype C, 5 were serotype A, while the remaining 6 isolates could not be assigned to a serotype.

Indole spot tests using isolated, nonhemolytic colonies of Haemophilus species were positive for 90 of 151 (60%) respiratory isolates of Haemophilus influenzae, whereas 67 to 72 (93%) isolates of H. influenzae from cerebrospinal fluid and blood specimens were indole positive. Only 4 of 117 (3%) Haemophilus parainfluenzae isolates were positive for indole spot tests. Thus, indole-positive, nonhemolytic Haemophilus isolates in respiratory cultures can be presumptively identified as H. influenzae.

The current classification of recognized actinobacilli and pasteurellas does not allow differentiation of the two genera by their phenotypic features. Recent investigations of their genetic relationships have shown that several species hitherto assigned to the genus Pasteurella are more closely related to the actinobacilli. Moreover, some recently described taxa were located by DNA-DNA hybridization in one or the other of the two genera. On the basis of the genetic system, improved identification keys have been devised which separate the taxonomic groups on the genus and species levels according to an appropriate set of biochemical characteristics.

This report provides a description and evaluation of the indirect fluorescent-antibody technique for serotyping and detecting Haemophilus pleuropneumoniae. The indirect fluorescent-antibody technique was serotype-specific when reference strains and sera were tested. Sixty-five field isolates were serotyped by indirect fluorescent-antibody technique and belonged to types 1,2 an 5. Two isolates did not fit any of the five established types. Twenty-three of the 65 isolates were also typed by immunodiffusion and the two tests agreed completely. Haemophilus pleuropneumoniae was detected in freshly-fixed impression smears from 12 lungs having acute pleuropneumonia. The identity of the bacteria was subsequently confirmed by cultural procedures. Fixed smears of cultured Haemophilus bacteria can be stored or mailed at -20 degrees to +24 degrees C without losing stainability.

Haemophilus aegyptius should be considered a junior subjective synonym of Haemophilus influenzae. Both nomenspecies are indistinguishable by DNA/DNA hybridization (S1 nuclease method). No single phenotypic test can unambigously separate H. aegyptius from the other biotypes of H. influenzae.

Four hundred and forty-three Dutch field isolates of Haemophilus pleuropneumoniae were serotyped by rapid slide agglutination (RSA) using specific antisera against serotypes 1 to 5 and against the recently proposed types 6 to 9. The predominant serotypes were 9 (49%) and 2 (32%). Serotypes 1, 3, 5, 7 and 8 were isolated in small numbers: together they accounted for 3% of the total. Five percent of the isolates were not typable either due to autoagglutination or because they were not agglutinated by any of the available antisera. The remaining 49 strains (11%) agglutinated in more than one antiserum and could therefore not be properly classified. Forty-four of these 49 strains agglutinated in both anti type 1 and anti type 9 serum. Antigenic relationships between serotype 1, serotype 9 and isolates reacting with both antisera were studied using immunodiffusion and RSA with adsorbed sera. Serotype 9 strains appeared not to be a homogenous group. Isolates agglutinating exclusively in anti type 9 serum can be divided into two groups: one closely related and another hardly related to serotype 1. Serotype 9 reference strain 13261 belongs to the latter. Type 1 + 9 strains have antigens in common with serotypes 1 and 9, but they also have their own specific antigenic material. Such strains are proposed as a new serotype 10.

Cellular fatty acid compositions of contagious equine metritis isolates and three reference Haemophilus equigenitalis cultures were determined by gas chromatography. The chromatographic data were standardised and normalised fatty acid methyl ester (FAME) profiles were produced. The profiles were compared visually and similarity indices were determined using a computer peak matching method. There was little difference between the profiles of the three reference strains, each strain being characterised by three major fatty acids; C 18:1, C 16:0 and 30H-C 14:0. Variations in cultural conditions had no significant effect on the FAME profiles. The identification of laboratory isolates using the technique was in agreement with the presumptive identification based on the currently recommended tests and an improvement on the confirmatory serological identification. The FAME profiles provided confirmation of identity where it was not possible to use the presently recommended serological procedures. The authors recommend the gas chromatography technique for use in the diagnostic laboratory as an adjunct to the presently accepted identification methods.

Haemophilus somnus was isolated from 40 of 79 unprocessed bovine semen samples, 14 of 23 preputial washings of bulls and three of eight preputial washings of steers. The results indicate nonvenereal colonization of the male urogenital tract. It is suggested that dissemination of H. somnus from the urogenital tract may be of significance in the epizootiology of H. somnus associated diseases.

Corynebacterium vaginale is a sexually transmitted organism which was first recognized in 1953. It appears to utilize glycogen stored in vaginal epithelial cells, causing a malodorous vaginal discharge characterized by an abnormally high pH (5.0 to 5.5) and composed mainly of epithelial cells and hordes of bacilli. Infected men are asymptomatic, carry the organism for an unknown period of time, and transmit it through intercourse. The organism requires five B-vitamins, purines, pyrimidines, and a fermentable carbohydrate; neither factors X nor V are required. It is not a member of genus Haemophilus and is not likely to be a Corynebacterium. Appearing mainly Gram-negative, it has many characteristics of Gram-positive organisms including its pattern of sensitivity to antibiotics and the possession of certain enzyme systems. As the cause of bacterial vaginitis, C. vaginale may be the most prevalent sexually-transmitted organism.

One hundred and eighty eight isolates of Haemophilus influenzae and 187 isolates of H parainfluenzae from patients with cystic fibrosis, patients with respiratory infections but without cystic fibrosis, and patients with neither cystic fibrosis nor respiratory infections were biotyped. Biotype I of H influenzae were found significantly more often in patients with cystic fibrosis compared with those with normal respiratory tracts. On the other hand, biotype II strains of H influenzae were found less often in the cystic fibrosis group. Half of the biotype V strains produced beta-lactamase.

Over a period of 6 years, 114 strains of Haemophilus influenzae and Haemophilus parainfluenzae were isolated from genital, mother-infant, or neonatal infections. Their serotypes, biotypes, antibiotic resistance phenotypes, and outer membrane protein (OMP) electrophoretic patterns were characterized and correlated with the various clinical outcomes. Genital H. influenzae and H. parainfluenzae appeared to behave mostly as opportunistic pathogens; for instance, 62% of the cases of endometritis or pelvic inflammatory disease were related to the presence of an intrauterine device. However, as seen clearly in one case, the strains may be sexually transmitted. The analysis of OMP patterns proved to be a very convenient method to seek evidence for the sexual origin of the infection. H. influenzae was more often involved in complicated genital infections than was H. parainfluenzae. Nontypeable and biotype II H. influenzae strains were the more frequent isolates, except in pelvic inflammatory diseases, in which biotype I prevailed, and in mother-infant infections, in which one-fourth of the cases were due to biotype IV. Characterization of H. influenzae isolates did not support a general concept of specific genital strains. However, strains of biotype IV clearly stood out with two characteristics: (i) a peritrichous fimbriation and (ii) a very peculiar homogeneous OMP pattern comprising an OMP of molecular weight approximately 18,000 unique to this biotype. These characteristics were also found in H. influenzae biotype IV strains isolated from genital infections in the United States and used as controls. H. influenzae biotype IV strains may thus correspond to a group somewhat adapted to the genital tract.

The thermogenesis of 17 strains belonging to 12 species of the Enterobacteriaceae family was measured at 30 degrees C with an ampoule microcalorimeter. It was analyzed qualitatively (aspect of profiles) and quantitatively (total heat evolved, thermogenesis duration, maximum thermal power). The value of these criteria is discussed with respect to their discriminating value in the classification of bacteria. The information obtained may concur with the identification of species; it gives ground to reconsider actual phenotypes and genotypes and taxonomy generally.

One hundred and ninety-nine strains of Haemophilus isolates were biotyped by Kilian's method(1) and a modified API 10S strip and the results compared. One hundred percent correlation was found between the two systems. The ONPG test proved of value in differentiating between Haemophilus influenzae and Haemophilus parainfluenzae when there was growth factor disc failure.

Bacterial meningitis remains a major cause of mortality and morbidity in many countries of the world despite effective antimicrobial therapy. Studies of the etiology and some laboratory characteristics of bacterial meningitis in Egypt were conducted during 1977-1978. All patients suspected of having bacterial meningitis were studied at the time of admission to the two fever hospitals of Cairo. Direct culture, serological identification of the capsular type, and countercurrent-immunoelectrophoresis of 1627 CSF specimens were done. Of these, 276 had bacteria identified either by culture or Gram stain. Pneumococci were the most common and the serotype distribution was similar to that reported from other parts of Africa; second were meningococci with groups C and B predominating; in third place was Haemophilus influenzae type b which caused the highest mortality and had an unusually young age distribution. There were 77 bacterial isolates (22%), including 11 species, designated as "other" because there was no predominant species. There were many "clear" CSF specimens that were found to contain pneumococci, meningococci or H. influenzae type b, confirming the need for more comprehensive laboratory facilities for accurate diagnosis of the etiology of bacterial meningitis.

Haemophilus pleuropneumonia (Matthews and Pattison, 1961; Shope, 1964) should now be accepted as the valid name of the pathogen of hemophilus pleuropneumoniae of swine, according to studies and the description by Kilian and co-workers (1978), while the name of Haemophilus parahaemolyticus should be considered a synonym. On top of Haemophilus pleuropneumoniae, Haemophilus parasuis and Haemophilus spec. "minor group" are also important to pneumonia, serositis, and arthritis (Glässer's disease) in swine, whereas Haemophilus suis is hardly any longer isolated. Evidence as to dependence on growth factors and to haemolysin activity is not sufficient in all cases for high-accuracy differentiation between those four haemophilus species. Such tests have to be supplemented with evidence as to separation of urea, xylose, lactose, and mannite or, in extraordinary cases, even with evidence to fermentation of melibiose and D-ribose, Serological typing is another tool for further differentiation. Pathologico-anatomic changes in terms of haemorrhagic-necrotising pleuropneumonia are caused by what is called Pasteurella haemolytica-like organism (Bertschinger and co-workers, 1978; Pohlenz and co-workers, 1978) rather than by haemophilic bacteria only. The authors, using the above pathogens, succeeded in causing in specific-pathogen-free pigs (SPF) manifest pleuropneumonia which could not be distinguished from haemophilus pleuropneumonia. Pathologically and bacteriologically, those germs are of great relevance to differential diagnosis, in the context of haemophilus pleuropneumonia.

The antimicrobial susceptibility of 73 Actinobacillus (Haemophilus) pleuropneumoniae isolates from swine in Missouri was determined with a microdilution minimal inhibitory concentration test system. Serotyping was accomplished by means of co-agglutination. Serotype 1 (39/73) and serotype 5 (30/73) were commonly found, whereas serotype 7 (4/73) was infrequently encountered. Most isolates (MIC90) were found susceptible to ampicillin (amoxicillin), cephalothin, penicillin, erythromycin, gentamicin, and kanamycin. Marked resistance was found with oxytetracycline, tylosin, and sulfadimethoxine. The data indicate that use of ampicillin (amoxicillin) or penicillin may correlate well with the favorable outcome of treatment.

A 4-h Micro-ID technique for direct identification of oxidase-negative gram-negative rods from positive blood cultures was compared to subculture and species identification of single colonies by API 20E and Micro-ID, using standardized inocula. A total of 127 patients (220 positive cultures) were studied. Isolates included 96 Escherichia coli, 46 Klebsiella pneumoniae, 7 Klebsiella oxytoca, 8 Enterobacter aerogenes, 17 Enterobacter cloacae, 19 Serratia marcescens, 2 Serratia liquefaciens, 8 Proteus mirabilis, 1 Salmonella species, 1 Morganella morganii, 6 Haemophilus influenzae, 2 Haemophilus parainfluenzae, 3 Bacteroides fragilis, 3 Acinetobacter calcoaceticus biotype anitratus, and 1 Pseudomonas maltophilia. In 90% of the cultures, identification by Micro-ID was identical to that obtained after subculture; if the 15 non-enterobacterial isolates were excluded, the corresponding figure was 96.6%. Enterobacteria identified incorrectly by direct Micro-ID were three S. marcescens (two identified as S. liquefaciens, one as Hafnia alvei), two S. liquefaciens (both identified as E. cloacae), and two K. pneumoniae (one identified as Klebsiella ozaenae, the other as Serratia rubidaea). None of the 15 non-enterobacterial cultures were correctly identified by Micro-ID (non-identifiable, or classified as Providencia/Yersinia/Klebsiella species). Although biochemical discrepancies between direct and final Micro-ID tests occurred in 41% of the enterobacterial cultures, this did not seriously interfere with identification. Direct species identification of Enterobacteriaceae from blood cultures by direct Micro-ID is accurate and easily performed and identified organisms within 4 h compared to at least 24 h by most other methods; the direct Micro-ID technique would be rendered even more valuable by the additional capability of identifying non-enterobacterial gram-negative isolates.

The biochemical characteristics of 464 strains of Haemophilus influenzae and 83 strains of Haemophilus parainfluenzae isolated over an 18-month period are described. Of 22 characteristics obtained, only 6 were necessary to biochemically identify and biotype the isolates. The key substrates or tests were urease, ornithine, indole, o-nitrophenyl-beta-D-galactopyranoside, sucrose, and xylose. Five biotypes of H. influenzae and four of H. parainfluenzae were commonly recognized. Some strains were encountered which could not be accommodated in the recognized taxa but which constituted separate biotypes of the two species, H. influenzae biotype I was recovered principally from blood, cerebrospinal fluid, and upper respiratory secretion, and biotypes II and III were recovered from eye and sputum cultures. Biotype I was recovered primarily from children less than 1 year of age, whereas biotypes II and III were from persons 1 to 5 years old and from those over 20 years of age. Multiple isolates recovered from the same patient were almost always of the same biotype. Strains of H. parainfluenzae were isolated primarily from sputum, with others being isolated from body sources such as dental abscesses, gastric aspirates, and peritoneal fluid. An inverse relationship was noticed between hemolysis and mannose fermentation among H. parainfluenzae biotype III strains, whereas the relationship was absent among the other biotypes.

The capsular polysaccharide of Haemophilus pleuropneumoniae serotype 1 (ATCC 27088) was found to be a teichoic acid type polysaccharide of a repeating disaccharide unit composed of 2-acetamido-2-deoxy-D-glucose and D-galactose units. By composition analysis, methylation, partial hydrolysis, dephosphorylation, and one- and two-dimensional 500-MHz proton nuclear magnetic resonance experiments, together with 13C nuclear magnetic resonance studies, it was concluded that the capsular polysaccharide is a high molecular weight linear polymer having the structure: (Formula: see text)

Counterimmunoelectrophoresis, direct immunofluorescence and immunodiffusion procedures were used to serotype 15 strains of Haemophilus pleuropneumoniae isolated from the respiratory tract of pigs in southern Brazil. Antigens were prepared by extracting cultures with a saline solution or by the phenol-water method. Antisera were prepared in rabbits against serotypes 1, 2, 3 and 5. Thirteen of the isolates were type 5 and two were type 3. No differences were observed between the results obtained in serotyping with counter immunoelectrophoresis and direct immunodiffusion, but both procedures were significantly better than immunodiffusion except with the saline extracted antigen. Counterimmunoelectrophoresis was quicker, more sensitive and more easily performed than the other techniques.

The commercially available API ZYM microbiological identification system was evaluated for the rapid identification of Haemophilus somnus. Eighty-seven isolates of the organism had API ZYM profiles which were characteristic. The API ZYM profiles demonstrate clear differences between H. somnus and other genera but suggest a close association to three related organisms. Enzyme activity of H. somnus isolates were similar to organisms identified as Histophilus ovis, Haemophilus agni and strains UQV of Actinobacillus actinoides and Actinobacillus seminis but was clearly different from isolates of Pasteurella haemolytica, Pasteurella multocida, Bordetella bronchiseptica and group EF4. The API ZYM system allowed more rapid identification of H. somnus than conventional biochemical tests and may be a useful adjunct to conventional methods used for identification of H. somnus isolates. The test did not reveal obvious differences between isolates from various anatomic locations.

The banding profiles generated by Bam H1 restriction endonuclease cleavage of bacterial DNA from clinical and reference isolates of Histophilus ovis, Haemophilus somnus and related bacteria were compared. H. ovis, H. somnus and Haemophilus agni isolates were found to have distinct similarities in banding profiles characterised by 10 common bands between 2.0 and 9.6 kilobases (kb). The close taxonomic relationship of these isolates was reinforced by these findings. The reference isolates examined in this study--Actinobacillus lignieresii, Actinobacillus seminis, H. agni, H. somnus, H. ovis, Haemophilus influenzae, Haemophilus parainfluenzae, Haemophilus parahaemolyticus--could be distinguished from each other on the basis of their characteristic banding profiles. Actinobacillus sp were observed to have more bands between 2 and 23 kb compared with the H. ovis and Haemophilus sp isolates studied. Analysis of isolates from an experimental infection trial illustrated the potential of restriction endonuclease analysis in molecular epidemiological applications. It was possible to demonstrate by this means that the post-challenge isolates had identical banding profiles to the challenge (or infecting) isolate which had a distinctly different banding profile from that of pre-challenge H. ovis isolates. Furthermore, restriction endonuclease analysis of H. ovis isolates obtained from follow-up investigations of a recurrent problem of epididymitis in unmated rams, indicated that the H. ovis isolates implicated in epididymitis, were present as a single strain in a number of sheep over a period of time. This suggested that the mechanism of transmission was by perinatal perputial contamination.

Twenty-two Haemophilus cultures of types prevalent in swine and of different geographic origins were subjected to biochemical and cultural examinations. Three subgroups were identified: One was unrease-positive, produced porphyrin from delta-aminolevulinic acid, and grew on infusion mediums supplemented only with V factor; the 2nd was unrease-negative, porphyrin-positive, and grew only on serum-enriched mediums with added V factor; and the 3rd was unrease-negative, porphyrin-negative, and grew only on serum-enriched mediums with added V and X factors. The groups generally corresponded to Haemophilus parahaemolyticus, Haemophilus parasuis, and Haemophilus suis, respectively. By means of the unrease and porphyrin tests, it was possible to assign, presumptively, porcine haemophilus cultures to 1 of the 3 species. Other tests, such as beta-galactosidase, hemolysis, and fermentation of carbohydrates were of secondary value in differentiating between these species.

Crystal violet, lincomycin, spectinomycin and bacitracin were evaluated as selective agents in media for isolation of Haemophilus pleuropneumoniae. No single antimicrobial agent or combination of two or more inhibited all non-Haemophilus strains (Escherichia coli, Pasteurella haemolytica, Pasteurella multocida, Streptococcus faecalis, Streptococcus equisimilis and Staphylococcus aureus) without marked suppression of 16 H. pleuropneumoniae strains. A medium containing 1 micrograms/mL of crystal violet, 1 microgram/mL of lincomycin, 8 micrograms/mL of spectinomycin and 128 micrograms/mL of bacitracin inhibited one E. coli strain and the Gram-positive strains while H. pleuropneumoniae strains were suppressed to a minor degree only. Haemophilus pleuropneumoniae was isolated on the selective medium on three occasions from the nose or pharynx of two out of eight experimentally inoculated pigs. Haemophilus pleuropneumoniae was recovered from the nose of only two pigs at necropsy and from tonsil of one, whereas the lower airways in most pigs and the lung lesions in all pigs were positive. There was no advantage to using the selective medium for the recovery of H. pleuropneumoniae at necropsy from these eight experimentally infected pigs, probably because other bacteria were absent or present in very low numbers in the tissues with H. pleuropneumoniae. The isolation rate on selective medium was higher than the rate on non-selective medium (p less than or equal to 0.1; chi 2 test) when the airways of slaughtered pigs were cultured. This was likely due to a high degree of contamination. Dry swabs placed in tryptone yeast extract with nicotinamide-adenine-dinucleotide gave a significantly higher recovery rate than commercial Culturette swabs in modified Stuart's transport medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Methods and their evaluation are described for the routine characterisation of Haemophilus spp isolates using a test for dependence on V factor and a test for the conversion of delta-amino-laevulinic acid (ALA) to porphyrin in which the ALA is incorporated into a solid medium. A method is also described whereby the difference in the size of the inhibition zones around discs of ampicillin and of amoxycillin/clavulanate can be used to detect the production of beta-lactamase.

Reference strains of serotypes 1 to 12 of Actinobacillus (Haemophilus) pleuropneumoniae were cultured in Eagle minimal essential medium with 10% Serum Plus. Culture supernatants were examined for cytotoxicity to alveolar macrophages and for the ability to hemolyze sheep erythrocytes. All strains except the reference strain of serotype 6 produced cytotoxin, whereas only serotypes 1, 5, 9, 10, and 11 produced hemolysin. Both cytotoxin and hemolysin appeared to be heat labile. Antisera were raised against cytotoxin- and hemolysin-containing culture supernatants of serotypes 1 to 11. Cross-neutralization studies revealed that the hemolysins were serologically homogeneous. In contrast, four serologically different cytotoxins were distinguished. One cytotoxin was produced by serotypes 1, 5, 9, and 11, and a second was produced by serotypes 2, 3, 4, and 8. A third cytotoxin was produced by serotypes 7 and 12; this cytotoxin was related to the cytotoxins of serotypes 1, 2, 4, 5, 9, and 11. A fourth cytotoxin, produced by serotype 10, was related to the cytotoxin of serotypes 1, 5, 9, and 11. Seventy field strains belonging to serotypes 2, 3, 7, 8, 9, and 11 were also tested for production of cytotoxin and hemolysin. All strains belonging to serotypes 9 and 11 produced hemolysin and cytotoxin, whereas all strains of serotypes 2, 3, 7, and 8 produced only cytotoxin. Hemolysins and cytotoxins of both the field strains and the corresponding serotype reference strains were comparably neutralized. These findings strongly suggest that the observed differences in production and type of hemolysin and cytotoxin were related to serotype and not to strain.

Biologic and serologic relationships between Page's serotypes A, B, and C and Sawata's serotypes 1 and 2 of Haemophilus paragallinarum were investigated. Of the 7 Page's serotype strains testes (which were biologically confirmed as H paragallinarum), serotype B strains Spross and 0222 (which produced a low-iridescent smooth-type colony) required V factor, but did not require chicken serum for growth. Serotype A strains 083, W, Georgia, and Germany and serotype C strain Modesto, as well as Sawata's serotype 1 strain 221 and serotype 2 strain H-18 (which produced a high-iridescent smooth-type colony) required V factor and chicken serum for growth. Strains 083, W, Georgia, and Germany of Page's serotype A and Modesto strain of serotype C, as well as strain 221 of Sawata's serotype 1 and H-18 of serotype 2, had a heat-labile, trypsin-sensitive L antigen. Because serotype A strains had serotype 1-specific antigen and common L antigen, which is shared by serotypes 1 and 2, these strains corresponded to Sawata's serotype 1. Serotype C strain Modesto, which had serotype 2-specific antigen and common L antigen, corresponded to serotype 2. However, serotype B strains Spross and 0222 lacked serotype B-specific L antigen, and were not typeable.