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This study was undertaken in an effort to improve the identification scheme of catalase-negative, non-beta-hemolytic, gram-positive cocci isolated from milk samples obtained from cows. First, the sensitivity and specificity of the identification procedure currently in use in our laboratory were compared to the results obtained with API 20 STREP strips which were set as the gold standard. Second, a number of other identification tests, which could contribute to increase the sensitivity and specificity of the identification procedure of these microorganisms, were evaluated and selected. The data have shown that there is a necessity to review the identification procedure. Some modifications are suggested to laboratories doing milk sample analyses. A standardized procedure, using the CAMP test, esculin and sodium hippurate hydrolysis, the presence of the enzymes pyrolidonyl arylaminase and leucine aminopeptidase, and acid production from 1% inulin and raffinose broth, would not only improve the results of the identification process of gram-positive cocci isolated from milk samples but also ensure greater uniformity of the epidemiological data.

Streptococcus agalactiae is considered one of the major causes of bovine intramammary infections. It is also found in the vaginas of women without any apparent clinical symptoms, but reports of neonatal infections, causing significant morbidity, are relatively frequent. The aim of this study was to evaluate the genetic diversity of S. agalactiae strains isolated from bovine milk and from asymptomatic women in Québec, Canada, by randomly amplified polymorphic DNA (RAPD) analysis. A total of 185 bovine isolates and 38 human isolates were first serotyped for capsular polysaccharide by double diffusion in agarose gel (bovine isolates) and coagglutination (human isolates). Strains were then studied by RAPD using 3 primers, designated OPS11, OPB17, and OPB18, which were selected from 12 primers. Thirty-eight percent of bovine isolates and 82% of human isolates could be serotyped. Prevalent serotypes were type III (28%) for bovine isolates and types V (26%) and III (24%) for human isolates. RAPD results showed that, taken together, all isolates (of bovine and human origin) shared 58% similarity. Ninety-four percent of these isolates were clustered in four groups (I, II, III, and IV) with 70% similarity among them. Three clusters, A (48 isolates), B (14 isolates), and C (32 isolates), with 79 to 80% similarity were identified within group IV, whereas the three other groups did not present any clusters. Despite some clustering of human isolates, relatively high diversity was seen among them. Relatively high heterogeneity was observed with the RAPD profiles, not only for field strains belonging to different serotypes but also for those within a given serotype.

The genetic diversity of 88 Streptococcus suis serotype 2 isolates which were recovered from various countries was examined by randomly amplified polymorphic DNA (RAPD) analysis with three primers. This bacterial collection included 80 isolates of porcine origin and 8 of human origin. This investigation allowed the identification of 23 RAPD types containing 1 to 30 isolates originating from one to six countries. Common RAPD patterns were found between human and pig isolates. The isolates were also tested for the production of virulent factors such as hemolysin, muramidase-released protein (MRP), and extracellular factor (EF). All isolates exhibiting the virulent phenotype hemolysin+ MRP+ EF+ clearly clustered on the basis of fingerprinting by RAPD analysis. In a similar way, most of isolates with the hemolysin- MRP- EF- phenotype were assigned to one RAPD cluster. Therefore, RAPD clusters are more related to the phenotype defined with hemolysin, MRP, and EF than to the geographic origin of the isolates. These data indicate that RAPD analysis used in conjunction with phenotypic methods provides a reliable method for the assessment of the clonal relationship between S. suis isolates responsible for infections in pigs or humans, especially for those exhibiting the classic "virulent" phenotype hemolysin+ MRP+ EF+.

On the basis of biochemical, phenotypic, and 16S rRNA analysis, a novel gram-negative bacterium, isolated from normal and diarrheic dogs as well as humans with gastroenteritis, has been recently named Helicobacter canis. A 2-month-old female crossbred puppy was submitted to necropsy with a history of weakness and vomiting for several hours prior to death. The liver had multiple and slightly irregular yellowish foci up to 1.5 cm in diameter. Histologically, the liver parenchyma contained randomly distributed, occasionally coalescing hepatocellular necrosis, often accompanied by large numbers of mononuclear cells and neutrophils. Sections of liver stained by the Warthin-Starry silver impregnation technique revealed spiral- to curve-shaped bacteria predominantly located in bile canaliculi and occasionally in bile ducts. Aerobic culture of liver was negative, whereas small colonies were noted on Campylobacter selective media after 5 days of microaerobic incubation. The bacteria were gram negative and oxidase positive but catalase, urease, and indoxyl acetate negative; nitrate was not reduced to nitrite, and the organism did not hydrolyze hippurate. The bacteria were also resistant to 1.5% bile. Electron microscopy revealed spiral-shaped bacteria with bipolar sheathed flagella. By 16S rRNA analysis, the organism was determined to be H. canis. This is the first observation of H. canis in active hepatitis in a dog and correlates with recent findings of Helicobacter hepaticus- and Helicobacter bilis-related hepatic disease in mice. Further studies are clearly warranted to ascertain whether H. canis-associated hepatitis is more widespread in canines as well as a cause of previously classified idiopathic liver disease in humans.

Polyvalent coagglutination reagents (PRs) have been evaluated for the serotyping of Streptococcus suis. Monovalent antisera produced against 28 S. suis reference strains have been grouped to obtain five different pools. A total of 249 field isolates previously identified and belonging to different serotypes were tested with PRs prepared by two different procedures: (i) monovalent coagglutination reagents were individually prepared and mixed in equal proportions, and (ii) antisera were mixed in equal proportions before the addition of the Staphylococcus aureus suspension. Only antisera tested by a tube agglutination test with 2-mercaptoethanol and presenting titers of 1:32 or higher were used. Results obtained with PRs prepared by both procedures were similar, and there was a very good correlation between the capsular type of the isolate and the reaction obtained with PRs. Thus, from a practical viewpoint, it is suggested that PRs be prepared by the first procedure. To isolates, were tested in parallel with both the PRs and the monovalent coagglutination reagents over a 1-year period. Ninety-nine percent of the typeable and all of the untypeable isolates were correctly identified. Serotyping with PRs is suggested to be a very useful and reliable screening procedure, particularly when a large number of S. suis isolates have to be serotyped. In addition, the choice of antisera to be included in a given pool is facultative and should be oriented to the needs of a region or a country.

Six new capsular types of Streptococcus suis (types 23 to 28) are described. All reference strains were isolated from diseased pigs and were morphologically and biochemically similar to previously described capsular types 1 to 22. Clear and specific reactions were obtained for each of the new capsular types with three different typing techniques; no cross-reactions were detected among them or with other S. suis capsular types. Their capsular material presented similar ultrastructural characteristics, as shown by electron microscopy, and fimbriae similar to those described for other capsular types of S. suis were observed. When untypeable field isolates were tested with antisera raised against the six new capsular types, capsular type 23 appeared to be the most prevalent, representing more than 50% of all these isolates. Most isolates were recovered from cases of pneumonia, septicemia, and meningitis. Presumptive biochemical identification described for S. suis capsular types 1 to 22 may also be used for capsular types 23 to 28.

Sixty-two isolates of Campylobacter cryaerophila were recovered from aborted porcine and bovine fetuses, from porcine, bovine, and equine feces, and from different tissues of a dead piglet. Phenotypic characterization was carried out on all isolates, and the results were compared with those obtained with the reference strains of C. cryaerophila, C. jejuni, C. coli, C. laridis, and C. hyointestinalis. The ability of C. cryaerophila strains to grow under aerobic conditions at 16 degrees C was found to be most useful in differentiating them from strains of other Campylobacter species. Studies were undertaken to develop a serotyping system for C. cryaerophila on the basis of the Lior serotyping system for C. jejuni and C. coli by use of a tube agglutination test with formalinized whole-cell (FWC) and boiled whole-cell (BWC) antigens. Antisera against 18 strains of C. cryaerophila were produced in rabbits. Thirty-five percent of C. cryaerophila strains were typed with the FWC suspension as an antigen, and 61% were typed with the BWC suspension as an antigen. None of the C. cryaerophila strains tested autoagglutinated in saline. BWC antigens of C. jejuni, C. coli, and C. laridis cross-reacted with C. cryaerophila, whereas FWC antigens did not cross-react. Neither FWC nor BWC antigens of C. hyointestinalis reacted with C. cryaerophila antisera.

A total of 49 strains (23 reference strains and 26 field isolates) of Streptococcus suis were tested for their ability to agglutinate erythrocytes from different animal species. Ten different hemagglutination patterns were established. Thirty-three strains (67%) did not agglutinate any of the erythrocytes tested; sixteen strains (33%) agglutinated erythrocytes from one or more animal species. Different strains belonging to the same capsular type presented different hemagglutination patterns. No correlation was found between the tissue origin and/or the virulence (evaluated in 4-week-old mice) of different field isolates and their hemagglutination activity. Hydrophobic surface properties were also evaluated. All S. suis strains studied appeared to possess a hydrophilic cell surface. Morphologically similar fimbriae were observed on hemagglutinating as well as on nonhemagglutinating strains of S. suis. This study provides evidence that certain strains of S. suis possess hemagglutinating properties which do not appear to involve hydrophobic interactions. The possible role of fimbriae in hemagglutination remains unclear.

Of 24 isolates of nutritionally variant streptococci recovered from equine corneal ulcers, 22 were tested for growth requirements, physiological and biochemical reactions, and susceptibility to different antimicrobial agents. Satisfactory growth was obtained by supplementing blood agar and Todd-Hewitt broth with pyridoxal hydrochloride, and all of the media for the culture and the biochemical testing were supplemented with 0.002% of this substance. Biochemical patterns of 12 of the isolates resembled those of two viridans streptococcal species, Streptococcus intermedius and Streptococcus constellatus. Patterns of 10 isolates did not resemble those of any recognized viridans species. All of the isolates were inhibited by less than or equal to 0.25 microgram of erythromycin per ml, less than or equal to 4 micrograms of chloramphenicol per ml, and less than or equal to 4 micrograms of gentamicin per ml, and all but two were inhibited by less than or equal to 0.1 microgram of penicillin per ml.

Rapid slide agglutination (RSA), quantitative plate agglutination, slow tube agglutination (STA), and ring precipitation (RP) tests were performed on 200 isolates of Haemophilus pleuropneumoniae by using the type sera produced in rabbits against five known serotype strains and one strain 202. RSA and RP tests both yielded the same results as those by STA. None of the agglutination procedures could be used for serotyping isolates that autoagglutinated in saline. The RP test was successfully used for serotyping such strains. The specificity of the RSA and RP tests was confirmed by cross-absorption studies. All of the isolates except two had strong serotype-specific activities. The most common serotype isolated in Quebec was serotype 1, followed by serotypes 5 and 2. None of the isolates belonged to serotypes 3 and 4. Only two isolates were found to be untypable; they could possibly belong to serotype(s) not yet defined. The RSA and RP tests may be at least as reliable as the STA test, but easier to perform, less expensive, and much more rapid than any of the other methods reported. Of all the procedures studied by us, the RP test proved to be the method of choice for serotyping H. pleuropneumoniae; hence, it should replace the STA test for serotyping H. pleuropneumoniae.

Specific diagnosis of Haemophilus pleuropneumoniae infection by the conventional culture and identification method usually requires 3 to 4 days. Since H. pleuropneumoniae may be disseminated from infected individuals during this period, this amount of time required for diagnosis may be too slow to aid in epidemic control. To obtain an earlier diagnosis, a coagglutination test was successfully used to detect serotype-specific antigens in lung extracts of infected pigs. A total of 19 lung tissues from experimentally infected pigs, 240 lung tissues from naturally infected pigs that died of pleuropneumonia, and 571 lung specimens from an apparently healthy pig population were examined for culture isolation as well as for antigen detection. The results showed that detection of antigens in lung tissues by the coagglutination test is an extremely rapid, simple, quite specific, and highly sensitive procedure for the diagnosis of H. pleuropneumoniae infection. Further, detection of antigens in lung tissues was found to be a much simpler and much more rapid method than culture isolation. The coagglutination test seems to be especially useful for detecting H. pleuropneumoniae in pigs exposed to chronic infection as well as those in which multiple serotypes are involved.

Serological properties of antigens extracted from strains of Haemophilus pleuropneumoniae belonging to seven different serotypes were investigated. Antisera were prepared in rabbits against Formalin-treated whole cell suspensions as well as autoclaved cell suspensions. Saline and heat extracts and their alcohol precipitate antigens of H. pleuropneumoniae were used in the indirect hemagglutination test. All the antigens used were easily adsorbed directly onto sheep erythrocytes. Saline extract antigen showed maximum type specificity. Heating of the whole cell suspension revealed the cross-reactive minor antigenic determinants. Thus, the heat extract preparations had both type-specific and species-specific antigens. It is suggested that the indirect hemagglutination test may be useful for both serotyping and serodiagnosis of H. pleuropneumoniae infections in pigs.

Specimens of colonic mucosa from two pigs with diarrhea were examined by light and electron microscopy. The epithelial surfaces of both pigs were extensively colonized by large spirochetes morphologically compatible with Treponema hyodysenteriae or Treponema innocens. The microorganisms were intimately attached end-on to the luminal cells. A weakly beta-hemolytic spirochete similar to T. innocens was isolated from the colon of one of the pigs.

Fourteen new capsular types of Streptococcus suis (types 9 to 22) are described. All reference strains are morphologically and biochemically similar to types previously described. Reference strain types 9 to 13, 15, 16, and 22 were isolated from diseased pigs, whereas types 17 to 19 and 21 came from clinically healthy pigs; type 14 was isolated from a human case of meningitis, and type 20 was isolated from a diseased calf. The group T streptococcus of de Moor has been included in the typing system as type 15. Two-way cross-reactions between types 6 and 16 and a one-way cross-reaction between types 2 and 22 have been demonstrated. In addition, several cross-reactions probably not due to capsular material were detected among different types by using the coagglutination test. This test should not be used alone; weak or multiple positive reactions must be confirmed by the capsular reaction test or the capillary precipitation test.

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.

There are strong cross-reactions among strains of Actinobacillus pleuropneumoniae belonging to serotypes 3, 6, and 8. Various serological tests were used to differentiate these serotypes from each other. Tube agglutination, coagglutination, and indirect hemagglutination tests were not sufficiently sensitive to differentiate strains of serotypes 3, 6, and 8. However, higher antibody titers were obtained with a 2-mercaptoethanol agglutination test in homologous rabbit antisera. Absorption of immune sera with homologous and heterologous serotypes as well as quantitative estimation of antigenic activity in the unheated and heat-treated bacterial cell suspensions of reference strains with rabbit homologous and heterologous antisera revealed serotype-specific and cross-reacting group-specific antigens. Usually, serotype-specific antigens were major and dominant over group-specific antigens. The coagglutination test could be used quantitatively to measure the ratio of serotype-specific and group-specific antigens with rabbit hyperimmune sera against serotypes 3, 6, and 8. The highest antigen content for a particular serotype reflected serotype-specific antigen. For strains showing equal amounts of antigen for two or more serotypes in the coagglutination test, the immunodiffusion test with boiled cell-saline extract as the antigen and rabbit antisera against whole-cell suspensions of serotypes 3, 6, and 8 clearly revealed the serotype-specific antigen. It is suggested that coagglutination and immunodiffusion tests could be used successfully to determine the exact serotype of strains belonging to serotypes 3, 6, and 8.