An evaluation of agglutination and coagglutination techniques for serotyping of Haemophilus pleuropneumoniae isolates

A comparative evaluation of rapid slide agglutination, tube agglutination, 2-mercaptoethanol tube agglutination, and coagglutination tests was made for serotyping isolates of Haemophilus pleuropneumoniae. The results indicated that a majority of the isolates could be serotyped by any of these tests. But, it was not uncommon to find isolates which were inagglutinable or poorly agglutinable in homologous sera. Heat treatment of whole-cell suspensions of such isolates was essential to unmask the serotype-specific antigenic determinants; however, in the process of heat treatment, cross-reactive common antigens of minor nature were also exposed. The antibodies involved in such cross-reactions were mainly of immunoglobulin M type, because the cross-reactivities were completely abolished in coagglutination and 2-mercaptoethanol agglutination tests. Thus, both these tests were satisfactory for serotyping inagglutinable mucoid strains. For serotyping strains which were either polyagglutinating or autoagglutinating, agglutination tests could not be used, but the coagglutination test proved to be satisfactory. The coagglutination test was serotype-specific, sensitive, simple, rapid, reproducible, and easier to read and interpret than rapid slide or tube agglutination tests. This test could be used to serotype mucoid, smooth, or rough isolates.     

Detection of strains of Haemophilus pleuropneumoniae using the coagglutination test

Three tests were used for the serological identification of the strains of Haemophilus pleuropneumoniae (Actinobacillus pleuropneumoniae) isolated from pigs and coming from 66 sites where pleuropneumonia, caused by Haemophilus, occurred in pigs. The coagglutination test was found to be the best for the identification of the causal agent; the ring precipitation test was somewhat less sensitive, and worse results were obtained when rapid slide agglutination was used. Of all the field isolates of H. pleuropneumoniae, serovar 2 occurred most frequently (56%), followed by serovar 1 (39%); one strain was identified as serovar 7. Two strains have remained unidentified. The serological identification of the strains was performed on the basis of their comparison with eight type serovars of H. pleuropneumoniae.     

Experimental infection of mares with Haemophilus equigenitalis

Inoculation of Haemophilus equigenitalis into the uterus of 7 mares caused a disease clinically indistinguishable from contagious equine metritis. The duration of clinical signs varied from 4 to 11 days. The causative organism persisted for a relatively short time (2 to 10 weeks) in 5 mares, but in 2 others it established a carrier status and persisted until they were killed 6 and 10 months after infection. H. equigenitalis was recovered from the vestibule of the vagina and from a combined swab of the clitoral fossa and sinuses throughout the course of the infection. In some mares there were extended periods (2 weeks) when it could not be reisolated. All mares experienced a transitory serological response to infection. The complement fixation test was generally negative 12 weeks after infection whereas antibodies detected by the passive haemagglutination test and serum agglutination test were more persistent. In some animals the PHT and SAT titres increased during the breeding season following infection. The serological response did not appear to be related to the duration of infection.     

Serotyping of Haemophilus pleuropneumoniae by rapid slide agglutination and indirect fluorescent antibody tests in swine

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.     

Serological classification of Japanese isolates of Haemophilus paragallinarum using two serovar-specific monoclonal antibodies

A serological classification of 106 Japanese isolates of Haemophilus paragalinarum recovered from 1960 to 1984 was performed by dot-blotting and hemagglutination-inhibition (HI) tests using two serovar-specific monoclonal antibodies (MAbs), E5C12D10 and F2E6. By the dot-blotting test, 49 of the isolates were serovar A and 55 isolates were serovar C, and the two remaining isolates did not react with either MAb. These two nontypable strains had no hemagglutinating activity against chicken erythrocytes and were nonpathogenic to chickens. Although 49 serovar A isolates were serotyped by the HI test, only 23 of the 55 serovar C isolates could be serotyped. The remaining 32 isolates could not be serotyped because no or low hemagglutinating activity could be detected. Our results indicate that H. paragallinarum serovars A and C have both been present in Japan since 1960, with serovar A isolates being dominant before 1970 and serovar C isolates more prevalent than serovar A since 1970.     

The causative organism of contagious equine metritis 1977: proposal for a new species to be known as Haemophilus equigenitalis.

The aetiological agent of contagious equine metritis (CEM) has been investigated bacteriologically in a wide range of cultural and conventional biochemical tests, in the eletron microscope, for DNA base composition (36.1 per cent GC), for susceptibility to various antimicrobial agents and antigenically by means of tube and slide agglutination tests. The organism is a fastidious, Gramnegative, non acid-fast coccobacillus which in biochemical tests is very unreactive. In conventional tests, only the oxidase, catalase and phosphatase tests were positive. Dependance on neither X nor V factors could be demonstrated, but some stimulation of growth by X factor was observed. The organism could not be identified with any known species and even allocation to an appropriate characters, we propose the organism as a new species of the genus Haemophilus: H. equigenitalis, type strain NCTC 11184 (61717/77).     

A rapid agglutination assay for canine brucellosis using antigen coated beads

Brucella canis is the causative agent of canine brucellosis and facultative intracellular pathogen. The diagnosis of canine brucellosis is based on bacteriological examination and serological methods including agglutination and gel diffusion tests. In this study, crude antigens were extracted from B. canis using hot saline, coated on to latex beads and their usefulness in the serological diagnosis of canine brucellosis was examined. Mixing the antigen coated latex beads with the sera of dogs infected with B. canis produced clear agglutination, but this was not so for B. canis free dog sera. N-terminal amino acid sequence analysis of the crude hot saline extracts, showed that they contained copper-zinc superoxide dismutase, ribose ABC transporter and hypothetical protein of Brucella as antigens. A serological survey of canine serum samples conducted by means of an agglutination test using the antigen coated latex beads, showed that this method was more specific than the tube agglutination test using whole bacterial cell antigens. Although these results suggest that our method in which crude hot saline extracted antigens are coated on to latex beads would be useful in the serological diagnosis of canine brucellosis, we need further investigation using more serum samples to confirm the usefulness of our method.     

Serological characterization of Danish Haemophilus parasuis isolates

A total of 103 Danish Haemophilus parasuis field isolates was collected from diseased pigs in connection with routine diagnostics. The isolates were serotyped using indirect haemagglutination (IHA) and for 57 of the isolates the serotyping was also performed by immunodiffusion. Serovar 5 was the most prevalent (36%), followed by serovar 4 (13%) and serovar 13 (22%), whereas 15% of the strains were nontypeable by IHA. Serovars 1, 2, 6, 7, 9, 12, 14, and 15 were only represented by a small number of isolates. Most of the Danish isolates belong to serovars, which earlier have been shown to be virulent. The strains could be divided into two groups depending on whether they were isolated from cases with systemic disease (polyserositis, arthritis or meningitis) or if they only were found in the lower respiratory tract. The most marked differences were observed for serovar 4, which had a higher prevalence in respiratory disease compared to systemic infection, and for the nontypeable isolates, which were mainly found in cases of systemic infection.     

Serological classification of Australian isolates of Haemophilus paragallinarum

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.