Typing of heat-stable and heat-labile antigens of Campylobacter jejuni and Campylobacter coli by coagglutination.

A coagglutination system has been devised for typing heat-stable and heat-labile antigens of Campylobacter jejuni and C. coli. The use of protein A-positive Staphylococcus aureus cells carrying Campylobacter sp. serotype antibody and the treatment of Campylobacter sp. cells with DNase in the antigen suspension permitted rapid and specific coagglutination of rough (autoagglutinable) as well as smooth cultures. Cells of S. aureus were sensitized with Campylobacter sp. serotype antisera. Four to five types of sensitized S. aureus cells were pooled. A strain of Campylobacter sp. was first tested with the pools and then typed with the individual reagents of the reactive pool. After the described procedures, 68 serotype strains tested blindly as unknowns were correctly typed according to their heat-stable or heat-labile antigens. The two most commonly used typing schemes which are based separately on the heat-stable or the heat-labile antigens as assayed by passive hemagglutination and slide agglutination, respectively, can be utilized simultaneously in the coagglutination system for strain characterization. The coagglutination system is simple, yields results rapidly, conserves typing reagents, and offers the flexibility of formulating the pools of reagents according to the experimental design or the prevalence of serotypes in a geographic location. It should be a practical system for the typing of Campylobacter spp. in public health or clinical laboratories.     

Common somatic O and heat-labile serotypes among Campylobacter strains from sporadic infections in the United States.

Somatic O (formerly heat-stable) and heat-labile (HL) serotyping methods are commonly used to type Campylobacter jejuni and Campylobacter coli isolates. Although both systems are effective, the labor and time required for each have limited their application. These systems can be simplified by reducing the number of antisera used. To find an appropriate panel of antisera, we determined the distribution of common serotypes in the United States among a representative sample of 298 Campylobacter isolates. The strains, obtained between July 1989 and June 1990 from persons with sporadic cases of diarrhea, were collected from 19 randomly chosen counties in all geographic (census) regions of the United States. All strains were serotyped by the O and HL systems. By phenotypic methods, 288 C. jejuni, 9 hippurate-negative C. jejuni/C. coli, and 1 Campylobacter lari were identified. Of 57 O antisera, 24 typed 252 (84.6%) strains. Of the 55 HL antisera, 23 serotyped 253 (84.9%) strains. All strains were typeable in the unabsorbed O antisera. In the absorbed HL antisera, four strains were nontypeable and 14 were rough and untypeable. In each geographic region, 9 or more O and HL serotypes were found. Serotypes O:1, O:4, and O:13,16,43,50 and HL 1 were identified in all regions. The combination of both schemes gave greater discrimination than either system alone, but the maintenance of both requires a large resource investment. A serotyping scheme incorporating the 24 most prevalent O and 23 most prevalent HL serotypes could be useful for outbreak support and for surveillance. In the near future, we anticipate using a molecular subtyping method in combination with limited serotyping to distinguish Campylobacter strains.     

Comparison of the Penner and Lior methods for serotyping Campylobacter spp.

We compared two Campylobacter serotyping systems by using 1,405 isolates of Campylobacter collected from human, animal, and environmental sources during epidemiologic investigations and special studies. We found 96.1% of isolates to be typable by the Penner method for heat-stable antigens, which involved the use of an indirect hemagglutination technique, and 92.1% of isolates to be typable by the Lior method for heat-labile antigens, which involved the use of a slide agglutination technique and absorbed antisera. Absorbed antisera were not required for the Penner method, making that method less difficult to implement. The Lior method was simpler to perform and gave more rapid results than did the Penner method. Cultures frequently reacted in multiple antisera with the Penner method, whereas multiple reactions were rare with the Lior method. Thus, results were easier to interpret with the Lior system. Strains of a single serotype in one system were sometimes found to be multiple serotypes in the other system; hence, the two methods have the potential to be complementary. Both systems were comparable in serotyping isolates from human and nonhuman sources and for evaluating the relationship of strains collected during outbreak investigations.     

Serotyping and biotyping of Campylobacter jejuni and Campylobacter coli from sporadic cases and outbreaks in Norway

Of 172 thermophilic campylobacters isolated from human cases of gastroenteritis in Norway, 149 (86.6%) were classified as Campylobacter jejuni, whereas 23 isolates (13.4%) belonged to Campylobacter coli. C. jejuni biotype 1 comprised 66.3% and C. jejuni biotype 2 comprised 20.3% of the total number. Using 50 unabsorbed antisera, we were able to serotype 109 (80.1%) of 136 campylobacters on the basis of heat-stable antigens identified by means of passive hemagglutination. The typable strains fell into 36 different serotypes. A large proportion of the strains were isolated from travellers returning from abroad, a state of affairs which may have influenced the serotype and biotype distribution. Two family outbreaks were found to be caused by a bio-serotype common to all diseased members of the particular families. A third family outbreak and an outbreak among employees at a poultry processing plant each involved two distinct strains.     

Campylobacter jejuni and Campylobacter coli serotypes isolated from chickens, cattle, and pigs

A total of 191 Campylobacter jejuni and 125 Campylobacter coli were isolated from the intestinal content of 398 chickens, 421 cattle, and 203 pigs. All 108 chicken isolates and 73 of 80 cattle isolates were C. jejuni, but 115 of the 118 pig isolates were C. coli. A total of 84% of the C. jejuni and 64% of the C. coli isolates were typed on the basis of thermostable antigens with 20 antisera prepared against frequently occurring serotypes in Campylobacter enteritis in man (15 C. jejuni, 6 C. coli serotypes). A total of 96% of the chicken isolates and 67% of the cattle isolates belonged to 11 C. jejuni serotypes that occur most frequently in human cases of enteritis (serotypes 1, 2, 3, 4, 5, 13/16, 18, 21, 23, 31, and 36). Serotype 8, a relatively common human isolate, was not recovered. The C. coli isolates from pigs belonged to serotypes uncommon among human isolates.     

Serotypes of 'Pasteurella' anatipestifer isolates from poultry in different countries

'Pasteurella' anatipestifer (PA) isolates from ducks, turkeys and other birds were typed by agglutination tests using antisera against representative strains of existing serotypes. The majority of the isolates were from the USA, but some were from Singapore, England and Germany. Five new serotypes were identified, three from ducks in the USA and two from Singapore. Four isolates received from Germany did not react with any of the available antisera. The representative strain of serotype 4 (H) was excluded from this study because it grew on MacConkey agar and has been reported to have a cell-protein profile unlike that of other PA isolates. A revised serotype classification was proposed to include existing and new serotypes.     

Serotyping of Canadian isolates of Treponema hyodysenteriae and description of two new serotypes.

A total of 30 isolates of Treponema hyodysenteriae collected in the Saint-Hyacinthe (Quebec, Canada) area were serotyped by agar gel double immunodiffusion by using extracted lipopolysaccharide and hyperimmune rabbit antisera. Only 17% (5 of 30) of the isolates were typed with antisera specific for each of the seven known serotypes of T. hyodysenteriae. Antisera raised against 11 untypeable local isolates were then produced and tested against each lipopolysaccharide extract. Results showed two serologically distinct groups among 21 of the 25 untypeable isolates. The isolates in each group shared identical antigens. No detectable reactions could be observed between antisera raised against these 11 isolates and the antigens extracted from 7 reference serotype strains. On the basis of these results, two new serotypes of T. hyodysenteriae, serotypes 8 and 9, are proposed. We also propose isolate FM 88-90 as the reference strain for serotype 8 and isolate FMV 89-3323 as the reference strain for serotype 9. These two new serotypes, which represented 70% of the isolates tested, seem to be the major serotypes found in the province of Quebec.     

Investigation of a waterborne outbreak of Campylobacter jejuni enteritis with a serotyping scheme based on thermostable antigens

Serotyping of 11 human and 2 water isolates of Campylobacter jejuni associated with a waterborne outbreak revealed two serotypes among the human isolates. One of these (serotype 58) was a new serotype and was added to the serotyping scheme. Serotypes were defined by using extracted thermostable antigens and passive hemagglutination titrations of both unabsorbed and cross-absorbed antisera. Two water isolates of the same serotype as six human isolates provided evidence to link a contaminated water supply to the outbreak.     

DNA fingerprinting and serotyping of Campylobacter jejuni isolates from epidemic outbreaks.

The aim of the present investigation was to compare DNA fingerprinting and serotyping (heat-stabile and heat-labile antigens) of isolates from epidemic outbreaks as well as of solitary isolates. Campylobacter jejuni isolates from two epidemic outbreaks in Sweden, one milkborne (35 isolates) and one waterborne (17 isolates), and one waterborne outbreak in Norway (11 isolates), as well as 30 solitary isolates from Swedish patients with gastroenteritis, were analyzed. A total of 93 isolates were analyzed. In the waterborne outbreak in Norway, only one serotype with one DNA pattern was found. In the milkborne outbreak in Sweden, two serotypes (HS2:HL4 and HSNT:HL4) with two different DNA patterns were found. The isolates from the waterborne outbreak in Sweden were different serotypes. For two isolates of the same serotype, different DNA patterns were seen. This was also recorded for isolates from solitary cases. It was concluded that serotyping is a useful tool in most epidemiological situations but sometimes lacks sufficient discriminatory power. DNA fingerprinting can add valuable epidemiological information to that supplied by serotyping and can in some situations provide sufficient epidemiological information when used alone.     

High-resolution genotyping of Campylobacter upsaliensis strains originating from three continents

Ninety-six Campylobacter upsaliensis strains that originated from Australia, Canada, and Europe (Germany) and that were isolated from humans, dogs, and cats were serotyped for their heat-stable surface antigens. All of them were genotyped by enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) profiling, and 83 strains were genotyped by macrorestriction analysis with the endonuclease XhoI. Eighty-four percent of the strains belonged to five different serotypes (serotypes OI, OII, OIII, OIV, and OVI), with the proportions of strains in each serotype being comparable among the groups of strains from all three continents. Two serotypes, OIII and OIV, were prevalent at rates of 35 to 40%. Serotypes OI, OII, and OVI were detected at rates of 1.5 to 15%. Between 10 and 17.7% of the strains did not react with the available antisera. Analysis of the ERIC-PCR profiles revealed two distinct genotypic clusters, which represented the German and the non-European strains, respectively. XhoI macrorestriction yielded two genotypic clusters; one of them contained 80.2% of the German strains and 34.6% of the non-European strains, and the second cluster consisted of 65.4% of the non-European strains and 19.8% of the German strains. Fourteen strains from all three continents were analyzed for their 16S rRNA gene sequences. Only two minor variations were detected in four of the strains. In conclusion, C. upsaliensis has undergone diverging processes of genome arrangement on different continents during evolution without segregating into different subspecies.     

Serotyping of Campylobacter jejuni isolated from sporadic cases and outbreaks in British Columbia.

Campylobacter jejuni from sporadic cases and outbreaks of gastroenteritis were serotyped on the basis of heat-extracted soluble thermostable antigens identified with the use of the passive hemagglutination technique. A total of 168 isolates were separated into 45 different types. The largest proportion of the isolates fell into three serotypes, each with 11 to 12.5% of the total number. Three less frequently occurring serotypes each included approximately 5%, and the remaining 50% of the isolates were distributed among 39 other serotypes. In most cases, serotyping demonstrated that epidemiologically linked isolates were of the same serotype, but the outbreak strains could belong either to frequently or to infrequently isolated serotypes. The high correlation between clinical findings and serotyping results confirmed the applicability of the serotyping scheme in epidemiological investigations of C. jejuni infections.     

Evaluation of a simplified procedure for serotyping Campylobacter jejuni and Campylobacter coli which is based on the O antigen.

A simplified procedure for serotyping Campylobacter jejuni and Campylobacter coli on the basis of thermostable antigens was developed and tested for its applicability as a routine typing method. The assay involves the sensitization of erythrocytes with an antigenic extract and performance of a slide agglutination assay with specific antisera. In order to simplify the typing system to a greater extent, the standard typing antisera were pooled into nine groups for C. jejuni and four groups for C. coli. The five antiserum samples allocated to each pool were selected so that pairs or groups of cross-reacting antisera were included in the same pool. When this system was tested with the serotype reference strains, it was found that, in most cases, a strain reacted in only one pool. The specific serotype of that strain could then be further defined by typing in each of the antisera belonging to that pool. To evaluate the specificity of the simplified method, 246 clinical isolates of C. jejuni and 57 clinical isolates of C. coli were typed at the same time by the standard passive hemagglutination assay and by the rapid slide agglutination system. Although both schemes effectively differentiated isolates and results from both schemes were generally very similar, differences were noted for a few isolates. On the basis of these findings, the simplified procedure may be recommended as an alternative means for serotyping these species for epidemiological purposes.     

Structural and antigenic properties of lipopolysaccharides from serotype reference strains of Campylobacter jejuni.

To investigate the molecular basis for heat-stable antigenic diversity in Campylobacter jejuni, lipopolysaccharides (LPSs) from serotype reference strains and serotyped isolates were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis coupled with silver staining and immunoblotting. By silver staining, only low-Mr components, consisting of one major band and as many as three minor bands ranging in Mr from 4,500 to 5,000, were detected. However, by immunoblotting with homologous antisera, 10 of 34 strains were shown to have a series of high-Mr LPS components characteristic of molecules with O side chains of various lengths. Isolates of the same serotype as the reference strain that had high-Mr LPS molecules were also found to have high-Mr LPS and in one case of cross-reacting strains it was found that the cross-reaction was associated with antibodies against high-Mr LPS. The reactions of LPSs with homologous and heterologous antisera indicated that both high- and low-Mr-type LPSs were strain-specific antigens, but in some cases cross-reactions were noted. Evidently, all C. jejuni strains possess low-Mr LPS that is readily detectable by silver staining, but some serotypes also possess high-Mr LPS components that can be visualized by immunoblotting.     

Serotypes of 'Pasteurella' anatipestifer isolates from ducks in Singapore: a proposal of new serotypes

'Pasteurella' anatipestifer (Pa) isolates from local ducks were typed by slide and tube agglutination tests using antisera against representative strains of existing serotypes. As the strains of serotype 13 and serotype 17 were found to be serologically identical, it is proposed that they be jointly designated as serotype 13. It is also proposed that the English serotype P, represented by strain HPRS 2565, be adopted as a replacement for the existing serotype 4 which has been excluded as Pa. Three new serotypes were identified among the 352 isolates from ducks in Singapore. It is proposed that these new serotypes be designated as serotypes 17, 18 and 19 under the present classification system.     

Comparison of a commercial test for serotyping heat-stable antigens of Campylobacter jejuni with genotyping by pulsed-field gel electrophoresis.

A new commercial serotyping set based on heat-stable Penner's antigens was compared with pulsed-field gel electrophoresis (PFGE) with SmaI and SacII restriction endonucleases. Among 50 isolates of Campylobacter jejuni from Finnish patients, which represented predominant PFGE patterns selected from isolates from sporadic cases and isolates associated with small outbreaks, 11 different serotypes were demonstrated from 43 typable isolates. Several PFGE patterns could be found within one serotype; on the other hand, several serotypes could be demonstrated within one PFGE type. Most isolates originated from sporadic cases; however, some isolates were epidemiologically associated and showed identical serotypes and PFGE patterns. Although the new serotyping set would have been useful in the few epidemic cases studied, several isolates (14%) representing the major PFGE patterns remained untypable or gave weakly positive agglutination reactions only suggesting a plausible serotype (18%). This might restrict the use of the novel serotyping set, at least in Finland.     

Comprehensive ribotyping scheme for heat-stable serotypes of Campylobacter jejuni.

Strains from diverse sources belonging to all 47 heat-stable Penner serotypes of Campylobacter jejuni were examined for polymorphism around the 16S rRNA genes. Penner serotype reference strains and a group of nonserotypeable isolates were included in the study. Complete typeability was obtained; 30 distinct PstI and 42 HaeIII polymorphisms were found. Three bands were detected in almost all strains with these enzymes, confirming that three copies of the 16S rRNA gene are typical for C.jejuni. By combination of the two enzyme polymorphisms, 77 16S ribotypes were defined among the 261 strains analyzed. With two exceptions, no specific association was observed between these ribotypes and heat-stable serotypes. Nine serotypes were homogeneous with respect to the 16S ribotype. Most nonserotypeable strains belonged to ribotypes defined elsewhere in the study. The 16S ribotypes of C.jejuni described here were not found in strains of Campylobacter coli, and vice versa.     

Naturally occurring auxotrophs of Campylobacter jejuni and Campylobacter coli.

The nutritional requirements for 439 Campylobacter jejuni isolates and 46 Campylobacter coli isolates were determined by using a previously described chemically defined medium, campylobacter defined medium. With this medium, 45% of both human and nonhuman C. jejuni isolates demonstrated auxotrophic requirements. None of the 46 C. coli isolates studied demonstrated requirements for amino acids on campylobacter defined medium. The most common auxotrophic requirement among C. jejuni isolates was for methionine, which was present as a single requirement or in combination with other markers in 21% of human and 28% of nonhuman isolates. There was no correlation between plasmid carriage and auxotype, and a comparison of the Lior serotypes of 472 of the strains showed a correlation only between proline auxotrophs and Lior serotype 11 for strains isolated in the Seattle-King County region.     

Lectin typing of Campylobacter isolates.

Isolates of Campylobacter jejuni, C coli, C fetus and C laridis were tested for agglutination reactions with a panel of five lectins: Arachis hypogaea, Bauhinia purpurea, Solanum tuberosum, Triticum vulgaris and Wisteria floribunda. Twenty three patterns of agglutination (lectin types) were recorded among 376 isolates. Patterns were consistent and reproducible. Only 4.5% of isolates were untypable because of autoagglutination. Some lectin types were found exclusively or predominantly in a species, but others were shared between species. Forty two per cent of C jejuni and 35% of C coli isolates belonged to lectin type 4. There was no apparent correlation between lectin type and serotype; different lectin types were found among strains of single Penner and Lior serotypes. Lectin typing is a simple and economical procedure suitable for use in non-specialist laboratories, either as an adjunct to serogrouping or, after further development, as a sole typing scheme.     

New serotypes of Riemerella anatipestifer isolated from ducks in Thailand

Thirty-two Riemerella anatipestifer isolates from ducks were serotyped by agar gel precepitin test using chicken antisera against serotypes 1 to 19 R. anatipestifer reference strains. The heat stable saline extracts from 29 field isolates reacted with the antisera of serotypes 1, 6, 7, 10, 11, 14, or 19. The isolates belonging to serotype 1 were the most prevalent (56.3%). Antigens from the remaining three isolates did not react with any of the available antisera. Additional investigations showed that they represent two new serotypes, serotypes 20 and 21.     

Serotype distribution of Campylobacter jejuni and Campylobacter coli isolated from hospitalized patients with diarrhea in central Australia.

Campylobacter jejuni and/or Campylobacter coli was cultured from 218 of 1,078 patients of all age groups admitted to Alice Springs Hospital, Alice Springs, central Australia, between July 1988 and June 1989 for treatment of diarrhea. One hundred sixty-six Campylobacter colonies from 127 patients were subjected to O serotyping by using the Penner typing scheme. All except 29 colonies could be serotyped. A total of 46 serotypes were identified, and the predominant serotypes were O:8, 17, O:22, O:1,44, and O:19. A large proportion of colonies reacted with more than one antiserum, and nine serotypes had antigenic compositions not observed previously. Several patients had multiple infections with more than one serotype, and some patients were shown for the first time to be infected with up to three different serotypes. Repeated reinfections with different serotypes were seen in some patients. In some patients, provided it was not due to reinfection with the same serotype, long-term excretion of the same serotype was seen, and for the first time, one patient showed evidence of excretion of the same serotype for up to 73 days.