Eighty isolates of Riemerella anatipestifer representing 71 outbreaks of riemerellosis in Thailand between 1994 and 1999 were serotyped using the gel diffusion precipitin test. Based on the precipitation patterns, 25 serological profiles containing one to three antigenic determinants were recognized. Heat-stable antigens of the organism reacted with antisera raised against 16 known serotypes and an untypable strain 698/95. The most prevalent serotype appeared to be serotype 7, followed by serotypes 5, 10, 21 and 1. Further study demonstrated that the untypable strain probably represents a new serotype. Analysis of the polymerase chain reaction-amplified rrs genes for restriction fragment length polymorphisms verified the inclusion of strain 698/95 within the species R. anatipestifer and supported earlier work excluding strain 670/89, which had originally been designated the reference strain of serotype 20. Therefore, it is suggested that the strain 698/95 could be adopted as a replacement for the reference strain of serotype 20. Attention should be paid to strains with multiple antigenic factors as they may be useful for the preparation of vaccines.

SUMMARYThirty-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.

Strains belonging to Riemerella anatipestifer serotype 1 are responsible for most of the major disease outbreaks caused by this organism in ducks in Thailand. Therefore, the immunogenicity induced by a broth culture bacterin prepared from a local strain (1081) of this serotype was studied in ducks and a protective index (PI) was derived. The inactivated vaccine contained the equivalent of 5 X 10(9) colony-forming units of bacteria per 0.5-ml dose. A single subcutaneous inoculation of the broth culture bacterin in 2-week-old Khaki Campbell ducklings gave adequate protection (PI = 88) against challenge with the homologous strain 7 days after vaccination, while the highest protection (PI = 95.6) was obtained 2 weeks after vaccination. The duration of immunity was at least 6 months. Inoculation of the vaccine at both 1 and 5 weeks of age gave the greatest protection. The vaccine was found to be safe in one-week-old ducklings and it also gave significant protection (PI values from 66.7 to 100) against challenge with virulent strains of the homologous serotype. The immunogenicity of broth culture bacterin prepared from local strains of various serotypes was also studied. Strains of serotypes 1, 6, 11, 14 and 19 gave no significant protection against challenge with strains of heterologous serotypes. Concentrated cell-free culture filtrates prepared from strain 1081 of serotype 1 and strain 328 of serotype 19 induced highly significant protection against homologous challenge (P < 0.01), but cross-protection between the two strains was not significant.

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