Nonsuppurative encephalitis in calves aged 4-12 months, cow abortion and fetal deformities were endemic in dairy farms in Taiwan in recent years. A virological investigation emphasizing on Arthpodborn virus (Arbovirus) was conducted. Total of 11 strains of Akabane virus were isolated from endemic districts between June and July of 1992. Among them, seven viruses were isolated from blood samples of 15 calves showing nervous signs. Another 4 Akabane viruses were isolated from clinically healthy calves from three of six dairy farms investigated. All the six investigated farms had a recent history of abortion and fetal deformities. The isolates caused prominent cytopathic effects in HmLu-1 cells and could reach a high virus titers (5 x 10(6) TCID50/ml). As demonstrated by a cross neutralization test, the isolates had identical antigenicity to Iriki strain of Akabane virus, but were antigenically more distant to JaGar-39 and OBE-1 strain of Akabane virus. This is the first report on the isolation of Akabane virus in Taiwan, and also the second report on the isolation of Iriki virus in the world.

Hyperimmune rabbit sera directed to the KSCN extract of 3:A Pasteurella multocida were characterized by enzyme-iinked immunosorbent assay (ELISA), presolubilized cell radioimmunoprecipitation, and immunoblotting analysis. The results showed that the hyperimmïne serum had a very high titer of immunoglobulin G ELISA antibody and a negligible immunoglobulin A ELISA antibody, precipitated 10 different outer membrane protein antigens by radioimmunoprecipitation, and reacted to 10 different membrane vesicle antigens of P. multocida by immunoblotting analysis. The hyperimmune rabbit sera were also evaluated for protective efficacy against experimental rabbit pasteurellosis by homologous challenge. Thirty-six rabbits were divided into four groups. Group 1, 2, and 3 rabbits were inoculated intranasally with hyperimmune rabbit serum, phosphate-buffered saline, or normal rabbit serpm, respectively, at 24 h prior to and 24, 48, and 72 h after iptranasal challenge with the virulent homologous P. multocida strain. Group 4 rabbits were inoculated with normal rabbit serum without challenge. Necropsies of surviving rabbits were performed 2 weeks postinfection. The mortality rates for groups 1 through 4 were 25% (3 of 12), 67% (8 of 12), 75% (6 of 8), and 0% (0 of 4), respectively. The prevalence and severity of pneumonia were significantly lower in the hyperimmune serum-treated rabbits. The prevalence of P. multocida colonization in lungs was significantly lower in group 1 rabbits, and the geometric mean CFU of P. multocida in lungs was 59,166-fold less in group 1 rabbits than in group 3 rabbits. The geometric mean CFU of P. multocida in nasal cavities of group 1 rabbits was significantly lower than that of group 3 rabbits. All challenged rabbits (groups 1, 2, and 3) had elevated nasal immunoglobulin A and pulmonary (lung lavage) immunoglobulin A antibody levels at necropsy (day 14 postinfection). Similarly, all challenged rabbits had elevated levels of ELISA immunoglobulin G antibody in serum at day 14 but not at day 7 postinfection, indicating that rabbits receiving hyperimmune serum can moVnt a specific humoral immune response against the homologous challenge P. multocida organisms. We concluded that hyperimmune serum directed to the KSCN extract of 3:A P. multocida provides significant protection against homologous challenge in rabbits.

In October 1989, an epizootic duckling disease with high mortality occurred in Taiwan. The disease was characterized by droopiness, inappetence, ataxia, ruffled feathers, and watery diarrhea. Affected ducklings were lame, were unable to stand, showed opisthotonos, and often died 3 or 4 days after the onset of the disease. Tolerant maturing ducklings displayed atrophic upper bills with a protruding tongue and became stunted as they reached maturity. No diagnostic histopathologic lesions were found in these ducklings. Fourteen parvovirus isolates, 33 duck viral hepatitis virus (DVHV) isolates, two adenovirus isolates, and two reovirus isolates were obtained and identified from more than 500 sick ducklings in the epizootic. The epizootic was diagnosed as a co-outbreak of duck parvovirus infections and duck viral hepatitis. The high mortality in ducklings and the bill atrophy syndrome were reproduced in ducklings by inoculating the parvovirus isolates alone. The epizootic was controlled by an emergency immunization program of ducklings with sera collected from recovered ducks or a bivalent inactivated vaccine composed of local DVHV and parvovirus isolates.

Monoclonal antibodies (MAbs) directed against the 37.5-kDa outer membrane protein were produced by fusing myeloma cells with spleen cells obtained from mice immunized with a pathogenic strain of Pasteurella multocida isolated from a rabbit. Desirable MAbs were selected by enzyme-linked immunosorbent assay, whole-cell radioimmunoprecipitation (WC-RIP), and Western blot (immunoblot) analysis. WC-RIP and Western blot analyses, using MAb 1608 adsorbed with intact P. multocida cells and the eluted MAb, demonstrated that the antigen recognized by this MAb is exposed on the cell surface, is antibody accessible, and has an estimated molecular mass of 37.5 kDa. Treatment of outer membrane vesicles of P. multocida with proteinase K totally abrogated the MAb 1608 activity, indicating that this MAb binds to a protein antigenic determinant. Furthermore, MAb 1608 was nonreactive to purified lipopolysaccharide in Western blot analysis. Passive transfer studies showed that nine rabbits inoculated intranasally with MAb 1608 and homologously challenged intranasally had significantly reduced mortality, severity of pneumonia, prevalence of P. multocida colonization in nonrespiratory organs, and numbers of P. multocida in nasal cavities compared with the controls. Furthermore, the number of P. multocida in lungs was reduced 84,750-fold. Similarly, passive transfer experiments indicated that MAb 1608 protected mice against homologous and heterologous challenges with P. multocida strains bearing the antigenic determinant recognized by MAb 1608. However, no protection was afforded by MAb 1608 when mice were challenged with a P. multocida strain lacking the antigenic determinant recognized by MAb 1608. This study establishes that the 37.5-kDa outer membrane protein is the target for a protective MAb.

Potassium thiocyanate extracts of a virulent Pasteurella multocida 3:A rabbit isolate were prepared and used as a vaccine in rabbits. The extract contained protein, carbohydrate, hyaluronic acid, lipopolysaccharide, DNA, and RNA. The protein and lipopolysaccharide profiles of the extract were similar to those of the P. multocida cell membrane. Rabbits were vaccinated intranasally (i.n.) or intramuscularly (i.m.) four times at 1- or 3-week intervals and challenged i.n. with the homologous P. multocida 2 weeks after the last vaccination. Rabbits vaccinated with the extract by the i.n. route developed persisting serum immunoglobulin G (IgG) and nasal IgA antibodies, whereas rabbits immunized by the i.m. route produced persisting serum IgG and transient nasal IgA antibodies. The extract prevents the death of rabbits which were vaccinated by either route and challenged. Vaccination by the i.n. route in rabbits reduced the numbers of virulent P. multocida in nasal cavities and lungs and the prevalence and severity of rhinitis and pneumonia. These i.n.-vaccinated rabbits were also resistant to virulent P. multocida colonization in liver, spleen, uterus, and tympanic bullae. Similarly, i.m. vaccination in rabbits resulted in a reduction in the severity of rhinitis; the numbers of virulent P. multocida in lungs; and the prevalence of colonization in liver, spleen, uterus, and tympanic bullae. Vaccination by the i.n. route was superior to that by the i.m. route in that there was a significant reduction in the severity of pneumonia and numbers of virulent P. multocida in nasal cavities and lungs. Rabbits vaccinated with the extract without challenge showed no lesions.