Shizuo Sato

Evaluation of the efficacy of a bacterin against Salmonella enteritidis infection and the effect of stress after vaccination.

An oil-emulsion vaccine was prepared from phage type 4 Salmonella enteritidis to assess the potential protective efficacy of S. enteritidis vaccine and the effect of stress after vaccination. Hens were vaccinated at 14 and 18 weeks of age and challenged orally at 24 weeks of age with 10(6) or 10(3) cells of a homologous S. enteritidis strain. Pullets were vaccinated at 8 and 12 weeks of age and challenged with 10(9) cells. The O antibody titers of vaccinated hens rose quickly and were unchanged after challenge. S. enteritidis was isolated from cecal droppings of significantly fewer vaccinated hens than unvaccinated control hens for 6-21 days and 2-15 days after challenge with 10(6) and 10(3) cells, respectively. S. enteritidis was also isolated from livers and spleens of significantly fewer vaccinated pullets for 1 week. Vaccinated and control hens were equally affected by exposure to environmental stress, the result being that the shedding rate of S. enteritidis in cecal droppings increased equally in both groups.

Horizontal Transmission of Salmonella enteritidis and Effect of Stress on Shedding in Laying Hens

Horizontal transmission of Salmonella enteritidis in laying hens and the short-term effect of stress on shedding were examined in 32 seven-month-old laying hens. Half were inoculated with 10(5) colony-forming units of S. enteritidis phage type 4, and the remaining half were left uninoculated to study horizontal transmission. Isolation of S. enteritidis from cecal droppings of all hens was attempted every morning. Uninoculated hens rapidly became infected through contaminated drinking water. Introduction of young chickens to the same rearing room and withdrawal of water and feed for 2 days coincided with a rapid increase in the shedding rate of S. enteritidis for a short period of time. The results showed that a short-term increase in the shedding rate of S. enteritidis is associated with short-term exposure to environmental stress.

DETECTION OF INTERLEUKIN-6 AND PROSTAGLANDIN E2 IN BRONCHOALVEOLAR LAVAGE FLUIDS OF PIGS EXPERIMENTALLY INFECTED WITH MYCOPLASMA HYOPNEUMONIAE

In this study, interleukin-6 (IL-6) and prostaglandin E2 (PGE2) were detected in the bronchoalveolar lavage fluids (BALF) from pigs experimentally infected with Mycoplasma hyopneumoniae. IL-6 was detected at 2 weeks post-inoculation (PI), and significantly increased levels of PGE2 were observed at 4 weeks PI. In the BALF collected from infected pigs at 4 weeks PI, the levels of IL-6 increased significantly in the pigs with pneumonic lesions. However, increased levels of PGE2 were observed in all the infected pigs.

Epizootic outbreaks of gizzard erosion associated with adenovirus infection in chickens.

Two outbreaks of gizzard erosion in slaughtered broiler chickens in Japan were examined pathologically and microbiologically. The prevalences of such lesions were 9%-11% and 4%-50% in the affected flocks. Affected chickens had no clinical signs. Group I fowl adenovirus (FAV) serotype 1 was isolated from gizzard lesions. Histologically, gizzard mucosa were necrotic. Intranuclear inclusion bodies were seen in the enlarged nuclei of degenerating epithelial cells of the gizzard. The keratinoid layer in the erosion was edematous and desquamated and contained degenerative cells. Moderate to marked inflammatory cell infiltration was observed in the lamina propria and perivascular connective tissue in the submucosa and muscle layer. Immunohistochemical staining showed evidence of FAV antigens in the intranuclear inclusion bodies within degenerating epithelial cells. Ultrastructurally, numerous viral particles were demonstrated in the inclusions.

Immunoblotting analysis of antibody response in swine experimentally inoculated with Mycoplasma hyopneumoniae

Serological cross-reactivity among swine mycoplasmas, and porcine antibody response to Mycoplasma hyopneumoniae antigens during experimental infection were investigated by enzyme-linked immunosorbent assay (ELISA) and the immunoblotting technique. Mycoplasmas solubilized with Tween 20 were used as antigen. Cross-reactivity between M. hyopneumoniae and M. flocculare was indicated by ELISA and it was demonstrated by immunoblotting, which revealed that the predominant cross-reactive antigens were components of M. hyopneumoniae with molecular weights of 74,000 (74 K) and 53 K. Further, it was indicated that 96 K, 70 K, 46 K and 38 K antigenic components of M. hyopneumoniae and 67 K, 56 K and 23 K antigens of M. flocculare were specific for the respective species. Antibodies to 96 K, 76 K, 70 K, 53 K, 46 K and 38 K antigens were detected in serum from pigs experimentally infected with M. hyopneumoniae. Of these antibodies, those against the 46 K antigen reacted early and were most consistently detected during experimental infection. Therefore, the antibodies against the 46 K antigen of M. hyopneumoniae proved to be excellent markers of mycoplasmal pneumonia of swine.

The Effect of the Flow of Air on Horizontal Transmission of Salmonella enteritidis in Chickens

Horizontal transmission of Salmonella enteritidis and the effect of airflow on spreading were examined in 80 5-wk-old chickens divided into five groups. Sixteen chickens in each group were placed in four cages in a row separated by wire. One among four chickens placed in a cage at the downwind end of the row was inoculated orally with 10(9) colony-forming units of S. enteritidis. Cecal droppings, drinking water, and feed were cultured every day. Horizontal transmission was rapid in the row with low air velocity but slow in the row with high air velocity. However, in another experiment, where the inoculated chicken was situated in a cage upstream in the airflow, horizontal transmission was equally rapid whether the airflow was rapid or slow. Contamination of feed and water never preceded the appearance of positive cecal droppings. These findings suggest that the rapidity of horizontal transmission of S. enteritidis may be affected by airflow patterns.

Comparison of the Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Pattern of the Fiber Gene and Pathogenicity of Serotype-1 Fowl Adenovirus Isolates from Gizzard Erosions and from Feces of Clinically Healthy Chickens in Japan

The fiber gene sequence and pathogenicity of the serotype-1 fowl adenovirus (FAdV-1) isolated from gizzard erosions and from clinically normal chickens were compared among isolates. The FAdV-99ZH strain, which induced gizzard erosions, had a nucleotide sequence of the long fiber gene that was different from that of the Ote strain, which did not induce gizzard erosions. The differences could be distinguished by use of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. The long fiber gene of 16 FAdV-1 isolates from gizzard erosions and 10 FAdV-1 isolates from the feces of clinically normal chickens was examined by use of PCR-RFLP analysis. All 16 FAdV-1 isolates from gizzard erosions had the same restriction patterns as those of strain 99ZH; however, 10 FAdV-1 isolates from normal chickens were classified into 3 groups. Specific-pathogen-free (SPF) chickens were inoculated orally with 2 FAdV-1 isolates from gizzard erosions or 3 FAdV-1 isolates from clinically normal chickens to determine the pathogenicity of each strain. Two of 2 FAdV-1 isolates from gizzard erosions induced gizzard erosions. Two of 3 FAdV-1 isolates from normal chickens had the same PCR-RFLP patterns as those of the Ote strain, but did not induce any gizzard erosions. However, 1 FAdV-1 isolate from clinically normal chickens had the same PCR-RFLP pattern as that of strain 99ZH and induced gizzard erosions. These results indicate that there are FAdV-1 strains that have different pathogenicity; one strain induces gizzard erosions, and the other does not. Use of PCR-RFLP analysis of long fiber genes may be able to distinguish between these two strains.

Outbreaks of adenoviral gizzard erosion in slaughtered broiler chickens in Japan

Gizzard erosion in broiler chickens was investigated at 18 slaughterhouses in Japan. The condition was observed in 13 of them, and adenoviral gizzard erosion (AGE) was diagnosed histologically, immunohistochemically and virologically in the eroded gizzards from nine of these 13. The antigen-positive intranuclear inclusion body of group 1 fowl adenovirus was observed in the epithelial cells of the affected gizzards, and fowl adenoviruses were isolated from the lesions. In two of the slaughterhouses the total weights of the gizzards disposed of in three years were 3590 kg (0.40 per cent of the gizzards inspected) and 2880 kg (0.19 per cent). Sixteen of the 19 outbreaks of gizzard erosion on 15 farms that were confirmed in three of the slaughterhouses, including the previous two slaughterhouses, were diagnosed as AGE, and the condition was suspected in the other three outbreaks. Most of the adenoviruses isolated were identified as serotype-1 by PCR-restriction fragment length polymorphism. No apparent clinical signs were observed in any of the affected flocks.

Experimental Infection of Specific-Pathogen-Free Chickens with Serotype-1 Fowl Adenovirus Isolated from a Broiler Chicken with Gizzard Erosions

Gizzard lesions were formed in specific-pathogen-free (SPF) white leghorn chickens inoculated with fowl adenovirus (FAV). The virus, serotype 1 FAV 99ZH strain (FAV-99ZH), was originally isolated from the gizzard mucosa of commercial broiler chickens exhibiting gizzard erosion with intranuclear inclusion bodies. Five-day-old and 53-day-old SPF white leghorn chickens were inoculated with FAV-99ZH by both oral and ocular routes and then examined at necropsy on days 3, 5, 7, 10, 14, and 21 postinoculation (PI). There were no clinical signs in any of the chickens after the inoculation. Focal gizzard lesions occurred macroscopically, however, in inoculated chickens at several experimental periods. FAV was recovered from tissue samples of the proventriculus, gizzard, pancreas, and rectum by day 10 or 7 PI but was not recovered from liver samples of any of the chickens. These results indicate that FAV isolated from gizzard erosion is able to reproduce gizzard lesions as necrosis and erosion in SPF white leghorn chickens and that it may have a greater degree of tissue tropism in gizzards and other digestive organs than in the liver.

Cytological and immunological changes in bronchoalveolar lavage fluid and histological observation of lung lesions in pigs immunized with Mycoplasma hyopneumoniae inactivated vaccine prepared from broth culture supernate

We have compared the cytology of bronchoalveolar lavage fluid (BALF) and the pathology of lung lesions in pigs immunized with/without Mycoplasma hyopneumoniae inactivated vaccine prepared from broth culture supernate on experimental infection. Numbers of total cells, macrophages, neutrophils and lymphocytes have decreased in BALF of vaccinated pigs following infection. The mean percentage of lung lesions, inflammatory cell infiltration into the airways and T cells accumulation around the bronchi were reduced in vaccinated pigs. The levels of tumor necrosis factor (TNF)-alpha also decreased in vaccinated pigs. These results suggest that the vaccination may contribute to decrease TNF-alpha production, and therefore, inflammatory cell responses in the lung due to M. hyopneumoniae infection were suppressed, resulting in fewer lung lesions.