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Establishment of Murine Norovirus S7 Infection System for Vaccine Development
Takeyama, N.1, 2), Yingju, C.1), Tohya, Y.3), Oroku, K.2), Kiyono, H.1)and Yuki, Y.1)
1) Division of Mucosal Immunology, The Institute of Medical Science, The University of Tokyo
2) Nippon Institute for Biological Science
3) Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University
International Congress of Immunology 2013, Milano
Abstract： Norovirus (NV) belongs to the Caliciviridae family with non-enveloped, positive-strand RNA. Over 60% of non-bacterial gastroenteritis outbreaks are due to human NV (HNV), which is a major public health concern. The lack of HNV cell culture system and animal infection model hampers the studies in HNV vaccine development. The discovery of murine NV (MNV) has provided a great volume of biological information of NV. In this study, we aimed to establish the MNV infection mouse model by using MNV-S7 isolated in Japan, as an alternative approach for HNV vaccine development. When Balb/cA and C57BL/6J were orally inoculated with 7.5log10 TCID50/dose of MNV-S7, MNV-RNA and the viral titers could be detected in fecal samples from 1 day post infection (dpi). The excretion of MNV-S7 in feces persisted for at least 2 weeks, with peaks between 1 to 5 dpi. After 14 dpi, MNV-RNA and viral titers decreased, which might be attributed to antigen-specific host immune responses. Using newly established MNV-S7 infection model, we vaccinated Balb/cA nasally with MVN-S7 recombinant VP1 P-domain [rVP1(P)], together with or without cholera toxin. VP1 specific antibodies in both systemic and mucosal compartments, particularly with high levels of serum IgG, were induced in both groups. After MNV-S7 challenge, vaccinated mice showed a slight decrease in MNV-RNA and the viral titers at 5 and 7 dpi, indicating that rVP1(P) exhibited a limited protection. These results will be a support in improving the researches in host infection and immunity on NV and the vaccine development of NV.
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Characterization of the Recently Isolated Erysipelothrix rhusiopathiae and the Effect of Commercial Erysipelas Vaccines.
Tsutsumi, N., To, H., Tazumi, T., Kamada, T., Nagai, S., Nagano, T., Nunoya, T. and Iwata, A.
Nippon Institute for Biological Science
The 6th Asian Pig Veterinary Society Congress 2013, Vietnam
Abstract： 【Introduction】 Since swine erysipelas reappeared as a clinical problem in pig populations in Japan and in the Midwerstern United States, it has been considered as a reemerging disease that contributes substantially to economic losses in swine production. During 2008-2011 many outbreaks of erysipelas have been reported among vaccinated and nonvaccinated swine herds in Japan. The object of the present work was to determine genetic and biological characteristics of Erysipelothrix rhusiopathiae strains from Japan; and also to evaluate the protective effect of commercial erysipelas vaccines. 【Materials and Methods】 Eighty-three Erysipelothrix sp. strains isolated from pigs with erysipelas were used to determine serotype and nucleotide sequence of a 432-bp hypervariable region in spaA gene. The 15 field and 2 reference strains were used to determine the pathogenicity in mice and the sensitivity to acriflavine. A total of 250 female mice were used for immunizing with “Nisseiken” Swine erysipelas inactivated vaccine (SER, aluminum-adsorbed vaccine containing killed whole culture of E. rhusiopathiae strain Tama 96 of serotype 2) or “Nisseiken” ARBP/Swine erysipelas combined inactivated vaccine (BPSER, formulated with the killed cells of Bordetella bronchiseptica, E. rhusiopathiae strain Tama 96, and Pasteurella multocida toxoid), and “Nisseiken” Swine erysipelas live vaccine C (SEL, reconstituted live vaccine containing approximately 108 CFU of E. rhusiopathiae strain Koganei 65-0.15, serotype 1a, per ml). The control and immunized mice were challenged with the reference and field strains of E. rhusiopathiae.Ten conventional pigs, known to be free from PRRS and APP, were used for immunizing with SER or ARBP. The control and immunized pigs were challenged with the field strains. SpaA-specific antibody in pig serum was detected using the indirect ELISA with the recombinant SpaA protein as an antigen. 【Results】 Eighty-three (100%) strains were identified as E. rhusiopathiae, based on serotyping and spaA PCR. Fifty (60.3%), 5 (6.0%), and 28 (33.7%) strains were isolated from animals with acute, subacute and chronic outbreaks, respectively, of which 79 (95.2%), 1 (1.2%), and 3 (3.6%) belonged to serotypes 1a, 2a, and untypeable, respectively. Fifteen strains (including 3, 2, and 10 from acute, subacute, and chronic cases, respectively) were sensitive to acriflavine, and showed high levels of virulence in mice; of which strains from acute cases, and from subacute and chronic cases killed 100%, and 80 to 100% mice, respectively at challenge doses of 102 CFU per mouse. Based on sequence analysis of a 432-bp hypervariable region in spaA gene, 83 strains could be divided into 3 groups: (i) group 1 (3 strains of serotype 1a) had Ala-195 and Ile-203; (ii) group 2 (76 strains of serotype 1, and 3 of untypeable) had Asp-195 and 203-Met; and (iii) group 3 (one strain of serotype 2a) had Asn-195 and Ile-203. A total of 150 mice immunized subcutaneously with live or inactivated vaccines were protected against challenge exposure to one reference strain (Fujisawa, 1a) and four field strains of serotype 1a (Kumamoto-S1, Gunma-657, Gunma-649, Nagano-16). Ten conventional pigs immunized intramuscularly with inactivated vaccines (SER or BPSER) developed specific antibodies against the SpaA protein of E. rhusiopathiae and were protected against challenge with two field strains (Gunma-657, Gunma-649). 【Discussion and Conclusions】 The results show that sequence analysis also parallelled the mouse pathogenicity and the acriflavine resistance tests for discrimination of the live vaccine strain from E. rhusiopathiae field strains. It has been reported that the sequence analysis is easy to perform and prevent any ambiguity in the results obtained, affords the same results irrespective of the laboratory involved, and can screen more samples at the same time. Our findings together with data reported by other investigators support the idea that the majority of chronic strains not related to the live vaccine strain have high levels of virulence in mice. The present study indicates that the nucleotide and amino acid sequences of strains isolated from 2008 to 2011 were different from those of strains reported previously, and also suggests that the serotype 1a strains belonging to the group 2 might be widespread in pig populations in Japan. This is the first study to demonstrate that the commercially available vaccines could protect animals against challenge with the most recently isolated SpaA-type strains of E. rhusiopathiae. |
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Genetic Characterization and protective immunity of APXIIA and APXIIIA of Actinobacillus pleuroneumoniae.
To, H., Tsutsumi, N., Tazumi, A., Nagai, S., Nagano, T., Nunoya, T. and Iwata, A.
Nippon Institute for Biological Science
The 6th Asian Pig Veterinary Society Congress 2013 , Vietnam
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