| Swine erysipelas(SE),also called "diamond skin disease" or "red fever",is an acute and highly contagious disease caused by Erysipelothrix rhusiopathiae(E.rhusiopathiae).SE,classical swine fever and swine pasteurellosis are the three most important infectious diseases causing huge economic loss to swine industries in China many years ago.Later,SE was effectively controlled by the widely used antibiotics and vaccines.Few outbreaks of acute SE were reported for last decades,so little was known about its pathogenic characteristics,virulence changes and prevalence.However,seven isolates from different places were identified as E.rhusiopathiae strains by PCR since July,2013.Meanwhile,several outbreaks of SE were reported in other regions of China.To monitor the infection status of E.rhusiopathiae in the pig population and detect if any changes in virulence or resistance occurred,the spaA sequence analysis in combination with serotyping,PFGE,acriflavine resistance,mouse pathogenicity and antimicrobial susceptibility tests were used to identify and characterize recent field strains of E.rhusiopathiae from acute SE.Also,to study the pathogenic mechanism of E.rhusiopathiae strains,one attenuated strain HX130709a was obtained by passing its parent strain HX130709 on agar media containing acriflavine dye for 55 passages.Based on the virulent/avirulent strains,murine cytokine detection,phagocytosis test using 3D4/21 cells and study on immunological characteristics of different forms of vaccines against SE were carried out,hoping to provide new ideas for research on pathogenic mechanism and new vaccine development of SE.The contents of this research contain seven parts as following:1 Isolation and characterization of E.rhusiopathiae strainsFrom July to November in 2013,seven tissue samples were collected from Nanzhang,Hexian,Xuancheng,Sihong and Yancheng.BHI-T 80 agar/broth,TSA and 5%sheep blood agar plates were used for isolation of bacterial strains from tissue samples.The strains were identified and confirmed as E.rhusiopathiae by polymerase chain reaction(PCR)after Gram staining and microscopic examination.All strains were isolated from cases of acute swine erysipelas causing sudden death of fattening pigs and postpartum sows.In order to explore the cause of the outbreak,the spaA sequence analysis in combination with serotyping,PFGE,acriflavine resistance,mouse pathogenicity and antimicrobial susceptibility tests were used to identify and characterize recent field strains of E.rhusiopathiae from acute swine erysipelas including one strain isolated in 2009 and one vaccine strain.Eight strains isolated from cases of septicemia were determined as serotype 1a,and 4 of the isolates were resistant to acriflavine.The 432-bp hypervariable region in spaA gene of the field strains were amplified and sequenced.It was further compared with the vaccine strain G4T10,and thus,the eight field strains can be divided into four spaA-types.Results of pulsed-field gel electrophoresis showed that eight distinct patterns with 22 to 30 DNA fragment bands were produced from field strains.Mouse pathogenicity test showed that the mortality of the mice infected with 104 CFU field strains was 100%.All the field strains were highly sensitive to P-lactam antibiotics,tetracyclines and macrolides but resistant to kanamycin and sulfisoxazole.2 Attenuation of an E.rhusiopathiae isolate and characterization of the attenuated StrainOne isolate strain named HX130709 was attenuated on agar media containing a gradually increasing concentration of acriflavine dye(0.0025%-0.03%).Strains were stored every 5 passages under-70℃.The partial spaA gene analysis also showed that no point mutations occurred among different archived passages of HX130709 during the attenuation.Results of pulsed-field gel electrophoresis showed twelve distinct patterns with 23 to 27 DNA fragment bands were produced from different passages of the attenuated strains.Mouse pathogenicity test showed that the attenuation of strain HX 130709 occurred between 46 and 50 passages.The attenuated strain was more sensitive to most antibiotics than its parent strain,but still resistant to kanamycin and sulfisoxazole.3 Study on pathogenic characteristics of Erysipelothrix rhusiopathiaeTo study the pathogenic characteristics of Erysipelothrix rhusiopathiae,mice were infected with E.rhusiopathiae virulent strain HX130709 and attenuated strain HX130709a,respectively.Total mRNAs were extracted from murine splenic lymphocytes and reverse transcripted to cDNAs at 6h,12h,24h,36h and 48h post-infection,respectively.The expression level of IL-2,IL-4,IL-6,IL-10 and IFN-y was detected through real-time fluorescence quantitative PCR.Meanwhile,E.rhusiopathiae strains HX130709 and HX130709a were interacted with 3D4/21 cells for 30min,60min,90min and 120min,respectively.Interactions were observed by confocal laser scanning microscope to see if any phagocytosis happened.Results showed that mice infected with HX130709a produced high levels of IL-10 in the early stage while productions of IL-2,IL-4,IL-6 and IFN-γ were not induced obviously.Mice infected with HX130709 produced high levels of IFN-γ in the early stage,followed by higher-level production of IL-2,IL-4 and IL-6.Then,the expression of all the above cytokines declined to low levels.The virulent strain HX130709 was more resistant to phagocytosis against 3D4/21 cells than the attenuated strain HX130709a.Differences of cytokine production levels and resistance to phagocytosis between the virulent and attenuated E.rhusiopathiae strains provided a reference for studies on pathogenic characteristics of E.rhusiopathiae strains.4 Immunological characteristics of different forms of vaccines against swine erysipelasSwine erysipelas is an acute and highly contagious disease that can cause huge economic loss in pig industries.In order to choose a safe and highly effective vaccine form,bacteria killed by 1%formaldehyde and 0.4%Acridine orange,autoclaved extract of bacteria,supernatant from bacteria culture(inactivated forms vaccines)emulsified with freund’s adjuvant and attenuated swine erysipelas strains were used to immunize mice.Each group of mice received an subcutaneous injection of 1.5×109 CFU/0.1mL inactivated E.rhusiopathiae strains,1×107 CFU/0.1mL strain HX130709a,autoclaved extract and supernatant from the same amount of bacteria,and PBS as control,respectively.Two weeks after the first immunization,mice were enhanced by second immunization.Four weeks after the first immunization,each mouse were challenged with a subcutaneous injection of 1.5×109 CFU/0.1mL strain HX130709.The antibody levels were detected with indirect ELISA coated with recombinant SpaA after 7,14,28 and 42 days,respectively.Though the inactivated forms of vaccines could induce antibody production,the antibody level was very low.By contrast,the antibody level induced by the attenuated strain HX130709a increased obviously at the 7th day after the first immunization and kept high levels till the 14 th and 28 th day.Challenge results showed that the inactivated forms of vaccines could only provide partial protection.By contrast,the attenuated vaccine could offer complete protection against swine erysipelas with fewer doses than inactivated vaccines. |
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