Construction And Characterization Of BscN Deleted Mutants Of Swine Bordetella Bronchiseptica Strain

Bordetella bronchiseptica can cause respiratory infections in swine and other mammals.A genetically-defined,rationally attenuated live B. bronchiseptica vaccine may have been shown to function as safe and effective live intranasal vaccines,can efficiently and rapidly colonize the ciliated respiratory epithelium and stimulate local mucosal and systemic humoral and Cellular anti-B. bronchiseptica immunity. Such attenuated strains of B. bronchiseptica can be easily produce and administer as the intranasally.The efficiency with which B. bronchiseptica colonize at respiratory tract efficiently means that such vaccine strains may also function as superior live vectors for delivering heterologous antigens to the respiratory tract of a range of animals.In order to develop an attenuated Bordetella bronchiseptica strain and exploit it as a live vaccine vector for mucosal immunization, based on the mutant aroA of Bb QH0814 in our laboratory,we conducted the construction and characterization of the mutant QH09 (QH0814AaroA\AbscN),and studied on its biological characteristics,test its pathogencity and protection efficiency in mice and swines.The more detailed information of this study was described as follows:1. Construction of recombination suicide plasmids and identification of the mutantAccording to sequences of bscN of RB50 (Genbank accession No.:BX470250),the two flanks of bscN genes were amplified and cloned from HH0809 genome.The upstream and downstream of bscN gene were respectively subcloned into suicidal plasmid pRE112, which contained chloramphenicol resistance (Cm) gene.The recombination suicide plasmid was designated as pREAbscN which contained 541bp-deleted bscN gene. The E. coil donor strain X7213 transformed with plasmid pREAbscN as conjugated with the recipient QH0814AaroA.Finally, the Cms colonies were further confirmed by PCR, the aroA/bscN double mutant was named QH09.2.Biological characteristics of mutantHeredity stability of this B.brochiseptica mutant was confirmed by PCR test in 50 passages. In order to confirm whether the deletion in bscN gene affected the growth ability of B.brochiseptica.The growth curves were drawn, the results showed that the growth rate of the mutants QH09 were significantly slower than the HH0809.The biochemistry chatacterristic test showed no difference between mutant and WT strain. 3. Virulence in mice and the time course of mouse respiratory tract colonizationThe degree of attenuation of the double deleted mutant QH09 in mice was investigated by comparing with the single mutant QH0814△aroA and WT HH0809 using a murine intranasal infection model.Data indicated that deletion in bscN caused a 225-fold attenuation of HH0809.To determine the ability of QH09 to colonize the respiratory tracts of mice, the mice were inoculated intranasally with 50 uL of PBS containing 1×106 CFU of either QH09, QH0814△aroA and QH0809.The numbers of CFU present in the respiratory organs were determined at the indicated time.The data showed that the bacterial numbers of QH09 was much less than QH0814△aroA and WT strain QH0809 in nasal cavity,tracheal and lung respectively.4. Protection in mice4-week-old Balb/C mice were vaccinated twice intranasally (i.n.) with approximately 1×106 CFU of QH09 or 200uL inactived vaccine by subcutaneous injection. Two weeks after the second immunization, all groups of mice were challenged intranasally with 15×LD50(1.9×107CFU) of HH0809 in 50 uL PBS. The clinical signs were observed and the number of the dead mice was recorded for 14 days post-challenge. Both QH09 and inactived vaccine have a protective efficiency of 100% against challenge. Mice were blooded at day 0,14 and 28, serum samples were detected by ELISA. Results suggested that QH09 can elicit higher IgA antibodies than inactived vaccine.5. Virulence in pigsThe virulence of the QH09 and HH0809 in pigs was assessed by an intratracheally (i.t.) infection model. Pigs were monitored carefully, clinical signs and death were calculated according to admitted methods.The results indicated that deletion of bscN led to the attenuation of the virulence of B.bronchiseptica.6. Protection in pigsThe whole 14 pigs were divided into four groups randomly.The 4 pigs in group 1 were intranasally (i.n.) with approximately 2×1010 CFU of QH09, the 4 pigs in group 2 were intramuscularly with 2×1010CFU inactived vaccine and the 4 pigs in group 3 intranasally (i.n.) with 2ml PBS as a negative control group, the last 2 pigs were without any treatment. Two weeks after the booster immunization, all the pigs in group 1,2 and 3 were challenged intratracheally with WT virulence strain HH0809.Pigs were blooded at day 0,14 and 28 to detect antibody.The pigs were monitored daily for clinical signs of turbinal born and pleuropneumonia for 30 days after challenge.At day 30 post-challenge, all surviving pigs and the unchallenged control pigs were euthanized and the scores of turbinal born and lung lesions were recorded.Bloods samples were collected and examined by ELISA,results indicated that QH09 vaccinated pigs elicited more higher IgA antibodies titer against HH0809 and provide more better protection than inactived vaccine,suggesting that QH09 can induce local mucosal and cell-mediated immune responses to aganst the WT chanllenge.