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Pasteurella multocida vaccine

A Pasteurella, bloody technology for use in diagnostic assays to address issues of unknown nature of attenuating behavior, outbreaks of pasteurellosis, etc.

Inactive Publication Date: 2007-12-12
INTERVET INT BV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, these vaccines would be the vaccine of choice, but the use of live vaccines has two serious disadvantages: first, the currently used live attenuated Pasteurella multocida vaccine strains are not well-defined (ill-defined ): the nature of their attenuating behavior is unknown
Therefore, there is always a danger of reversion to toxicity
[0010] Second, there have been outbreaks of pasteurillosis due to the use of live vaccine strains, a possible cause of which may be insufficient levels of reversion to virulence or attenuation of the vaccine strains used

Method used

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  • Pasteurella multocida vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Selection of spontaneous nalidixic acid-resistant mutants of P-1059

[0082] Pasteurella multocida strains were grown overnight at 37°C in Luria-Bertani (LB) broth. 0.2 ml of the culture was sprayed onto LB agar plates containing 10 mg / ml nalidixic acid and incubated at 37°C for 48 hours. A pair of resistant colonies were picked and streaked on LB agar plates containing nalidixic acid. After further incubation for 48 hours, a resistant colony was picked and inoculated in 10 ml LB broth containing 20 mg / ml nalidixic acid and grown overnight. The culture was mixed with 5ml glycerol, aliquoted into 1.8ml tubes (1ml / tube) and stored at -70°C. The nalidixic acid resistant strain was designated P-1059NR.

Embodiment 2

[0084] Selection of spontaneous thyA-mutants of P-1059NR

[0085] P-1059NR was cultured at 37°C in LB medium containing 20 mg / ml nalidixic acid and 10 mg / ml thymine. 0.2 ml of the culture was spread onto LB plates containing 20 mg / ml nalidixic acid, 10 mg / ml trimethoprim and 50 mg / ml thymine and incubated at 37°C for 24-48 hours. Ten colonies were transferred to the same type of plate and the LB plate contained only 20 mg / ml nalidixic acid and 10 mg / ml trimethoprim. Colonies that grew on the first plate but not on the second plate were thyA-mutants. One of the thyA-mutant mutants was inoculated into a culture solution of 10 ml LB containing 20 mg / ml nalidixic acid and 150 mg / ml thymine and cultured overnight at 37°C. The medium was mixed with 5ml of glycerol, aliquoted into 1ml / tube and stored at -70°C. The conserved thyA-mutant is called P9818.

Embodiment 3

[0087] Construction of vector pYL1.3

[0088] The E. coli thyA gene was amplified from genomic DNA of E. coli K-12 by polymerase chain reaction (PCR) using primers 5'-AAGCTTGGCTGTCTCAGGTTTGTTCC-3' and 5'-TAGCTTGGCCAGTTTCTATTTCTTCG-3'. The PCR fragment was trimmed with T4 DNA polymerase plus dGTP. pLOF / Ptt (Herreno, M., de Lorenzo, V. Timmis, K.N., J. Bacteriology, 172, 1990, 6557-6567) was digested with Xba I and Sf to remove the Ptt gene and partially filled in with Klenow enzyme and dCTP. The trimmed PCR fragment (one end) was ligated into a partially filled XbaI end of the digested plasmid. The other end of the PCR fragment and the Sfi end of the digested plasmid were blunt-ended with T4 DNA polymerase and dNTPs. The plasmid treated with the ligated PCR fragment was self-ligated and transformed into E. coli SM10. One transformant containing the correct size plasmid was purified, aliquoted and stored at -70°C. The plasmid in this transformant was named pYL1.3.

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Abstract

The present invention relates to live attenuated bacteria of the species Pasteurella multocida, to live attenuated vaccines comprising such live attenuated bacteria, to the use of such bacteria for the manufacture of such vaccines, to methods for the preparation of such vaccines and to diagnostic tests for the detection of such bacteria.

Description

technical field [0001] The present invention relates to live attenuated bacteria of Pasteurella multocida (pasteurella multocida) species, to live attenuated vaccines comprising such live attenuated bacteria, to the use of such bacteria in the preparation of such vaccines Applications, methods relating to the preparation of such vaccines and diagnostic assays for the detection of such bacteria. Background technique [0002] The Gram-negative bacterium Pasteurella multocida has been known for a century as the causative agent of disease in several animal species. Pasteurella multocida is a known infectious agent causing fowl cholera in poultry, hemorrhagic septicemia in cattle and atrophic rhinitis in swine. Furthermore, its importance as a human pathogen has become increasingly clear over the past 60 years. [0003] There is only one species of Pasteurella multocida and no subspecies exist. However, subdivisions can be based on differences in capsular and LPS antigens. Fi...

Claims

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Application Information

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IPC IPC(8): C07K14/285A61K35/74C12N1/21C12Q1/68A61K39/102
CPCC12Q1/689A61P31/12A61K35/74A61K39/102C12N1/20
Inventor 罗玉刚P·韦尔梅杰A·A·C·雅各布斯
Owner INTERVET INT BV
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