ABSTRACT
BACKGROUND: Caseous lymphadenitis (CLA) is a disease that affects sheep, goats and occasionally humans. The etiologic agent is the Corynebacterium pseudotuberculosis bacillus. The objective of this study was to build a gene expression library from C. pseudotuberculosis and use immunoscreening to identify genes that encode potential antigenic proteins for the development of DNA and subunit vaccines against CLA. RESULTS: A wild strain of C. pseudotuberculosis was used for extraction and partial digestion of genomic DNA. Sequences between 1000 and 5000 base pairs (bp) were excised from the gel, purified, and the digested DNA fragments were joined to bacteriophage vector ZAP Express, packaged into phage and transfected into Escherichia coli. For immunoscreening a positive sheep sera pool and a negative sera pool for CLA were used. Four clones were identified that strongly reacted to sera. The clones were confirmed by polymerase chain reaction (PCR) followed by sequencing for genomic comparison of C. pseudotuberculosis in GenBank. The genes identified were dak2, fagA, fagB, NlpC/P60 protein family and LPxTG putative protein family. CONCLUSION: Proteins of this type can be antigenic which could aid in the development of subunit or DNA vaccines against CLA as well as in the development of serological tests for diagnosis. Immunoscreening of the gene expression library was shown to be a sensitive and efficient technique to identify probable immunodominant genes.
Subject(s)
Antigens, Bacterial/genetics , Antigens, Bacterial/immunology , Antigens, Bacterial/isolation & purification , Corynebacterium Infections/immunology , Corynebacterium Infections/veterinary , Corynebacterium pseudotuberculosis/genetics , Corynebacterium pseudotuberculosis/immunology , Lymphadenitis/veterinary , Animals , Antigens, Bacterial/blood , Bacteriophages/genetics , Base Sequence , Corynebacterium Infections/microbiology , Corynebacterium Infections/prevention & control , Corynebacterium pseudotuberculosis/pathogenicity , Databases, Nucleic Acid , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Gene Library , Genes, Bacterial/genetics , Genome, Bacterial , Goat Diseases/blood , Goat Diseases/immunology , Goat Diseases/microbiology , Goats , Lymphadenitis/immunology , Lymphadenitis/microbiology , Polymerase Chain Reaction/veterinary , Sheep , Sheep Diseases/blood , Sheep Diseases/immunology , Sheep Diseases/microbiology , Vaccines, DNA/therapeutic useABSTRACT
Objetivou-se com este estudo comparar genotipicamente 35 isolados de Corynebacterium pseudotuberculosis recuperados de conteúdo de abscessos de caprinos e ovinos com linfadenite caseosa, procedentes de cinco municípios localizados no Sertão de Pernambuco, Brasil. Utilizou-se a técnica de fingerprint RFLP-PCR com as enzimas de restrição Hpy-Ch4 e Msp1 aplicada ao gene rpoB e as enzimas Pst I e Msp I para o gene pld. Não houve diferença nos padrões de fragmentos de bandas entre os isolados, independente da espécie hospedeira ou da área geográfica estudada, definindo-se um padrão genotípico homogêneo de C. pseudotuberculosis responsável por abscessos superficiais na região.(AU)
The objective was to genotypically compare 35 samples of Corynebacterium pseudotuberculosis obtained from abscesses of sheep and goats diagnosed with caseous lymphadenitis originated from 5 different municipalities in the semi-arid region of Pernambuco, Brazil. The RFLP-PCR technique with Hpy-Ch4 and Msp I and Pst I Msp I restriction enzimes was used to fingerprint the genes rpoB and pld, respectively. The results demonstrate that there was no difference on the fragments banding pattern among samples, independently of the host species or geographic area studied, defining a homogeneous profile of C. pseudotuberculosis responsible for superficial abscesses for the region.(AU)
