Detection of mastitis-associated bacteria can be accomplished by culturing or by molecular techniques. On the other hand, rapid and inexpensive methods to enumerate bacterial load without culturing can be better achieved by molecular methods. Staphylococcus aureus and Escherichia coli are the predominant bacterial pathogens associated with bovine mastitis. Here, we describe the application of conventional PCR for the limit of detection (LOD) of genomic DNA of S. aureus and E. coli based on single-copy genes. The selected genes were thermonuclease (nuc), aureolysin (aur), and staphopain A (scpA) for S. aureus and β-D-glucuronidase A (uidA), cytochrome d oxidase (cyd), and rodA (a gene affecting cell shape and methicillin sensitivity) for E. coli. The LOD was 5.3, 15.9, and 143 pg for aur, nuc, and scpA genes, corresponding to S. aureus genomic copies of 1.75 × 10(3), 5.16 × 10(3), and 4.71 × 10(4), respectively. The LOD was 0.45, 12.3 and 109 pg for uidA, rodA and cyd genes, corresponding to E. coli genome copies of 8.91 × 10(1), 2.43 × 10(3), and 2.16 × 10(4), respectively. Application of uidA and aur PCRs to field strains revealed that as low as approximately 100 genome copies of E. coli and 1000-10,000 copies of S. aureus could be detected. This study is the first to report LOD of genomic DNA using conventional PCR for aur and scpA genes of S. aureus, and rodA and cyd genes of E. coli. The results should be useful for developing assays to assess bacterial load in milk and to determine the load that contributes to subclinical or clinical mastitis.

• MeSH
• bakteriální proteiny genetika   MeSH
• DNA bakterií genetika   MeSH
• Escherichia coli genetika   izolace a purifikace   MeSH
• infekce vyvolané Escherichia coli diagnóza   mikrobiologie   veterinární   MeSH
• limita detekce MeSH
• mastitida skotu diagnóza   mikrobiologie   MeSH
• polymerázová řetězová reakce metody   MeSH
• skot MeSH
• stafylokokové infekce diagnóza   mikrobiologie   veterinární   MeSH
• Staphylococcus aureus genetika   izolace a purifikace   MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• DNA bakterií MeSH

A multiplex PCR method was developed to characterize the potential variability within the sip gene from bovine isolates of Streptococcus agalactiae. An incomplete sip gene (ncosip) was found in four isolates of S. agalactiae. According to the in silico analysis of the missing region at amino acid level, the N-terminal of surface immunogenic protein (Sip) was found to contain a LysM domain motif; whilst the incomplete Sip (NcoSip) lacked a part of this motif. Immunity elicitation against Sip was confirmed by immunization of mice. This response was partially observed also with NcoSip.

• MeSH
• aminokyselinové motivy MeSH
• antigeny bakteriální chemie   genetika   imunologie   MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• protilátky bakteriální krev   MeSH
• sekvence aminokyselin MeSH
• Streptococcus agalactiae chemie   genetika   imunologie   MeSH
• streptokokové infekce imunologie   mikrobiologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny bakteriální MeSH
• protilátky bakteriální MeSH
• SIP protein, Streptococcus group B MeSH Prohlížeč

Bacterial 16S rDNA from fecal samples of two calves were amplified by PCR and analyzed by denaturing gradient gel electrophoresis; selected bands were sequenced. Escherichia coli and Bifidobacterium animalis were the initial colonizers, followed by species closely related to the genera Bacteroides, Clostridium and Faecalibacterium. Change of diet was connected with shifts of bacterial population and with the occurrence of many bacterial species that have not been cultured up to now. The diet change corresponded with an alteration in a volatile-fatty-acid concentration in fecal samples.

• MeSH
• Bacteria klasifikace   genetika   růst a vývoj   izolace a purifikace   MeSH
• Bifidobacterium genetika   růst a vývoj   izolace a purifikace   MeSH
• DNA bakterií analýza   MeSH
• ekosystém MeSH
• elektroforéza metody   MeSH
• Escherichia coli genetika   růst a vývoj   izolace a purifikace   MeSH
• feces mikrobiologie   MeSH
• gastrointestinální trakt mikrobiologie   MeSH
• novorozená zvířata * MeSH
• polymerázová řetězová reakce metody   MeSH
• ribozomální DNA analýza   MeSH
• RNA ribozomální 16S genetika   MeSH
• sekvenční analýza DNA MeSH
• skot * MeSH
• zvířata MeSH
• Check Tag
• skot * MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA bakterií MeSH
• ribozomální DNA MeSH
• RNA ribozomální 16S MeSH