ABSTRACT
Salmonella enterica serovar Gallinarum causes a severe systemic disease, fowl typhoid, primarily in chickens and turkeys, and it remains a disease of worldwide significance. Multilocus variable-number tandem-repeat analysis (MLVA) has proved to be very useful for subtyping other Salmonella serovars. We describe the development of a simple MLVA assay for S. enterica serovar Gallinarum that is comparable with pulsed-field gel electrophoresis (PFGE) in resolution. The genome sequence of S. enterica serovar Gallinarum strain 287/91 was analysed for potential variable-number tandem repeats (VNTRs) and then polymerase chain reaction assays were developed to assess the variability of the loci. Four VNTR markers were selected and used in a multiplex fragment analysis assay. The stability of the VNTR markers was assessed by conducting in vitro passage experiments with two strains (95 clones per strain) over a 30-day period. A MLVA of 68 strains of S. enterica serovar Gallinarum based on the four VNTR loci distinguished 26 allelic profiles. The MLVA assay showed a Simpson's diversity index of 0.918, whereas PFGE analysis produced 23 patterns and had a diversity index of 0.874. Most importantly, the MLVA further discriminated strains having the same PFGE pattern. The MLVA assay is a highly discriminatory genotyping method for S. enterica serovar Gallinarum. Therefore, MLVA can be a useful addition to routine PFGE analysis for molecular epidemiological investigation of fowl typhoid.
Subject(s)
Bacterial Typing Techniques/methods , Genetic Markers/genetics , Genetic Variation , Minisatellite Repeats/genetics , Poultry/microbiology , Salmonella enterica/genetics , Animals , Electrophoresis, Gel, Pulsed-Field/methods , Polymerase Chain Reaction , Salmonella enterica/classificationABSTRACT
Salmonella enterica serovar Gallinarum isolates (n=105) from chickens in South Korea between 2002 and 2007 were tested for antimicrobial susceptibility by determining minimum inhibitory concentrations of 16 antimicrobials, and their predominant resistance profiles were genetically characterized. Most isolates (99/105; 94.3%) were resistant to nalidixic acid and resistant/intermediately resistant to fluoroquinolones, and 63.8% (67/105) of the isolates were resistant to three or more antimicrobials. Forty-two quinolone-resistant isolates, of which the quinolone resistance-determining regions of the gyrA genes were sequenced, contained a substitution of a Ser to a Phe or Tyr at position 83 (71.4%), or a substitution of an Asp to an Asn, Gly, or Tyr at position 87 (28.6%). Fifty-seven sulphamethoxazole-resistant isolates were tested for the presence of class 1 integrons by polymerase chain reaction, and their resistance gene cassettes were analysed by sequencing. Three different class 1 integrons containing the resistance-gene insert aadA (52.6%; n=30), aadB (12.3%; n=7), or aadB-aadA (12.3%; n=7) were identified. Most isolates harbouring the integron containing aadB-aadA displayed resistance to all three aminoglycosides tested and also showed increased resistance to fluoroquinolones. These findings suggest that fluoroquinolone resistance may be epidemiologically linked to multiple aminoglycoside resistance.
Subject(s)
Chickens/microbiology , Salmonella Infections, Animal/microbiology , Salmonella enterica/drug effects , Amino Acid Substitution , Animals , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , DNA Gyrase/chemistry , DNA Gyrase/genetics , DNA Primers , Drug Resistance, Bacterial , Glycine , Microbial Sensitivity Tests , Phenotype , Poultry Diseases/drug therapy , Poultry Diseases/genetics , Poultry Diseases/microbiology , Quinolones/pharmacology , Republic of Korea , Salmonella Infections, Animal/drug therapy , Salmonella Infections, Animal/genetics , Salmonella enterica/genetics , Salmonella enterica/isolation & purification , Serine , Sulfamethoxazole/pharmacology , TyrosineABSTRACT
Epidemiology of Eimeria species in poultry flocks is important to increase the effectiveness of vaccinations and prophylactic strategies on chicken farms. In this study, fecal samples from 356 chicken farms were collected randomly and examined for the prevalence of Eimeria species. Through microscopic examination, it was determined that 78.7% of the tested farms were positive in Eimeria-infection. Seven Eimeria species were detected in all the positive farms by PCR amplification of the internal transcribed spacer 1 (ITS-1) region with species-specific primers. E. acervulina and E. tenella were the most prevalent, followed by E. brunetti and E. praecox (87.5, 62.5, 59.3, and 37.5% of the farms, respectively). Each of E. maxima, E. mitis, and E. necatrix was identified in 31.3% of the farms. Individual positive fecal samples contained multiple Eimeria species (mean=3.4). Since E. maxima is known to generate antigenic variants, cross-immunity was investigated for four isolates of E. maxima from the poultry farms in different regions of Korea. The extent of cross-protection varied from 54.3 to 100% against the heterologous isolates. The results obtained from this large-scale survey will be a useful reference for controlling coccidiosis in the poultry industry.