A. Casamayor

Multiplex PCR Assay for Detection of Bacterial Pathogens Associated with Warm-Water Streptococcosis in Fish

ABSTRACT A multiplex PCR-based method was designed for the simultaneous detection of the main pathogens involved in warm-water streptococcosis in fish (Streptococcus iniae, Streptococcus difficilis, Streptococcus parauberis, and Lactococcus garvieae). Each of the four pairs of oligonucleotide primers exclusively amplified the targeted gene of the specific microorganism. The sensitivity of the multiplex PCR using purified DNA was 25 pg for S. iniae, 12.5 pg for S. difficilis, 50 pg for S. parauberis, and 30 pg for L. garvieae. The multiplex PCR assay was useful for the specific detection of the four species of bacteria not only in pure culture but also in inoculated fish tissue homogenates and naturally infected fish. Therefore, this method could be a useful alternative to the culture-based method for the routine diagnosis of warm-water streptococcal infections in fish.

First identification of Streptococcus phocae isolated from atlantic salmon (Salmo salar)

Different genera and species of gram-positive, catalase-negative cocci are pathogenic to fish. The numbers of infections caused by these microorganisms have increased during the last decade, and such infections are responsible for significant economic losses in the fish farm industry ([9][1]).

A genetic comparison of pig, cow and trout isolates of Lactococcus garvieae by PFGE analysis.

Aims:  Genetic comparison of Lactococcus garvieae isolated from mammals and fish.

Streptococcus plurextorum sp. nov., isolated from pigs.

Biochemical and molecular genetic studies were performed on an unknown Gram-positive, catalase-negative, coccus-shaped organism isolated from clinical samples from pigs. On the basis of the results of cellular morphological and biochemical tests, the organism was identified as a streptococcal species. 16S rRNA gene sequence comparisons confirmed its identification as a member of the genus Streptococcus, but the organism was distinct from any recognized species of this genus. The closest phylogenetic relative of the unknown organism corresponded to Streptococcus suis NCTC 10234(T) (97.2 % 16S rRNA gene sequence similarity) and this phylogenetic position was confirmed by analysis of rpoB and sodA sequences. DNA-DNA hybridization studies showed that the unidentified organism produced a DNA reassociation value of 36.6 % with respect to S. suis NCTC 10234(T). The novel bacterium was distinguished from S. suis and other Streptococcus species using biochemical tests. On the basis of phenotypic and phylogenetic data, the unidentified organism represents a novel species of the genus Streptococcus, for which the name Streptococcus plurextorum sp. nov. is proposed. The type strain is 1956-02(T) (=CECT 7308(T)=CCUG 52972(T)).

Comparison of two biochemical methods for identifying Corynebacterium pseudotuberculosis isolated from sheep and goats

The biochemical pattern of Cowan and Steel (BPCS) was compared with a commercial biochemical strip for the identification of Corynebacterium pseudotuberculosis isolated from small ruminants. On 16S rRNA gene sequencing, 40/78 coryneform isolates from the lymph nodes of sheep and goats with lesions resembling caseous lymphadenitis were identified as C. pseudotuberculosis. The sensitivities of the BPCS and the commercial biochemical strip relative to 16S rRNA sequencing were 80% and 85%, and their specificities were 92.1% and 94.7%, respectively; the level of agreement between the BPCS and the commercial biochemical strip was high (κ=0.82). Likelihood ratios for positive and negative results were 10.0 and 0.22 for the BPCS, and 16.0 and 0.16 for the commercial biochemical strip, respectively. These results indicate that the BPCS and the commercial biochemical strip are both useful for identifying C. pseudotuberculosis in veterinary microbiology laboratories.

Isolation of Enterococcus hirae from suckling rabbits with diarrhoea.

ENTEROCOCCI are commensal microorganisms of the intestinal tract of human beings and animals, and can also be found in a wide range of environments ([Klein 2003][1], [Donskey 2004][2]). Enterococci were considered to be non-pathogenic for many years, but several species, most notably Enterococcus

Streptococcus cuniculi sp. nov., isolated from the respiratory tract of wild rabbits

Biochemical and molecular genetic studies were performed on four unknown Gram-stain-positive, catalase-negative, coccus-shaped organisms isolated from tonsils (n = 3) and nasal samples (n = 1) of four wild rabbits. The micro-organism was identified as a streptococcal species based on its cellular morphological and biochemical tests. Comparative 16S rRNA gene sequencing confirmed its identification as a member of the genus Streptococcus, but the organism did not correspond to any recognized species of this genus. The closest phylogenetic relative of the unknown cocci from wild rabbits was Streptococcus acidominimus NCIMB 702025(T) (97.9% 16S rRNA gene sequence similarity). rpoB and sodA sequence analysis of the novel isolate showed interspecies divergence of 16.2% and 20.3%, respectively, from the type strain of its closest 16S rRNA gene phylogenetic relative, S. acidominimus. The novel bacterial isolate could be distinguished from the type strain of S. acidominimus by several biochemical characteristics, such as the production of esterase C4, acid phosphatase and naphthol-AS-BI-phosphohydrolase and acidification of different sugars. Based on both phenotypic and phylogenetic findings, it is proposed that the unknown bacterium be classified as a novel species of the genus Streptococcus, Streptococcus cuniculi sp. nov. The type strain is NED12-00049-6B(T) ( = CECT 8498(T) = CCUG 65085(T)).

DNA macrorestriction analysis by pulsed-field gel electrophoresis of Pseudomonas aeruginosa isolates from mastitis in dairy sheep.

nosa are also responsible for a significant proportion of cases of mastitis in dairy sheep (Lafi and others 1998, Scott and Jones 1998, Rapoport and others 1999). P aeruginosa represents less than 10 per cent of the isolates from subclinical mastitis (Lafi and others 1998), but it is responsible for outbreaks of clinical mastitis with significant sanitary and economic repercussions (Las Heras and others 1999b, Rapoport and others 1999). Molecular characterisation studies provide relevant information towards greater knowledge of the epidemiology of the different bacterial pathogens which cause mastitis, as well as for the establishment of rational control measures. These studies have generally been carried out in dairy cows with bacteria which are of higher clinical significance, mainly with S aureus (Fitzgerald and others 1997, Lange and others 1999, Buzzola and others 2001, Joo and oth-