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 culturebased method for the routine diagnosis of warm-water streptococcal infections in fish.

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Aims:  Genetic comparison of Lactococcus garvieae isolated from mammals and fish. Methods and Results:  One hundred and ninety‐seven L. garvieae isolates obtained from trout (n = 153), cow (n = 7) and pigs (n = 37) were genetically characterized by determining their pulsed‐field gel electrophoresis (PFGE) profiles after macrorestriction with Bsp120I. Overall, L. garvieae isolates from pigs, cow and trout exhibited distinct PFGE patterns, with a low genetic relationship between them. Isolates from trout generated two pulsotypes [Genetic diversity (GD) 0·01] showing that the fish isolates were more genetically homogenous than the others. The L. garvieae isolates from cows displayed five (GD 0·71) different pulsotypes, while the swine isolates displayed 13 different pulsotypes (GD 0·35). Twenty‐one of the 37 swine strains (56·8%) were grouped in a single cluster that included two closely related (93% similarity) pulsotypes. These pulsotypes exhibited a high frequency of isolation from different organs of the animals, and they were also broadly distributed among herds, suggesting a wide distribution across the swine population. This suggests that L. garvieae might be able to colonize different organs of the swine cardio‐respiratory system. Conclusions:  Results indicate that most L. garvieae isolates from pigs and trout exhibited a distinct genetic background. Significance and Impact of the Study:  The present study describes the isolation of L. garvieae from both diseased and healthy pigs for the first time, and the findings suggest that pigs could be a previously unknown reservoir of this pathogen.

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Biochemical and molecular genetic studies were performed for five isolates of unknown Gram-positive, catalase-negative, cocci-shaped micro-organisms obtained from clinical samples from pigs. The micro-organisms were tentatively identified as Aerococcus species on the basis of the results from cellular morphological and biochemical tests. 16S rRNA gene sequencing studies confirmed the provisional identification of the isolates as members of the genus Aerococcus, but the micro-organism did not correspond to any recognized species of this genus. The nearest phylogenetic relatives of these unknown cocci isolated from pigs were Aerococcus viridans (95.9 % 16S rRNA gene sequence similarity) and Aerococcus urinaeequi (95.8 %). The unknown bacterium, however, was distinguishable from these two species and from other animal aerococci by using biochemical tests. On the basis of both phenotypic and phylogenetic findings, the isolates represent a novel species of the genus Aerococcus, for which the name Aerococcus suis sp. nov. is proposed. The type strain is 1821/02 T (=CECT 7139 T =CCUG 52530 T ).The genus Aerococcus, when originally described by Williams et al. (1953), comprises Gram-positive, microaerophilic, catalase-negative cocci arranged in tetrads and clusters. Initially, only one species, Aerococcus viridans, was included in this genus. In the last few decades, improved taxonomic methods, such as chemotaxonomic and molecular-based approaches (especially 16S rRNA gene sequencing), have contributed to the reclassification of Pediococcus urinaeequi as Aerococcus urinaeequi (Felis et al., 2005) and the description of four novel species, namely Aerococcus urinae, Aerococcus sanguinicola, Aerococcus christensenii and Aerococcus urinaehominis (Aguirre & Collins, 1992; Collins et al., 1999; Lawson et al., 2001a, b). Aerococci have been isolated from air, vegetation, dust and in the indigenous microbiota of humans and animals. Moreover, some Aerococcus species have been involved as infrequent causes of infection in humans. A. viridans has been associated with endocarditis, meningitis, urinary tract infections and arthritis (Colman, 1967;Janosek et al., 1980;Nathavitharana et al., 1983;Taylor & Trueblood, 1985) and A. urinae has been documented as an agent of urinary tract infection and endocarditis in immunocompromised patients (Christensen et al., 1991;Kristensen & Nielsen, 1995). However, as far as we know, only one report has shown the isolation of aerococci from clinical specimens from animals (Devriese et al., 1999). In this article, we report the phenotypic and phylogenetic characterization of five strains of an unusual Aerococcus-like species isolated from various clinical specimens from pigs.The bacterial strains were isolated in different years from different clinical specimens from pigs located at different farms. The pigs were kept under intensive management conditions. The bacterial strains (strain no./year of isolation) were isolated from a lung (803/04), a bronchial lymph node (926/04), a joint (2189/02), the...