Molecular serotyping, virulence gene profiling and pathogenicity of Streptococcus agalactiae isolated from tilapia farms in Thailand by multiplex PCR.

AIMS: This study aimed to biotype Streptococcus agalactiae isolated from tilapia farms in Thailand based on molecular biotyping methods and to determine the correlation between the serotype and virulence of bacteria. In addition to a biotyping (serotyping) technique based on multiplex PCR of cps genes, in this study, we developed multiplex PCR typing of Group B streptococcus (GBS) virulence genes to examine three clusters of virulence genes and their correlation with the pathogenicity of S. agalactiae. The epidemiology of S. agalactiae in Thailand was analysed to provide bacterial genetic information towards a future rational vaccine strategy for tilapia culture systems. METHODS AND RESULTS: Streptococcus agalactiae were isolated from diseased tilapia from different areas of Thailand. A total of 124 S. agalactiae isolates were identified by phenotypic analysis and confirmed by 16S rRNA PCR. Bacterial genotyping was conducted based on (i) molecular serotyping of the capsular polysaccharide (cps) gene cluster and (ii) virulence gene profiling using multiplex PCR analysis of 14 virulence genes (lmb, scpB, pavA, cspA, spb1, cyl, bca, rib, fbsA, fbsB, cfb, hylB, bac and pbp1A/ponA). Only serotypes Ia and III were found in this study; serotype Ia lacks the lmb, scpB and spb1 genes, whereas serotype III lacks only the bac gene. Virulence tests in juvenile Nile tilapia demonstrated a correlation between the pathogenicity of the bacteria and their virulence gene profile, with serotype III showing higher virulence than serotype Ia. Epidemiological analysis showed an almost equal distribution in all regions of Thailand, except serotype III was found predominantly in the southern areas. CONCLUSIONS: Only two serotypes of S. agalactiae were isolated from diseased tilapia in Thailand. Serotype Ia showed fewer virulence genes and lower virulence than serotype III. Both serotypes showed a similar distribution throughout Thailand. SIGNIFICANCE AND IMPACT OF THE STUDY: We identified two major serotypes of S. agalactiae isolates associated with the outbreak in tilapia culture in Thailand. We developed multiplex PCR assays for 14 virulence genes, which may be used to predict the pathogenicity of the isolates and track future infections. Multiplex PCR typing of the GBS virulence genes was developed and might be further used to predict the pathogenicity of S. agalactiae.

Effect of the microsporidian Enterocytozoon salmonis on the immune response of chinook salmon.

The protozoan parasite Enterocytozoon salmonis is an intranuclear microsporidian of salmonid mononuclear leukocytes. Experimental infections were initiated in chinook salmon to determine the effects of parasitism on selected host immune functions. The humoral antibody response to dinitrophenylated-keyhole limpet hemocyanin and the in vitro blastogenic responses of isolated mononuclear leukocytes to mitogens (concanavalin A, lipopolysaccharide and phytohemagglutinin-P) were evaluated. The humoral response as detected by enzyme-linked immunosorbent assays was suppressed following infection. The degree of suppression increased as the severity of the infection progressed. Additionally, the response to mitogen-induced lymphoproliferation was impaired. These results suggest that infection with E. salmonis may cause suppression of host cell immune functions, thus increasing the susceptibly of infected fish to other pathogens.

In vitro characteristics of the microsporidian: Enterocytozoon salmonis.

Enterocytozoon salmonis, as intranuclear microsporidian of salmonid fish, was propagated in vitro using chinook salmon mononuclear leukocytes. Characteristic morphology and infectivity of the cultured parasites were evaluated to determine the effect of in vitro maintenance and passage on the parasites. Cultured parasites developed through several stages from meronts to infectious spores. Parasites obtained from in vitro passages tested up to the 17th subculture, retained their morphological characteristics and pathogenicity for chinook salmon (Oncorhynchus tshawytscha). The disease induced by experimental infections with parasites from in vitro cultures was identical to that observed in naturally infected chinook salmon. An examination of supernatants obtained from the infected cultures revealed evidence of soluble factor(s) produced by E. salmonis-infected cells that stimulated uninfected target cells in vitro. This observation may explain in part the proliferative disease of hematopoietic tissues which characterizes the disease in infected chinook salmon.