Diagnosis of flavobacteriosis by direct amplification of rRNA genes

Abstract

A broad-range bacterial PCR method with universal 16S rDNA targeting primers and bacterial cultivation was used to identify the putative pathogen in flavobacterial outbreaks. Restriction fragment length polymorphism (PCR-RFLP) analysis and sequencing of the partial 16S rDNA PCR products of 10 skin samples and 10 representative isolates derived from the same fish specimens revealed differences between direct molecular and cultivation-based analysis. Flavobacterium columnare-like sequences dominated in the direct molecular analysis in most cases, whereas most of the isolates belonged to a phylogenetically heterogeneous group of flavobacteria clustering with F. hibernum. F. columnare was isolated in only 1 outbreak. The possible explanations for the different results may be attributable to difficulties in the plate cultivation procedure of external flavobacterial samples. During plate cultivation, the dominating Flavobacterium species can be masked by saprophytic species of the same genus or other genera, or the growth of flavobacteria can be completely inhibited by antagonistic bacteria such as Pseudomonas. Direct analysis of the prevailing 16S rDNA sequences avoids the problems with cultivation and may thus be preferable for the diagnosis of flavobacterial diseases. When isolating flavobacteria from external samples, serial dilution of the sample before plating can improve the results.

Effect of Pseudomonas sp. MT5 baths on Flavobacterium columnare infection of rainbow trout and on microbial diversity on fish skin and gills

“…In the case of Flavobacterium columnare, cultivation is a timeconsuming process and can easily be biased by competitive and opportunistic bacteria present in the sample (Tiirola et al 2002). Thus, there is a need for the development of reliable and fast tools to diagnose fish bacterial pathogens.…”

“…Isolation was made from only 31% of the fish that tested positive for the F. columnare fragment in LH-PCR. Based on these results, we propose that culture-based methods are not sufficiently reliable for diagnosing flavobacterial diseases (see Tiirola et al 2002). In fish with clinical signs of columnaris disease, the pathogen usually dominates the microbial community of the tissue sample (Table 1, Fig.…”

“…Screening of microbial communities in fish for pathogens by universal PCR is cost effective because the presence of the pathogen can be revealed before clinical disease occurs, and carrier fish can be detected in cases where the pathogen itself cannot be isolated. However, because LH-PCR separates sequences only by their length, several taxonomical bacterial groups can be included within 1 fragment size (Tiirola et al 2002), which can be considered a drawback if precise information about the bacterial diversity of the sample is needed. …”

“…PCR amplification of rRNA genes has been shown to be a reliable method for diagnosing bacterial pathogens in humans (e.g. Relman et al 1992, Rantakokko-Jalava et al 2000, Rogers et al 2003) and in fish (Tiirola et al 2002). Species-specific primers are used when samples are screened for known pathogens and such primers have also been developed for Flavobacterium columnare (Bader & Shotts 1998, Bader et al 2003b).…”

Identification and Phylogenetic Analysis of Bacterial Isolates from Litopenaeus vannamei Shrimp Culture System and Gut Environment Based on 16SrRNA Gene Sequence Data

“…The method can be used for identification of microorganism identity to the genus or species level (Tiirola et al 2002) and construct phylogenetic analysis which plays on important role in taxonomic positioning. It can be undertaken by comparing the gene sequences with other bacteria sequences in the GenBank database using basic local alignment search tool (BLAST) search and continue with method of neighbor-joining to construct a phylogenetic tree (Saitou and Nei 1987;Holmes 2003).…”