Identification of Cytophaga psychrophila by PCR Targeted 16S Ribosomal RNA.

Physical changes in Flavobacterium psychrophilum, the causative agent of rainbow trout fry syndrome (RTFS), were examined over a 19 wk period of starvation. Bacteria were maintained in either Cytophaga broth, filtered stream water, or filtered distilled water, or were maintained in broth after disinfection as a negative control for dead bacteria. Culturability and viability of the bacterium were assessed using colony-forming units (CFUs) and a commercially available live/dead kit. Antigenic profiles and general morphology of the bacterium were also examined using Western blot analysis and electron microscopy, respectively. The bacterium appeared to stop multiplying and became smaller and rounded when maintained in stream water. Its culturability declined until it was no longer possible to obtain colonies on agar plates at the end of the trial at 19 wk, and results from the live/dead kit did not correspond with the viability obtained as CFUs in culture. However, it was still possible to obtain growth of the bacterium after 36 wk with a resuscitation step in Cytophaga broth. Bacteria maintained in distilled water or treated with a disinfectant appeared non-viable using the live/dead kit and were unable to grow on agar 1 h after setting up the experiment; no morphological changes were observed in the bacteria maintained under these conditions. Bacteria maintained in broth were present as long, slim rods, some of which developed into 'ring' formations. Small differences were observed in the antigen profiles of the bacteria maintained under the different treatments, possibly due to a reduction in the size and metabolism of the bacteria. There was also a marked decline in the sensitivity of the PCR with bacteria maintained under the different treatments 14 wk from the onset of the study. KEY WORDS: Flavobacterium psychrophilum · Rainbow trout fry syndrome · Starvation · Viability · Detection Resale or republication not permitted without written consent of the publisherDis Aquat Org 56: [115][116][117][118][119][120][121][122][123][124][125][126] 2003 water column or in sediment also cannot be excluded. However, for this latter mode of transmission, F. psychrophilum must be able to survive outside the fish for an extended period of time.Flavobacterium psychrophilum is considered a very fastidious organism, and problems with the culture of the bacterium have been reported (Daskalov et al. 1999, Michel et al. 1999. Under adverse conditions, it is possible that the bacterium enters a viable but nonculturable state, as in the case of many other bacterial species, such as Salmonella sp. (Dupray et al. 1997) and Yersinia ruckeri (Thorsen et al. 1992). This characteristic results in an inability to detect the pathogen in environmental samples when culture is used as the only means of identification.The ability of Flavobacterium psychrophilum to survive under conditions of starvation was examined in this study, and changes in the organism as an adaptation to its environment are described. F. psychrophilum cells we...

mentioning

confidence: 99%

Starvation of Flavobacterium psychrophilum in broth, stream water and distilled water

Vatsos

Thompson

Adams³

2003

“…damselae implies that any n~olecular tool intended to serve as a diagnosis procedure for fish pasteurellosis must be tested with an array of strains of these subspecies, in order to make sure that no cross-reaction occurs. Though 16s rRNA genes have been widely used as a target for PCR-based detection procedures of an important number of fish pathogens (Magnkson et al 1994, Toyama et al 1994, Hiney & Smith 1998, Gibello et al 1999, in the case of fish pasteurellosis there is a limitation imposed by the total homology at the 16s rRNA gene sequence level found between P. damselae subsp. piscicida and P. damselae subsp.…”

Section: Discussionmentioning

confidence: 99%

Multiplex PCR assay for ureC and 16S rRNA genes clearly discriminates between both subspecies of Photobacterium damselae

Osorio¹,

Toranzo²,

Romalde³

et al. 2000

A multiplex-PCR approach, employing 2 primer pairs directed to internal regions of the 16s rRNA and ureC genes, was utilized to analyze a collection of Photobacterium damselae strains, including 25 isolates of subspecies piscicida and 15 isolates of subspecies darnselae. With this procedure, all the P. darnselae subsp. damselae strains yielded 2 amplification products, one of 267 bp and the other of 448 bp, corresponding to internal fragments of the 16s rRNA and ureCgenes, respectively. However, P. damselae subsp. piscicida isolates only showed the PCR product of 267 bp (16s rRNA fragment), indicating the absence of the urease gene in its genome. We have constructed a DNA probe directed to an internal region of the ureC gene, and corroborated by dot blot hybridization that the P. darnselae subsp. piscicida lacks this gene, whereas it is present in the subspecies darnselae. This constitutes the first successful discrimination between both subspecies using a PCR procedure, which could become a useful tool for diagnosis of pasteurellosis in the field. In addition, since these 2 subspecies have been shown to share nearly the same rrn operon sequence, our results provided evidence that one of the steps in the P, damselae speciation proccess included gainfloss events associated with the ure operon.

“…In our study, [ills method was rapid, hlghly sensitive, specific and able to detect viable, but not necessarily culturable bacteria (Morgan et al 1993). These techniques have been extensively developed for a wide range of pathogenic bacteria, especially those considered to be human health hazards (Wright et al 1993, Leon et al 1994, Martinez-Picado et al 1994, Toyama et al 1994, Arias et al 1995, Miyata et al 1996, Venkateswaran et al 1998.…”

Section: Discussionmentioning

confidence: 99%

Rapid and sensitive PCR detection of Vibrio penaeicida, the putative etiological agent of Syndrome 93 in New Caledonia

Saulnier

Avarre²,

Moullac³

et al. 2000

Experimental infections of Penaeus (Litopenaeus) stylirostris were performed with a Vibrio penaeicida strain (AM101) isolated in New Caledonia from Syndrome 93 dseased shrimp. Cumulative mortalities resulting from intramuscular injection or immersion of shrimp in bacterial suspensions demonstrated high virulence for this bacterial strain and suggested that V. penaeicida could be the etiological agent of Syndrome 93. The median lethal dose (L&) for AM101 was 1.3 X 104 CFU (colony forming units) ml-' by immersion and less than 5 CFU shrimp-' by intramuscular challenge, with mortality outbreaks at 48 and 22 h after challenge, respectively. A polymerase chain reaction (PCR) detection assay using a primer set designed from the 16s riboson~al RNA gene of V. penaeicida was developed. It gave an expected amplicon of approximately 310 bp in ethidium bromide-stained agarose gels. The specificity of these primers was assessed with different Vibrio species. Furthermore, DNA extracted by the chelexTM method could be used to detect fewer than 20 cultured Vibrio cells in seawater or shrimp hemolymph by this assay. It appears to be a reliable screening method for detecting V penaejcida in shrimp and from the aquatic environment.