New attenuated vaccine against columnaris disease in fish: choosing the right parental strain is critical for vaccine efficacy.

Flavobacterium columnare, the causative agent of columnaris disease, is a highly diverse species comprised by three genomovars. Genomovar II strains are more virulent toward catfishes than genomovar I isolates. The objective of this study was to compare the vaccine efficacy of avirulent mutants derived from genomovars I and II using a rifampicin-resistance strategy. First, we compared the efficacy of 13 genomovar II mutants in channel catfish (Ictalurus punctatus) fingerlings and identified mutant 17-23 as the best vaccine candidate based on their relative percent survival (RPS) against a highly virulent genomovar II strain (BGFS-27). In the second experiment, we vaccinated zebrafish (Danio rerio) with two genomovar II mutants (17-23 and 16-534) and FCRR (genomovar I mutant) followed by exposure to BGFS-27 strain. RPS values were 28.4, 20.3 and 8.1% for 17-23, 16-534, and FCRR, respectively. For experiments 3 and 4, we tested both 17-23 and FCRR in channel catfish fry and Nile tilapia (Oreochromis niloticus). In both experiments, vaccinated fish were divided in two groups and each challenged with either a genomovar I (ARS-1) or a II (BGFS-27) strain. Channel catfish fry vaccinated with 17-23 and FCRR followed by challenge with BGFS-27 resulted in RPS values of 37.0% and 4.4%. When fish were challenged with ARS-1, RPS values were 90.9% and 72.7% for fish vaccinated with 17-23 and FCRR, respectively. Nile tilapia vaccinated with 17-23 and FCRR followed by challenged with BGFS-27 had RPS values of 82.1% and 16.1%, respectively. When fish were challenged with strain ARS-1, RPS values were 86.9% and 75.5%. Overall, our results demonstrated that vaccination with genomovar II mutant 17-23 confers better protection in channel catfish and Nile tilapia than FCRR against columnaris disease caused by genomovar II. Both mutants were equally protective against columnaris caused by genomovar I showing that 17-23 mutant cross-protected against both genomovars.

Adaptive response to starvation in the fish pathogen Flavobacterium columnare: cell viability and ultrastructural changes.

BACKGROUND: The ecology of columnaris disease, caused by Flavobacterium columnare, is poorly understood despite the economic losses that this disease inflicts on aquaculture farms worldwide. Currently, the natural reservoir for this pathogen is unknown but limited data have shown its ability to survive in water for extended periods of time. The objective of this study was to describe the ultrastructural changes that F. columnare cells undergo under starvation conditions. Four genetically distinct strains of this pathogen were monitored for 14 days in media without nutrients. Culturability and cell viability was assessed throughout the study. In addition, cell morphology and ultrastructure was analyzed using light microscopy, scanning electron microscopy, and transmission electron microscopy. Revival of starved cells under different nutrient conditions and the virulence potential of the starved cells were also investigated. RESULTS: Starvation induced unique and consistent morphological changes in all strains studied. Cells maintained their length and did not transition into a shortened, coccus shape as observed in many other Gram negative bacteria. Flavobacterium columnare cells modified their shape by morphing into coiled forms that comprised more than 80% of all the cells after 2 weeks of starvation. Coiled cells remained culturable as determined by using a dilution to extinction strategy. Statistically significant differences in cell viability were found between strains although all were able to survive in absence of nutrients for at least 14 days. In later stages of starvation, an extracellular matrix was observed covering the coiled cells. A difference in growth curves between fresh and starved cultures was evident when cultures were 3-months old but not when cultures were starved for only 1 month. Revival of starved cultures under different nutrients revealed that cells return back to their original elongated rod shape upon encountering nutrients. Challenge experiments shown that starved cells were avirulent for a fish host model. CONCLUSIONS: Specific morphological and ultrastructural changes allowed F. columnare cells to remain viable under adverse conditions. Those changes were reversed by the addition of nutrients. This bacterium can survive in water without nutrients for extended periods of time although long-term starvation appears to decrease cell fitness and resulted in loss of virulence.

Adhesion dynamics of Flavobacterium columnare to channel catfish Ictalurus punctatus and zebrafish Danio rerio after immersion challenge.

The adhesion dynamics of Flavobacterium columnare to fish tissues were evaluated in vivo by immersion challenge followed by bacterial plate count and confirmatory observations of gill-adhered bacterial cells using scanning electron microscopy. Adhesion of F. columnare genomovar I (ARS-1) and II (BGFS-27) strains to skin and gill of channel catfish Ictalurus punctactus and gill of zebrafish Danio rerio was compared. At 0.5 h post-challenge, both strains adhered to gill of channel catfish at comparable levels (10(6) colony forming units [CFU] g(-1)), but significant differences in adhesion were found later in the time course. Channel catfish was able to effectively reduce ARS-1 cells on gill, whereas BGFS-27 persisted in gill beyond the first 24 h post-challenge. No significant difference was found between both strains when adhered to skin, but adhered cell numbers were lower (10(3) CFU g(-1)) than those found in gill and were not detectable at 6 h post-challenge. Adhesion of BGFS-27 cells to gill of zebrafish also occurred at high numbers (> 10(6) CFU g(-1)), while only < 10(2) CFU g(-1) of ARS-1 cells were detected in this fish. The results of the present study show that particular strains of F. columnare exhibit different levels of specificity to their fish hosts and that adhesion to fish tissues is not sufficient to cause columnaris disease.

Molecules infer origins of ectoparasite infrapopulations on tuna.

Infrapopulation genetic variation of the oioxenous, hermaphroditic flatworm Nasicola klawei (Monogenea: Capsalidae) infecting the nasal cavities of nine yellowfin tuna, Thunnus albacares, from the Gulf of Mexico was analyzed using the first internal transcribed spacer (ITS1) single strand conformation polymorphism (SSCP), ITS1 sequencing, and amplified fragment length polymorphism (AFLP). Of a total of 32 worms, six had unique ITS1-SSCP types and the rest was grouped by three types. Two worms of the same infrapopulation shared an ITS1-SSCP type in nine instances but no infrapopulation was monophyletic by ITS1-SSCP analysis. ITS1 sequences (420 bp) varied by 1-11 (0.2-2.6%) nucleotides. Twenty-three AFLP profiles of 80-110 bands failed to support genomic monophyly of any N. klawei infrapopulation. 28S rDNA (990 bp) sequences from four worms representing four infrapopulations were identical and matched conspecific GenBank sequences. Concordant ITS1-SSCP and AFLP analyses indicated that these N. klawei infrapopulations principally resulted from tuna being repeatedly colonized by planktonic, infective larvae (oncomiracidia) rather than by a single host colonization followed by parasite maturation, self-fertilization, and production of auto-infecting progeny.

High intragenomic heterogeneity of 16S rRNAgenes in a subset of Vibrio vulnificus strains from the western Mediterranean coast

Heterogeneity among ribosomal operons in Vibrio vulnificus is purported as a probabilistic indicator of strain virulence and classifies V. vulnificus strains as 16S rRNA genes type A and B. In this study, 16S rRNA genes typing of V. vulnificus strains isolated from the Valencia city coast, in the western Mediterranean, showed that 24 out of 30 isolates were type A, one was type B and five could not be typed. Single strand conformation polymorphism (SSCP) analysis of this gene region revealed complex patterns indicative of intragenomic ribosomal operon sequence heterogeneity. The 16S rRNA genes of three untypeable isolates C27, C30, and C34, along with type A (ATCC 27562) and B (C7184) reference strains, were amplified, cloned and sequenced. The number of unique 16S rRNA gene sequences was 4, 3, and 4 for the environmental isolates. The type strain of the species (ATCC 27562) presented only two 16S rRNA gene types, while the reference isolate C7184 of clinical origin had only one 16S rRNA gene type. Sequences differed from five to 35 bp (99.6% to 97.6% sequence similarity). Areas of variability concentrated in helices 10, 18, and 37 and included variants with short intervening sequences in helix 10. Most of the substitutions showed compensatory mutations suggesting ancient sequence divergence generated by lateral gene transfer (AU)

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Cloning, expression, and immunogenicity of Flavobacterium columnare heat shock protein dnaJ.

The Flavobacterium columnare heat shock protein (HSP) gene dnaJ* was isolated, cloned, expressed, and used as an antigen in a recombinant vaccine strategy for channel catfish Ictalurus punctatus. The F. columnare dnaJ* sequence was obtained from genomovars I and II and showed intraspecies variability. Recombinant protein was expressed and purified from Escherichia coli cultures and injected intraperitoneally (12 microg of purified DnaJ/fish) into fingerling channel catfish. In addition, induced (expressing the recombinant DnaJ) and uninduced (no recombinant protein being produced) E. coli cultures were also used to immunize fish. At 28 d postimmunization, antibody response was evaluated and the fish were challenged with F. columnare. A specific immune response against DnaJ was observed in fish immunized with DnaJ or E. coli cultures expressing DnaJ. No protection against the disease, however, was observed in F. columnare-challenged fish that had been immunized with DnaJ. Some level of protection was observed in fish immunized with uninduced and induced E. coli lysates. Although HSPs have been shown to be immunodominant and good candidates for subunit vaccines in other animals, DnaJ failed to protect against columnaris disease in channel catfish.

High intragenomic heterogeneity of 16S rRNA genes in a subset of Vibrio vulnificus strains from the western Mediterranean coast.

Heterogeneity among ribosomal operons in Vibrio vulnificus is purported as a probabilistic indicator of strain virulence and classifies V. vulnificus strains as 16S rRNA genes type A and B. In this study, 16S rRNA genes typing of V. vulnificus strains isolated from the Valencia city coast, in the western Mediterranean, showed that 24 out of 30 isolates were type A, one was type B and five could not be typed. Single strand conformation polymorphism (SSCP) analysis of this gene region revealed complex patterns indicative of intragenomic ribosomal operon sequence heterogeneity. The 16S rRNA genes of three untypeable isolates C27, C30, and C34, along with type A (ATCC 27562) and B (C7184) reference strains, were amplified, cloned and sequenced. The number of unique 16S rRNA gene sequences was 4, 3, and 4 for the environmental isolates. The type strain of the species (ATCC 27562) presented only two 16S rRNA gene types, while the reference isolate C7184 of clinical origin had only one 16S rRNA gene type. Sequences differed from five to 35 bp (99.6% to 97.6% sequence similarity). Areas of variability concentrated in helices 10, 18, and 37 and included variants with short intervening sequences in helix 10. Most of the substitutions showed compensatory mutations suggesting ancient sequence divergence generated by lateral gene transfer.

Host-specific association between Flavobacterium columnare genomovars and fish species.

A total of 90 Flavobacterium columnare isolates were recovered from predominant wild fish species in the Mobile River, Alabama, USA. Isolates were identified and confirmed by fatty acid methyl ester analysis and specific PCR amplification. Genomovar ascription was performed using 16S-restriction fragment length polymorphism (RFLP) analysis. The majority of genomovar I isolates were recovered from threadfin shad while genomovar II isolates came from catfish (including channel and blue catfish). Additional genotyping methods, including multilocus sequence analysis (MLSA), internal spacer region-single strand conformation polymorphism analysis (ISR-SSCP) and amplified fragment length polymorphism (AFLP), confirmed a clear division of the isolates into two groups that matched genomovar ascription. Fingerprinting methods revealed a higher genetic diversity within genomovar II isolates. Our data confirmed the coexistence of F. columnare genomovars I and II in a natural environment. A statistically significant association between genomovar I and threadfin shad was demonstrated while genomovar II strains were mainly recovered from catfish species.

Plant somatic hybrid cytoplasmic DNA characterization by single-strand conformation polymorphism.

Unlike maternal inheritance in sexual hybridization, plant somatic hybridization allows transfer, mixing and recombination of cytoplasmic genomes. In addition to the use of somatic hybridization in plant breeding programs, application of this unique tool should lead to a better understanding of the roles played by the chloroplastic and mitochondrial genomes in determining agronomically important traits. The nucleotide sequences of cytoplasmic genomes are much more conserved than those of nuclear genomes. Cytoplasmic DNA composition in somatic hybrids is commonly elucidated either by length polymorphism analysis of restricted genome regions amplified with universal primers (PCR-RF) or by hybridization of total DNA using universal cytoplasmic probes. In this study, we demonstrate that single-stranded conformational polymorphism (SSCP) analysis is a powerful, quick and easy alternative method for cytoplasmic DNA characterization of somatic hybrids, especially for mitochondrial DNA. The technique allows detection of polymorphisms based on both size and sequence of amplified targets. Twenty-two species of the subfamily Aurantioideae were analyzed with eight universal primers (four from chloroplastic and four from mitochondrial regions). Differences in chloroplastic DNA composition were scored in 98% of all possible two-parent combinations, and different mitochondrial DNA profiles were found in 87% of them. Analysis by SSCP was also successfully used to characterize somatic hybrids and cybrids obtained by fusion of Citrus sinensis (L.) Osb. and C. excelsa Wester protoplasts.

Molecular typing of isolates of the fish pathogen, Flavobacterium columnare, by single-strand conformation polymorphism analysis.

Flavobacterium columnare intraspecies diversity was revealed by analyzing the 16S rRNA gene and the 16S-23S internal spacer region (ISR). Standard restriction fragment length polymorphism (RFLP) of these sequences was compared with single-strand conformation polymorphism (SSCP). Diversity indexes showed that both 16S-SSCP and ISR-SSCP improved resolution (D>or=0.9) when compared with standard RFLP. ISR-SSCP offered a simpler banding pattern than 16S-SSCP while providing high discrimination between isolates. SSCP analysis of rRNA genes proved to be a simple, rapid, and cost-effective method for routine fingerprinting of F. columnare.

First report of Yersinia ruckeri biotype 2 in the USA.

A polyphasic characterization of atypical isolates of Yersinia ruckeri (causative agent of enteric redmouth disease in trout) obtained from hatchery-reared brown trout Salmo trutta in South Carolina was performed. The Y. ruckeri isolates were biochemically and genetically distinct from reference cultures, including the type strain, but were unequivocally ascribed to the species Y. ruckeri, based on API 20E, VITEK, fatty acid methyl ester profiles, and 16S rRNA gene sequencing analysis. These isolates were nonmotile and unable to hydrolyze Tween 20/80 and were therefore classified as Y. ruckeri biotype 2. Genetic fingerprint typing of the isolates via enterobacterial repetitive intergenic consensus (amplified by polymerase chain reaction) and fragment length polymorphism showed biotype 2 as a homogeneous group distinguishable from other Y. ruckeri isolates. This is the first report of Y. ruckeri biotype 2 in the USA.

Protoplast transformation and regeneration of transgenic Valencia sweet orange plants containing a juice quality-related pectin methylesterase gene.

Valencia orange [Citrus sinensis (L.) Osbeck] is the leading commercial citrus species in the world for processed juice products; however, the presence of thermostable pectin methylesterase (TSPME) reduces its juice quality. A long-term strategy of this work is to eliminate or greatly reduce TSPME activity in Valencia orange. Previous work resulted in the isolation of a putative TSPME gene, CsPME4, associated with a thermostable protein fraction of Valencia orange juice. To begin research designed to overexpress CsPME4 to verify the thermostability of the protein product and/or to downregulate the gene, a sense gene cassette containing a gene-specific sequence from a putative TSPME cDNA and the enhanced green fluorescent protein (GFP) as a selectable marker was constructed (M2.1). In the work reported here, M2.1 plasmid DNA was transformed (polyethylene glycol-mediated) into protoplasts isolated from an embryogenic suspension culture of Valencia somaclone line B6-68, in an effort to obtain transgenic Valencia lines. A vigorous transformed line was identified via GFP expression, physically separated from non-transformed tissue, and cultured on somatic embryogenesis induction medium. One transgenic proembryo expressing GFP was recovered and multiple shoots were regenerated. The recovery of multiple transgenic plants was expedited by in vitro grafting. Polymerase chain reaction analysis revealed the presence of the PME gene in transgenic plants, and subsequent Southern blot analysis confirmed the presence of the eGFP gene. These transgenic plants show normal growth and minor morphological variation. The thermostability of PME in these plants will be assessed after flowering and fruit set. This is the first successful transfer of a target fruit-quality gene by protoplast transformation with recovery of transgenic plants in citrus. This method of transformation has the advantage over Agrobacterium-mediated transformation in that it requires no antibiotic-resistance genes.