Development of a PCR assay for the specific identification of the fish pathogen Tenacibaculum piscium.

Tenacibaculosis is an emerging disease that severely affects salmonid farming in Chile, producing high mortalities and causing great economic losses. This work describes a novel PCR assay for the specific detection of Tenacibaculum piscium, a species recently described and identified in tenacibaculosis outbreaks in Norway and Chile. The designed primers amplified a 678-bp fragment of the peptidase gene (peptidase M23 family) from T. piscium. This method is specific for T. piscium; no other chromosomal DNA amplification products were obtained for other Tenacibaculum species. In pure cultures, the PCR assay detected up to 500 pg of DNA, or the equivalent of 2.44 ± 0.06 × 104 CFU/ml. For seeded fish samples (i.e., gills, liver, kidney, and mucus), the sensitivity limit was 4.88 ± 0.11 × 106 CFU/g, sufficient to detect T. piscium in acute infections in fish. Notably, this sensitivity level was 100-fold lower for DNA extracted from mucus samples. As compared to other existing methodologies (e.g., gene sequencing), the PCR approach described in this work allowed for the easiest detection of T. piscium in mucus samples obtained from challenged fish, an important outcome considering that the identification of this bacterium is difficult. Our results indicate that the designed specific primers and PCR method provide a rapid and specific diagnosis of T. piscium.

Draft Genome Sequence of Tenacibaculum haliotis Strain RA3-2T, Isolated from Korean Wild Abalone (Haliotis discus hannai).

Here, we present the draft genome sequence of Tenacibaculum haliotis strain RA3-2T (i.e., KCTC 52419T and NBRC 112382T), isolated from Korean wild abalone (Haliotis discus hannai). As the only strain for this Tenacibaculum species worldwide, the information is of use for comparative genomic analyses delineating Tenacibaculum species.

Method for lineage typing of epidemic Renibacterium salmoninarum in Chilean salmon farms.

Renibacterium salmoninarum (Rs) is the etiological agent of bacterial kidney disease (BKD), which significantly affects farmed and wild salmonids worldwide. Although the whole genome of Rs (~3.1 million nucleotides) is highly conserved, genomic epidemiology analyses have identified four sub-lineages from Chilean isolates. A total of 94 Rs genomes from the BIGSdb aquaculture database were aligned and compared using bioinformatics tools, identifying 2199 independent single-nucleotide polymorphisms (SNPs) spread along the genome. A detailed analysis of the distribution of the SNPs showed five local zones of a length in the range of 10-15 kbp that should be used to unambiguously identify a specific sub-lineage. Based on the Rs type strain DSM 20767T , we designed multiplex PCR primers that produce specific amplification products which were further sequenced by the Sanger method to obtain the genotype of the sub-lineage. For the genetic typing, we evaluated 27 Rs isolates recovered from BKD outbreaks from different fish species and regions of Chile. Based on the findings reported here, we propose the PCR approach as a valuable tool for the rapid and reliable studying of the relationships between Rs isolates and the different sub-lineages without requiring the sequencing of the entire genome.

Co-existence of two Yersinia ruckeri biotypes and serotype O1a retrieved from rainbow trout (Oncorhynchus mykiss) farmed in Puno, Peru.

Yersinia ruckeri causes important economic losses for rainbow trout (Oncorhynchus mykiss) farms worldwide. This bacterial disease is likely the most common among trout in Peru; however, no commercial vaccine is available nationally, which is, in part, due to a lack of information on the bacterium. The aim of the current study was to characterize 29 Y. ruckeri isolates sampled from seven cage-reared farms in the Puno Region, the focal point for aquaculture activities in Peru. For this, samples were taken from fish with clinical signs (i.e. haemorrhages, uni- or bilateral exophthalmia, hyphaemia and/or melanosis). Notable among our findings was the existence of both Y. ruckeri biotype 1 (9 isolates) and biotype 2 (20 isolates; negative for sorbitol and Tween 80). The isolates further differed in API profiles 5307100 (21 isolates), 1307100 (4 isolates), 1305100 (2 isolates), 1307120 (1 isolate) and 5305100 (1 isolate), with the main differences being in the tests for lysine decarboxylase, gelatine hydrolysis and D-saccharose fermentation. Despite these differences, all isolates shared identical ERIC-PCR and REP-PCR profiles and belonged to the O1a serotype. Fingerprints were identical to the reference strain CECT 955 (serotype O1a). The information obtained will be used for epidemiological purposes by health authorities and for the development of a vaccine against Y. ruckeri, a prominent request made by fish farmers in Peru.

Collective behavior and virulence arsenal of the fish pathogen Piscirickettsia salmonis in the biofilm realm.

Piscirickettsiosis is a fish disease caused by the Gram-negative bacterium Piscirickettsia salmonis. This disease has a high socio-economic impact on the Chilean salmonid aquaculture industry. The bacterium has a cryptic character in the environment and their main reservoirs are yet unknown. Bacterial biofilms represent a ubiquitous mechanism of cell persistence in diverse natural environments and a risk factor for the pathogenesis of several infectious diseases, but their microbiological significance for waterborne veterinary diseases, including piscirickettsiosis, have seldom been evaluated. This study analyzed the in vitro biofilm behavior of P. salmonis LF-89T (genogroup LF-89) and CA5 (genogroup EM-90) using a multi-method approach and elucidated the potential arsenal of virulence of the P. salmonis LF-89T type strain in its biofilm state. P. salmonis exhibited a quick kinetics of biofilm formation that followed a multi-step and highly strain-dependent process. There were no major differences in enzymatic profiles or significant differences in cytotoxicity (as tested on the Chinook salmon embryo cell line) between biofilm-derived bacteria and planktonic equivalents. The potential arsenal of virulence of P. salmonis LF-89T in biofilms, as determined by whole-transcriptome sequencing and differential gene expression analysis, consisted of genes involved in cell adhesion, polysaccharide biosynthesis, transcriptional regulation, and gene mobility, among others. Importantly, the global gene expression profiles of P. salmonis LF-89T were not enriched with virulence-related genes upregulated in biofilm development stages at 24 and 48 h. An enrichment in virulence-related genes exclusively expressed in biofilms was also undetected. These results indicate that early and mature biofilm development stages of P. salmonis LF-89T were transcriptionally no more virulent than their planktonic counterparts, which was supported by cytotoxic trials, which, in turn, revealed that both modes of growth induced important and very similar levels of cytotoxicity on the salmon cell line. Our results suggest that the aforementioned biofilm development stages do not represent hot spots of virulence compared with planktonic counterparts. This study provides the first transcriptomic catalogue to select specific genes that could be useful to prevent or control the (in vitro and/or in vivo) adherence and/or biofilm formation by P. salmonis and gain further insights into piscirickettsiosis pathogenesis.