Co-occurrence of heterogeneous Flavobacterium psychrophilum isolates within the same Chilean farm and during the same infectious outbreak.

Flavobacterium psychrophilum is a pathogenic bacterium affecting Chilean salmonid farms. High antigenic and genetic diversity exists among Chilean F. psychrophilum isolates, but the distribution thereof among farms is poorly understood. These epidemiological data are key for developing isolate-specific vaccines. The present study isolated F. psychrophilum in diseased Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) from five freshwater farms between 2018 and 2019. Each farm only raised one salmonid species and was geographically separated from and did not share culturing water with the other farms. Antigenic and genetic analyses were conducted to shed light on the possibility of isolates coexisting within the same farm during outbreaks. A total of 68 Chilean F. psychrophilum isolates were recovered from skin lesions, gills, fins, kidney and spleen of moribund and live fish. Among the 68 Chilean isolates, mPCR-serotyping indicated three major serotypes (i.e. 23.5% type 0; 47.1% type 2; and 26.5% type 4) and, to a lesser degree, serotype 1 (2.9%). Sixteen antigenic groups were detected by slide agglutination. Genetic characterizations by 16S rRNA alleles identified 71% of the isolates as the virulent genogroup CSF259-93 allele. A predominant serotype was associated with each farm, with types 0 and 4 related to Atlantic salmon and types 1 and 2 to rainbow trout. Notwithstanding, several antigenic groups coexisted within some farms. Likewise, the experimental intramuscular challenges (n = 20) demonstrated that the type-2 isolates from rainbow trout were the most pathogenic among isolates recovered from infectious outbreaks in Atlantic salmon, especially as compared to those from types-0 and -4. These results allow us to suggest that prevention measures, specifically vaccines, should be developed according to dominant isolates and with specificity to each farm, that is the use of autogenous or site-specific vaccines.

First report and characterization of Tenacibaculum maritimum isolates recovered from rainbow trout (Oncorhynchus mykiss) farmed in Chile.

The present study reports on the first isolation of Tenacibaculum maritimum in rainbow trout (Oncorhynchus mykiss) farmed in Chile. In March 2020, two cages raising rainbow trout (~250 g) in the Los Lagos Region suffered a disease outbreak. In total, 17,554 fish died (3.5%-4.8% accumulated mortality). Microbiological analysis of the diseased fish obtained two representative isolates (i.e. Tm-035 and Tm-036). These were obtained from the external gross skin lesions-typical of tenacibaculosis-of two fish. Phenotyping, PCR tests and sequencing of the 16S rRNA and housekeeping genes confirmed the isolates as T. maritimum. The pathogenic potential of Tm-035 was further assessed by bath challenging Atlantic salmon (Salmo salar), which killed 70 ± 15% of fish within 11 days. Dead fish presented the same external clinical signs as did the farmed rainbow trout specimens. This research further broadens the known host distribution of this pathogen. Furthermore, the virulence experiments demonstrated that T. maritimum does not have a specific host. Additional studies are needed to evaluate the risk of T. maritimum for the O. mykiss farming industry.

Variable Number of Tandem Repeats (VNTR) analysis of Flavobacterium psychrophilum from salmonids in Chile and Norway.

BACKGROUND: Flavobacterium psychrophilum causes serious fish diseases such RTFS and BCWD, affecting the aquaculture industry worldwide. Commercial vaccines are not available and control of the disease depends on the use of antibiotics. Reliable methods for detection and identification of different isolates of this bacterium could play an important role in the development of good management strategies. The aim of this study was to identify genetic markers for discrimination between isolates. A selection of eight VNTRs from 53 F. psychrophilum isolates from Norway, Chile, Denmark and Scotland were analyzed. The results were compared with previous work on the same pathogen using MLST for genetic differentiation. RESULTS: The VNTR analysis gave a separation between the F. psychrophilum isolates supporting the results of previous MLST work. A higher diversity was found among the Chilean isolates compared to those from Norway, which suggests a more homogenous reservoir in Norway. Transgenerational transmission of F. psychrophilum from other countries, exporting salmon embryos to Chile, may explain the differences in diversity. The same transmission mechanisms could also explain the wide geographical distribution of identical isolates in Norway. But, this could also be a result of movement of smolts and embryos. The selected VNTRs are stable genetic markers and no variation was observed after several passages on agar plates at different temperatures. CONCLUSIONS: These VNTRs are important additions for genotyping of F. psychrophilum isolates. Future studies on VNTRs of F. psychrophilum should include isolates from more host species from a wider geographical area. To get a more robust genotyping the VNTRs should be used in concert with MLST. Future studies of isolates with high and low virulence should focus on identifying virulence markers using VTNRs and MLST.

Draft genome for Flavobacterium psychrophilum isolates from diseased coho salmon (Oncorhynchus kisutch) in Chile.

We present the draft genome sequences of six Flavobacterium psychrophilum isolates recovered from diseased coho salmon (Oncorhynchus kisutch) cultured by two farms in Chile. This study provides the first detailed insights into the genomic characteristics of this fish pathogen recovered from a host with limited information and cultured in Chile.

A Proof-of-Concept Study to Develop a Peptide-Based Vaccine against Salmon Lice Infestation in Atlantic Salmon (Salmo salar L.).

Proteins present in blood samples from Atlantic salmon (Salmo salar) infected with salmon lice (Lepeophtheirus salmonis) were analyzed using liquid chromatography-high-resolution mass spectrometry. Bioinformatic analyses revealed 1820 proteins, of which 58 were assigned to lice. Among these, peroxiredoxin-2, an antioxidant protein, was found relevant with respect to blood feeding of the parasite. The three-dimensional structure analysis of the protein revealed a surface amino acid sequence of interest. A 13-amino-acid peptide was selected as a potential antigen due to its predicted solubility, antigenicity, probable non-allergenic, and non-toxic nature. This peroxiredoxin-2-derived peptide was synthesized, combined with a commercially available adjuvant, and used for vaccination. The test vaccine demonstrated a 60-70% protection rate against early-stage Lepeophtheirus salmonis infection in a challenge trial in Norway. Additionally, the vaccine was tested against salmon lice (Caligus rogercresseyi) in Chile, where a remarkable 92% reduction in the number of adult lice was observed. Thus, in combination with the selected adjuvant, the peptide showed antigenic potential, making it a suitable candidate for future vaccine development. The approach described holds promise for the development of peptide vaccines against various ectoparasites feeding on blood or skin secretions of their hosts.

Case Report: Strawberry Disease in Farmed Chilean Rainbow Trout.

Strawberry disease is a chronic, nonlethal skin condition that affects Rainbow Trout Oncorhynchus mykiss in the United States and several European countries, where it is also known as red-mark syndrome. We provide the first identification and characterization of three strawberry disease outbreaks occurring at two aquaculture farms in southern Chile. Clinically affected fish weighing an average of 400 g presented multiple bright-red, usually raised, skin lesions on the flank, ventral surface, and dorsal surface. A PCR using Rickettsia-like-organism (RLO)-specific primers was performed on nine affected fish, and all skin samples were positive for the RLO 16S ribosomal RNA sequence. All PCR results for Flavobacterium psychrophilum and other bacterial and viral pathogens were negative. Histopathological examination of the skin lesions revealed extensive dermatitis, with severe lymphocytic infiltration in advanced cases. This report is the first to describe strawberry disease in farmed Chilean Rainbow Trout. Additional studies are needed to evaluate the risk for Rainbow Trout culture; fish challenge experiments should be performed to fulfill Koch's postulates and to demonstrate that RLO is the cause of this disease. Received December 27, 2014; accepted October 23, 2015.

Pseudo-membranes on internal organs associated with Rhodococcus qingshengii infection in Atlantic salmon (Salmo salar).

This paper describes a pathological condition in intensive reared Atlantic salmon (Salmo salar), restricted to the appearance of pseudo-membranes covering internal organs (i.e. spleen, liver, heart and others) associated with the presence of large numbers of a Gram-positive bacteria. Isolate 79043-3, obtained as pure culture from affected fish, was subjected to a polyphasic taxonomic study in order to determine its exact taxonomic position, as well as to experimental challenges leading to determine its pathogenic potential for cultured fish. Based on this characterization, we report the first isolation of Rhodococcus qingshengii, from a farmed population of Atlantic salmon in Chile. Virulence studies demonstrated that the isolate fulfilled the Koch's postulates, suggesting that this bacterial species could be considered as an opportunistic pathogen for Atlantic salmon.

Chryseobacterium piscicola sp. nov., isolated from diseased salmonid fish.

Eight bacterial strains isolated from diseased rainbow trout (n=5) and Atlantic salmon (n=3) were characterized by phenotypic and molecular taxonomic methods. The isolates were negative for the Gram-reaction, non-motile, rod-shaped and catalase- and oxidase-positive. Colonies on solid media were yellow, smooth, shiny and circular with regular edges. Growth occurred at 4-28 degrees C (optimum, 15 degrees C) and with 0-3 % NaCl (optimum, 0.5 %). Analysis of the 16S rRNA gene sequence allocated the micro-organisms to the genus Chryseobacterium, with Chryseobacterium soldanellicola PSD1-4(T) and Chryseobacterium soli JS6-6(T) as their closest relatives (96.9 and 97.1 % sequence similarity, respectively). The levels of DNA-DNA hybridization towards these nearest phylogenetic neighbours were below 17.1 %. The DNA G+C contents of strains VQ-6316s(T) and VQ-4836s were 32.5 and 32.3 mol%, respectively. The predominant menaquinone was MK-6 and the major fatty acids were iso-C(15 : 0), anteiso-C(15 : 0), iso-C(17 : 1)omega9c, iso-C(17 : 0) 3-OH and summed feature 3 (comprising C(16 : 1)omega7c and/or C(16 : 1)omega7t and/or iso-C(15 : 0) 2-OH). The eight isolates were classified as representatives of a novel species, Chryseobacterium piscicola sp. nov., with strain VQ-6316s(T) (=CECT 7357(T)=DSM 21068(T)) as the type strain.