Horizontal transmission of Piscirickettsia salmonis from the wild sub-Antarctic notothenioid fish Eleginops maclovinus to rainbow trout (Oncorhynchus mykiss) under experimental conditions.

Piscirickettsia salmonis is the aetiological agent of piscirickettsiosis, a bacterial disease that affects farmed salmonids, causing high mortalities and significant economic losses in the Chilean salmon farm industry. Given the Chilean native fish species Patagonian blenny, Eleginops maclovinus, lives in the vicinity of salmon farms, it is relevant to clarify the epidemiological role that this species could play in the transmission and/or dissemination of this pathogen. This study aimed to evaluate the bidirectional transmission of P. salmonis between the Patagonian blenny and Oncorhynchus mykiss (rainbow trout), via a cohabitation challenge model. The results of this study demonstrated the transmission of the bacteria from Patagonian blennies to rainbow trout, considering the specific mortality in cohabitant rainbow trout, reaching 46%: the necropsy of these specimens, evidencing the characteristic pathological lesions of the disease and the positive results of the qPCR analysis for P. salmonis, in the same individuals. In contrast, no mortalities of Patagonian blenny specimens were recorded in the challenged experimental groups. This study is the first report showing the horizontal transmission of P. salmonis from a native non-salmonid species, such as the Patagonian blenny, to a salmonid species, generating the disease and specific mortality in rainbow trout, using a cohabitation challenge.

Identification and genetic characterization of Piscirickettsia salmonis in native fish from southern Chile.

Piscirickettsia salmonis is the etiological agent of piscirickettsiosis, a severe disease causing high mortalities in salmonids. This bacterium has been previously identified and isolated in all cultivated salmonids in Chile and worldwide, including Salmo salar, Oncorhynchus kisutch, and O. mykiss, in addition to being found in non-salmonid species such as Dicentrarchus labrax and Atractoscion nobilis. In this study, the 16S rRNA gene and intergenic spacer ITS-1 of P. salmonis were amplified by PCR from DNA samples extracted from the native Chilean fish species Eleginops maclovinus, Odontesthes regia, Sebastes capensis, and Salilota australis. Analysis of the 16S rRNA sequences from O. regia demonstrated a close phylogenetic relationship with the 16S rRNA gene in the Chilean EM-90 strain. The 16S rRNA sequences from E. maclovinus, S. capensis, and S. australis were related to the Chilean LF-89 sequence and Scottish strains. To confirm these findings, analysis of P. salmonis ITS-1 sequences obtained from the 4 sampled native species demonstrated a high degree of identity and a close phylogenetic relationship with Chilean P. salmonis sequences, including LF-89 and EM-90. These results suggest a strong relationship between the nucleotide sequences from the 16S rRNA and ITS-1 genes amplified from native fish with those sequences described in the first P. salmonis strains to be identified and isolated in Chile.

A Novel and Validated Protocol for Performing MIC Tests to Determine the Susceptibility of Piscirickettsia salmonis Isolates to Florfenicol and Oxytetracycline.

This paper presents a validated protocol, using a novel, specifically formulated medium, to perform broth microdilution antimicrobial susceptibility assays of the salmonid bacterial pathogen Piscirickettsia salmonis. The minimum inhibitory concentrations (MIC) for florfenicol and oxytetracycline against 58 P. salmonis isolates recovered from various outbreaks occurred in Chilean salmonid farms were determined using this protocol. Normalized resistance interpretation (NRI) analysis was applied to these data to calculate appropriate protocol-specific epidemiological cut-off values. These cut-off values allow the isolates to be categorized as either fully susceptible wild type (WT) members of this species, or as manifesting reduced susceptibility non-wild type (NWT). The distribution of MIC values of florfenicol was bimodal and the distribution of the normalized values for the putative WT observation had a standard deviation of 0.896 log2 µg mL-1. This analysis calculated a cut-off value of ≤0.25 µg mL-1 and categorized 33 (56%) of the isolates as manifesting reduced susceptibility to florfenicol. For the oxytetracycline MIC data the NRI analysis also treated the distribution as bimodal. The distribution of the normalized values for the putative WT observation had a standard deviation of 0.951 log2 µg mL-1. This analysis gave a cut-off value of ≤0.5 µg mL-1 and categorized five isolates (9%) as manifesting reduced susceptibility to oxytetracycline. The susceptibility testing protocol developed in this study was capable of generating MIC data from all the isolates tested. On the basis of the precision of the data it generated, and the degree of separation of values for WT and NWT it achieved, it is argued that this protocol has the performance characteristics necessary for it to be considered as a standard protocol.

Antimicrobial Susceptibility of Flavobacterium psychrophilum from Chilean Salmon Farms and Their Epidemiological Cut-Off Values Using Agar Dilution and Disk Diffusion Methods.

Flavobacterium psychrophilum is the most important bacterial pathogen for freshwater farmed salmonids in Chile. The aims of this study were to determine the susceptibility to antimicrobials used in fish farming of Chilean isolates and to calculate their epidemiological cut-off (COWT) values. A number of 125 Chilean isolates of F. psychrophilum were isolated from reared salmonids presenting clinical symptoms indicative of flavobacteriosis and their identities were confirmed by 16S rRNA polymerase chain reaction. Susceptibility to antibacterials was tested on diluted Mueller-Hinton by using an agar dilution MIC method and a disk diffusion method. The COWT values calculated by Normalized Resistance Interpretation (NRI) analysis allow isolates to be categorized either as wild-type fully susceptible (WT) or as manifesting reduced susceptibility (NWT). When MIC data was used, NRI analysis calculated a COWT of ≤0.125, ≤2, and ≤0.5 µg mL-1 for amoxicillin, florfenicol, and oxytetracycline, respectively. For the quinolones, the COWT were ≤1, ≤0.5, and ≤0.125 µg mL-1 for oxolinic acid, flumequine, and enrofloxacin, respectively. The disk diffusion data sets obtained in this work were extremely diverse and were spread over a wide range. For the quinolones there was a close agreement between the frequencies of NWT isolates calculated using MIC and disk data. For oxolinic acid, flumequine, and enrofloxacin the frequencies were 45, 39, and 38% using MIC data, and 42, 41, and 44%, when disk data were used. There was less agreement with the other antimicrobials, because NWT frequencies obtained using MIC and disk data, respectively, were 24 and 10% for amoxicillin, 8 and 2% for florfenicol, and 70 and 64% for oxytetracycline. Considering that the MIC data was more precise than the disk diffusion data, MIC determination would be the preferred method for susceptibility testing for this species and the NWT frequencies derived from the MIC data sets should be considered as the more authoritative. Despite the high frequency of isolates showing full susceptibility to florfenicol, the significant frequencies of isolates exhibiting reduced susceptibility to oxytetracycline and quinolones may result in treatment failures when these agents are used.