Cost-effectiveness of longitudinal surveillance for Piscirickettsia salmonis using qPCR in Atlantic salmon farms (Salmo salar) in Chile.

Costs of diagnostic testing including sample collection, sampling frequency and sample size are an important consideration in the evaluation of the economic feasibility of alternative surveillance strategies for detection of infectious diseases in aquatic animals. In Chile, Piscirickettsia salmonis is the primary reason for antibiotic treatments in farmed Atlantic salmon. In 2012, a surveillance and control programme for piscirickettsiosis was established with an overall goal of reducing antibiotic use. The present study estimated the cost-effectiveness of different sampling frequencies and sample sizes to achieve at least 95% confidence of early detection of P. salmonis at the netpen and farm levels using a validated qPCR test. We developed a stochastic model that incorporated variability in test accuracy, within-pen prevalence and sampling costs. Our findings indicated that the current piscirickettsiosis surveillance programme based on risk-based sampling of five moribund or dead fish from 2 to 3 netpens is cost-effective and gives a high probability of detection of P. salmonis in Atlantic salmon farms in Chile at both the netpen and farm levels. Results from this study should incentivize salmon farmers to establish cost-effective strategies for early detection of P. salmonis infection and the application of this approach to other highly infectious diseases.

Bayesian estimation of diagnostic sensitivity and specificity of a qPCR and a bacteriological culture method for Piscirickettsia salmonis in farmed Atlantic salmon (Salmo salar L.) in Chile.

Early detection of piscirickettsiosis is an important purpose of government- and industry-based surveillance for the disease in Atlantic salmon farms in Chile. Real-time qPCRs are currently used for surveillance because bacterial isolation is inadequately sensitive or rapid enough for routine use. Since no perfect tests exist, we used Bayesian latent class models to estimate diagnostic sensitivity (DSe) and specificity (DSp) of qPCR and culture using separate two-test, single-population models for three farms (n = 148, 151, 44). Informative priors were used for DSp (culture (beta(999,1); qPCR (beta(98,2)), and flat priors (beta 1,1) for DSe and prevalence. Models were run for liver and kidney tissues combined and separately, based on the presence of selected gross-pathological signs. Across all models, qPCR DSe was 5- to 30-fold greater than for culture. Combined-tissue qPCR median DSe was highest in Farm 3 (sampled during P. salmonis outbreak (DSe = 97.6%)) versus Farm 1 (DSe = 85.6%) or Farm 2 (DSe = 83.5%), both sampled before clinical disease. Median DSe of qPCR was similar for liver and kidney, but higher when gross-pathological signs were evident at necropsy. High DSe and DSp and rapid turnaround-time indicate that the qPCR is fit for surveillance programmes and diagnosis during an outbreak. Targeted testing of salmon with gross-pathological signs can enhance DSe.

Análisis epidemiológico del programa de vigilancia activa de Piscirickettsia salmonis del Servicio Nacional de Pesca y Acuicultura de Chile.

Piscirickettsia salmonis is the causative agent of piscirickettsiosis, a disease that causes significant economic losses in salmonid sea farms in Chile. The objective of this study was to determine and describe the geographical distribution, seasonality and time period when P. salmonis was first detected in farms studied under the active surveillance programme for piscirickettsiosis of the National Fisheries and Aquaculture Service of Chile (SERNAPESCA), which was conducted from January 2013 to March 2017. A 0.28% prevalence of piscirickettsiosis was determined in freshwater fish and one of 58.1% in sea farms. The prevalence of P. salmonis was 61.1% in the Aysén region, 59.8% in the Los Lagos region, 5.1% in the Los Ríos region and 3.0% in the Magallanes region. In Los Lagos and Aysén, eight clusters of sea farms were identified, in space and time, as having a positive diagnosis of P. salmonis, whereas, in Magallanes, none was identified, confirming the absence of horizontal transmission or spread of the agent in this geographical area. A seasonal variation was found in the monthly prevalence of P. salmonis, with increases in Salmo salar and Oncorhynchus mykiss in summer and autumn, and in Oncorhynchus kisutch in winter, spring and summer. It was determined that the average time required to detect the agent after fish had been transferred to the sea was 105 days (minimum, 7 days; maximum, 351 days), and no differences were found either between regions or species. Thus the results obtained from the active surveillance programme have helped to increase knowledge of the epidemiology of P. salmonis.

Piscirickettsia salmonis est l'agent étiologique de la piscirickettsiose, une maladie à l'origine de lourdes pertes économiques pour la filière de la salmoniculture marine du Chili. Les auteurs présentent les résultats d'une étude visant à déterminer et à décrire la distribution géographique, les variations saisonnières et le moment où P. salmonis est détectée pour la première fois dans les fermes salmonicoles couvertes par le programme de surveillance active de la piscirickettsiose mis en oeuvre par le Service national de la pêche et de l'aquaculture (Sernapesca) du Chili de janvier 2013 à mars 2017. Les taux de prévalence de la piscirickettsiose étaient de 0,28 % chez les poissons d'eau douce et de 58,1% dans les sites marins. Au niveau des régions, le taux de prévalence de P. salmonis était de 61,1 % à Aysén, de 59,8 % à Los Lagos, de 5,1 % à Los Ríos et de 3,0 % à Magallanes. À Los Lagos et à Aysén huit groupements de fermes salmonicoles marines ont été identifiés dans l'espace et le temps comme ayant été infectés par l'agent pathogène, tandis qu'à Magallanes aucune détection n'a eu lieu, ce qui confirme l'absence de transmission horizontale et de dissémination de l'agent pathogène dans cette zone géographique. La prévalence mensuelle de P. salmonis fait ressortir une variation saisonnière, avec une prévalence accrue en été et en automne chez Salmo salar et Oncorhynchus mykiss, et en hiver, au printemps et en été chez O. kisutch. Il a été établi que le laps de temps nécessaire pour détecter l'agent pathogène après le transfert en mer des poissons était de 105 jours en moyenne (minimum 7 jours, maximum 351 jours), moyenne non affectée par la région ou l'espèce. Ces résultats ont donc permis de mieux appréhender l'épidémiologie de l'agent pathogène grâce au programme de surveillance active.

Piscirickettsia salmonis es el agente causal de la piscirickettsiosis, enfermedad que causa importantes pérdidas económicas en los centros marinos de cultivos de salmónidos de Chile. Este estudio tuvo como objetivo determinar y describir la distribución geográfica, la estacionalidad y momento de la primera detección de P. salmonis en los centros de cultivo estudiados en el programa de vigilancia activa de la piscirickettsiosis del Servicio Nacional de Pesca y Acuicultura (Sernapesca) de Chile, que se llevó a cabo entre enero de 2013 y marzo de 2017. Se determinó una prevalencia de piscicrickettsiosis del 0,28% en peces de agua dulce y del 58,1% en centros marinos. En la región de Aysén, la prevalencia de P. salmonis fue del 61,1%, en Los Lagos, del 59,8%, en Los Ríos, del 5,1%, y en Magallanes, del 3,0%. En Los Lagos y Aysén, se identificaron ocho conglomerados de centros marinos, en el espacio y en el tiempo, con diagnóstico positivo del agente, en cambio, en Magallanes no se detectó, lo cual confirma la inexistencia de transmisión horizontal y de diseminación del agente en esta área geográfica. Se observó una variación estacional en la prevalencia mensual de P. salmonis, en la cual se comprueba un alza en verano y otoño en el caso de Salmo salar y Oncorhynchus mykiss, y en invierno, primavera y verano en el caso de O. kisutch. Se determinó que la media de tiempo necesario para la detección del agente desde la transferencia de los peces al mar era de 105 días (mínimo, 7; máximo, 351 días), y no se observaron diferencias entre regiones o especies. Así los resultados contribuyen a conocer la epidemiología del agente a través del programa de vigilancia activa.

Electrochemical detection of Piscirickettsia salmonis genomic DNA from salmon samples using solid-phase recombinase polymerase amplification.

Electrochemical detection of solid-phase isothermal recombinase polymerase amplification (RPA) of Piscirickettsia salmonis in salmon genomic DNA is reported. The electrochemical biosensor was constructed by surface functionalization of gold electrodes with a thiolated forward primer specific to the genomic region of interest. Solid-phase RPA and primer elongation were achieved in the presence of the specific target sequence and biotinylated reverse primers. The formation of the subsequent surface-tethered duplex amplicons was electrochemically monitored via addition of streptavidin-linked HRP upon completion of solid-phase RPA. Successful quantitative amplification and detection were achieved in less than 1 h at 37 °C, calibrating with PCR-amplified genomic DNA standards and achieving a limit of detection of 5 · 10-8 µg ml-1 (3 · 103 copies in 10 µl). The presented system was applied to the analysis of eight real salmon samples, and the method was also compared to qPCR analysis, observing an excellent degree of correlation. Graphical abstract Schematic of use of electrochemical RPA for detection of Psiricketessia salmonis in salmon liver.

Real time PCR detection of Piscirickettsia salmonis from formalin-fixed paraffin-embedded tissues.

Piscirickettsia salmonis is the causative agent of piscirickettsiosis, a transmissible disease of salmonid fish. Diagnosis of piscirickettsiosis has traditionally been based upon identification of typical pathological changes by histological investigation, with confirmation by immunohistochemistry on formalin-fixed, paraffin-embedded tissues. However, implementation of more rapid confirmatory techniques, preferably with higher levels of sensitivity and possibilities for quantification, is desirable. A real-time polymerase chain reaction (PCR) assay was designed for specific detection of P. salmonis and tested on samples extracted from formalin-fixed paraffin-embedded material. Construction of a PCR-target mimic allowed determination of detection limits, linearity of the real-time PCR and quantitative detection of P. salmonis. The present study demonstrates the capability of the described real time PCR assay for detection of P. salmonis from paraffin-embedded material with a high degree of sensitivity and specificity. Implementation of this assay constitutes an important development for a rapid and secure diagnosis of piscirickettsiosis.

Occurrence of Piscirickettsiosis-like syndrome in tilapia in the continental United States.

From 2001 to 2003, tilapia (Oreochromis sp.) farms in Florida, California, and South Carolina experienced epizootics of a systemic disease causing mortality. The fish exhibited lethargy, occasional exophthalmia, and skin petechia. The gills were often necrotic, with a patchy white and red appearance. Grossly, the spleen and kidneys were granular with whitish irregular nodules throughout. Granulomatous infiltrates were observed in kidney, spleen, testes, and ovary tissues, but not in the liver. The granulomas contained pleomorphic coccoid bacteria, measuring 0.57 +/- 0.1 x 0.8 +/- 0.2 microm, that were Giemsa-positive, acid-fast-negative, and Gram-negative. The bacteria had a double cell wall, variable electron-dense and -lucent areas, and were present in the cytoplasm and within phagolysosomes. The syndrome was associated with cold stress and poor water conditions. These findings are consistent with an infectious process caused by a Piscirickettsia-like bacterium described previously in tilapia in Taiwan and Hawaii. This report involves the first identified cases of a piscirickettsiosis-like syndrome affecting tilapia in the continental United States.