The intracellular facultative bacteria Piscirickettsia salmonis is one of the most important pathogens of the Chilean aquaculture. However, there is a lack of information regarding the whole genomic transcriptional response according to different extracellular environments. We used next generation sequencing (NGS) of RNA (RNA-seq) to study the whole transcriptome of an isolate of P. salmonis (FAVET-INBIOGEN) using a cell line culture and a modified cell-free liquid medium, with or without iron supplementation. This was done in order to obtain information about the factors there are involved in virulence and iron acquisition. First, the isolate was grown in the Sf21 cell line; then, the bacteria were cultured into a cell-free liquid medium supplemented or not with iron. We identified in the transcriptome, genes associated with type IV secretion systems, genes related to flagellar structure assembly, several proteases and sigma factors, and genes related to the development of drug resistance. Additionally, we identified for the first time several iron-metabolism associated genes including at least two iron uptake pathways (ferrous iron and ferric iron uptake) that are actually expressed in the different conditions analyzed. We further describe putative genes that are related with the use and storage of iron in the bacteria, which have not been previously described. Several sets of genes related to virulence were expressed in both the cell line and cell-free culture media (for example those related to flagellar structure; such as basal body, MS-ring, C-ring, proximal and distal rod, and filament), which may play roles in other basic processes rather than been restricted to virulence.

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Piscirickettsia salmonis is the aetiological agent of piscirickettsiosis, a disease which affects a variety of teleost species and that is particularly severe in salmonid fish. Bacterial-free supernatants, obtained from cultures of three isolates of Piscirickettsia salmonis, were inoculated in Atlantic salmon, Salmo salar L., and in three continuous cell lines in an effort to determine the presence of secretion of extracellular products (ECPs) by this microorganism. Although steatosis was found in some liver samples, no mortalities or clinical signs occurred in the inoculated fish. Clear cytotoxicity was observed after inoculation in the cell lines CHSE-214 and ASK, derived from salmonid tissues, but not in MDBK, which is of mammalian origin. The degree of cytotoxicity of the ECPs was different among the P. salmonis isolates tested. The isolate that evidenced the highest cytotoxicity in its ECPs exhibited only an intermediate virulence level after challenging fish with bacterial suspensions of the three P. salmonis isolates. Almost complete inhibition of the cytotoxic activity of ECPs was seen after proteinase K treatment, indicating their peptidic nature, and a total preclusion of the cytotoxicity was shown after their incubation at 50 °C for 30 min. Results show that P. salmonis can produce ECPs and at least some of them are thermolabile exotoxins that probably play a role in the pathogenesis of piscirickettsiosis.

The main objective of this work was to determine the presence of anisakid larvae in fresh Chilean hake Merluccius gayi gayi coming from the coastal area near the city of Talca, Chile, commonly commercialized by a regional fish supply center located in the same city. Flesh and viscera of 214 fish were analyzed via direct observation, and the prevalence of parasitism was calculated. The results showed a prevalence of 100% and 24.3% in viscera and flesh, respectively. Morphologic characterization of 138 randomly selected larvae was performed via diaphanization technique, observing 106 larvae of Anisakis spp., 13 larvae of Pseudoterranova spp. and 19 nematodes classified as “unidentified.” All larvae here analyzed were viable based on the spontaneous movement ability of parasites. Overall, the high prevalence of fish infected with Anisakidae larvae highlights the importance of taking early intervention actions in order to prevent the occurrence of anisakidosis in the human Chilean population; particularly critical given the current culinary preference for raw fish dishes such as ceviche and sushi, which poses a public health issue.

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Zoonotic larvae of the family Anisakidae found in several fish species represent a serious risk in public health since they may cause food-borne anisakidosis in humans. Chile has culinary preferences including eating raw fish in many traditional preparations. In the present study, a total of 180 fish specimens representing three different fish species, i.e., Chilean hake (Merluccius gayi), snoek (Thyrsites atun), and sea bream (Brama australis), were caught at central coast of Chile. Parasitological examination was performed on musculature and abdominal cavity for subsequent extraction and quantification of anisakid larvae. Estimation of infection parameters, such as prevalence, was performed indicating 100% (CI: 0.94–1.0) prevalence of anisakid L3 in Chilean hakes and snoeks. Moreover, sea breams reached a prevalence of 35% (CI: 0.23–0.48). Prevalence of anisakid larvae in muscle was also analyzed showing values of 18.6% (CI: 0.097–0.309) in Chilean hakes, 15% (CI: 0.07–0.26) in snoeks, and 1.7% (CI: 0–0.089) in sea breams. Meanwhile, prevalence of anisakid larvae in internal organs showed highest values for peritoneum (100% and 83.3%) for snoeks and Chilean hakes, respectively, for liver (96.7%) and gonads (86.6%) in Chilean hakes, and for intestine (98.3%) in snoeks. Molecular analysis of collected anisakid L3 unveiled presence of two potentially zoonotic nematode species, i.e., Pseudoterranova cattani and Anisakis pegreffii. P. cattani was found in Chilean hakes and snoeks being the first molecular host species report for Chilean snoeks. Besides, A. pegreffii was also identified in these species being the first molecular report on this regard. These findings are relevant for better understanding of epidemiology of anisakiasis in Chilean coasts and for public health issues considering potential risk of human population due to its culinary preferences in eating raw fish.

El objetivo de este trabajo fue determinar la presencia de larvas de parásitos de importancia zoonótica en reinetas (Brama australis) frescas y en ceviches preparados con esta especie que son vendidos en el centro-sur de Chile (ciudad de Talca). Se adquirieron 27 reinetas enteras sin eviscerar, obtenidas desde la zona costera cerca de la ciudad de Talca y vendidas en la misma ciudad, así como 48 porciones de ceviche de reineta obtenidas desde el comercio formal e informal. Todas las muestras fueron analizadas macroscópicamente y luego mediante digestión enzimática. No se detectaron larvas de anisákidos en las porciones de ceviche ni en los pescados frescos; sin embargo, se detectó la presencia de larvas de cestodos del género Hepatoxylon en 24 reinetas (88.9%), con una abundancia media de 1.9 larvas.

SUMMARYAn immunohistochemstry (IHC) method to detect scrapie in sheep third eyelid lymphoid tissue was applied in post mortem animals. This method would allow to carry out the diagnosis in Chile, including the preclinical phase of the disease in sheep flock. Eighty samples from two year old sheep were obtained. Fourty two showed neurological signs consistent with scrapie and thirty eight came from slaughterhouse. Another thirteen blind samples were provided by a Reference Center (RC) in addition to sixteen obex used as controls. Samples were evaluated in sections with hematoxilin-eosin and classified as suitable if presenting at least four lymphoid follicles in the tissue. Based on this, seventy four out of ninety samples were considered suitable. The IHC results showed that all samples were negative to scrapie and only seven provided by the RC were positive. All obex samples were positive to immunostaining by PrP Sc , unlike negative controls. The results demosntrate that this diagnostic tool may be considered for monitoring the sanitary condition related to scrapie in the ovine flock in Chile.Key words: scrapie, prion, TSE, IHC. RESUMENEn este trabajo se implementó y aplicó un método para detectar el scrapie, utilizando la técnica de inmunohistoquímica (IHQ) en tejido linfoide de tercer párpado ovino. Este método permitiría realizar el diagnóstico en animales vivos, incluso en fases preclínicas de esta enfermedad en la masa ovina nacional. Se obtuvieron y procesaron 80 muestras de tercer párpado de ovinos nacionales mayores de 2 años, 42 con sintomatología nerviosa compatible con scrapie y 38 que fueron obtenidos de mataderos. Otras 13 muestras ciegas de validación de tercer párpado y sus respectivos óbex, además de 16 muestras de óbex utilizadas como controles, fueron aportadas por un Centro de Referencia (CR). Las muestras fueron evaluadas en cortes teñidos con Hematoxilina-Eosina y clasificadas como aptas cuando presentaban al menos 4 folículos linfoides en el tejido obtenido. Sobre esta base, 74 de las 93 muestras totales de tercer párpado fueron consideradas como aptas. Los resultados de IHQ mostraron que todas las muestras nacionales fueron negativas a scrapie y sólo 7 de las proporcionadas por el CR fueron positivas. Además, en todos los controles positivos de óbex se observó inmunomarcaje de PrP Sc , lo cual no se evidenció en los controles negativos. Los resultados descritos anteriormente permiten demostrar la aplicabilidad y capacidad para utilizar esta herramienta diagnóstica en la masa ovina, lo cual permitiría establecer la condición del país respecto al scrapie y obtener los beneficios de esta situación sanitaria.