ABSTRACT The aim of this study was to characterize the microbiota of the intestinal tract of robalo (Centropomus sp), from natural environment and captivity, and to isolate and identify bacteria with probiotic potential. The intestinal microbiota was characterized by metagenomics directed to the 16S rRNA gene. In addition, samples of intestinal mucus were cultured in MRS medium for bacterial isolation. Molecular identification was done by sequencing the 16S rRNA gene. Probiotic capabilities were evidenced by proteolytic and bacterial antagonism assays against Plesiomonas shigelloides, Aeromonas hydrophila, Aeromonas veronii and Vibrio harveyi. The most abundant bacterial genera were Clostridium, Photobacterium, Cetobacterium and Rubritepida. Bacterial species, including Klebsiella sp, two strains of Weisiella cibaria and Lactococcus sp were isolated on MRS agar from the intestinal tract. W. cibaria strains showed broad antagonistic spectrum and positive proteolytic activity.

RESUMEN El objetivo del estudio fue caracterizar la microbiota del tracto intestinal de robalo (Centropomus sp), proveniente de medio natural y cautiverio, además de aislar e identificar bacterias con potencial probiótico. La microbiota intestinal se caracterizó mediante metagenómica dirigida al gen 16S ARNr. Además, se sembraron muestras de mucus intestinal en medio de cultivo MRS para el aislamiento bacteriano. La identificación molecular se hizo mediante la secuenciación del gen 16S ARNr. Las capacidades probióticas se evidenciaron mediante ensayos proteolíticos y de antagonismo bacteriano frente a Plesiomonas shigelloides, Aeromonas hydrophila, Aeromonas veronii y Vibrio harveyi. Los géneros bacterianos con mayor abundancia fueron Clostridium, Photobacterium, Cetobacterium y Rubritepida. Las especies bacterianas, incluyendo Klebsiella sp, dos cepas de Weisiella cibaria y Lactococcus sp fueron aisladas en agar MRS a partir del tracto intestinal. Las cepas W. cibaria mostraron amplio espectro antagonista y actividad proteolítica positiva.

Tilapia Lake Virus (TiLV) is a pathogen that causes massive mortalities in both cultured and wild tilapia populations around the world. The development of an effective vaccine against this emerging pathogen is imperative to prevent economic losses. In this work an expression vector was designed and evaluated as a potential DNA vaccine against this virus. Initially, a threading analysis was done to predict the threedimensional structures and functions of the TiLV proteins. Structural homologies were found between the TiLV proteins corresponding to the genomic segment 1 and the genomic segment 4, with the RNA-dependent RNA polymerase proteins of the influenza B virus (56%) and the neuraminidase protein belonging to the influenza A virus capsid (12%), respectively. The PCR product of the viral neuraminidase gene was inserted into the expression plasmid vector pCMV. Finally, the plasmid construct was injected into juveniles of the Nile tilapia Oreochromis niloticus and its expression was measured by real time RT-PCR at 8h, 16h, 24h, and 72h after the second immunizing injection. It was possible to detect gene expression in the four evaluated times and greater expression at 16 hours post injection. These results are the first step in the development of an effective vaccine for the protection of tilapia stocks around the world.

El Virus de la Tilapia del Lago (TiLV), es un patógeno causante de mortalidades masivas tanto en poblaciones de tilapias cultivadas y silvestres alrededor del mundo. El desarrollo de una vacuna efectiva contra este patógeno emergente es imperativo para prevenir pérdidas económicas. En este trabajo se diseñó y evaluó un vector de expresión como una potencial vacuna de ADN contra este virus. Inicialmente, se realizó un análisis de enhebramiento para predecir las estructuras tridimensionales y las funciones de las proteínas del TiLV. Se encontraron homologías estructurales entre las proteínas correspondientes al segmento genómico 1 y al segmento genómico 4 del TiLV, con las proteínas de ARN polimerasa dependiente de ARN del virus de la influenza B (56%) y la proteína neuraminidasa que pertenece a la cápside del virus de la influenza A (12%), respectivamente. Se insertó el producto de PCR del gen neuraminidasa viral en el vector plasmídico de expresión pCMV. Finalmente, se inyectó el constructo plasmídico en juveniles de la tilapia del Nilo Oreochromis niloticus y se midió su expresión mediante RT-PCR en tiempo real a las 8h, 16h, 24h, 72h después de la segunda inyección inmunizante. Se logró detectar expresión génica en los cuatro tiempos evaluados, con mayor expresión a las 16 horas post inyección. Estos resultados constituyen el primer paso para el desarrollo de una vacuna efectiva para la protección de los stocks de tilapias alrededor del mundo.

The aim of this study was to characterize the PepT1 of pirarucu Arapaima gigas through genomic and proteomic tools. A transcriptomic and proteomic analysis was made from sections of the intestine, spleen, liver and kidney of fingerlings. The transcriptomic analysis allowed obtaining two consensus sequences of 357 and 459 bp. Both fragments showed a high degree of homology (78 and 80%), mainly with nucleotide sequences coding for the PepT1 of Scleropages formosus of the same Order as A. gigas. The proteomic analysis by double mass spectrometry MALDI TOF/TOF allowed to identify 15 peptide sequences of PepT1 in pirarucu. In conclusion, PepT1 was partially characterized in the intestine of pirarucu, which could be used for further studies on the evaluation of its expression.

El objetivo del estudio fue caracterizar el PepT1 del paiche Arapaima gigas mediante herramientas genómicas y proteómicas. Se realizó un análisis transcriptómico y proteómico a partir de secciones del intestino, bazo, hígado y riñón de alevines. El análisis transcriptómico permitió obtener dos secuencias consenso de 357 y 459 pb. Ambos fragmentos presentaron un alto grado de homología (78 y 80%), principalmente con secuencias de nucleótidos codificantes del PepT1 de Scleropages formosus del mismo Orden que el paiche. El análisis proteómico por espectrometría de doble masa MALDI TOF/TOF permitió identificar quince secuencias peptídicas del PepT1 en paiche. Como conclusión se logró caracterizar parcialmente el PepT1 en el intestino del paiche, las cuales podrán ser usadas para posteriores estudios sobre la evaluación de su expresión.

O objetivo deste trabalho foi selecionar reprodutores de Litopenaeus vannamei sobreviventes de um surto do vírus da síndrome da mancha-branca (WSSV), adaptados às condições locais e diagnosticados negativamente para WSSV e para o vírus da necrose infecciosa hipodermal e hematopoiética (IHHNV), e avaliar se esta extratégia é uma alternativa viável para produção em Santa Catarina. Foram selecionados fenotipicamente 800 machos e 800 fêmeas, de um viveiro. Análises de nested-PCR de 487 fêmeas e de 231 machos, sexualmente maduros, mostraram que 63% das fêmeas e 55% dos machos estavam infectados com IHHNV. Os animais livres de IHHNV foram testados para WSSV, e os considerados duplo negativos destinados à reprodução. As pós-larvas produzidas foram estocadas em nove berçários, para análise. Das 45 amostras, com 50 pós-larvas cada, apenas duas foram positivas para IHHNV e nenhuma para WSSV. Os lotes de pós-larvas diagnosticadas livres de vírus por nested-PCR foram encaminhados para seis fazendas. Foi realizada análise comparativa em viveiros de engorda, entre pós-larvas locais e pós-larvas do Nordeste do Brasil. Também foram analisados caranguejos (Chasmagnathus granulata), siris (Callinectes sapidus) e lebres do mar (Aplysia brasiliana), que são possíveis vetores dos vírus. A média de sobrevivência foi de 55% para as pós-larvas locais e de 23,4% para as pós-larvas do Nordeste. As lebres do mar apresentaram prevalência de 50% e os caranguejos de 67% de WSSV.

The objective of this work was to select surviving breeders of Litopenaeus vannamei from white spot syndrome virus (WSSV) outbreak, adapted to local climatic conditions and negatively diagnosed for WSSV and infectious hypodermal and hematopoietic necrosis virus (IHHNV), and to evaluate if this strategy is a viable alternative for production in Santa Catarina, Brazil. A total of 800 males and 800 females were phenotypically selected in a farm pond. Nested-PCR analyses of 487 sexually mature females and 231 sexually mature males showed that 63% of the females and 55% of the males were infected with IHHNV. Animals free of IHHNV were tested for WSSV, and those considered double negative were used for breeding. The post-larvae produced were stocked in nine nursery tanks for analysis. From the 45 samples, with 50 post-larvae each, only two were positive for IHHNV and none for WSSV. Batches of larvae diagnosed free of virus by nested-PCR were sent to six farms. A comparative analysis was carried out in growth ponds, between local post-larvae and post-larvae from Northeast Brazil. Crabs (Chasmagnathus granulata), blue crabs (Callinectes sapidus), and sea hares (Aplysia brasiliana), which are possible vectors of these viruses, were also evaluated. The mean survival was 55% for local post-larvae against 23.4% for post-larvae from the Northeast. Sea hares showed prevalence of 50% and crabs of 67% of WSSV.