Utilização comparativa de enrofloxacino, probiótico (Lactobacilus plantarum) e propionato de sódio na larvicultura de camarão branco do pacífico / Comparative use of enrofloxacin, probiotic (Lactobacillus plantarum) and sodium propionate in pacific white shrimp larvae / Utilización comparativa de enrofloxacino, probiótico (Lactobacillus plantarum) y propionato de sodio en larvicultura de camarón blanco del pacífico

O objetivo do presente trabalho foi avaliar métodos de controle e tratamento de bacterioses na larvicultura de Litopenaeus vannamei, avaliando o uso contínuo de probiótico e o uso pontual de enrofloxacino 15mg.L-1 e propionato de sódio 0,5 mM.L-1 nos momentos de metamorfose sobre os, parâmetros zootécnicos e microbiológicos (larvas e da água). Foram utilizadas 16 unidades de 60L, povoadas na densidade de 325 náuplios.5L-1, divididos em três tratamentos: enrofloxacino, probiótico, propionato de sódio e controle. O enrofloxacino e o propionato foram ministrados em protozoea 3, misis 3 e pós-larva 4 e o probiótico foi ministrado na ração ao longo de todo o experimento. O probiótico aumentou as contagens de bactérias ácido-láticas em relação aos demais tratamentos na água de cultivo (p=0,00001) e em relação ao enrofloxacino e o proprionato nas larvas (p=0,0048). A água do tratamento com probiótico apresentou menor contagem de Vibrio spp. que o enrofloxacino (p=0,0011) e as larvas tratadas com probiótico apresentaram menor contagem de Vibrio spp. que o propionato (p=0,0158). Contudo, não foi observada diferença nos índices zootécnicos avaliados. Assim, os aditivos na dose utilizada não alteram parâmetros zootécnicos da larvicultura do camarão L. vannamei.

The purpose of this study was to assess control and treatment methods for bacterial diseases in Litopenaeus vannamei, evaluating the continuous use of probiotics and occasional use of 15mg-L-1 enrofloxacin and 0.5-mM.L-1 sodium propionate at the morphological change moments on the performance and microbiological parameters of larvae and water. A total of 16 60-L units were used, stocked with 325 nauplii/5L-1, divided into three treatments and one control. Enrofloxacin and propionate were administered into protozoea 3, misis 3 and 4, and post-larvae 4, while the probiotic was administered in the feed throughout the experiment. The probiotic increased the counts of lactic acid bacteria in relation to the other treatments in the culture water (p = 0.00001) and in relation to enrofloxacin and propionate in larvae (p = 0.0048). The treatment water with probiotic had lower counts of Vibrio ssp. than enrofloxacin (p = 0.0011) and larvae treated with probiotic showed lower counts of Vibrio ssp. that propionate (p = 0.0158). However, no difference was observed in the performance indexes assessed. Thus, it can be concluded that additives in the assessed doses did not influence the performance parameters of L. vannamei.

El objetivo del presente trabajo fue evaluar métodos de control y tratamiento de enfermedades bacterianas en larvicultura de Litopenaeus vannamei, evaluando el uso continuo de probiótico y el uso puntual de 15 mg.L-1 de enrofloxacino y 0,5 mM.L-1 de propionato de sodio en momentos de metamorfosis sobre los parámetros zootécnicos y microbiológicos (larvas y del agua). Se han utilizado 16 unidades de 60L, pobladas en la densidad de 325 nauplios.5L-1, divididos en tres tratamientos: enrofloxacino, probiótico, propionato de sodio y control. El enrofloxacino y el propionato fueron suministrados en protozoea 3, misis 3 y postlarva 4 y el probiótico suministrado en alimento durante el transcurso del experimento. El probiótico aumentó el contaje de bacterias ácido-lácticas en correlación a los demás tratamientos en agua de cultivo (p=0,0001) y las larvas en relación al enrofloxacino y el propionato en las larvas (p=0,0048). El agua del tratamiento con probiótico presentó menor contaje de Vibrio spp. que el enrofloxacino (p=0,0011) y las larvas tratadas con probiótico presentaron menor contaje de Vibrio spp. que el propionato (p=0,0158). Sin embargo, no se ha observado diferencia en los índices zootécnicos de larvicultura de camarón L. vannamei.

Bioaccumulation of animal adenoviruses in the pink shrimp

Adenoviruses are among the most promising viral markers of fecal contamination. They are frequently found in the water, sediment and soil of regions impacted by human activity. Studies of the bioaccumulation of enteric viruses in shrimp are scarce. The cities located in the northern coast of the lake systems in Southern Brazil have high urbanization and intensive farming rates, and poor sewage collection and treatment. One hundred (n = 100) Farfantepenaeus paulensis pink-shrimp specimens and 48 water samples were collected from coastal lagoons between June 2012 and May 2013. Water samples were concentrated and the shrimp, mashed. After DNA extraction, samples were analyzed by real time polymerase chain reaction (qPCR) in order to detect and quantify viral genomes. Thirty-five percent of shrimp samples were positive for contamination, predominantly by avian adenoviruses. A total of 91.7% of water samples contained adenoviruses DNA, with the human form being the most frequent. Our results provided evidence of significant bioaccumulation of adenoviruses in shrimp, showing the extent of the impact of fecal pollution on aquatic ecosystems..

Bioaccumulation of animal adenoviruses in the pink shrimp

Adenoviruses are among the most promising viral markers of fecal contamination. They are frequently found in the water, sediment and soil of regions impacted by human activity. Studies of the bioaccumulation of enteric viruses in shrimp are scarce. The cities located in the northern coast of the lake systems in Southern Brazil have high urbanization and intensive farming rates, and poor sewage collection and treatment. One hundred (n = 100) Farfantepenaeus paulensis pink-shrimp specimens and 48 water samples were collected from coastal lagoons between June 2012 and May 2013. Water samples were concentrated and the shrimp, mashed. After DNA extraction, samples were analyzed by real time polymerase chain reaction (qPCR) in order to detect and quantify viral genomes. Thirty-five percent of shrimp samples were positive for contamination, predominantly by avian adenoviruses. A total of 91.7% of water samples contained adenoviruses DNA, with the human form being the most frequent. Our results provided evidence of significant bioaccumulation of adenoviruses in shrimp, showing the extent of the impact of fecal pollution on aquatic ecosystems.

Comparative analysis of conventional PCR and real-time PCR to diagnose shrimp WSD

The aims of this study were to standard and optimize a qPCR protocol with FAM-BHQ1 probe, and to compare its sensitivity against TaqMan qPCR and PCR methods to diagnose shrimp WSD. The FAM-BHQ1 qPCR presented higher clinical sensitivity and showed to be a robust alternative to detect WSSV in clinical samples.

A ten-month diseases survey on wild Litopenaeus setiferus (Decapoda: Penaeidae) from Southern Gulf of Mexico / Diez meses de análisis de las enfermedades en especímenes silvestres de Litopenaeus setiferus en el Sur del Golfo de México

The development of shrimp aquaculture in Mexican coasts of the Gulf of Mexico began to be explored using the Pacific white shrimp Litopenaeus vannamei in the mid 90´s. Many concerns over the risk of disease transmission to the economically important native penaeids, have been the main deterrent for the aquaculture of L. vannamei in the region. Concurrently, more than 10 years of research experience on the aquaculture suitability of the native Litopenaeus setiferus from the Terminos Lagoon, in the Yucatán Peninsula, have been accumulated. The aim of this study was then to determine the seasonal variations of the naturally acquired diseases and the possible detection of exotic pathogens. For this, random subsamples (n~60) of juveniles L. setiferus were collected from monthly captures. In order to detect the widest range of pathogens, including infectious hypodermal and hematopoietic necrosis (IHHNv) and white spot syndrome (WSSv) viruses, both histopathological and molecular methods were employed. Monthly prevalence (%) was calculated for every finding. We were able to detect a total of 16 distinct histological anomalies, most of which the presump- tive aetiological agent was readily identified. PCR results for viruses were negative. For some pathogens and symbionts, the prevalence was significantly different between the adult and juvenile populations. Prevalence of diseases tended to be higher in juvenile shrimp than in adults. The results of this study indicated that L. setiferus carry a wide variety of pathogens and symbionts that seem to be endemic to penaeids of the Gulf of Mexico, and those juveniles were more conspicuous to acquire pathogens and symbionts than adults.

Durante la década de los 90´s se introdujo el camarón blanco del Pacífico Litopenaeus vannamei a los Estados costeros mexicanos del Golfo de México con fines acuícolas, por lo que desde entonces existe preocupación por la posible introducción de enfermedades que puedan afectar a las poblaciones de camarones nativos. La investigación sobre la domesticación de especies nativas para una acuacultura sustentable se ha realizado por más de 10 años, sin embargo, aún existe escasa información sobre las enfermedades que se presentan de manera natural en estas poblaciones y posible trasfaunación. El presente estudio aborda el problema de las enfermedades encontradas en subpoblaciones de jóvenes y adultos de Litopenaeus setiferus del Área natural protegida Laguna de Términos, estado de Campeche, México. Técnicas de histología y biología molecular fueron utilizadas como herramientas de diagnóstico. Se encontró que L. setiferus es portador de patógenos y simbiontes endémicos del Golfo de México, y comparativamente, los jóvenes son más susceptibles en adquirir estas infecciones que los adultos, como probable respuesta al ambiente lacustre que ocupan. No se encontró evidencia de los virus IHHNv y WSSv, aunque en trabajos más recientes en algunos Estados del Norte ya se han detectado en poblaciones silvestres.

Detection of white spot syndrome virus in Brazil using negative staining, immunoelectron microscopy and immunocytochemistry techniques / Detección del virus del síndrome de mancha blanca en el Brasil utilizando inmunomicroscopía e inmunomarcación con partículas de oro coloidal

In this study thirty shrimp samples from commercial marine shrimp (L. vannamei) farms of southern region of Brazil were obtained. Hepatopancreas and shell scrapings fragments collected in these animals were processed by transmission electron microscopy using negative staining (rapid preparation), immunoelectron microscopy and immunocytochemistry (immunolabelling with colloidal gold particles) techniques. On the transmission electron microscopy a great number of white spot virus particles, ovoid or bacilliform-to-ellipsoid, measured 230-290 nm in length and 80-160 nm in diameter with intra-nuclear projections were visualized by the negative staining technique in 27 (90 percent) out of 30 samples examined. Using immunoelectron microscopy technique, the anti-VP 664 serum agllutinated a large number of particles formed by antigen-antibody interaction. In the immunocytochemistry technique, the antigen-antibody reaction was styrongly marked by the particles of colloidal gold over the virus. Notably, this is the first report, to our knowledge, describing use of these microscopy techniques to study Brazilian L. vannamei marine shrimp samples; moreover, this methodology also appears to be a viable complementary tool for diagnosing the presence of the white spot virus within shrimp tissues. Importantly, these are the first photoelectron micrographs of the WSSV in Brazil.

Se obtuvieron para el estudio 30 muestras de camarones marinos comerciales (L. vannamei) de las granjas de la región sur de Brasil. Fueron procesados fragmentos de hepatopáncreas y raspados internos del cefalotórax recogidos en estos animales por microscopía electrónica de transmisión con tinción negativa (preparación rápida), inmunomicroscopía y técnicas de inmunocitoquímica (inmunomarcación con partículas de oro coloidal). En la microscopía electrónica de transmisión de un gran número de partículas de virus de la mancha blanca, ovoide o elipsoidal a baciliformes, medían 230-290 nm de longitud y 80-160 nm de diámetro. En 27 (90 por ciento) de las 30 muestras examinadas intra-nuclear proyecciones se visualizaron mediante la técnica de tinción negativa. Utilizando una técnica de inmunomicroscopía electrónica, el anti-suero VP 664 reunió a un gran número de partículas formadas por la interacción antígeno-anticuerpo. En la técnica de inmunocitoquímica, la reacción antígeno-anticuerpo fue fuertemente reforzada por las partículas de oro coloidal en los virus. En particular, en Brasil este es el primer informe, a nuestro entender, que describe el uso de estas técnicas de microscopía en muestras de camarón marino L. vanamei. Además, esta metodología también parece ser una herramienta complementaria viable para diagnosticar la presencia del virus de la mancha blanca en tejidos de camarón. Es importante destacar que estas son las primeras fotos en microscopia electrónica del WSSV obtenidas en Brasil.