Development and application of a real-time polymerase chain reaction method for quantification of Escherichia coli in oysters (Crassostrea gigas).

Oysters are important mariculture species worldwide. Because of their filter-feeding behaviors, oysters can accumulate microorganisms, including pathogens, from surrounding water and concentrate bacteria in high numbers. Rapid and suitable methods for quantification of Escherichia coli in oysters are necessary considering that oysters are perishable foods often consumed raw and some countries use E. coli as the regulatory limit. The objective of this study was to develop a qPCR method for quantification of E. coli in oysters. Additionally, different methods were evaluated for DNA extraction from oyster samples and the more reliable method was chosen. Primers and probe were designed targeting uidA gene of E. coli and shown to specifically amplify DNA from E. coli. Standard curves with bacterial DNA extracted from oysters samples artificially inoculated with E. coli were conducted. A good correlation was noticed when the qPCR method was compared to a culture method in oyster samples. This is the first report of a method exclusively developed for direct quantification of E. coli in oyster, the method showed to be suitable for quantification of E. coli in oysters and could be useful in routine analyses, as it requires less time than the culture method.

Development of a droplet digital RT-PCR for the quantification of foot-and-mouth virus RNA.

Foot-and-mouth-disease (FMD) is a highly contagious disease of domestic animals which can result in substantial economic losses, caused by the FMD virus (FMDV). The aim of this study was to develop and standardize a novel reverse transcriptase droplet digital PCR (RT-ddPCR) assay for the quantification of FMDV RNA. This assay was based upon an OIE-recognized real-time RT-PCR that detects the 3D-encoding region of FMDV. The limit of detection at 101.4 TCID50/mL and 26.5 copies was determined using FMDV-A24-Cruzeiro-virus and a plasmid containing the 3D-FMDV sequences, respectively. FMDV O, A and C serotypes and 11 species of non-FMDV were used to confirm the sensitivity and specificity of the assay. The RT-ddPCR was standardized using 60 bovine samples (representing negative and positive samples of epithelium and/or oesophageal-pharyngeal [OP] fluid) from animals suspected of vesicular diseases and previously tested by RT-qPCR. The RT-ddPCR showed robustness, sensitivity, specificity and accuracy, with similar results to the RT-qPCR. Moreover, the new RT-ddPCR diagnostic tool allowed the absolute quantification of FMDV RNA from epithelium and OP-fluid samples, as well as having the advantages of direct quantification by endpoint, eliminating the need for a calibration standard curve required in quantitative real-time RT-PCR.

Molecular epidemiology of senecavirus A associated with vesicular disease in pigs in Brazil.

Senecavirus A (SV-A) may cause vesicular disease and neonatal mortality in pigs, and was first detected in Brazil in 2015. Samples including tissues and serum from pigs with suspected vesicular diseases were collected from January to August in 2015 from farms in the states of Minas Gerais, Santa Catarina, Goiás and Rio Grande do Sul, Brazil, and tested for the presence of SV-A by reverse transcriptase PCR. All samples were negative for foot and mouth disease virus, as well as 13 other infectious agents associated with vesicular diseases in pigs. SV-A was detected by PCR in 65/265 (24.5%) specimens. A 530 base pair fragment sequenced from the VP1 protein coding region indicated a high genetic distance from SV-A in other countries, but a common origin among the Brazilian isolates.

Absence of Bovine leukemia virus (BLV) infection in buffaloes from Amazon and southeast region in Brazil.

Enzootic bovine leucosis is an infectious disease caused by Bovine leukemia virus (BLV) and is well described in bovines. The majority of infected animals are asymptomatic, one to five percent develop lymphoma and from 30 to 50% present a persistent lymphocytosis. The virus occurs naturally in cattle and experimentally in buffaloes, capybaras and rabbits. The occurrence of lymphoma in buffaloes has been attributed to BLV infection by some authors in India and Venezuela, but not confirmed by other studies and little information on natural BLV infection in buffaloes is available. The aim of this study was to evaluate the occurrence of BLV in a sub-sample of buffalo from Amazon and southeast regions in Brazil. Three hundred and fifteen serum samples were negative using commercial AGID and ELISA (ELISA-gp51) which detect anti-BLV glycoprotein gp51 antibodies. The same samples were also evaluated for antibodies to whole virus through a commercial ELISA (ELISA-BLV) in which 77 (24.44%) were found seropositive and two (0.63%) inconclusive. On the other hand, all animals were negative by PCR to BLV targeted to the env and tax genes. These results suggest that ELISA-BLV produces false positive results in buffalo serum (p<0.001). In addition, one buffalo lymphoma sample was negative in both PCR assays used in this study. BLV was not detected in buffaloes from the Amazon basin and the southeast region of Brazil. Serological tests, like ELISA-BLV, usually used for cattle may produce false-positive results for BLV in buffaloes and direct detection tests such as PCR should be chosen in these surveys. The occurrence of lymphoma in buffalo was not associated with BLV infection in the one case analyzed in this work and the etiology and pathogenesis of this disease should be clarified.

Direct detection of Mycobacterium tuberculosis complex in bovine and bubaline tissues through nested-PCR.

Post-mortem bacterial culture and specific biochemical tests are currently performed to characterize the etiologic agent of bovine tuberculosis. Cultures take up to 90 days to develop. A diagnosis by molecular tests such as PCR can provide fast and reliable results while significantly decreasing the time of confirmation. In the present study, a nested-PCR system, targeting rv2807, with conventional PCR followed by real-time PCR, was developed to detect Mycobacterium tuberculosis complex (MTC) organisms directly from bovine and bubaline tissue homogenates. The sensitivity and specificity of the reactions were assessed with DNA samples extracted from tuberculous and non-tuberculous mycobacteria, as well as other Actinomycetales species and DNA samples extracted directly from bovine and bubaline tissue homogenates. Regarding the analytical sensitivity, DNA of the M. bovis AN5 strain was detected up to 1.5 pg by nested-PCR, whereas DNA of M. tuberculosis H37Rv strain was detected up to 6.1 pg. The nested-PCR system showed 100% analytical specificity for MTC when tested with DNA of reference strains of non-tuberculous mycobacteria and closely-related Actinomycetales. A clinical sensitivity level of 76.7% was detected with tissues samples positive for MTC by means of the culture and conventional PCR. A clinical specificity of 100% was detected with DNA from tissue samples of cattle with negative results in the comparative intradermal tuberculin test. These cattle exhibited no visible lesions and were negative in the culture for MTC. The use of the nested-PCR assay to detect M. tuberculosis complex in tissue homogenates provided a rapid diagnosis of bovine and bubaline tuberculosis.

Physicochemical characteristics and sensory profile of honey samples from stingless bees (Apidae: Meliponinae) submitted to a dehumidification process.

This study was conducted to evaluate the effect of a dehumidification process on the physicochemical and sensory characteristics of stingless-bee honey. Melipona scutellaris and M. quadrifasciata honey samples were submitted to a dehumidification process and to physicochemical (reducing sugars, apparent sucrose, moisture, diastatic activity, hydroxymethylfurfural, ash, pH, acidity, and electric conductivity) and sensory evaluations (fluidity, color, aroma, crystallization,flavor,and acceptability). The results indicated that the dehumidification process does not interfere with honey quality and acceptability.

Physicochemical characteristics and sensory profile of honey samples from stingless bees (Apidae: Meliponinae) submitted to a dehumidification process

This study was conducted to evaluate the effect of a dehumidification process on the physicochemical and sensory characteristics of stingless-bee honey. Melipona scutellaris and M. quadrifasciata honey samples were submitted to a dehumidification process and to physicochemical (reducing sugars, apparent sucrose, moisture, diastatic activity, hydroxymethylfurfural, ash, pH, acidity, and electric conductivity) and sensory evaluations (fluidity, color, aroma, crystallization,flavor,and acceptability). The results indicated that the dehumidification process does not interfere with honey quality and acceptability.

Este estudo foi conduzido com o objetivo de avaliar o efeito do processo de desumidificação sobre as características físico-químicas e sensoriais do mel das abelhas sem ferrão. Amostras de méis de Melipona scutellaris e M. quadrifasciata foram submetidas ao processo de desumidificação, passando em seguida por avaliações físico-químicas (açúcares redutores, sacarose aparente, umidade, atividade diastásica, hidroximetilfurfural, cinzas, pH, acidez e condutividade elétrica) e sensoriais (fluidez, cor, aroma, cristalização, sabor e aceitabilidade). Os resultados indicaram que o processo de desumidificação não interfere na qualidade e aceitabilidade do mel.

Infestação natural de Anastrepha spp. (Diptera: Tephritidae) em umbu-cajá no Município de Cruz das Almas, Recôncavo Baiano / Natural Infestation of Anastrepha spp. (Diptera: Tephritidae) in Spondias sp. in the Municipality of Cruz das Almas, Recôncavo Baiano, Brazil

Este trabalho teve como objetivo conhecer os índices de infestação natural, o percentual de emergência dos adultos e as espécies de moscas-das-frutas associadas ao umbu-cajá (Spondias sp.) no Recôncavo Baiano. Foram coletadas 49 amostras dos frutos entre os meses de março a julho de 2002, totalizando 4.095 frutos (74,4 kg), dos quais obtiveram-se 30.579 pupários, com emergência de 37,4 por cento de tefritídeos. Anastrepha obliqua (Macquart) (99,59 por cento) foi a espécie dominante, seguida por A. fraterculus (Wied.) (0,24 por cento) e A. sororcula Zucchi (0,17 por cento). O índice de infestação foi de 410,7 pupários por quilograma de frutos e 7,5 pupários por fruto.

The objective of this work was to quantify the natural infestation, pupal survival and fruit fly species associated to fruits of Spondias sp. in the Recôncavo Baiano, State of Bahia, Brazil. From March to July, 2002, 49 samples of fruits were collected with the total of 4,095 fruits and a biomass of 74.4kg. About 30,579 fruit fly puparia were obtained from which 37.4% yielded adults. Anastrepha obliqua (Macquart) (99.6%) was the dominant species, followed by A. fraterculus (Wied.) (0.24%) and A. sororcula Zucchi (0.17%). The infestation index was 410,7 pupae for kilogram of fruits and 7.5 pupae per fruit.