Direct detection of Salmonella from poultry samples by DNA isothermal amplification.

1. Salmonellosis is one of the most important diseases in public health and it is usually associated with poultry product consumption. This study aimed to validate rapid methods to detect Salmonella spp. from poultry samples. 2. A DNA isothermal amplification method, previously developed for other matrices, was applied for the specific detection of Salmonella spp. from various samples, including poultry tissues, drag and boot swabs, faeces and feed. A new procedure was validated with Salmonella spp. serotypes and isolates from other enteric bacterial species, as well as naturally contaminated poultry samples. 3. The study demonstrated the successful development and implementation of a procedure, including a DNA isothermal amplification method, for the detection of Salmonella spp. directly from tissues, drag and boot swabs, faeces and feed. The whole procedure can be performed in less than 24 hours and it has been successfully used in a veterinary diagnostic laboratory.

Molecular detection of Salmonella serovars Enteritidis, Heidelberg and Typhimurium directly from pre-enriched poultry samples.

1. Salmonella is one of the most important pathogens in public health and it is usually associated with food-borne diseases. Salmonella serovars Enteritidis and Typhimurium are widespread in the world with outbreaks frequently associated with consumption of poultry products; furthermore, there is an increasing public health concern with the wide dissemination of the serovar Heidelberg in poultry flocks. 2. The aim of the experiment was to develop and to validate rapid methods to detect Salmonella serovars Enteritidis, Typhimurium, and Heidelberg by real-time PCRs and test isolates from pre-enriched poultry samples. 3. Three real-time PCRs were developed and used in combination to detect the serovars Enteritidis, Typhimurium and Heidelberg. These assays were validated by the analysis of 126 Salmonella isolates, eight other enteric bacterial species and 34 naturally contaminated poultry samples after pre-enrichment with buffered peptone water (BPW). 4. Real-time PCRs detected the isolates of the most important poultry serovars (Enteritidis, Typhimurium and Heidelberg) with 100% inclusivity and exclusivity in each assay. The PCR identified monophasic variants of the serovars Typhimurium and Heidelberg. All PCRs were validated in detecting these specific serovars directly from pre-enriched poultry samples. The whole analytical procedure was performed in less than 24 h in a veterinary diagnostic laboratory.

Detection of genetically modified maize in processed products, dry grains, and corn ears intended for fresh consumption in South Brazil.

Conventional and genetically modified (GM) maize cultivars have been widely planted in Brazil to produce grains for processed food, feed, or to be consumed fresh as corn ears. This study used real-time PCR to detect GM maize in processed products and fresh commercial corn ears produced in the last two years in South Brazil. Eighteen conventional and GM maize cultivars were obtained from seed production companies and 50 commercial samples (including canned corn, corn flour, dry grains, and fresh corn ears) were purchased in small local stores and supermarkets. All samples were analyzed by real time TaqMan PCR to detect one constitutive maize gene (hmg) and three genetic regions present in GM plants (p-35S promoter, major gene cry 1A.105, and t-Nos terminator). Each commercial sample was classified as conventional or GM based on the PCR results. PCR targeting the hmg gene generated positive results from all DNA samples, which were further tested with the GM targets. These targets were not detected in the five conventional maize cultivars, but were detected in the GM seeds hosting these fragments. Analysis of processed foods identified four cultivars as conventional and six as GM, which were mostly correctly labeled. Seven (53.8%) dry grain samples were classified as conventional, while six (46.2%) were classified as GM. Three (11.1%) corn ear samples were identified as conventional, and the remaining 24 (88.9%) were GM maize. These results demonstrate the high frequency of GM maize in processed products, including fresh corn ears intended for consumption in South Brazil.

Direct Detection and Quantification of Bacterial Pathogens from Broiler Cecal Samples in the Slaughter Line by Real-Time PCR

Chicken meat is an important source of foodborne pathogens, including Salmonella, Campylobacter, and Clostridium perfringens. These bacteria can occur in the intestinal microbiota of broilers and contaminate chicken carcasses in industrial meat processing. This study aimed to develop and evaluate a procedure based on real-time PCRs for the direct detection and quantification of these three bacteria in broilers' ceca collected in poultry slaughter houses and demonstrate the occurrence of these important foodborne pathogens in Brazilian poultry production flocks. Cecal contents were collected from 45 different broiler flocks in three different slaughterhouses in the state of Paraná, Brazil, totaling 45 samples (in pools of 10 different ceca/chickens per broiler flock). Then, these samples were tested for the detection and quantification of Salmonella, Campylobacter, and Clostridium perfringens by real-time PCRs. The results demonstrated the occurrence of three (6.7%) positive pools for Salmonella, 20 (44.4%) for Campylobacter, and 32 (71.1%) for C. perfringens. Mean bacterial concentrations in the positive samples were 4.3log10 cells/g for Salmonella, 6.4 log10 cells/g for Campylobacter, and 5.5 log10 cells/g for C. perfringens. In conclusion, Salmonella, Campylobacter, and C. perfringens could be detected and quantified directly from the broilers cecal contents collected in the slaughter line. This procedure will be certainly useful to more quickly detect these foodborne pathogens and prevent their occurrence in chicken meat and other poultry food products.(AU)

Comparison of CVI and HVT vaccine strains replication in feather tips in different Marek´s disease vaccination programs / Comparação da replicação da estirpe vacinal CVI e HVT no folículo da pena em diferentes programas vacinais para a doença de Marek

Marek's disease is an important neoplastic disease in birds caused by a serotype 1 specific herpesvirus; it is controlled by vaccination. In commercial breeders and layers in Brazil, current vaccination programs use the combination of attenuated or non-pathogenic strains of the HVT virus (turkey herpesvirus - serotype 3) and CVI 988 (Rispens - serotype 1). The combination of serotype 3 and 1 it has been an important and effective control strategy through the vaccination of long-lived birds. In addition, more recently the recombinant rHVT strain (vectorized vaccines) has been used in some vaccine programs. This studys main objective was to compare CVI and HVT components replication in feather tips in three different Marek's disease (MD) immunoprophylactic programs (T01 program A, T02 program B and T03 program C). Quantification of these two vaccine strains was performed by real-time PCR in samples collected at the ages of 14, 21, and 28 days. At 14 days, mean of log[cvi] in program B was significantly higher than C (p<0.05). For mean of log[hvt], at 28 days, program B was significantly higher than A (p<0.05). For proportion of positives, at 14 days, program B had 2.7 times more risk to be positive in CVI than program C (p<0.001). At 21 days, program B had 1.7 times more risk to be positive in CVI than program C (p=0.005). For HVT, at 28 days, program B had 3.2 times more risk to be positive than program A (p=0,009). Results showed significant differences between the treatments evaluated. In general the conventional combination Mareks vaccine containing CVI+HVT (program B) showed higher replication rate and percentage of vaccine coverage than the programs with rHVT vector vaccines (program A and C).(AU)

A doença de Marek é uma importante doença neoplásica das aves causada por um herpesvírus específico do sorotipo 1 e seu controle se faz por vacinação. Em reprodutoras e poedeiras comerciais do Brasil, os programas de vacinação utilizam a combinação de estirpes atenuadas ou não patogênicas do vírus HVT (turkey herpesvirus - sorotipo 3) e CVI 988 (Rispens - sorotipo 1). A combinação do sorotipo 3 e 1 tem sido uma importante e efetiva estratégia de controle para aves de vida longa. Além disso, mais recentemente a fração rHVT recombinante (vacinas vetorizadas) vem sendo utilizada em alguns programas vacinais. O objetivo deste estudo foi comparar a replicação das frações CVI e HVT no folículo da pena em três programas imunoprofiláticos distintos (T01 programa A, T02 programa B e T03 programa C). A quantificação das duas estirpes vacinais foi realizada por PCR em tempo real nas amostras colhidas nas idades de 14, 21 e 28 dias. Aos 14 dias, em média, log[cvi] do programa B foi significativamente maior do que C (p<0.05). Aos 21 e 28 dias, a média do log[cvi] do programa C foi significativamente menor do que A e B (p<0.05). Para log[hvt], aos 28 dias, a média do programa B foi significativamente maior do que A (p<0.05). Para proporção de positivos, aos 14 dias, o programa B teve 2,7 vezes mais risco de ter positivos no CVI do que C (p<0.001). Aos 21 dias, o programa B teve 1,7 vezes mais risco de ter positivos no CVI do que C (p=0.005). Para HVT, aos 28 dias, o programa B teve 3,2 vezes mais risco de ter positivos do que A (p=0,009). Os resultados obtidos evidenciam diferenças significativas entre os tratamentos. De maneira geral a vacina convencional de Marek combinada com o CVI e HVT (programa B) apresentou maior taxa de replicação, velocidade e percentual de cobertura vacinal do que os programas compostos com vacinas vetorizadas com rHVT (programa A e C).(AU)

Comparison of CVI and HVT vaccine strains replication in feather tips in different Marek´s disease vaccination programs / Comparação da replicação da estirpe vacinal CVI e HVT no folículo da pena em diferentes programas vacinais para a doença de Marek

Marek's disease is an important neoplastic disease in birds caused by a serotype 1 specific herpesvirus; it is controlled by vaccination. In commercial breeders and layers in Brazil, current vaccination programs use the combination of attenuated or non-pathogenic strains of the HVT virus (turkey herpesvirus - serotype 3) and CVI 988 (Rispens - serotype 1). The combination of serotype 3 and 1 it has been an important and effective control strategy through the vaccination of long-lived birds. In addition, more recently the recombinant rHVT strain (vectorized vaccines) has been used in some vaccine programs. This study’s main objective was to compare CVI and HVT components’ replication in feather tips in three different Marek's disease (MD) immunoprophylactic programs (T01 – program A, T02 – program B and T03 – program C). Quantification of these two vaccine strains was performed by real-time PCR in samples collected at the ages of 14, 21, and 28 days. At 14 days, mean of log[cvi] in program B was significantly higher than C (p<0.05). For mean of log[hvt], at 28 days, program B was significantly higher than A (p<0.05). For proportion of positives, at 14 days, program B had 2.7 times more risk to be positive in CVI than program C (p<0.001). At 21 days, program B had 1.7 times more risk to be positive in CVI than program C (p=0.005). For HVT, at 28 days, program B had 3.2 times more risk to be positive than program A (p=0,009). Results showed significant differences between the treatments evaluated. In general the conventional combination Marek’s vaccine containing CVI+HVT (program B) showed higher replication rate and percentage of vaccine coverage than the programs with rHVT vector vaccines (program A and C).

A doença de Marek é uma importante doença neoplásica das aves causada por um herpesvírus específico do sorotipo 1 e seu controle se faz por vacinação. Em reprodutoras e poedeiras comerciais do Brasil, os programas de vacinação utilizam a combinação de estirpes atenuadas ou não patogênicas do vírus HVT (turkey herpesvirus - sorotipo 3) e CVI 988 (Rispens - sorotipo 1). A combinação do sorotipo 3 e 1 tem sido uma importante e efetiva estratégia de controle para aves de vida longa. Além disso, mais recentemente a fração rHVT recombinante (vacinas vetorizadas) vem sendo utilizada em alguns programas vacinais. O objetivo deste estudo foi comparar a replicação das frações CVI e HVT no folículo da pena em três programas imunoprofiláticos distintos (T01 – programa A, T02 – programa B e T03 – programa C). A quantificação das duas estirpes vacinais foi realizada por PCR em tempo real nas amostras colhidas nas idades de 14, 21 e 28 dias. Aos 14 dias, em média, log[cvi] do programa B foi significativamente maior do que C (p<0.05). Aos 21 e 28 dias, a média do log[cvi] do programa C foi significativamente menor do que A e B (p<0.05). Para log[hvt], aos 28 dias, a média do programa B foi significativamente maior do que A (p<0.05). Para proporção de positivos, aos 14 dias, o programa B teve 2,7 vezes mais risco de ter positivos no CVI do que C (p<0.001). Aos 21 dias, o programa B teve 1,7 vezes mais risco de ter positivos no CVI do que C (p=0.005). Para HVT, aos 28 dias, o programa B teve 3,2 vezes mais risco de ter positivos do que A (p=0,009). Os resultados obtidos evidenciam diferenças significativas entre os tratamentos. De maneira geral a vacina convencional de Marek combinada com o CVI e HVT (programa B) apresentou maior taxa de replicação, velocidade e percentual de cobertura vacinal do que os programas compostos com vacinas vetorizadas com rHVT (programa A e C).

Hepatitis C virus genotypes in Southern Brazil

The prevalence of hepatitis C virus (HCV) genotypes in Southern Brazil was studied in the plasma of 100 HCV-RNA-positive patients attended in Porto Alegre, South of Brazil. Reverse transcriptionpolymerase chain reaction (RT-PCR) products from the 5' noncoding region were double digested with RsaI-HaeIII and BstNI-HinfI and analyzed by restriction fragment length polymorphism (RFLP). Three genotypes (1, 2 and 3) were demonstrable, the most prevalent being HCV type 1 (55 of 100 patients, 55 per cent), followed by HCV type 3 (37 of 100 patients, 37 per cent) and HCV type 2 (8 of 100 patients, 8 per cent). There was an unusual high prevalence of genotype 3, in contrast to the majority of published data from the Southeast region.