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.

Lack of association between human leukocyte antigen polymorphisms rs9277535 and rs7453920 and chronic hepatitis B in a Brazilian population.

Hepatitis B virus (HBV) infection is a serious public health problem worldwide. The progression of the disease depends on several host and viral factors and may result in fulminant hepatitis (very rare), acute hepatitis with spontaneous clearance, and chronic hepatitis B infection. Previous studies demonstrated that variations in the human leukocyte antigen (HLA) class II (HLA-DPB1 and HLA-DQB2 genes) are related to the chronic HBV infection. This study aimed to investigate the association of two single nucleotide polymorphism (SNPs), one in the HLA-DPB1 (rs9277535) and one in the HLA-DQB2 (rs7453920), with chronic hepatitis B infection in a southern Brazilian sample. This case-control study included 260 HBV patients attended in a Specialized Center for Health in Caxias do Sul (Brazil) between 2014 and 2016. The same number of controls (matching for age, gender, and ethnicity) was obtained in a University Hospital in the same city and period. Blood samples were collected and genomic DNA was extracted. Genotyping were performed by real-time Taqman PCR method. Odds ratios with 95% confidence intervals and significance level of 5% (P < 0.05) were calculated. Allele frequencies in the SNP rs9277535 were 72.6% for A and 27.4% for G nucleotides in cases and 75.0% for A and 25.0% for G in controls. Allele frequencies in the SNP rs7453920 were of 25.7% for A and 74.3% for G in cases and 28.8% for A and 71.2% for G in controls. No statistically significant association was found between both SNPs and chronic hepatitis B (P > 0.05).

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.

Alcohol misuse and illicit drug use are associated with HCV/HIV co-infection.

We studied hepatitis C virus (HCV) prevalence and risk factors for HCV infection in a sample of Brazilian HIV-positive patients. A cross-sectional study was conducted with 580 HIV-positive patients from a specialized HIV/AIDS diagnosis and treatment centre in southern Brazil. All patients were interviewed for socio-demographic and risk factors and tested for HCV antibodies and HCV-RNA detection. A multivariate analysis was performed to identify risk factors for HCV infection. A total of 138 (24%) patients had past or chronic hepatitis C. The following risk factors were associated with HCV infection for each gender: alcohol misuse and injecting drug use in women (P < 0·001) and low educational level, smoking drug use, and injecting drug use in men (P < 0·01). These results suggest that alcohol misuse, low educational level, smoking drug use, and injecting drug use are probable risk factors for HCV infection in HIV-positive patients. This information contributes to an understanding of the epidemiology of HIV/HCV co-infection in Brazil.

Prevalence and genotypic diversity of cervical human papillomavirus infection among women from an urban center in Brazil.

Human papillomavirus (HPV) infection is a common viral sexually transmitted infection and the main cause of cervical cancer in women worldwide. Epidemiological data on the prevalence of HPV in a given population is essential for the establishment of effective prevention strategies. The aim of this study was to determine HPV prevalence in women who attended a public health service within an urban center in Brazil. Cervical samples were collected from 337 women recruited from a primary public health care clinic in the city of Cruz Alta located in Rio Grande do Sul, the southernmost State of Brazil. Samples were analyzed for HPV DNA and with Pap smear screening tests. HPV was detected in 114 (34%) women. HPV type analysis revealed that 95 (83.3%) of those represented infections with a single genotype, while 19 (16.7%) were mixed genotype infections. High- and low-risk HPV genotypes were detected in 83 (72.8%) and 48 (42.1%) samples, respectively. Furthermore, a great diversity of HPV genotypes was observed (18 high-risk, 12 low-risk, and 1 indeterminate). The most commonly identified low-risk types were candHPV62 (7.9%) and 61 (5.3%), while the most common high-risk types were 16 and 33 (8.8% each). Abnormal cytology was observed in 10 (3.0%) women, 9 of which were infected with HPV. Of the remaining 327 women with normal cytology results, 107 (32.7%) were positive for HPV DNA. HPV infection was correlated with younger age (less than 40 years), a first Pap smear, and other vaginal infections.

Factors affecting the performance of 5' nuclease PCR assays for Listeria monocytogenes detection.

The design and operating parameters affecting the performance of 5' nuclease PCR (TaqMan) assays for the detection of Listeria monocytogenes was investigated. A system previously developed and based on the hlyA gene was used as a model [Appl. Environ. Microbiol. 61 (1995) 3724]. A series of fluorogenic probes labeled with a reporter and a quencher dye was synthesized to explore the effect of probe position and sequence content on the efficiency of probe hydrolysis. In addition, a series of PCR primer pairs that altered the distance between the upstream primer and the interceding probe was examined. The effects of various assay parameters were evaluated by measuring the ratio of the fluorescence intensity of the reporter dye over the quencher dye (deltaRQ). For a given probe sequence, the deltaRQ was typically lower if the 5' terminus was a G residue. Decreasing the probe concentration increased the deltaRQ, although this was at the expense of reproducibility in the assay readout. The distance between the upstream primer and the interceding probe has a significant effect on probe hydrolysis. Reducing the primer-probe distance from, for example, 127 to 4 nt increased the deltaRQ from 2.87 to 5.00. These general rules were used to develop a 5' nuclease PCR (TaqMan) assay with enhanced signal output, providing higher and more reproducible deltaRQ values for L. monocytogenes detection.

Identification of porcine intestinal spirochetes by PCR-restriction fragment length polymorphism analysis of ribosomal DNA encoding 23S rRNA.

The Brachyspira (formerly Serpulina) species rrl gene encoding 23S ribosomal RNA (rRNA) was used as a target for amplification of a 517bp DNA fragment by polymerase chain reaction (PCR). The primers for PCR amplification had sequences that were conserved among Brachyspira 23S rRNA gene and were designed from nucleotide sequences of Brachyspira hyodysenteriae, Serpulina intermedia, Brachyspira innocens and Brachyspira pilosicoli available from the GenBank database. Digestion of PCR-generated products from reference and field isolates of swine intestinal spirochetes with restriction enzymes Taq I and Alu I revealed five restriction fragment length polymorphism (RFLP) patterns. Each RFLP pattern corresponded to previously established genetic groups including B. hyodysenteriae (I), S. intermedia/B. innocens (II), Brachyspira murdochii (III), B. pilosicoli (IV) and B. alvinipulli (V). The 23S rRNA PCR/RFLP provided a relatively simple genotypic method for identification of porcine pathogenic B. hyodysenteriae and B. pilosicoli.

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 transcription-polymerase 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%), followed by HCV type 3 (37 of 100 patients, 37%) and HCV type 2 (8 of 100 patients, 8%). There was an unusual high prevalence of genotype 3, in contrast to the majority of published data from the Southeast region.

Detection of Babesia equi (Laveran, 1901) by nested polymerase chain reaction.

We describe a nested polymerase chain reaction (PCR) for the detection of Babesia equi in equine infected erythrocytes using oligonucleotides designed on the published sequence of a B. equi merozoite antigen gene (ema-1). A 102bp DNA fragment is specifically amplified from B. equi but not from Babesia caballi, Babesia bovis or Babesia bigemina DNA. In a mock infection we were able to detect down to six infected cells in 10(8) equine erythrocytes or to detect the parasite in blood with an equivalent parasitemia of 0.000006%. Furthermore, gene polymorphism was found by performing a PCR-RFLP (PCR combined with restriction fragment length polymorphism) on both the 102bp and the entire ema-1 gene DNA amplified from two B. equi isolates, Florida (USA) and Pelotas (Southern Brazil) isolates. The polymorphism was confirmed by sequencing the entire ema-1 gene from the B. equi isolate Pelotas. Our results demonstrate that the ema-1 based nested PCR is a valuable technique for routine detection of B. equi in chronically infected horses. It may be used for epidemiological and phylogenetic studies of the parasite as well as monitoring B. equi infected horses in chemotherapeutic trials.

Molecular characterization of Brazilian infectious bursal disease viruses.

A reverse transcriptase-polymerase chain reaction (RT-PCR) procedure was used to amplify a VP2 gene fragment (248 bp) from infectious bursal disease virus (IBDV). The procedure allowed the detection of known IBDV strains from the United States, along with field isolates and commercial vaccines produced in Brazil. Amplified VP2 fragments were further characterized by restriction fragment length polymorphism (RFLP) analysis. From 55 Brazilian commercial flocks, 48 field samples were IBDV positive by RT-TCR. Vaccine RFLP patterns were found in 12 flocks, a pattern compatible with classic IBDV in one flock, four new patterns in 31 flocks, and a pattern compatible with very virulent (vv) IBDV in four flocks. Sequence analysis showed that the vvIBDV RFLP patterns were closely related to the vvIBDVs described in Europe and Asia. Phylogenetic analysis of the four new RFLP patterns showed that they were closely related to but distinct from other classic, variant, and vvIBDVs, suggesting a high prevalence of different IBDV strains in Brazilian commercial flocks.

Bovine alphaherpesvirus 1 and 5 in semen from bulls presenting genital lesions under field conditions in Brazil / Alfaherpesvírus bovino 1 e 5 em sêmen de touros com lesões genitais, sob condições a campo no Brasil

Bovine alphaherpesviruses 1 and 5 (BoHV-1/5) are main pathogens of respiratory, reproductive and neurological diseases in cattle. The aim of this study was to investigate the frequency of neutralizing antibodies against BoHV-1/5 in serum samples and to detect viral DNA in semen of bulls from beef cattle farms located in RS. A total of 372 serum and semen sample from bulls were collected in eighteen farms. Serum samples were submitted to virus neutralization (VN) assay, while semen samples were used to detect BoHV-1 and BoHV-5 DNA by PCR. VN results showed that BoHV-1/5 antibodies were detected in bulls of 66.7% (12/18) of the farms, 295 (79.5%) BoHV positive bulls, 287 for BoHV-1 and 234 for BoHV-; at 43 vaccinated bulls 72.1% (31/43) showing serology negative. BoHV-1/5 DNA was detected in the semen of three bulls; one of the them presenting BoHV-1, one out three presenting BoHV-5 and one BoHV-1/5.co-infection All BoHV DNA positive samples came from animals presenting posthitis and other genital lesions at sampling. Results showed a high seroprevalence of BoHV-1/5 antibodies in bulls as well as strong evidence that these viruses are actively circulating in the cattle farms. A remarkable finding is that in the presence of clinically evident lesions in the genital tract, both BoHV-1 and 5 may found in semen.(AU)

Os alfa-herpesvírus bovinos 1 e 5 (BoHV-1/5) são importantes patógenos de doença respiratória, reprodutiva e neurológica em bovinos. O objetivo deste estudo foi investigar a frequência de detecção de anticorpos neutralizantes contra BoHV-1/5 em amostra de soro e detectar DNA viral em sêmen de touros do rebanho bovino localizado nas fazendas de gado de corte do RS. Um total de 371 amostras de soro e sêmen foi coletado de touros em 18 fazendas, 325 das quais são provenientes de touros não vacinados e 43 de vacinados. Amostras de soro foram submetidas à técnica de vírus-neutralização (VN), enquanto as amostras de sêmen foram submetidas à extração de DNA e posterior PCR (polymerase chain reaction) para detecção de BoHV-1 e 5. Os resultados da VN demostraram que anticorpos contra BoHV-1/5 foram detectados nos touros não vacinados em 66,7% (12/18) das fazendas, 295 (79,5%) touros mostraram-se positivos para BoHV, 287 para BoHV-1 e 234 para BoHV-5; e para 43 touros vacinados, observou-se que 72,1% (31/43) foram negativos na sorologia DNA de BoHV-1/5, detectado no sêmen de três touros: um deles apresentava BoHV-1, outro BoHV-5 e em um foi detectada coinfecção por BoHV-1/5. Todas as amostras positivas para o DNA viral eram provenientes de animais que apresentavam lesões de postite e outras lesões genitais. Esses resultados demonstram que há uma alta soroprevalência de BoHV-1/5 em touros, bem como uma forte evidência de que esses vírus estão circulando ativamente no rebanho bovino dessas fazendas. Um achado interessante foi a detecção de BoHV-1 e 5 em touros com lesões na região do trato genital.(AU)

Identification and inter-relationship analysis of Bradyrhizobium japonicum strains by restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD).

Genomic DNA of 13 Bradyrhizobium japonicum strains was prepared and analysed by restriction fragment length polymorphism (RFLP) with nif and nod probes, and by random amplified polymorphic DNA (RAPD) with 11 primers of arbitrary nucleotide sequence. Polymorphism was observed in both analyses. The RFLP and RAPD banding patterns of different strains were used to calculate genetic divergence and to construct phylogenetic trees, allowing studies on the relationships between the strains. RFLP with nif and nod probes permitted the separation of the strains into two divergent groups, whereas RAPD separated them into four main groups. RAPD allowed closely related strains to be distinguished.

Utilizaçäo de marcadores de DNA para o diagnóstico genômico de animais domésticos: 1. Detecçäo da mutaçäo pontual causadora da deficiência de adesäo de leucócitos bovinos (BLAD) em gado Holandês no Brasil / Use of DNA markers for genomic diagnosis of domestic animals: 1. Detection of point mutation causing bovine leukocyte adhesion deficiency (BLAD) in Brazilian Holstein cattle

Analisaram-se 10 bovinos da raça Holandesa, descendentes da linhagem Ivanhoe. Submeteram-se os DNAs, purificados a partir de leucócitos destes animais, à técnica de Polymerase Chain Reaction (PCR) e posterior digestäo com as enzimas de restriçäo Hae III e Taq I. Estabeleceu-se, desta maneira, o diagnóstico genômico da Deficiência de Adesäo de Leucócitos Bovinos (BLAD). Os exames revelaram que 2 animais eram portadores e 8, normais. A partir do desenvolvimento da metodologia de PCR, tornou-se disponível um método rápido, prático e eficiente para a seleçäo de touros em Centrais de Inseminaçäo Artificial, por meio da detecçäo de animais portadores e normais

Utilizaçäo de marcadores de DNA para o diagnóstico genômico de animais domésticos: 2. Detecçäo da mutaçäo pontual causadora da Paralisia Hipercalêmica Periódica (HYPP) em equinos da raça Quarto de Milha / Use of DNA markers for genomic diagnosis of domestic animals: 2. Detection of point mutation causing Hyperkalemic Periodic Paralysis (HYPP) in Quarter horses

Analisaram-se 12 equinos da raça Quarto de Milha, descendentes da linhagem Impressive. Submeteram-se os DNAs purificados a partir de leucócitos destes animais, à técnica de Polymerase Chain Reaction (PCR) e posterior digestäo com a enzima de restriçäo Taq I. Estabeleceu-se, dessa maneira, o diagnóstico genômico da Paralisia Hipercalcêmica Periódica (HYPP). Os exames revelaram que 9 animais eram portadores heterozigotos (N/H) e 3, normais homozigotos (N/N). A partir do desenvolvimento da metodologia de PCR tornou-se possível diagnosticar o problema e propor maneiras de controle do alastramento desse gene defectivo na populaçäo por meio da detecçäo de animais portadores e normais

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.