Genetic and antigenic characterization of Brazilian SRLV strains: Natural small ruminant interspecies transmission from mixed herds.

Cross-species transmission events and mixed infection of small ruminant lentiviruses (SRLVs) were studied in seven goats and two sheep from three small ruminant mixed flocks from Northeast and Southeast Brazil. Genetic and antigenic analyses with gag/env genes and ELISA multiepitope SU1/SU5 recombinant antigens were carried out, respectively. The genetic analysis of gag and env sequences showed high viral diversity in both species, MVV-like (subtype A1) and CAEV-like B1 in goats, and CAEV-like (subtype B1) in sheep, revealing SRLV interspecies transmission from sheep to goats and vice versa in Brazilian farms. Two Brazilian caprine lentiviruses were segregated in two new genetic clades based on gag analyses, which suggests a new classification into heterogenic genotype A. Furthermore, goat isolates were grouped into subtype A1 and B1 clusters. Cross-reactive antibodies were detected in goats using ELISA with a recombinant antigen carrying SU1 and SU5 immunodominant epitopes; the results showed anti-CAEV and MVV antibodies in goats and anti-CAEV antibodies in sheep. This result can be associated with the high divergence in the V4 region due to SRLV variability. All results confirm cross-species infection of SRLV in Brazilian mixed herds.

Diagnostic performance of a commercial ELISA used as a complementary test for bovine tuberculosis in two bovine herds with different disease status / Desempenho diagnóstico de um ELISA comercial usado como teste complementar para tuberculose bovina em dois rebanhos bovinos com diferentes estágios de controle da doença

Bovine tuberculosis is a worldwide spread zoonotic disease. Intradermal tuberculinizations are the most used diagnostic tests in the world. Serological tests can be an ancillary diagnosis for bovine tuberculosis. The objective of this study was to evaluate the diagnostic performance of the ELISA Mycobacterium Bovis Antibody Test Kit IDEXX ™ in infected herds, which were in different disease control stages. One hundred and twenty animals from two dairy herds of Minas Gerais state, Brazil, were subjected to the ELISA serological test and the comparative cervical tuberculin test (CCT). Diagnostic test parameters were estimated using Bayesian latent class models and concordance between tests estimated by the frequentist approach. The ELISA test presented lower sensitivity than CCT in both herds. Its sensitivity was higher in the herd in sanitation process. Specificity estimates were above 95% in both herds. Kappa index indicated low concordance or even disagreement between tests. According to the results, the ELISA IDEXX should not be used as substitution for CCT. The tests must not be associated in series. Parallel association increased diagnostic sensitivity in the herd which was in the process of sanitation.(AU)

A tuberculose bovina é uma zoonose de distribuição mundial cujos testes mais utilizados para o diagnóstico são as tuberculinizações intradérmicas, simples e compartivas. Contudo, testes sorológicos podem constituir diagnósticos auxiliares. O objetivo deste estudo foi avaliar o desempenho diagnóstico do teste ELISA Mycobacterium Bovis Antibody Test Kit IDEXX ® em rebanhos bovinos infectados, que se encontravam em diferentes estágios de controle da doença. Cento e vinte animais de dois rebanhos leiteiros provenientes do estado de Minas Geais-Brasil foram submetidos ao ELISA e à tuberculinização cervical compartiva (TCC). Avaliou-se o desempenho dos testes por meio de modelos Bayesianos de classe latente e a concordância entre os eles, por meio de estatística frequentista. Uma maior sensibilidade do teste foi observada no rebanho previamente tuberculinizado. Em ambos os rebanhos o TCC foi mais sensível que o ELISA. Especificidade acima de 95% foi encontrada em ambos os rebanhos. Foram observadas baixa concordância ou mesmo discordância entre os testes. De acordo com os resultados obtidos, o teste ELISA-IDEXX não deve ser utilizado em substituição à TCC, tampouco devem ser associados em série. Houve aumento da sensibilidade quando os testes foram associados em paralelo no rebanho que já se encontrava em processo de saneamento.(AU)

Molecular detection of bovine immunodeficiency virus (BIV) in bovines from the state of Minas Gerais, Brazil / Detecção molecular do vírus da imunodeficiência bovina (BIV) em bovinos do estado de Minas Gerais, Brasil

O vírus da imunodeficiência bovina é o agente causador da imunodeficiência viral bovina que é conhecido por infectar bovinos em todo o mundo. Como em outras infecções por retrovírus, os hospedeiros desenvolvem uma infecção de longo prazo e a maioria dos animais infectados permanece assintomática. O objetivo deste estudo foi detectar DNA proviral BIV em amostras de sangue de bovinos e estimar a ocorrência de infecção no estado de Minas Gerais, Brasil. Amostras de sangue de 391 bovinos foram coletadas de duas regiões do estado, Zona da Mata e Central. O DNA proviral foi detectado por reação em cadeia da polimerase semi-nested (SN-PCR). Os resultados de SN-PCR indicaram uma ocorrência de BIV de 12,5% no estado. Os produtos amplificados foram confirmados como BIV por sequenciamento e a similaridade da sequência de nucleotídeos com a estirpe de referência (R-29) foi de 99%. Este é o primeiro estudo que relata a presença do BIV em Minas Gerais, Brasil. Os resultados indicam a necessidade de realizar um estudo detalhado sobre a prevalência da infecção por BIV no Brasil.(AU)

Genetic variability and phylogeny of the 5' long terminal repeat from Brazilian bovine leukemia virus.

We conducted a phylogenetic analysis of 22 strains of bovine leukemia virus obtained by polymerase chain reaction to amplify a 582-base pair fragment of the transcriptional regulatory region 5' long terminal repeat (LTR). Twenty-two samples of proviral DNA from peripheral blood mononuclear cells containing bovine leukemia virus from naturally infected bovine from 4 distinct geographic regions in Brazil were investigated. The products obtained by polymerase chain reaction were subjected to direct sequencing and sequence alignment. Fragments of 422 nucleotides were obtained, located between positions -118 and +303 base pairs of the 5'LTR. These fragments corresponded to 80% of the LTR region and included 56% of sub-region U3, 100% of R, and 82.5% of U5. Phylogenetic analysis of these sequences showed a high conservation degree in the 5'LTR region, with 5 well defined groups. However, a hotspot occurrence in the R-U5 region was also observed, which contained 40% of all nucleotide variability observed.

Efeitos da umidade relativa do ar na incubação e da idade da matriz leve sobre a eclodibilidade, qualidade dos pintos recém-eclodidos e desempenho da progênie / Effects of relative air humidity in the incubator and breeder hen age on hatchability, hatchling quality and progeny performance

Objetivou-se avaliar os efeitos da umidade relativa do ar nas incubadoras (URI) e da idade da matriz leve sobre a eclodibilidade, qualidade dos pintos recém-eclodidos e o desempenho da progênie. Foram incubados 10.840 ovos, sendo os tratamentos definidos pela idade das matrizes Lohmann LSL(r) (26, 41 e 56 semanas) e pelos níveis de URI (48, 56 e 64%). Após a incubação, 1.620 pintos fêmeas foram alojados em galpão experimental convencional, de acordo com os tratamentos, até as 12 semanas de idade. Para a avaliação de eclodibilidade sobre ovos férteis e fêmeas refugos, o delineamento experimental foi em blocos ao acaso no arranjo em parcelas subdivididas. A parcela foi a URI (três níveis) e a subparcela foi a idade da matriz (três idades). Os blocos foram constituídos pelos andares dos carrinhos de incubação, em que cada andar continha uma repetição de cada idade da matriz. Os nove tratamentos foram constituídos por 14 repetições cada. As repetições foram bandejas de incubação, constituindo 1.204 ovos por tratamento. Para a avaliação do desempenho das frangas, o delineamento experimental foi inteiramente ao acaso, constituído por um esquema fatorial 3x3 (três níveis de URI e três idades), com seis repetições de 30 aves em cada tratamento. A eclodibilidade sobre ovos férteis foi maior (P<0,05) com a utilização de 56% de umidade relativa do ar durante a incubação, independentemente da idade da matriz; e ovos de matrizes mais velhas (56 semanas) apresentaram eclodibilidade menor (P<0,05), independentemente do nível de umidade relativa do ar no interior das incubadoras. Não foram constatados efeitos da idade da matriz e URI sobre o percentual de pintos refugos. A URI ou a idade da matriz leve não afetam o desempenho da progênie.

The aim of this study was to evaluate the effects of relative humidity in incubators (RHI) and breeder hen age on hatchability, hatchling quality and performance of progeny. 10,840 eggs were incubated, and the treatments were defined by the Lohmann LSL(r) breeder age (26, 41 and 56 weeks) and the levels of RHI (48, 56 and 64%). After incubation, 1,620 female chicks were raised in a conventional experimental shed, according to the treatments, until 12 weeks of age. To assess the hatchability of fertile eggs and unviable female chicks, the experimental design was randomized blocks in split plots design. The plot factor was the RHI and the subplot was the age of the breeder hens. The blocks were made by the floors of the incubation carriages, where each floor contained a repetition of each breeder age. The nine treatments consisted of 14 repetitions each. The repetitions were trays of incubation, with 1,204 eggs per treatment. To evaluate the performance of the progeny, the experimental design was completely randomized, consisting of a 3x3 factorial scheme with six replicates of 30 birds in each treatment. Hatchability of fertile eggs was higher (P<0.05) with the use of 56% relative humidity during incubation, regardless of breeder age, and the eggs from the oldest breeder hens (56 weeks) had lower hatchability (P<0.05), regardless of the level of relative humidity inside the incubator. No effects of breeder age and RHI on the percentage of unviable chicks were observed. The RHI or the age of the breeder hen does not affect the performance of progeny.

Characterization of Weissella ceti infections in Brazilian rainbow trout, Oncorhynchus mykiss (Walbaum), farms and development of an oil-adjuvanted vaccine.

Weissella ceti is an emerging bacterial pathogen that affects rainbow trout, Oncorhynchus mykiss (Walbaum), farms. The aims of this study were to genotype W. ceti strains isolated from distinct geographical origins and to determine the efficacy of an oil-adjuvanted vaccine against the disease. Between 2010 and 2012, outbreaks were recorded in five Brazilian farms, and 34 W. ceti isolates were genetically characterized by repetitive extragenic palindromic PCR, enterobacterial repetitive intergenic consensus sequences PCR and pulsed-field gel electrophoresis. Two different W. ceti vaccines were tested: an aqueous-based whole-cell inactivated vaccine (bacterin) and oil-adjuvanted vaccine. Their efficacy was evaluated in rainbow trout at 30 and 60 days post-vaccination (d.p.v.). W. ceti was found to be a highly homogeneous population in Brazil, with clonally related genotypes. Oil-adjuvanted vaccine exhibited the best (P < 0.05) protection against disease, reaching relative percentage survival (RPS)values of 92% at 30 and 60 d.p.v. Bacterin resulted in RPS values of 67% and 58% at day 30 and 60, respectively. The oil-adjuvanted vaccine provided effective protection against W. ceti infection in rainbow trout.

Equilíbrio ácido-básico de embriões e pintos de corte em momentos críticos da incubação e submetidos a diferentes períodos de transferência para o nascedouro / Acid-base balance of embryos and broiler chicks in critical moments of incubation and submitted to different periods of transfer to the hatcher

Foram analisados os níveis de pCO2, pO2, pH e HCO3 - de embriões e pintos de corte provenientes de matrizes novas e velhas, nos momentos anteriores à bicagem interna e externa da casca e ao nascimento, submetidos a diferentes períodos de transferência para as condições ambientais dos nascedouros. Os tratamentos foram definidos pela idade da matriz (33 e 63 semanas) e pela transferência para o nascedouro (aos 15, 17 e 19 dias). O delineamento experimental foi em parcelas subdivididas, sendo a parcela os três momentos de transferência e a subparcela as duas idades das matrizes. Foram utilizadas seis repetições para cada tratamento, e o embrião ou o pinto foi considerado uma repetição. Os dados normais e homogêneos foram submetidos às análises de variância, e as médias comparadas pelos testes F, de Tukey e SNK. Embriões cujos ovos foram transferidos aos 15 dias para o nascedouro apresentaram melhor equilíbrio ácido-básico durante as fases pré-natal e perinatal. Durante o último terço da incubação, independentemente das diferentes condições ambientais às quais os embriões são submetidos e da idade da matriz, há capacidade de resposta fisiológica que permite uma estabilidade no equilíbrio ácido-básico dos pintos após a eclosão...

We analyzed the levels of pCO2, pO2, pH and HCO3 - in embryos and chicks from young and older breeders, in the moments before internal pipping, external pipping and at hatch and submitted to different periods in the environmental conditions of hatchers. The treatments were defined by breeder age (33 and 63 weeks) and transfer to the hatcher (at 15, 17 and 19 days). The experimental design was split plots with three periods in the hatcher and the plots were the two breeder ages. We used six replicates per treatment and the embryo or chick was considered a repetition. The normal data forms were submitted to analysis of variance and means were compared by F, Tukey and SNK tests. Embryos transferred at 15 days to the hatcher had better acid-base balance during the pre-and perinatal phases. During the last third of incubation, regardless of the different environmental conditions which the embryos are subjected and breeder age, there is physiological responsiveness that allows for stability in acid-base balance of the chicks after hatching...

Assessment of messenger RNA (mRNA) of Mycobacterium tuberculosis as a marker of cure in patients with pulmonary tuberculosis.

AIMS: To analyse the performance of RT-qPCR using 85B mRNA in the diagnosis of Mycobacterium tuberculosis infection and in the assessment of the response to treatment for pulmonary tuberculosis (TB). METHODS AND RESULTS: Ninety-eight patients with signs of pulmonary TB were selected: 56 were considered infected with Myco. tuberculosis and they had positive cultures or evident clinical response to anti-TB treatment. Patients with pulmonary tuberculosis were evaluated by culture and RT-qPCR for a 30-day specific treatment. It was found that both tests demonstrated a decline in viable bacilli at 15 and 30 days after the beginning of the therapy in most of the patients. The quantification of the 85B mRNA target was performed in 52 patients who had initially shown positive results by RT-qPCR and who were followed on the days 15 and 30 after the specific treatment. Thus 85B mRNA was detectable in sputum samples in 52 patients with a confirmed diagnosis of pulmonary tuberculosis on day 0. During the specific treatment the 85B mRNA was detectable in 13 patients on day 15 and in only three patients on day 30. CONCLUSIONS: Mycobacterium tuberculosis mRNA in the sputum is a useful prognostic marker and its quantification, an early and reliable indicator for monitoring response to treatment, drug resistance, re-infection and relapse. SIGNIFICANCE AND IMPACT OF THE STUDY: RT-qPCR is a tool that can be used in clinical and therapeutic monitoring as an indicator of bacterial resistance and indicator of the period of transmissibility of Myco. tuberculosis in patients with pulmonary TB undergoing treatment.

Genetic variation of bovine leukemia virus (BLV) after replication in cell culture and experimental animals.

This article reports the selection of bovine leukemia virus (BLV) variants after continuous passage in cell lines or experimental animals. Two wild BLV strains isolated from 2 naturally infected Holstein dairy cows in Brazil (cow codes: 485 and 141) were used for the experimental infection of 1 sheep and FLK cells, and 1 rabbit and CC81 cells. Viral DNA was isolated several months after infection, and env gene nucleotide and amino acid sequences of the "passaged" variants were compared against the 2 original infecting wild strains. The sequences of the original infecting wild strains were not recovered after their replication in the cell lines or experimental animals. These results indicate that genetic variation occurred after BLV replication in vivo and in vitro, with new variants being selected.

Genotyping of Streptococcus dysgalactiae strains isolated from Nile tilapia, Oreochromis niloticus (L.).

Streptococcus dysgalactiae is an emerging fish pathogen that is responsible for outbreaks of disease on fish farms around the world. Recently, this bacterium was associated with an outbreak at a Nile tilapia, Oreochromis niloticus (L.), farm in Brazil. The aim of this study was to evaluate the genetic diversity, best genotyping method and aspects of molecular epidemiology of S. dysgalactiae infections in Nile tilapia farms in Brazil. Twenty-one isolates from four farms located in different Brazilian states were characterized genetically using pulsed-field gel electrophoresis (PFGE), ERIC-PCR, REP-PCR and sodA gene sequencing. The discriminatory power of the different typing methods was compared using Simpson's index of diversity. Identical sodA gene sequences were obtained from all isolates, and ERIC-PCR and REP-PCR were unable to discriminate among the isolates. PFGE typing detected three different genetic patterns between the 21 strains evaluated; thus, it was the best genotyping method for use with this pathogen. The strains from Ceará State were genetically divergent from those from Alagoas State. The S. dysgalactiae isolates analysed in this study constituted a genetically diverse population with a clear association between geographical origin and genotype.

Efeitos do momento de transferência dos ovos para o nascedouro e da idade da matriz pesada sobre o rendimento de incubação / Effects of the moment of egg transfer to the hatcher and broiler breeder age on the incubation yield

Foram avaliados os efeitos do momento de transferência dos ovos da incubadora para o nascedouro e da idade da matriz pesada sobre o rendimento de incubação. Os tratamentos foram definidos pela idade das matrizes Cobb®, sendo considerados ovos de matrizes novas (33 semanas) e ovos de matrizes velhas (63 semanas), e pelos momentos de transferência da câmara de incubação para os nascedouros (aos 15, 17 e 19 dias). O delineamento experimental foi inteiramente ao acaso em parcelas subdivididas, sendo o fator da parcela os três momentos das transferências e as subparcelas as duas idades das matrizes, com 18 repetições por tratamento. A bandeja com 96 ovos foi considerada a repetição, totalizando 1.728 ovos para cada tratamento. Ovos de matrizes velhas são mais pesados, perdem mais peso durante a incubação e têm percentual de fertilidade menor (P<0,05), qualquer que seja o momento de transferência dos ovos. Independentemente da idade das matrizes, a transferência dos ovos da incubadora para o nascedouro aos 15, 17 ou 19 dias de incubação não interfere na mortalidade embrionária, eclodibilidade e peso dos pintos ao nascimento. É possível adaptar o manejo de transferência à necessidade logística do incubatório ou reduzir o período dos ovos na máquina de incubação para aumentar o fluxo de produção, sem prejuízo ao rendimento de incubação.

We evaluated the effects of the moment of egg transfer to the hatcher and broiler breeder age on the incubation yield. The treatments were defined by Cobb ® broiler breeder age, separating young breeders (33 weeks old) from older breeders (63 weeks old) and the moment of transfer from incubation chamber to the hatcher (at 15, 17 and 19 days). The experimental design was completely randomized split plot. The plots factor was the three times of transfers and the subplots the two breeder ages, with 18 replicates per treatment. A tray of 96 eggs was considered replay, totaling 1728 eggs for each treatment. Eggs from older breeders are heavier, lose more weight during incubation and fertility rate is lower (p<0.05), regardless of the moments of eggs transfer. Regardless of the breeder age, the transfer of eggs from the incubator to the hatcher at 15, 17 or 19 days of incubation does not interfere in embryonic mortality, hatchability and chick weight at hatch. You can adapt the transfer management to the logistic needs of the hatchery or reduce the period of egg incubation in the machine to increase the flow of production with no losses in incubation yield.

Comparação dos índices de eficiência reprodutiva por diferentes métodos em rebanhos bovinos leiteiros / Comparison of reproductive efficiency indexes by different methods in dairy cattle herds

O presente estudo teve como objetivo promover o levantamento de índices zootécnicos para calcular e comparar a eficiência reprodutiva de rebanhos bovinos leiteiros por diferentes métodos. O intervalo de partos dos rebanhos estudados apresentou uma variação de 13,3 a 15,2 meses por meio dos diferentes métodos utilizados para sua obtenção, não se observando diferença significativa (P>0,05) entre eles. As avaliações pelos métodos de Johnson et al. (1964) e Butendieck et al. (1972) mostraram, respectivamente, eficiência reprodutiva de 79,5 e 77,8%. Embora considerados métodos mais eficientes que o de Willcox et al. (1957), cuja média encontrada para os mesmos rebanhos foi de 90,2%, não se observou diferença significativa entre os resultados obtidos pelos três métodos.

The study aimed to promote the lifting of indexes to calculate and compare the reproductive efficiency of dairy cattle herds by different methods. The parturition interval studied showed a variation from 13.3 to 15.2 months by means of different methods for obtaining it are not observing a significant difference (P> 0.05) between them. Evaluations by the methods of Johnson et al (1964) and Butendieck et al. (1972) showed, respectively, reproductive efficiency of 79.5 and 77.8%. Although regarded as the most efficient methods of Willcox et al. (1957), whose average found in the same herd was 90.2%, there was no significant difference between the results obtained by three methods.

Economic impact of Rhipicephalus (Boophilus) microplus: estimate of decreased milk production on a dairy farm / Impacto econômico de Rhipicephalus (Boophilus) microplus: estimativa de redução de produção de leite

A redução da produção de leite em bovinos é um dos aspectos majoritários na composição total do valor de impacto econômico provocado por R. (B.) microplus. Portanto, o objetivo deste estudo foi estimar a perda decorrente da carga parasitária, observada em uma propriedade produtora de leite na região central de Minas Gerais. Para tal, foram consideradas 30 contagens de carrapatos, distribuídas de maneira uniforme durante o período de um ano, em um rebanho bovino com 74 vacas de composição racial 3/4 Holandês x Zebu, mantidas sob sistema de produção intensivo em pastoreio. A partir dos dados observados, foi possível estimar que 937.968 teleóginas completaram o repasto sanguíneo e foram responsáveis pela redução de 6.678 litros de leite durante o período avaliado. Esse valor corresponde a uma redução de 2,7% da produção, média de 90,24 litros por vaca em lactação.

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.

Hepatic photosensitization in buffaloes intoxicated by Brachiaria decumbens in Minas Gerais state, Brazil.

The aim of this paper is to report the study of hepatogenous photosensitization in buffaloes during two outbreaks provoked by ingestion of Brachiaria decumbens in Minas Gerais state, Brazil. Ten young buffaloes in outbreak 1 and seven buffaloes in outbreak 2 were intoxicated by B. decumbens. Nine clinically healthy buffaloes raised under the same conditions as the sick animals served as the control group. All animals were subjected to clinical examination, and serum was collected to measure gamma-glutamyl transferase (GGT), aspartate aminotransferase (AST), direct bilirubin (DB), indirect bilirubin (IB) and total bilirubin (TB) as indicators of liver function and urea and creatinine as indicators of renal function. Histopathology of liver fragments from five different animals was carried out. During the outbreaks and every two months for one year, samples of grass from paddocks where the animals got sick were collected for quantitative evaluation of the saponin protodioscin, combined with observations of pasture characteristics and daily rainfall. Clinical signs included apathy, weight loss, restlessness, scar retraction of the ears and intense itching at the skin lesions, mainly on the rump, the tail head, neck and hindlimbs, similar to the signs observed in other ruminants. Only the GGT enzyme presented significantly different (P < 0.01) serum levels between intoxicated animals (n = 17) and healthy animals (n = 9), indicating liver damage in buffaloes bred in B. decumbens pastures. Microscopy of the liver showed foamy macrophages and lesions of liver disease associated with the presence of crystals in the bile ducts, which have also been found in sheep and cattle poisoned by grasses of the genus Brachiaria. During the outbreaks, protodioscin levels were higher than 3%, and shortly after, these levels were reduced to less than 0.80%, suggesting a hepatic injury etiology. The outbreaks took place at the beginning of the rainy season, and there was a positive correlation between saponin and the amount of rainfall, as well as between saponin and the amount of green leaves in the pasture. These findings indicate that the grass was more toxic in this period. This is the first report of photosensitization by B. decumbens in buffalo.

Genetic diversity and new genotyping scheme for fish pathogenic Streptococcus agalactiae.

UNLABELLED: This study aimed to assess the genetic diversity of fish isolates of Streptococcus agalactiae by capsular serotyping, MLST and the pattern of selected virulence genes. Forty-six isolates from Nile tilapia and Amazon catfish were screened by PCR for the twelve virulence genes. The molecular capsular type and sequence type (ST) were determined. Two capsular types (Ia and Ib) and four STs (103, 260, 552 and 553) were identified. The ST-552 and ST-553 represent new allelic combinations. Variable results were found for the genes gbs2018-6, lmb, hylB and cylE. The combined evaluation of serotype, sequence type and pattern of the presence or absence of cylE and hylB allowed the classification of isolates into nine genetic profiles (I-IX). The proposed scheme showed higher discriminatory power and was able to detect evolutionary events missed by MLST analysis. This study provides new information about the genetic diversity of fish pathogenic Strep. agalactiae, and the proposed scheme was shown to be an improved approach to genotyping these strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This study showed that critical genetic events in Streptococcus agalactiae isolates pathogenic for fish have been missed by serotyping and multilocus sequence typing (MLST). A proposed genotyping scheme based on the evaluation of concatenated data from serotyping, MLST, and the presence/absence of virulence genes was created, and this was able to detect old and recent evolutionary events. It provided a better understanding of the genetic diversity of Strep. agalactiae populations from fish and will contribute to future studies of the molecular epidemiology, pathogenesis and evolutionary aspects of this pathogen.

PCR em tempo real para detecção do vírus da doença de Aujeszky / Real time PCR for detection of Aujeszky's disease virus

O objetivo deste trabalho foi desenvolver uma PCR em tempo real (qPCR) para o diagnóstico rápido e sensível da doença de Aujeszky. Os iniciadores amplificaram um fragmento de 123 pares de base do gene codificante da glicoproteína D. A qPCR foi testada em 25 amostras de cérebro de suíno positivas e 85 amostras negativas para DA no isolamento viral e na soroneutralização. A sensibilidade analítica foi calculada com acréscimo de um isolado brasileiro do SuHV-1 titulado em amostras de cérebro de suíno negativas na soroneutralização e na PCR. A técnica apresentou sensibilidade analítica de 10-0,5 TCID50/50µL. A qPCR foi capaz de distinguir reações inespecíficas devido a dímero de oligonucleotídeos iniciadores ou amplificações, além do alvo designado (evitando, assim, os falso-positivos), e de obter resultados rápidos.

The aim of this study was to validate a low-cost real-time PCR for a quick and sensitive diagnosis of the disease. The fluorofore used was a DNA intercalating agent, one of the cheaper detection systems. Primers amplified a 123 base pairs fragment of the gene coding for glycoprotein D. PCR was tested on 25 samples of pig brain positive for AD and 85 samples negative in viral isolation and serum neutralization. The detection limit was calculated on samples of pig brain contaminated with a Brazilian isolate of SuHV-1. The technique had a detection limit of 10-0,5 TCID50/50µL. PCR was able to distinguish nonspecific reactions due to primer dimers (thus avoiding false positives) and get faster results.

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.

Pseudorabies virus can be classified into five genotypes using partial sequences of UL44

Suid herpesvirus 1 (SuHV-1) is the causative agent of pseudorabies (PR), a disease of great importance due to the huge losses it causes in the swine industry. The aim of this study was to determine a method for genotyping SuHV-1 based on partial sequences of the gene coding for glycoprotein C (gC) and to elucidate the possible reasons for the variability of this region. A total of 109 gCsequences collected from GenBank were divided into five major groups after reconstruction of a phylogenetic tree by Bayesian inference. The analysis showed that a portion of gC (approximately 671 bp) is under selective pressure at various points that coincide with regions of protein disorder. It was also possible to divide SuHV-1 into five genotypes that evolved under different selective pressures. These genotypes are not specific to countries or continents, perhaps due to multiple introduction events related to the importation of swine.