BACKGROUND Leptospirosis is the most widespread zoonotic disease. It is caused by infection with pathogenic Leptospira species, of which over 300 serovars have been described. The accurate identification of the causative Leptospira spp. is required to ascertain the pathogenic status of the local isolates. OBJECTIVES This study aimed to obtain the complete genome sequence of a virulent Leptospira interrogans strain isolated from southern Brazil and to describe its genetic features. METHODS The whole genome was sequenced by next-generation sequencing (Ion Torrent). The genome was assembled, scaffolded, annotated, and manually reviewed. Mutations were identified based on a variant calling analysis using the genome of L. interrogans strain Fiocruz L1-130 as a reference. FINDINGS The entire genome had an average GC content of 35%. The variant calling analysis identified 119 single nucleotide polymorphisms (SNPs), from which 30 led to a missense mutation. The structural analyses identified potential evidence of genomic inversions, translocations, and deletions in both the chromosomes. MAIN CONCLUSIONS The genome properties provide comprehensive information about the local isolates of Leptospira spp., and thereby, could facilitate the identification of new targets for the development of diagnostic kits and vaccines.

A previous study by our group reported the isolation and characterisation of Leptospira borgpetersenii serogroup Ballum strain 4E. This strain is of particular interest because it is highly virulent in the hamster model. In this study, we performed whole-genome shotgun genome sequencing of the strain using the SOLiD sequencing platform. By assembling and analysing the new genome, we were able to identify novel features that have been previously overlooked in genome annotations of other strains belonging to the same species.

RESUMO: O objetivo desse estudo foi estimar a soroprevalência da neosporose e os possíveis fatores de risco em rebanhos (Bos taurus taurus) localizados na mesorregião Noroeste do Rio Grande do Sul, Brasil. Foram coletadas 322 amostras de sangue de bovinos leiteiros, em 18 propriedades localizadas em 17 munícipios desta mesorregião. Um questionário epidemiológico foi aplicado em cada propriedade, contendo questões relacionadas às características gerais dos rebanhos, dados reprodutivos e manejo animal. As amostras de soro foram testadas através do teste de imunoensaio enzimático (ELISA) para Neospora caninum. Os resultados demonstraram uma soroprevalência de Neospora de 88,9% (16/18) entre os rebanhos e 31,1% (100/322) entre os indivíduos. Entre os fatores de risco analisados foi observado que descarte por problemas reprodutivos (OR=0,6), presença de áreas alagadiças (OR=0,5) e venda comercial (OR=0,4) estavam associados a soroprevalência. No entanto, a compra de animais substituídos no rebanho desempenhou um papel significativo na ocorrência da doença (OR=2,2). Os resultados desse estudo sugerem que o Neospora caninum esteve presente nos rebanhos estudados, bem como, existem fatores associados com a soroprevalência nas propriedades da mesorregião do Noroeste do Rio Grande do Sul.

ABSTRACT: The aim of this study was to estimate neosporosis seroprevalence and its associated risk factors in milk herds (Bos taurus taurus) located in the northwestern region of Rio Grande do Sul State, Brazil. Three hundred twenty-two blood samples were collected from dairy cows on 18 farms in 17 cities of this region. An epidemiologic questionnaire was completed for each farm. It consisted of questions about the general characteristics of the herd, reproduction, and animal management. Serum samples were tested for Neospora caninum using a commercial enzyme-linked immunosorbent assay (ELISA) kit. Results indicated a seroprevalence of Neospora in 88.9% (16/18) of herds and 31.1% (100/322) of individuals. Risk factor analyses demonstrated that culling by reproductive disorder (OR = 0.6), flooding (OR = 0.5), and commercial sale (OR = 0.4) were associated with seroprevalence. Nevertheless, the purchase of replacement animals in the herd played an important role in disease occurrence (OR = 2.2). Results of this study suggested that Neospora caninum was present in the studied herds under investigation and that there are risk factors associated with its seroprevalence on the farms of the northwestern of Rio Grande do Sul.

BACKGROUND The B subunit of Escherichia coli heat-labile enterotoxin (LTB) is a potent mucosal immune adjuvant. However, there is little information about LTB's potential as a parenteral adjuvant. OBJECTIVES We aimed at evaluating and better understanding rLTB's potential as a parenteral adjuvant using the fused R1 repeat of Mycoplasma hyopneumoniae P97 adhesin as an antigen to characterise the humoral immune response induced by this construct and comparing it to that generated when aluminium hydroxide is used as adjuvant instead. METHODS BALB/c mice were immunised intraperitoneally with either rLTBR1 or recombinant R1 adsorbed onto aluminium hydroxide. The levels of systemic anti-rR1 antibodies (total Ig, IgG1, IgG2a, and IgA) were assessed by enzyme-linked immunosorbent assay (ELISA). The ratio of IgG1 and IgG2a was used to characterise a Th1, Th2, or mixed Th1/Th2 immune response. FINDINGS Western blot confirmed rR1, either alone or fused to LTB, remained antigenic; anti-cholera toxin ELISA confirmed that LTB retained its activity when expressed in a heterologous system. Mice immunised with the rLTBR1 fusion protein produced approximately twice as much anti-rR1 immunoglobulins as mice vaccinated with rR1 adsorbed onto aluminium hydroxide. Animals vaccinated with either rLTBR1 or rR1 adsorbed onto aluminium hydroxide presented a mixed Th1/Th2 immune response. We speculate this might be a result of rR1 immune modulation rather than adjuvant modulation. Mice immunised with rLTBR1 produced approximately 1.5-fold more serum IgA than animals immunised with rR1 and aluminium hydroxide. MAIN CONCLUSIONS The results suggest that rLTB is a more powerful parenteral adjuvant than aluminium hydroxide when administered intraperitoneally as it induced higher antibody titres. Therefore, we recommend that rLTB be considered an alternative adjuvant, even if different administration routes are employed.

BACKGROUND Bovine tuberculosis (TB) is a zoonotic disease caused by Mycobacterium bovis, responsible for causing major losses in livestock. A cost effective alternative to control the disease could be herd vaccination. The bacillus Calmette-Guérin (BCG) vaccine has a limited efficacy against bovine TB, but can improved by over-expression of protective antigens. The M. bovis antigen 85B demonstrates ability to induce protective immune response against bovine TB in animal models. However, current systems for the construction of recombinant BCG expressing multiple copies of the gene result in strains of low genetic stability that rapidly lose the plasmid in vivo. Employing antibiotic resistance as selective markers, these systems also compromise vaccine safety. We previously reported the construction of a stable BCG expression system using auxotrophic complementation as a selectable marker. OBJECTIVES The fundamental aim of this study was to construct strains of M. bovis BCG Pasteur and the auxotrophic M. bovis BCG ΔleuD expressing Ag85B and determine their stability in vivo. METHODS Employing the auxotrophic system, we constructed rBCG strains that expressed M. bovis Ag85B and compared their stability with a conventional BCG strain in mice. Stability was measured in terms of bacterial growth on the selective medium and retention of antigen expression. FINDINGS The auxotrophic complementation system was highly stable after 18 weeks, even during in vivo growth, as the selective pressure and expression of antigen were maintained comparing to the conventional vector. MAIN CONCLUSION The Ag85B continuous expression within the host may generate a stronger and long-lasting immune response compared to conventional systems.

Mycobacterium bovis is the main causative agent of animal tuberculosis (TB) and it may cause TB in humans. Molecular typing of M. bovis isolates provides precise epidemiological data on issues of inter- or intra-herd transmission and wildlife reservoirs. Techniques used for typing M. bovis have evolved over the last 2 decades, and PCR-based methods such as spoligotyping and mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) have been extensively used. These techniques can provide epidemiological information about isolates of M. Bovis that may help control bovine TB by indicating possible links between diseased animals, detecting and sampling outbreaks, and even demonstrating cases of laboratory cross-contamination between samples. This review will focus on techniques used for the molecular typing of M. bovis and discuss their general aspects and applications.

Bovine tuberculosis is a major infectious disease of the cattle. In this study, 85 M. bovis isolates from 162 lymph nodes, obtained from a herd of cattle on a farm in southern Brazil, were evaluated using spoligotyping and VNTR. The strains were grouped into five clusters and five orphans, showing a heterogenic genetic profile, what could represent diverse geographic origins of the introduced cows and/or the frequent movement of cattle between different properties.

Uma mutação no gene que codifica o receptor ryanodine 1 (RYR1), também conhecido como gene do halotano (hal) ou gene do estresse suíno, está associada à Síndrome do Estresse Suíno (PSS). A mutação é geralmente detectada por PCR, a partir da amplificação de um fragmento de 81pb do gene hal, seguida por digestão com a endonuclease de restrição HhaI. O alelo normal (N) é cortado em dois fragmentos, enquanto que o alelo mutado (n) não é digerido pela enzima de restrição. A eletroforese do DNA digerido em gel de agarose corado com brometo de etídio permite a leitura do resultado. A interpretação correta é difícil devido ao pequeno tamanho dos fragmentos. Neste estudo, foi projetado um novo par de iniciadores para a amplificação de um fragmento de 144pb, o que facilita a leitura do resultado. Adicionalmente, foi otimizada a reação de PCR para permitir a amplificação a partir de um único bulbo capilar, acrescentado diretamente na mistura de PCR, sem tratamento prévio. Esse método foi usado para genotipar 165 reprodutores utilizados em granjas produtoras de matrizes. Quarenta e nove porcento dos animais apresentaram genótipo NN, 50% Nn e apenas 1% nn.

A mutation in the gene coding for the ryanodine receptor 1 (RYR1), also known as halothane (hal) gene or swine stress gene, is associated to the porcine stress syndrome (PSS). Detection of the mutation is normally accomplished by PCR amplification of an 81bp fragment of the hal gene, followed by digestion with the HhaI restriction endonuclease. Wild-type allele (N) is cut in two fragments, whereas the mutant allele (n) is not digested by the restriction enzyme. Electrophoresis of the digested DNA on agarose gel and ethidium bromide staining allows the reading of the result. The correct interpretation is difficult due to the small size of the DNA fragments. In this study we designed a new set of primers for amplification of a 144bp fragment that facilitates the reading of the result. In addition, we optimized the PCR reaction to allow amplification from a single hair bulb, added directly into the PCR mix without previous treatment. This improved method was used to genotype 165 sows and boars used in a breeding program. Forty-nine percent of the animals had the NN genotype, whereas 50% were Nn and only 1% was nn.

A Pneumonia Enzoótica Suína (PES), causada pela bactéria fastidiosa Mycoplasma hyopneumoniae, é a doença respiratória mais importante dos suínos, responsável por enormes prejuízos à suinocultura brasileira e mundial. A elevada prevalência e o fato de pré-dispor os suínos à patógenos oportunistas tornam esta doença o alvo central de um programa de saúde de rebanho para doenças respiratórias. O conhecimento das características do agente etiológico bem como dos seus fatores de patogenicidade pode ajudar na elaboração de novas estratégias de controle da PES. O objetivo desta revisão foi discutir alguns aspectos da etiopatogenia da PES que têm implicação na imunoprofilaxia da doença e os principais resultados obtidos com vacinas de última geração avaliadas experimentalmente.

Swine Enzootic Pneumonia (SEP), caused by fastidious bacterium Mycoplasma hyopneumoniae, is the most important respiratory disease of swines, responsible for high losses to Brazilian and worldwide swine breeding. The high prevalence and the fact to predis-pose the swines to secondary pathogens make this disease the central target of a herd health program to respiratory diseases. The knowledge of the characteristics and pathogenicity factors of etiologic agent could help in the development of new strategies to control SEP. The aim of this review was to discuss some aspects of the etiopathogenesis of the SEP that have implications in the immunoprofilaxis of disease and the main results obtained with new generation vaccines evaluated experimentally.

A leptospirose canina é conhecida como enfermidade de Stuttgard desde 1898, sendo os cães, depois dos roedores, considerados como a segunda principal fonte de infecção para o homem. O isolamento de um sorovar patogênico da urina de um cão, laboratorial e clinicamente identificado como tendo leptospirose, e sua utilização para testar amostras de soro de casos de leptospirose humana e canina, evidenciou a sua importância no ecossistema da região sul do Brasil. Os resultados do teste de soroaglutinação microscópica indicaram que 100% das amostras de soro humano de 12 pacientes do banco de soro de 2001 do Centro de Controle de Zoonoses, que haviam reagido com títulos que variaram de 25 a 3.200 para o sorovar canicola, e 72% das amostras de 105 soros caninos do mesmo banco de soro, também reagiram contra o novo isolado. O título médio e mediana dos soros humanos testados com a bateria de antígenos recomendada pela OMS, foi respectivamente 630 e 100, ao passo que os testados com o isolado foi de 1.823 e 400. Nos soros caninos, os títulos foram respectivamente de 347 e 100 para a bateria e de 1.088 e 200 para o isolado.

Canine leptospirosis has been known as Stuttgart disease since 1898, and dogs are considered to be the second principal source of infection in man. The isolation of a pathogenic serovar from dog urine that was diagnosed clinically and laboratorial as having leptospirosis and its utilization to test serological samples of human and canine cases of leptospirosis, has demonstrated its importance to the ecosystem of the southern region of Brazil. The results of the serological microscopic agglutination test indicated that 100% of human serum samples from 12 patients from the serum bank of 2001 at the Center for Control of Zoonoses, that had titers between 25 and 3,200 with the canicola serovar, and 72% of 105 canine serum samples from the same serum bank, also reacted with the new isolate. The mean and median titers of the human serum samples tested with the battery of antigens recommended by WHO was 630 and 100 respectively, and when tested with the isolate it was 1,823 and 400. In the dog sera, the values were respectively 347 and 100 with the battery, and 1,088 and 200 with the isolate.

Colibacilose suína causada por Escherichia coli enterotoxigênica continua sendo um dos principais problemas sanitários na criação de suínos. A tecnologia do DNA recombinante proporciona a possibilidade de desenvolvimento de novas estratégias de imunização. Neste trabalho é descrito o desenvolvimento de uma vacina de subunidade através da produção e purificação da proteína FaeC da fímbria de E. coli K88. O gene que codifica este antígeno foi amplificado por PCR e clonado em um vetor de expressão em E. coli, fusionado a uma cauda de histidinas. A proteína recombinante expressa por esta bactéria foi purificada, e depois de quantificada foi utilizada para imunizar camundongos. Paralelamente a isso, o mesmo gene foi clonado no vetor de expressão em célula eucariótica, introduzindo a seqüência de Kozak para favorecer a tradução deste gene em células musculares. O plasmídio resultante, denominado pUP310, foi produzido em larga escala e também utilizado na imunização de camundongos. A resposta imune induzida por ambas formas de imunizações foi monitorada por ELISA, onde o antígeno utilizado foi a proteína FaeC purificada. Houve indução de resposta imune nos camundongos inoculados com pUP310 e FaeC purificada. Foi possível detectar anticorpos anti-FaeC 42 dias após a primeira inoculação e este título foi aumentando, sendo ainda detectável 7 meses após a primeira inoculação. Conclui-se que pUP310 e FaeC recombinante são candidatos potenciais para imunização de suínos contra E. coli K88.

Swine colibacillosis caused by enterotoxigenic Escherichia coli remains one of the main sanitary problems in pig farms. The recombinant DNA technology offers the possibility of developing new immunization strategies. This paper describes the development of a subunit vaccine through the expression and purification of the E. coli K88 FaeC fimbrial protein. The gene that codes for this antigen was amplified by PCR and cloned into an E. coli expression vector fused to a 6X histidine tag. The recombinant protein was purified by affinity chromatography and used for mice immunization. In parallel, the same gene was cloned into an eucariotic expression vector with the addition of the Kozak sequence for improving translation of this gene in muscle cells. The resulting plasmid named pUP310 was purified in large scale and used to immunize mice. The immune response afforded by both forms of immunization was monitored by ELISA. There was an immune response in mice inoculated with pUP310 and purified FaeC. It was possible to detect anti-FaeC antibodies 42 days after the first inoculation. The antibody titer increased with time, being still detectable 7 months after the first inoculation. It is concluded that recombinant FaeC and pUP310 are potential tools for immunization of swine against E. coli K88.

O gene do estresse suíno (hal) em homozigose recessiva (nn) ocasiona a síndrome do estresse porcino (PSS), e está relacionado com a ocorrência da carne pálida, mole e exudativa (PSE). Em heterozigose (Nn) está associado com diminuição da qualidade de carcaça. Um total de 179 suínos (86 Large White, 69 Landrace, 12 Duroc e 12 Pietrain) foram caracterizados como homozigotos normais (NN), heterozigotos e homozigotos recessivos por análise do DNA usando a reação de polimerização em cadeia (PCR), seguida de um ensaio com endonuclease de restrição. Foi utilizado folículo piloso como fonte de DNA genômico. O produto do PCR foi digerido com a enzima de restrição CfoI, seguindo-se análise dos fragmentos por eletroforese em gel de agarose. Dentre os 179 animais analisados, 126 (70,0%) foram caracterizados como NN, 50 (28,0%) como Nn, e 3 (2,0%) caracterizados como nn. A freqüência de heterozigotos foi maior (P < 0,05) em animais da raça Landrace do que em animais da raça Large White. Nove animais da raça Pietrain foram classificados como heterozigotos e tres como homozigotos recessivos. Estes resultados podem estar relacionados com a incidência de PSS e PSE nestas raças, as quais são largamente utilizadas em programas de cruzamento. A utilização de folículo piloso como fonte de DNA genômico foi um método rápido e não-invasivo que viabiliza a execução da caracterização do gene hal.

The swine stress gene (hal) in recessive homozygotes (nn) leads to porcine stress syndrome (PSS), and is associated with pale, soft, exudative pork (PSE). In heterozygosis (Nn) it is linked to poor carcass quality. A total of 179 pigs (86 Large White, 69 Landrace, 12 Duroc and 12 Pietrain) were characterized as normal homozygotes (NN), heterozygotes or recessive homozygotes following amplification of a target region of the hal gene using the polymerase chain reaction (PCR), followed by a restriction endonuclease assay. Plucked hair was used as a source of genomic DNA. The resulting PCR was digested with the restriction enzyme CfoI, followed by agarose gel electrophoresis. Of 179 animals tested, 70% were NN, 28% were Nn, and 2% were nn. The frequency of heterozygotes was higher (P < 0.05) in Landrace (0.43 for Nn) than in Large White pigs (0.09 for Nn). Nine of the 12 Pietrain animals were Nn and three were nn, suggesting a high frequency for the n allele in this breed. These results may be related to the incidence of PSS and PSE in these two breeds, both of which are widely used in breeding programs. The utilization of plucked hair as the source of genomic DNA was a non-invasive and quick method to screen farm animals.

O gene do estresse suíno foi caracterizado em 59 animais das raças Landrace, Large White, Duroc e cruzas destas raças com a Pietran, através da análise do ácido desoxirribonucléico (DNA) amplificado por reação em cadeia da polimerase (PCR) e clivado com endonuclease de restrição. Como fonte de DNA foi utilizado sangue, sêmen e folículo piloso, sendo que, este último revelou-se um modo rápido e não-invasivo de obtenção de amostra que viabiliza a execução da técnica a qualquer animal dentro do sistema de produção. A freqüência genotípica foi de 0,73 para NN, 0,26 para Nn e 0,01 para nn.

The porcine stress gene was characterized from 59 animals (Landrace, Large White, Duroc and Pietran) through analysis of deoxyribonucleic acid (DNA) amplified by polymerase chain reaction (PCR) and digested with a restriction endonuclease. Blood, semen and plucked hair were used as source of DNA. The use of plucked hair is fast and non-invasive method that can be used for any animal within the production system. The genotype frequency was 0.73 to NN, 0.26 to Nn and 0.01 to nn.