Leptospirosis in dairy cattle from southern Brazil - risk factors

Background: Cattle are susceptible to chronic leptospirosis infection, that results in reduced milk production and reproductive disorders such as abortions, stillbirths, fetal malformation, and mummified fetuses, causing significant economic losses.Commercially available vaccines against leptospirosis offer limited protection to cattle because they contain only the mostprevalent serovars worldwide, even though they are not prevalent in the specific region. This study aimed to evaluate theprevalence of specific antibodies against Leptospira serogroups, reproductive disorders and the risk factors in dairy herdsfrom different mesoregions of Rio Grande do Sul State, Southern Brazil.Materials, Methods & Results: An epidemiological survey was conducted, and serum samples from the bovine population representative of three mesoregions (MR1, MR2, and MR3) were studied; the samples were collected and tested forleptospirosis using the microscopic agglutination test (MAT) for 12 serogroups checking for the presence of agglutination.A total of 442 blood samples were collected from dairy cattle from November to December 2019 (MR1, 187; MR2, 88;and MR3, 167), including cows vaccinated with different commercial vaccines during the three months before sample collection (n = 295) and non-vaccinated against leptospirosis (n = 147). At the time of collection, an interview was conductedwith the owners with questions about the health of the animals, management, habitat, feeding and reproduction. Chi-squaretests univariate analysis with the SPSS® version 20.0 were performed to estimate the association of serogroup Djasimanseroreactivity with the occurrence of reproductive problems and related risk factors. The mean prevalence of antibodiesagainst leptospires was 78.7% (MR1, 74.9 %; MR2, 84.1 %; and MR3, 80.2 %). Serogroup prevalence was different in...

Occurrence of Mycoplasma hyopneumoniae in slaughter pigs from Southern Brazil / Ocorrência de Mycoplasma hyopneumoniae em amostras de pulmão de suínos obtidas de frigorífico do sul do Brasil

Mycoplasma hyopneumoniae is the causative agent of enzootic pneumonia (EP), a disease that is highly prevalent and globally distributed, causing significant economic losses to the swine industry. Disease progression is characterized by reduced feed conversion and the development of lung lesions. Considering the limited information about the epidemiology of EP in Southern Brazil, the main objective of this study was to determine the occurrence of M. hyopneumoniae in swine lung samples and to evaluate the scores of lung lesions caused by local strains. A total of 120 samples was randomly collected and processed. DNA was extracted from lung tissue to perform nested-PCR and lungs were inspected to evaluate the presence of the pneumonia-like gross lesions of M. hyopneumoniae. The results showed 95.8% positive samples, while the lung lesion score analysis showed suggestive lesions in 60% of samples. The detection of positive samples in nested-PCR was associated with the presence of pneumonia-like gross lesions (P < 0.01). The results demonstrate a high occurrence of EP in slaughter pigs from southern Brazil.(AU)

O Mycoplasma hyopneumoniae é o agente causador da Pneumonia Enzoótica Suína (PES), doença altamente prevalente e mundialmente distribuída, causando grandes perdas econômicas para a indústria suinícola. A progressão da doença é caracterizada pela redução das taxas de conversão alimentar e o desenvolvimento de lesões pulmonares. Visto que há informação limitada sobre a epidemiologia da PES no sul do Brasil, o objetivo do presente trabalho foi determinar a prevalência de M. hyopneumoniae em amostras de pulmão suíno e avaliar o score de lesões pulmonares causadas pelas cepas locais. Um total de 120 amostras foram coletadas aleatoriamente, processadas e analisadas. O DNA foi extraído do tecido pulmonar para realização de Nested-PCR e os pulmões foram inspecionados para presença de lesões macroscópicas sugestivas de M. hyopneumoniae. Os resultados demonstraram 95,8% das amostras positivas para o patógeno. A análise do score pulmonar mostrou lesões sugestivas da PES em 60% das amostras. A detecção de amostras positivas no Nested-PCR foi associada com a presença de lesões sugestivas (P < 0.01). Os dados obtidos neste trabalho demonstram a alta prevalência da PES em granjas do RS.(AU)

Occurrence of Mycoplasma hyopneumoniae in slaughter pigs from Southern Brazil / Ocorrência de Mycoplasma hyopneumoniae em amostras de pulmão de suínos obtidas de frigorífico do sul do Brasil

Mycoplasma hyopneumoniae is the causative agent of enzootic pneumonia (EP), a disease that is highly prevalent and globally distributed, causing significant economic losses to the swine industry. Disease progression is characterized by reduced feed conversion and the development of lung lesions. Considering the limited information about the epidemiology of EP in Southern Brazil, the main objective of this study was to determine the occurrence of M. hyopneumoniae in swine lung samples and to evaluate the scores of lung lesions caused by local strains. A total of 120 samples was randomly collected and processed. DNA was extracted from lung tissue to perform nested-PCR and lungs were inspected to evaluate the presence of the pneumonia-like gross lesions of M. hyopneumoniae. The results showed 95.8% positive samples, while the lung lesion score analysis showed suggestive lesions in 60% of samples. The detection of positive samples in nested-PCR was associated with the presence of pneumonia-like gross lesions (P < 0.01). The results demonstrate a high occurrence of EP in slaughter pigs from southern Brazil.(AU)

O Mycoplasma hyopneumoniae é o agente causador da Pneumonia Enzoótica Suína (PES), doença altamente prevalente e mundialmente distribuída, causando grandes perdas econômicas para a indústria suinícola. A progressão da doença é caracterizada pela redução das taxas de conversão alimentar e o desenvolvimento de lesões pulmonares. Visto que há informação limitada sobre a epidemiologia da PES no sul do Brasil, o objetivo do presente trabalho foi determinar a prevalência de M. hyopneumoniae em amostras de pulmão suíno e avaliar o score de lesões pulmonares causadas pelas cepas locais. Um total de 120 amostras foram coletadas aleatoriamente, processadas e analisadas. O DNA foi extraído do tecido pulmonar para realização de Nested-PCR e os pulmões foram inspecionados para presença de lesões macroscópicas sugestivas de M. hyopneumoniae. Os resultados demonstraram 95,8% das amostras positivas para o patógeno. A análise do score pulmonar mostrou lesões sugestivas da PES em 60% das amostras. A detecção de amostras positivas no Nested-PCR foi associada com a presença de lesões sugestivas (P < 0.01). Os dados obtidos neste trabalho demonstram a alta prevalência da PES em granjas do RS.(AU)

Challenges on the follow-up experimental leptospiral infection in sheep

Background: Leptospirosis is currently a source of significant economic losses in the agribusiness; as such, experimentalstudies on this infection are required to develop a better understanding of the pathogenesis, treatment, and immunoprophylaxisof the disease. Sheep may represent a good model for ruminants in such models. Despite the extent of the studies that hasbeen conducted thus far, researchers have yet to reach a consensus on the experimental practices to apply for leptospirosisin this animal species, and several gaps in understanding remain. To bridge these gaps, the present study aimed to assessthe usage of several tools for the monitoring of experimental leptospirosis in sheep.Materials, Methods & Results: Twelve Santa Ines sheep of different ages were each allocated to one of four groups (A, B,C, and D). The subjects in groups A, B, and C received different doses of Leptospira interrogans serogroup Icterohemorrhagiae by intraperitoneal route, 1x102, 1x105, and 1x108 respectively. Group D was the control. Hematological, biochemicaland clinical parameters were evaluated daily. Serology by microscopic agglutination test (MAT) and PCR were performedto evaluate the infection status. The most remarkable clinical signs were fever (41ºC) and dehydration, and acute pain(cub). Two animals from Group C presented leukocytosis. Only those in Group C exhibited positive results according toserology, while positivity in PCR was observed in animals in groups A and C. The results of the experiment indicated thatsheep may be experimentally infected and can, therefore, be used as a model for leptospirosis in ruminants. Clinical signscannot be considered to represent a reliable parameter for evaluating the development of leptospirosis in experimentallyinfected sheep. We recommend the use of urine PCR and serology to confirm the infection in...

Challenges on the follow-up experimental leptospiral infection in sheep

Background: Leptospirosis is currently a source of significant economic losses in the agribusiness; as such, experimentalstudies on this infection are required to develop a better understanding of the pathogenesis, treatment, and immunoprophylaxisof the disease. Sheep may represent a good model for ruminants in such models. Despite the extent of the studies that hasbeen conducted thus far, researchers have yet to reach a consensus on the experimental practices to apply for leptospirosisin this animal species, and several gaps in understanding remain. To bridge these gaps, the present study aimed to assessthe usage of several tools for the monitoring of experimental leptospirosis in sheep.Materials, Methods & Results: Twelve Santa Ines sheep of different ages were each allocated to one of four groups (A, B,C, and D). The subjects in groups A, B, and C received different doses of Leptospira interrogans serogroup Icterohemorrhagiae by intraperitoneal route, 1x102, 1x105, and 1x108 respectively. Group D was the control. Hematological, biochemicaland clinical parameters were evaluated daily. Serology by microscopic agglutination test (MAT) and PCR were performedto evaluate the infection status. The most remarkable clinical signs were fever (41ºC) and dehydration, and acute pain(cub). Two animals from Group C presented leukocytosis. Only those in Group C exhibited positive results according toserology, while positivity in PCR was observed in animals in groups A and C. The results of the experiment indicated thatsheep may be experimentally infected and can, therefore, be used as a model for leptospirosis in ruminants. Clinical signscannot be considered to represent a reliable parameter for evaluating the development of leptospirosis in experimentallyinfected sheep. We recommend the use of urine PCR and serology to confirm the infection in... (AU)

Atividade bactericida e bacteriostática da casca do pequi (Caryocar brasiliense) em bactérias deteriorantes e patogênicas isoladas de alimentos / Bactericidal and bacteriostatic activity of pequi (Caryocar brasiliense) against deteriorant and pathogenic bacteria from foods

O pequi é uma fruta nativa do Cerrado brasileiro que apresenta elevado teor de compostos antioxidantes e pode apresentar ação antimicrobiana. Dessa forma, objetivou-se com o presente trabalho avaliar a atividade bacteriostática e bactericida do extrato da casca do pequi. Das cepas estudadas 5 são padrões cedidos pela FIOCRUZ e 5 são bactérias de alta resistência a antimicrobianos isoladas de carne e frango. Foi determinada a concentração inibitória mínima (CIM) e concentração bactericida mínima do extrato da casca do pequi. O extrato da casca do pequi apresenta atividade antimicrobiana frente a bactérias patogênicas e deteriorantes. C. jejuni e C. colisão as mais sensíveis. S. aureus apresentou tolerância. E. coli O157:H7 e E. coli são menos sensíveis. O resíduo do pequi é uma potencial e importante fonte de antimicrobianos naturais.(AU)

Atividade bactericida e bacteriostática da casca do pequi (Caryocar brasiliense) em bactérias deteriorantes e patogênicas isoladas de alimentos / Bactericidal and bacteriostatic activity of pequi (Caryocar brasiliense) against deteriorant and pathogenic bacteria from foods

O pequi é uma fruta nativa do Cerrado brasileiro que apresenta elevado teor de compostos antioxidantes e pode apresentar ação antimicrobiana. Dessa forma, objetivou-se com o presente trabalho avaliar a atividade bacteriostática e bactericida do extrato da casca do pequi. Das cepas estudadas 5 são padrões cedidos pela FIOCRUZ e 5 são bactérias de alta resistência a antimicrobianos isoladas de carne e frango. Foi determinada a concentração inibitória mínima (CIM) e concentração bactericida mínima do extrato da casca do pequi. O extrato da casca do pequi apresenta atividade antimicrobiana frente a bactérias patogênicas e deteriorantes. C. jejuni e C. colisão as mais sensíveis. S. aureus apresentou tolerância. E. coli O157:H7 e E. coli são menos sensíveis. O resíduo do pequi é uma potencial e importante fonte de antimicrobianos naturais.

Protection efficacy of the rLTB-R1 chimera against experimental swine mycoplasmal pneumonia

Background: Mycoplasma hyopneumoniae is the etiological agent of the Swine Mycoplasmal Pneumonia (SMP), one ofthe most economically significant diseases in the swine industry worldwide. Commonly used vaccines for SMP controlconsist of inactivated whole cells (bacterins). These vaccines are efficacious against M. hyopneumoniae challenge, but donot prevent colonization by the pathogen or completely eliminate pneumonia. P97 adhesin is conserved in the M. pneumoniae virulent strains, therefore it is an attractive target to be used in recombinant vaccines against M. hyopneumoniae.The aim of the present study was to evaluate protection afforded by rLTB-R1, a recombinant chimera composed by LTBfused with the R1 repeat region of P97 adhesin of M. hyopneumoniae, in specific-pathogen-free (SPF) piglets vaccinatedby intranasal or intramuscular route and challenged with a pathogenic strain of M. hyopneumoniae.Materials, Methods & Results: PCR products of the LTB and R1 coding sequences were fused, then cloned into pETDEST42™ expression vector. The rLTB-R1 was expressed in Escherichia coli BL21 (DE3) Salt induction (SI). The pigletswere divided into three groups: four piglets were intranasally vaccinated with 1 mg of rLTB-R1 solubilized in 1 mL of PBSat 0 and 14 days (IN rLTB-R1 group); four piglets were intramuscularly vaccinated with 1 mg of rLTB-R1 solubilized in 1mL of PBS at 0 and 14 days (IM rLTB-R1 group); three piglets were intranasally and intramuscularly inoculated with 1 mLof PBS (control group). Two weeks after the last immunization (28 day), piglets were intratracheally challenged with 10 mLof a suspension containing 109 color-changing unit (CCU) of pathogenic M. hyopneumoniae 7448 strain on three consecutivedays. Until the challenge (28 days), intranasal and intramuscular vaccination with rLTB-R1 induced seroconversions of antiR1 systemic antibodies of 1.6 and 4.6 ×, respectively. The IN rLTB-R1...(AU)

Protection efficacy of the rLTB-R1 chimera against experimental swine mycoplasmal pneumonia

Background: Mycoplasma hyopneumoniae is the etiological agent of the Swine Mycoplasmal Pneumonia (SMP), one ofthe most economically significant diseases in the swine industry worldwide. Commonly used vaccines for SMP controlconsist of inactivated whole cells (bacterins). These vaccines are efficacious against M. hyopneumoniae challenge, but donot prevent colonization by the pathogen or completely eliminate pneumonia. P97 adhesin is conserved in the M. pneumoniae virulent strains, therefore it is an attractive target to be used in recombinant vaccines against M. hyopneumoniae.The aim of the present study was to evaluate protection afforded by rLTB-R1, a recombinant chimera composed by LTBfused with the R1 repeat region of P97 adhesin of M. hyopneumoniae, in specific-pathogen-free (SPF) piglets vaccinatedby intranasal or intramuscular route and challenged with a pathogenic strain of M. hyopneumoniae.Materials, Methods & Results: PCR products of the LTB and R1 coding sequences were fused, then cloned into pETDEST42™ expression vector. The rLTB-R1 was expressed in Escherichia coli BL21 (DE3) Salt induction (SI). The pigletswere divided into three groups: four piglets were intranasally vaccinated with 1 mg of rLTB-R1 solubilized in 1 mL of PBSat 0 and 14 days (IN rLTB-R1 group); four piglets were intramuscularly vaccinated with 1 mg of rLTB-R1 solubilized in 1mL of PBS at 0 and 14 days (IM rLTB-R1 group); three piglets were intranasally and intramuscularly inoculated with 1 mLof PBS (control group). Two weeks after the last immunization (28 day), piglets were intratracheally challenged with 10 mLof a suspension containing 109 color-changing unit (CCU) of pathogenic M. hyopneumoniae 7448 strain on three consecutivedays. Until the challenge (28 days), intranasal and intramuscular vaccination with rLTB-R1 induced seroconversions of antiR1 systemic antibodies of 1.6 and 4.6 ×, respectively. The IN rLTB-R1...

Seroprevalence estimate and associated risk factors for neosporosis in dairy cattle in the northwest region of Rio Grande do Sul State, Brazil / Estimativa da soroprevalência e fatores de risco para neosporose em rebanhos leiteiros na região Noroeste do estado do Rio Grande do Sul, Brasil

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.(AU)

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.(AU)

Molecular characterization of the first leptospires isolated from goats in Brazil

Two Leptospira sp. isolates were obtained by the first time from goats in Brazil and characterized by sequencing rrs, rpoB and secY genes, PFGE and typing with monoclonal antibodies. Both isolates are identical and belong to Leptospira santarosai. Analysis of the rrs and the rpoB genes sequences revealed 100% identity between the goat isolates and the Bananal reference strain. When secY sequences of the two isolates were compared to each other, it was observed that they had identical sequences. However, when compared to that of the Bananal reference strain, there were 15 mismatches along the 549 bp secY sequence. In conclusion, molecular methods are increasingly useful for the characterization of leptospires and allowed to identify those isolates of caprine origin as closely related but not identical to serovar Bananal, and constitute a new type named Carioca.

Molecular typing of Mycobacterium bovis isolates: a review

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.

Molecular typing of Mycobacterium bovis isolated in the south of Brazil

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.

Prokaryotic expression of a truncated form of bovine herpesvirus 1 glycoprotein E (gE) and its use in an ELISA for gE antibodies / Expressão procariota de uma forma truncada da glicoproteína E (gE) do herpesvírus bovino tipo 1 e uso em ELISA para anticorpos contra a gE

This article describes the expression of a truncated form of bovine herpesvirus 1 (BoHV-1) glycoprotein E (gE) for use as immunodiagnostic reagent. A 651 nucleotide fragment corresponding to the amino-terminal third (217 amino acids) of BoHV-1 gE - that shares a high identity with the homologous BoHV-5 counterpart - was cloned as a 6×His-tag fusion protein in an Escherichia coli expression vector. A soluble protein of approximately 25 kDa purified from lysates of transformed E. coli was recognized in Western blot (WB) by anti-6xHis-tag and anti-BoHV-1 gE monoclonal antibodies. In addition, the recombinant protein was specifically recognized in WB by antibodies present in the sera of cattle seropositive to BoHV-1 and BoHV-5. An indirect ELISA using the expressed protein as coating antigen performed comparably to a commercial anti-gE ELISA and was able to differentiate serologically calves vaccinated with a gE-deleted BoHV-5 strain from calves infected with BoHV-1. Thus, the truncated gE may be useful for serological tests designed to differentiate BoHV-1/BoHV-5 infected animals from those vaccinated with gE-negative marker vaccines.(AU)

Este trabalho relata a expressão de uma forma truncada da glicoproteína E (gE) do herpesvírus bovino tipo 1 (BoHV-1) para uso em imunodiagnóstico. Um fragmento de 651 pares de bases (pb) correspondente ao terço amino-terminal (217 aminoácidos) da gE do BoHV-1 - que compartilha uma alta identidade com a gE do BoHV-5 - foi clonada como proteína de fusão com cauda 6x de histidina em um vetor de expressão em Escherichia coli. Uma proteína solúvel de aproximadamente 25 kDa purificada de lisados de E.coli foi reconhecida em Western blot (WB) por anticorpos monoclonais anti-6xHis-tag e anti-gE. Além disso, a proteína recombinante purificada foi reconhecida em WB por anticorpos presentes no soro de animais soropositivos ao BoHV-1 e BoHV-5. Um ELISA indireto utilizando a proteína recombinante como antígeno apresentou performance comparável a um ELISA gE comercial e foi capaz e diferenciar sorologicamente animais vacinados com uma cepa gE-negativa de BoHV-5 de animais infectados com o BoHV-1. Portanto, a gE truncada pode ser útil em testes sorológicos diferenciais para uso conjunto com vacinas com marcador antigênico gE para o BoHV-1 e BoHV-5.(AU)

Characterization of a virulent Leptospira interrogans strain isolated from an abandoned swimming pool

Pathogenic Leptospira spp. are the etiological agents of leptospirosis, an important disease of both humans and animals. In urban settings, L. interrogans serovars are the predominant cause of disease in humans. The purpose of this study was to characterize a novel Leptospira isolate recovered from an abandoned swimming pool. Molecular characterization through sequencing of the rpoB gene revealed 100% identity with L. interrogans and variable-number tandem-repeat (VNTR) analysis resulted in a banding pattern identical to L. interrogans serogroup Icterohaemorrhagiae, serovar Copenhageni or Icterohaemorrhagiae. The virulence of the strain was determined in a hamster model of lethal leptospirosis. The lethal dose 50% (LD50) was calculated to be two leptospires in female hamsters and a histopathological examination of infected animals found typical lesions associated with severe leptospirosis, including renal epithelium degeneration, hepatic karyomegaly, liver-plate disarray and lymphocyte infiltration. This highly virulent strain is now available for use in further studies, especially evaluation of vaccine candidates.(AU)

An improved method for characterization of the mutation associated to porcine stress syndrome by PCR amplification followed by restriction analysis

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.

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.

An improved method for characterization of the mutation associated to porcine stress syndrome by PCR amplification followed by restriction analysis

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.

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.

PREVALENCE OF SHIGA TOXIN-PRODUCING Escherichia coli IN SOUTHERN BRAZIL ISOLATED FROM GROUND BEEF AND RAW MILK / PREVALÊNCIA DE Escherichia coli PRODUTORA DE SHIGA TOXINAS ISOLADA DE CARNE MOÍDA E LEITE CRU NO SUL DO BRASIL

The aim of this work was to investigate the occurrence of shiga toxin-producing Escherichia coli (STEC) in ground beef and raw milk in Southern Brazil, and to study the fate of STEC isolates from cattle faeces in ground beef and milk, and its resistance in acid and alcoholic media. Among 464 E. coli isolated from ground beef and raw milk there were no stx1 and stx2 genes. The population of STEC isolates from cattle faeces was quite stable when inoculated in ground beef and increased in inoculated milk along the 120 hours of storage at 8oC.  These STEC isolates were inactivated when exposed to pH 2.5 and 3.0, but they were viable after eight hours at pH 4.0. The STEC isolates did not survive 48 hours in medium containing 12% ethanol. At 6% ethanol, STEC O174:H21, O163:H19 and O112:H2 have shown an increase in population and STEC O91:H21 and O22:H8 did not resist beyond 24 and 48 hours of incubation, respectively. The low prevalence of STEC in foods together with the attributes of the STEC found in Brazilian cattle could be among the reasons for the low prevalence of foodborne diseases caused by STEC in Brazil.KEY WORDS: Acid, ethanol, ground beef, milk, Shiga toxin-producing Escherichia coli.

O trabalho teve como objetivo determinar a ocorrência de Escherichia coli produtora de toxina Shiga (STEC) em carne moída e leite cru no sul do Brasil e estudar o comportamento de STEC isoladas de fezes de bovinos de corte e leite, verificando sua resistência em meios ácido e alcoólico. Não foram identificados genes stx1 e stx2 nas 464 E. coli isoladas de carne moída e leite cru. STEC isoladas de fezes de bovinos mantiveram populações estáveis e apresentaram crescimento, respectivamente, em carne moída e em leite experimentalmente contaminados, durante 120 horas a 8ºC. Esses isolados foram inativados quando expostos a pH 2,5 e 3,0, mas permaneceram viáveis após oito horas em pH 4,0. Os isolados de STEC não sobreviveram 48 horas em meio contendo 12% de etanol. Em 6% de etanol, STEC O174:H21, O163:H19 e O112:H2 apresentaram crescimento, ao passo que STEC O91:H21 e O22:H8 não resistiram além de 24 e 48 horas de incubação, respectivamente. A baixa prevalência de STEC em alimentos e as características das cepas encontradas em bovinos podem estar relacionadas com a baixa prevalência de enfermidades de origem alimentar causadas por STEC no Brasil.PALAVRAS-CHAVES: Ácido, carne moída, etanol, leite, Escherichia coli produtora de toxina Shiga.

Soroprevalência da infecção leptospiral em capivaras (Hydrochoerus hydrochaeris) abatidas em um frigorífico do Rio Grande do Sul / Seroprevalence of leptospiral infection in capybaras (Hydrochoerus hydrochaeris) in a slaughterhouse of Rio Grande do Sul, Brazil

Capivaras (Hydrochoerus hydrochaeris) são roedores selvagens do continente americano com crescente importância comercial como fonte alternativa de carne para o consumo humano. Nessa espécie, os estudos sobre a soroprevalência da infecção leptospiral são escassos e restritos aos espécimes de vida livre. Relatamos aqui reações positivas para anticorpos aglutinantes anti-leptospiras em 27,3 por cento (6/22) das capivaras abatidas em um frigorífico do Rio Grande do Sul. Os níveis mais altos de anticorpos sugerem infecção pelo sorogrupo Australis devido à reação para uma cepa de referência do sorovar Bratislava e para um isolado canino local do sorovar Australis, caracterizado como Leptospira noguchii. Esses resultados ressaltam que considerável parcela de capivaras criadas em cativeiro podem funcionar como reservatório de leptospiras patogênicas e chamam atenção para o risco ocupacional dos trabalhos que envolvem a criação e o abate dessa espécie animal.(AU)

Capybaras (Hydrochoerus hydrochaeris) are wild rodents from the American Continent with increasing importance as a commercial alternative source of meat for human consumption. Studies on seroprevalence for leptospiral infection are scarce and restricted to free living capybaras. We report detection of agglutinating antibodies against leptospires in 27 percent (6/22) of all animals in a slaughterhouse from Rio Grande do Sul. The highest antibody titers predicted Australis as the infecting serogroup due to reactions against a reference strain of serovar Bratislava and a canine local isolate of serovar Australis, characterized as Leptospira noguchii. The data presented in this report highlight that a considerable fraction of capybaras in captivity may behave as reservoir for pathogenic leptospires emphasizing the occupational risk of those who deal with animal farming and slaughter.(AU)

Serum antileptospiral agglutinins in freshwater turtles from Southern Brazil

In this study, we observed the presence of antileptospiral agglutinins in freshwater turtles of two urban lakes of Pelotas, Southern Brazil. Forty animals (29 Trachemys dorbigny and 11 Phrynops hilarii) were captured and studied. Attempts to isolate leptospires from blood and urine samples were unsuccessful. Serum samples (titer > 100) reactive to pathogenic strains were observed in 11 animals. These data encourage surveys of pet turtles to evaluate the risk of transmission of pathogenic leptospires to humans.

Neste estudo, observamos a presença de aglutininas anti-Leptospira em tartarugas de água doce de dois lagos urbanos de Pelotas, Sul do Brasil. Quarenta animais (29 Trachemys dorbigny e 11 Phrynops hilarii) foram capturados e estudados. Esforços para isolar leptospiras do sangue e urina não foram bem sucedidos. Amostras de soro positivas (títulos > 100), reativas para cepas patogênicas, foram observadas em 11 animais. Estes dados encorajam inquéritos para avaliação de tartarugas como potenciais transmissoras de leptospiras patogênicas para humanos.