Mycobacterium avium subsp. paratuberculosis and Crohn's disease: characterization of the interaction with different aspects of the disease.

Crohn's disease (CD) is a chronic granulomatous inflammatory bowel disease with no fully understood etiology and cure. Mycobacterium avium subspecies paratuberculosis (MAP), the etiologic agent of paratuberculosis, is also isolated from samples from human patients with CD. Paratuberculosis is characterized by persistent diarrhea and progressive weight loss and primarily affects ruminants, which eliminate the agent via feces and milk. The involvement of MAP in the pathogenesis of CD and other intestinal diseases is unclear. Thus, the present study aimed to analyze immunological, socioepidemiological, biochemical, and therapeutic variables that may be related to the occurrence of MAP in blood samples and CD patients. The sampling was random, and the population of origin was the patients from the Bowel Outpatient Clinic of the Alpha Institute of Gastroenterology (IAG), Hospital das Clínicas, Universidade Federal de Minas Gerais (HC-UFMG). Blood samples were collected from 20 patients with CD, eight with ulcerative rectocolitis (UCR), and 10 control patients without inflammatory bowel diseases. Samples were subjected to real-time PCR for detection of MAP DNA, oxidative stress analyses, and socioepidemiological variables. MAP was detected in 10 (26.3%) of the patients, seven (70%) were CD patients, 2 (20%) were URC patients, and one (10%) was a non-IBD patient. MAP was found more frequently among CD patients, but not restricted to CD patients. The presence of MAP in the blood of these patients occurred simultaneously with an inflammatory response with an increase in neutrophils and significant alterations in the production of antioxidant enzymes such as catalase and GST.

Emergence of multidrug-resistant bacteria isolated from surgical site infection in dogs and cats / [Emergência de bactérias multirresistentes isoladas da infecção no sítio cirúrgico em cães e gatos]

Surgical site infections (SSIs) and antimicrobial resistance among pathogens causing SSI are a growing concern in veterinary hospitals. One major reason, the widespread use of antimicrobials, has led to increased incidence of SSIs. This study identified bacteria and resistance profiles to antimicrobials in the SSI cases diagnosed at the Surgical Clinic of Small Animals in the Veterinary Hospital, Federal University of Viçosa, Brazil. The main genus identified was Staphylococcus, followed by Escherichia, Enterococcus, Bacillus, Shigella, Citrobacter, Proteus, Morganella, Serratia, Enterobacter, Pseudomonas and Klebsiella were also found, but in small number. The results indicated the predominance of Gram-negative bacteria among the collected samples. Most of isolates identified were resistant to more than one of the following antimicrobials: ampicillin, tetracycline, enrofloxacin, amoxicillin/clavulanic acid and cephalotin. Of the 17 Staphylococcus sp. isolates, two (11.8%) were methicillin-resistant Staphylococcus aureus (MRSA) and 11 (64.7%) of them were methicillin-resistant Staphylococcus pseudintermedius (MRSP). There were bacterial genera identified with resistance to all tested antimicrobials in different proportions. This should alert veterinary hospitals to the emergence of multidrug-resistant bacteria and to the requirement for the revision of surgical protocols with regard to antimicrobial prophylaxis and therapy.(AU)

As infecções em sítio cirúrgico (ISCs) e a resistência bacteriana entre os patógenos relacionados constituem uma preocupação crescente nos hospitais veterinários. O aumento na incidência de ISCs possui forte relação com o uso amplo e disseminado de antibióticos. O presente estudo identificou bactérias e perfis de resistência a antibióticos nos casos de ISCs diagnosticados na Clínica Cirúrgica de Pequenos Animais do Hospital Veterinário da Universidade Federal de Viçosa, Brasil. O principal gênero identificado foi Staphylococcus, seguido pelos gêneros Escherichia, Enterococcus, Bacillus, Shigella, Citrobacter, Proteus, Morganella, Serratia, Enterobacter, Pseudomonas e Klebsiella, porém, em menor quantidade. Os resultados demonstraram a predominância de bactérias Gram-negativas entre as amostras coletadas. A maioria dos isolados identificados eram resistentes a um ou a mais de um dos seguintes antibióticos: ampicilina, tetraciclina, enrofloxacina, amoxicilina/ácido clavulânico e cefalotina. Entre os 17 isolados de Staphylococcus sp., dois (11,8%) eram Staphylococcus aureus resistentes à meticilina (SARM) e 11 (64,7%) eram Staphylococcus pseudintermedius resistentes à meticilina (SPRM). Houve identificação de gêneros bacterianos com diferentes proporções de resistência para todos os antibióticos avaliados. Esses achados devem alertar os hospitais veterinários para a emergência de bactérias multirresistentes e para a necessidade de revisar a profilaxia e a terapia antimicrobiana referente aos protocolos cirúrgicos.(AU)

Rapid baso-apical translocation of Mycobacterium avium ssp. paratuberculosis in mammary epithelial cells in the presence of Escherichia coli.

Infection of mammary gland cells with bacterial pathogens begins with adhesion, invasion, and persistence within the cells or systemic distribution. Some bacteria, such as Escherichia coli, are known to causes bovine mastitis, resulting in acute proinflammatory responses in the mammary tissue. Mycobacterium avium ssp. paratuberculosis (MAP), the etiological agent of paratuberculosis, is able to spread to distant organs after crossing intestinal cells, reaching the mammary gland and potentially being released in milk, infecting calves during suckling. Its exit from systemic sites may be influenced by preexisting inflammation such as that caused by E. coli mastitis. Interactions between E. coli and MAP in mammary epithelial cells have not yet been described. In this study, we posited that E. coli-infected bovine mammary epithelial cells would facilitate baso-apical translocation of MAP in an ex vivo model. We showed that the presence of E. coli in a bovine mammary epithelial cell line (MAC-T) increased baso-apical translocation of MAP to the apical side of the cells. Levels were significantly higher 30 min post-infection and decreased at 120 min post-infection. Cells previously infected with E. coli and MAP or with E. coli alone showed a significant increase in IL1B mRNA expression at 120 min. We detected no significant expression of p38 mitogen-activated protein kinase (mapkp38) or IL10, regardless of treatment. Thereby, the presence of E. coli in MAC-T cells alters the translocation of MAP through epithelial cells, enabling its rapid translocation to the cellular surface. Expression of IL1B was shown to influence the apical-basal translocation of MAP at 120 min. Findings from the current study suggest that MAP translocation into milk is likely enhanced by inflammatory states such as those induced during E. coli mastitis. This is the first report demonstrating the effect of E. coli under MAP coinfection in bovine mammary epithelial cells under experimental conditions.

Isolation and genetic stability of an infectious bronchitis virus strain (IBV) / Isolamento e estabilidade genética de uma cepa do vírus da bronquite infecciosa (IBV)

O vírus da bronquite infecciosa (IBV) é um importante patógeno respiratório presente na avicultura comercial e tem provocado grandes perdas econômicas em todo o mundo. A vacinação é realizada pela indústria produtora de aves, mas continuam surgindo novos sorotipos e variações antigênicas, dificultando o controle de IBV. Nós realizamos uma caracterização molecular de uma cepa de IBV obtida diretamente de tecidos e comparamos com a mesma cepa que havia sido passada três vezes em ovo embrionado. Nós mostramos uma variação significante na sequência viral depois de ter sido isolada em ovo embrionado.(AU)

Paratuberculosis in Latin America: a systematic review.

Latin America is the definition of the American group, where languages of Latin origin are spoken, including countries in South, Central, and North America. Paratuberculosis is a gastrointestinal contagious chronic disease that affects ruminants, whose etiological agent is the bacilli Mycobacterium avium subsp. paratuberculosis (MAP). Paratuberculosis is characterized by intermittent diarrhea, decreased milk production, dehydration, and progressive weight loss and is possibly involved in Crohn's disease, a human intestinal disease. MAP is resistant to environmental factors, pasteurization, and water disinfection, which coupled with the subclinical-clinical nature of the disease, and makes paratuberculosis a relevant socioeconomic and public health issue, justifying the descriptive review of research on the disease carried out in Latin American countries. A survey of articles, published until September 2016, on the Scopus database, PubMed, Agris, and Science Direct, about detection of the agent and the disease in Latin America, without restrictions to the date of the research was performed. The keywords were as follows: "paratuberculosis," "Mycobacterium avium subsp. paratuberculosis," "cattle," "milk," "wildlife," "goat," "ovine," "dairy," and the name of each country in English. Studies found from nine of the 20 Latin America countries, 31 related to Brazil, 17 to Argentina, 14 to Chile, eight to Colombia, six to Mexico, two to Peru, two to Venezuela, and one to Panama and to Bolivia, each. The agent was detected in cattle, goats, sheep, domesticated water buffalo, and wild animals. Microbiological culture, PCR, and ELISA were the frequent techniques. The small number of studies may result in overestimation or underestimation of the real scenario.

Short communication: Passive shedding of Mycobacterium avium ssp. paratuberculosis in commercial dairy goats in Brazil.

Goat farming is a low-cost alternative to dairy production in developing countries. In Brazil, goat production has increased in recent years due in part to the implementation of programs encouraging this activity. Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of paratuberculosis, a disease that causes chronic granulomatous enteritis in ruminants, but MAP transmission dynamics are still poorly understood in goats. In a previously published study of our research group, 10 dairy goat farms (467 animals) from Minas Gerais state were analyzed for MAP detection; 2 fecal cultures and 11 milk samples tested positive for MAP by conventional PCR and were confirmed by sequencing. Because no clinical signs were observed over 1 yr of monitoring, we hypothesized that these MAP-positive goats could be passive shedders. Thus, in the present study, 4 positive goats (4/13) from the previous study were purchased and feces and milk samples were collected for evaluation (twice, with an interval of 3 mo between tests) by culture of MAP, IS900 PCR, or both. All analyses were negative for MAP. At the last time point, blood samples were collected for ELISA, the animals were killed, and tissues collected for tissue culture and histopathology. At necropsy, no macroscopic lesions related to paratuberculosis were observed. Similarly, no histological changes were observed and MAP in samples stained by Ziehl-Neelsen was not detected. These animals were characterized as potential passive shedders with upward contamination of the teat canal by MAP. This is the first report of the passive shedding phenomenon in goats in Brazil and it highlights the importance of identifying these animals for control programs and to ensure the quality of dairy products.

Description of Methicillin-resistant Staphylococcus pseudintermedius from canine pyoderma in Minas Gerais state, Brazil / Descrição de Staphylococcus pseudintermedius meticilina-resistentes de piodermite canina no estado de Minas Gerais, Brasil

Methicillin-resistant Staphylococcus pseudintermedius (MRSP) is of worldwide concern in veterinary medicine. The identification of resistant strains is necessary for proper treatment and the prevention of its propagation among animals. This study aimed to identify S. pseudintermedius isolated from canine pyoderma and evaluate their resistance profiles. Lesions from 25 dogs with pyoderma were sampled. Bacterial isolates were subjected to phenotypic and genotypic analysis for identification of the causative agent. S. pseudintermedius isolates were subjected to SmaI macrorestriction analysis and PFGE for genetic grouping, and PCR to identify the presence of the mecA gene. Their resistance profiles against 12 antimicrobials were also assessed. According to the microbiological analysis, 70 of the 75 isolates obtained were S. pseudintermedius. The isolates presented PFGE patterns, with similarity varying between 84.6 and 100%, and were grouped into 19 clusters. Despite a high frequency of mecA-positive isolates (66 out 70), only 12 presented resistances to oxacillin. Multi-resistance was identified in 29 isolates. The high frequency of MRSP isolated in this study highlights the relevance of identifying resistant strains to lead proper clinical treatment.

Staphylococcus pseudintermedius meticilina-resistente (MRSP) é de preocupação mundial na medicina veterinária. A identificação de cepas resistentes é necessária a um tratamento adequado e à prevenção da sua propagação entre os animais. O objetivo do estudo foi identificar S. pseudintermedius isolados de piodermite canina e avaliar o perfil de resistência. Foram coletadas amostras de lesões de 25 cães com piodermite. Os isolados bacterianos foram submetidos a análises fenotípicas e genotípicas para identificação do agente causador. Isolados de S. pseudintermedius foram submetidos à análise de macrorrestrição por SmaI e PFGE para agrupamento genético e à PCR para identificar a presença do gene mecA. Seu perfil de resistência contra 12 antimicrobianos também foi avaliado. De acordo com a análise microbiológica, 70 dos 75 isolados obtidos foram identificados como S. pseudintermedius. Os isolados apresentaram padrões de PFGE com similaridade variando entre 84.6 e 100% e foram agrupados em 19 grupos genéticos. Apesar da frequência alta de isolados mecA positivos (66 em 70), apenas 12 apresentaram resistência à oxacilina. Multirresistência foi identificada em 29 isolados. A alta frequência de MRSP isolados neste estudo destaca a relevância de se identificarem cepas resistentes para se conduzir um tratamento clínico adequado.

Association of number of tandem repeats in two important adhesins in Mycoplasma hyopneumoniae / Associação entre os números de repetições em tandem em duas importantes adesinas de Mycoplasma hyopneumoniae

Diversidade genética de Mycoplasma hyopneumoniae tem sido relatada em análise múltipla de repetições em tandem em número variável (MLVA). O objetivo deste estudo foi descrever a distribuição espacial e a heterogeneidade genética de tipos de M. hyopneumoniae no Brasil, bem como investigar a correlação entre regiões de repetição 1 (RR1) e 3 (RR3) de duas adesinas importantes (P97 e P146). Foram identificados 39 tipos de MLVA baseados no número de repetições em tandem em P97 RR1 e RR3 P146. A correlação negativa significativa (Spearman's rho = -0,26; P = 0,022) entre P97 RR1 e RR3 P146 foi observada, o que sugere um possível mecanismo compensatório que permitiria a bactéria manter a sua capacidade de adesão. Os resultados contribuem para compreender a epidemiologia das M. hyopneumoniae no quarto maior país produtor de suínos do mundo.(AU)

Effect of the inhibitors phenylalanine arginyl ß-naphthylamide (PAßN) and 1-(1-naphthylmethyl)-piperazine (NMP) on expression of genes in multidrug efflux systems of Escherichia coli isolates from bovine mastitis.

The multidrug efflux system in bacteria can reduce antibiotic concentration inside the cell, leading to failure in the treatment of bacterial diseases. This study evaluated the influence of two efflux pump inhibitors (EPIs), phenylalanine arginyl ß-naphthylamide (PAßN) and 1-(1-naphthylmethyl)-piperazine (NMP), on the gene expression of three multidrug efflux systems, AcrAB, AcrEF and EmrAB in Escherichia coli bovine mastitis isolates resistant to ampicillin and sulfamethoxazole/trimethoprim simultaneously. Each isolate had at least three multidrug efflux system genes. The acrA and acrB had the lowest expression levels in all treatments, while the emrA or emrB showed the highest expression levels in the presence of ampicillin, sulfamethoxazole/trimethoprim, PAßN and NMP. EPIs also contributed to the decrease in arcF expression when used in combination with ampicillin treatment. Since PAßN showed stronger effects than NMP, it may serve as an alternative to assist in the antimicrobial therapy of mastitis.

Short communication: Viable Mycobacterium avium subspecies paratuberculosis in retail artisanal Coalho cheese from Northeastern Brazil.

Mycobacterium avium ssp. paratuberculosis (MAP) is the etiologic agent of paratuberculosis and it potentially plays a role in Crohn's disease. In humans, the main route of transmission of MAP might be the intake of contaminated milk and dairy products. Considering that MAP has already been detected in many types of cheese in different counties, and that Coalho cheese is an important dairy product in northeastern Brazil, the aim of this study was to report the first detection of MAP in retail Coalho cheese in Brazil by PCR and culture. Of 30 retail Coalho cheese samples, 3 (10%) amplified fragments of a similar size to that expected (626 bp) were obtained and viable MAP was recovered by culture from 1 (3.3%) sample. The DNA from the positive culture sample was sequenced and showed 99% identity with the insertion sequence IS900 deposited in GenBank. It was possible to identify the presence of MAP-specific DNA in the analyzed samples for the first time in Brazil, and to recover viable cells from retail Coalho cheese.

Sistemas de efluxo multidrogas em Escherichia coli e uso de inibidores como possíveis adjuvantes na terapia da mastite bovina / Multidrug efflux systems in Escherichia coli and the use of inhibitors as possible adjuvant therapy in bovine mastitis

Vinte e sete isolados de Escherichia coli provenientes de leite de bovinos com mastite clínica foram submetidos a teste de sensibilidade aos principais antimicrobianos usados no tratamento desta doença. Avaliou-se também a efetividade in vitro de dois inibidores de sistemas de efluxo multidrogas, fenilalanina arginyl ß naftilamida (PAβN) e 1-(1-Naphthylmethyl)-piperazine (NMP), utilizando-se a concentração inibitória mínima (CIM) como referência. A CIM e o sistema de efluxo foram detectados com base nas curvas de crescimento, utilizando-se a densidade óptica (D.O550), em diferentes concentrações da droga e na presença e ausência dos inibidores. Apenas quatro isolados apresentaram resistência à ampicilina e ao sulfametoxazol∕trimetoprim, simultaneamente, enquanto todos os 27 foram sensíveis aos demais antimicrobianos. Observaram-se valores para ampicilina variando de 6µg/mL a 250µg/mL e para sulfametoxazol∕trimetoprim de 12µg/mL a 1500µg/mL. Houve redução das CIMs desses antimicrobianos em todos os isolados na presença dos inibidores, exceto para sulfametoxazol∕trimetoprim na presença de NMP. Conclui-se que esses isolados possuem um estreito perfil de resistência e que PAßN apresentou melhor efeito inibitório em relação à ampicilina e ao sulfametoxazol∕trimetoprim, apresentando-se como um candidato a adjuvante no tratamento da mastite...

Twenty-seven isolates of Escherichia coli from cattle with clinical mastitis were subjected to sensitivity tests regarding main antimicrobials used in the treatment of this disease. We also evaluated in vitro effectiveness of two inhibitors of multidrug efflux systems, fenilalanina arginyl ß naftilamida (PAβN) and 1-(1-Naphthylmethyl)-piperazine (NMP), using the minimum inhibitory concentration (MIC) as a reference. MIC and multidrug efflux systems were detected in the growth curves, using optical density (D.O550) at different drug concentrations and the presence and absence of inhibitors. Only four isolates of E. coli were resistant to ampicillin and trimethoprim/sulfamethoxazole, simultaneously. However, all isolates were sensitive to the other antimicrobials. Were observed values ranging from 6mg to 250mg ampicillin/mL, and 12mg to 1500mg/mL trimethoprim/sulfamethoxazole. There was a reduction of the MIC of antimicrobials for all isolates in the presence of inhibitors, except for trimethoprim/sulfamethoxazole in the presence of NMP. In conclusion, these isolates have a narrow resistance profile and PAßN showed better inhibitory effect against ampicillin and trimethoprim/sulfamethoxazole, and is a candidate for the adjuvant treatment of mastitis...

Perfil de sensibilidade de células sésseis e planctônicas de Escherichia coli a antimicrobianos usados no tratamento da mastite bovina / Sensitivity profile of planktonic and sessile cells of Escherichia coli to antimicrobial used in the treatment of bovine mastitis

Escherichia coli é um micro-organismo altamente adaptativo e sua habilidade em formar biofilmes pode ser fundamental na resistência a tratamentos com antimicrobianos. A avaliação da concentração mínima inibitória (CMI) vem sendo utilizada para verificar a sensibilidade dos micro-organismos aos antimicrobianos. Entretanto, quando se avaliam células sésseis, a concentração do antimicrobiano requerido para erradicação do biofilme é maior do que a determinada pela CMI. Objetivou-se comparar as CMI com as concentrações mínimas de erradicação de biofilmes (CMEB) de antimicrobianos usados no tratamento da mastite em 27 isolados de E. coli produtores de biofilmes provenientes de mastite. Os isolados foram submetidos a testes de sensibilidade a antimicrobianos usados no tratamento da mastite, tanto para células planctônicas, por meio da CMI, quanto para células sésseis, pela avaliação da CMEB. Os resultados revelaram uma alta sensibilidade: apenas quatro (14,8%) isolados obtiveram valores da CMI elevados, variando de 4 a 10µg/mL, sendo classificados como resistentes. Para os demais isolados (85,2%), os valores foram menores, variando de 0,125 a 2µg/mL, classificados como sensíveis. A avaliação de CMEB indicou que a concentração dos antimicrobianos necessária para eliminar as células sésseis variou de 100µg/mL a 500µg/mL. Os valores de CMEB foram significativamente maiores nos isolados grandes e moderados produtores de biofilmes em relação aos isolados fracos produtores de biofilmes (p<0,001). Não houve correlação entre os valores de CMEB e CMI (p>0,05). A escolha da terapêutica antimicrobiana correta para o tratamento de infecções intramamárias em bovinos relacionadas com a produção de biofilmes parece exigir a aplicação de testes mais específicos. Testes de susceptibilidade antimicrobiana baseados apenas em valores de CMI mostraram-se ineficazes em determinar com precisão a susceptibilidade das células bacterianas sésseis.

Escherichia coli is a highly adaptive microorganism. Its ability to form biofilms may be critical for resistance to antimicrobial treatments. Evaluation of minimum inhibitory concentration (MIC) has been used to check the sensitivity of microorganisms to antibiotics, however, when evaluating sessile cells, the required antibiotic concentration to eradicate biofilm is greater than determined by MIC. This study aimed to compare MIC with minimum biofilm eradication concentration (MBEC) of antimicrobials used in mastitis treatment in 27 E. coli biofilm producers isolates from mastitis. Isolates were tested for sensitivity to antimicrobials used in mastitis treatment, for both planktonic cells (by CMI) and sessile cells (by MBEC). The results revealed high sensitivity: only four (14.8%) isolates showed high MIC values, ranging from 4 to 10g/mL and they were classified as resistant. All other isolates (85.2%) showed lower values, ranging from 0.125 to 2mg/mL, and they were classified as sensitive. Evaluation of MBEC indicated that concentration of antimicrobial needed to remove sessile cells ranged from 100mg/mL to 500mg/mL. MBEC values were significantly higher in large and moderate biofilm producers isolates regarding weak biofilm producers isolates (p<0.001). There was no correlation between MBEC and CMI values (p>0.05). The correct choice of antimicrobial therapy for treatment of mammary infections in cattle related to biofilm production seems to require application of more specific tests. Antimicrobial susceptibility testing based only on MIC values proved ineffectiveness to accurately determination the susceptibility of sessile bacterial cells.

Short communication: Recovery of viable Mycobacterium avium subspecies paratuberculosis from retail pasteurized whole milk in Brazil.

Mycobacterium avium ssp. paratuberculosis (MAP) is the etiological agent of paratuberculosis, a chronic granulomatous enteritis that affects all ruminants worldwide. Some researchers have indicated a possible role of MAP in Crohn's disease. Despite extensive research and large and important advances in the past few decades, the etiology of Crohn's disease remains indefinite. The most probable transmission route of MAP from animals to humans is milk and dairy products. Mycobacterium avium ssp. paratuberculosis has already been detected in milk samples worldwide, and some studies have reported that MAP is resistant to pasteurization. In Brazil, MAP has been reported in raw milk samples; however, Brazilian retail pasteurized milk has not yet been tested for viable MAP. The aim of this study was to investigate MAP in pasteurized milk in the region of Viçosa (Minas Gerais, Brazil). Thirty-seven samples were collected and processed for culture of MAP. One colony similar to MAP was observed and confirmed by IS900-nested PCR and sequencing. Analysis revealed 97 to 99% identity with the MAP K-10 strain. This study is the first report of the presence of MAP in retail pasteurized whole milk in Brazil.

Antimicrobial profile screening of two oils of Copaifera genus / Avaliação do perfil antimicrobiano de dois óleos do gênero Copaifera

O objetivo deste estudo foi identificar a atividade inibitória de óleos de copaíba sobre o crescimento dos micro-organismos: Shigella flexneri, Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Citrobacter freundi, Staphylococcus aureus, Actinobacillus pleuropneumoniae e Haemophilus parasuis. Foi realizado um teste de difusão em ágar com duas soluções a 10 por cento de óleos de copaíba obtidos de duas diferentes espécies de copaíba (Copaifera officinalis e C. langsdorffii) e um controle negativo com tween 80 e água. Os isolados clínicos de H. parasuis e A. pleuropneumoniae foram incubados em microaerofilia. Os resultados mostraram três espécies Gram-negativas inibidas por ambas as soluções de óleo de copaíba: E. coli, P. aeruginosa e S. flexneri. Na inibição de P. aeruginosa o óleo de C. officinalis foi superior ao de C. langsdorffii (P<0,05). Todas as cepas de S. aureus tiveram seu crescimento inibido pelas soluções no ensaio, sem diferença estatística entre os halos. Estes resultados sugerem que o óleo de copaíba pode ser uma fonte potencial de compostos inibitórios para ser utilizada como antimicrobianos no tratamento de infecções humanas e animais e conservação de alimentos.

Levantamento sorológico de Mycobacterium avium subesp. paratuberculosis em bovinos leiteiros no estado do Espírito Santo / Serological survey of Mycobacterium avium subsp. paratuberculosis in dairy cattle in Espírito Santo state

The occurrence of antibodies to Mycobacterium avium subsp. paratuberculosis (MAP) was verified in dairy cattle from Espírito Santo state. A total of 1,450 serum samples were analyzed for antibodies anti-MAP, using ELISA. Dairy cattle, males and females, from four regions of Espírito Santo state were used. One hundred sixty-five (11.4 percent) samples were positive for anti-MAP, 33 (2.3 percent) were considered suspicious, and 1,252 (86.3 percent) were negative. In all regions, seropositive animals were found, indicating that the agent is spread by the State, posing a threat to the local dairy farming and neighboring states, as well as public health, since MAP can be involved with Crohn's disease in humans. This result presents the first serologic anti-MAP survey in dairy cattle of Espírito Santo State.

Short communication: detection of Mycobacterium avium subspecies paratuberculosis by polymerase chain reaction in bovine milk in Brazil.

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of paratuberculosis, or Johne's disease, a chronic granulomatous enteritis that affects all ruminants worldwide. Since the isolation of MAP from intestinal tissue of human patients bearing Crohn's disease, there has been a debate on the possibility of this agent playing a role in the etiology of Crohn's disease. Milk could be the potential vehicle for transmission to humans. Mycobacterium avium ssp. paratuberculosis has already been detected in milk samples worldwide. In Brazil, detection of MAP is uncommon; however, it has already been detected by bacterial isolation and serological test. The aim of this study was to investigate the presence of MAP, by PCR, in raw milk samples in the region of Viçosa, Minas Gerais State, Brazil. Of 222 milk samples evaluated, 8 (3.6%) quarter milk samples amplified fragments of similar size to that expected of 626 bp. These fragments were cloned and sequenced. The genetic analysis revealed a 99% identity match between the sequences obtained in this study and the insertion sequence IS900 deposited in the GenBank. In the analyzed milk samples, MAP DNA was detected, confirming its presence in dairy cattle in the region of Viçosa. This is the first report of MAP presence in raw milk samples in Brazil.

Pontos críticos de controle de Salmonella spp. no abate de frangos / Critical control points for Salmonella spp. in poultry slaughter

Avaliou-se a presença de Salmonella spp. em esfregaços superficiais em 135 carcaças de frangos, coletadas em cinco diferentes fases do abate, utilizando os métodos de microbiologia convencional, reação em cadeia de polimerase (PCR) e imunoanálise. Os três métodos resultaram em tendência semelhante de detecção de Salmonella spp. nas carcaças de frango, ao longo da linha de abate. A maior frequência de Salmonella spp. foi determinada após o chuveiro de lavagem das carcaças, localizado entre a evisceração e o pré-resfriamento. Vinte (14,8 por cento) esfregaços foram determinados pela metodologia convencional, 52 (38,5 por cento) pela PCR e 66 (48,8 por cento) pela imunoanálise. A menor contaminação foi encontrada na saída do pré-resfriamento, com frequências de 3,7 por cento, 0 por cento e 16,7 por cento, respectivamente. Salmonella spp. foi encontrada em todas as fases de abate, mostrando a importância do monitoramento de diferentes pontos críticos de controle eventualmente identificados no abate de frangos.

The presence of Salmonella spp. in superficial swabs collected from 135 chicken carcasses in five different slaugther steps using the conventional microbiology, PCR, and immunoanalysis methods was evaluated. The three methods presented similar tendency to detect Salmonella spp. in the chicken carcasses along the slaugther line. The highest frequency of Salmonella spp. was found after the shower, located between the evisceration and the chiller. Twenty (14.8 percent) swabes were determined by the conventional methodology, 52 (38.9 percent) by the PCR, and 66 (48.9 percent) by the immunoanalysis. The lowest contamination was found after chiller tank, where the frequencies were 5 (3.7 percent), 0 (0 percent), and 24 (16.7 percent), respectively. Salmonella spp. was found in all the slaugther steps. This shows the importance for monitoring different CCP (Control Critical Point) in the poultry slaughterhouses.

Detection of Mycoplasma hyopneumoniae in lungs and nasal swabs of pigs by nested PCR / Detecção de Mycoplasma hyopneumoniae em pulmões e suabes nasais de suínos por nested PCR

Fifty-four samples were collected from growing and finishing pigs for the molecular diagnosis of enzootic porcine pneumonia. Nineteen lung fragments were obtained from pigs that showed signs of respiratory disease and 35 nasal swabs were obtained from clinically healthy pigs. For the detection of the bacterial genome in the samples, the nested PCR technique was used to amplify a fragment of 706bp. This fragment was subsequently cloned and sequenced. The sequence of obtained nucleotides was compared with six other sequences of Mycoplasma hyopneumoniae and 11 sequences of other bacteria available in the Genbank. To measure the sensitivity of the nested PCR, serial dilutions (10-1 to 10-15) of cloned fragments were conducted based on the concentration of 300ng. Ten lung fragments and eight nasal swabs showed positive for M. hyopneumoniae and the limit of detection was estimated to be 0.3fg DNA cloned. The sequence of nucleotides obtained showed 99.1 percent homology with the other sequences of M. hyopneumoniae, demonstrating that the nested PCR used in this study may provide an important diagnostic tool for the detection of this agent.

Foram coletadas 54 amostras de animais em fase de crescimento e terminação para o diagnóstico molecular da pneumonia enzoótica suína. Dezenove fragmentos de pulmão foram obtidos de suínos que apresentavam sinais de doença respiratória e 35 suabes nasais foram obtidas de suínos clinicamente saudáveis. Para a detecção do genoma bacteriano nas amostras, foi utilizada a técnica de nested PCR que originou um fragmento de 706pb, o qual foi, posteriormente, clonado e sequenciado. A sequência de nucleotídeos obtida foi comparada com outras seis sequências de Mycoplasma hyopneumoniae e 11 sequências de outras bactérias disponíveis no Genbank. Para medir a sensibilidade da nested PCR, foram realizadas diluições seriadas (10-1 a 10-15) do fragmento clonado, partindo da concentração de 300ng. Dez fragmentos de pulmões e oito suabes nasais apresentaram resultado positivo para M. hyopneumoniae e o limite de detecção foi estimado em 0,3fg de DNA clonado. A sequência de nucleotídeos obtida foi de 99.1 por cento de homologia com as outras sequências de M. hyopneumoniae, demonstrando que a nested PCR utilizada neste estudo pode ser uma importante ferramenta de diagnóstico para a detecção desse agente.

Resistência a antimicrobianos dependente do sistema de efluxo multidrogas em Escherichia coli isoladas de leite mastítico / Antimicrobial resistance dependent on multidrugs efflux in Escherichia coli isolated from the mastitic milk

Identificaram-se e caracterizaram-se a resistência e a multirresistência aos principais antimicrobianos usados no tratamento de mastite bovina causada por Escherichia coli. A concentração inibitória mínima (MIC) e o sistema de efluxo foram detectados pelas curvas de crescimento, com base na densidade óptica, em diferentes concentrações da droga e na presença e na ausência do desacoplador da força próton-motora (PMF). E. coli 1 foi resistente à neomicina e à gentamicina; E. coli 3 e 4, à tetraciclina e à estreptomicina; e E. coli 2 e 6 à gentamicina. E. coli 5 apresentou modelo de sensibilidade. Observou-se que MICs de todos os antimicrobianos dos multirresistentes (E. coli 1, 3 e 4) diminuíram na presença do desacoplador, o que sugere sistema de efluxo multidrogas. Após cura, apenas E. coli 1 apresentou modelo de sensibilidade, porém não houve alterações das MICs, antes e após adição do desacoplador. Os resultados indicam possível presença de mecanismo de resistência dependente da PMF codificado, ou parte dele, em plasmídeo.

Resistance and multiresistance to main antimicrobials used for treating bovine mastitis caused by Escherichia coli were identified and characterized. The minimal inhibitory concentration (MIC) and efflux systems were detected by the use of growth curves based on optical density at different drug concentrations and both presence and absence of uncoupler of the proton-motive force (PMF). E. coli 1 was resistant to neomycin and gentamycin, E. coli 3 and 4 were resistant to tetracycline and streptomycin, whereas E. coli 2 and 6 were resistant to gentamycin. E. coli 5 showed sensibility model. MICs of all antimicrobials of the multiresistant samples (E. coli 1, 3, and 4) were decreased in presence of the uncoupler, therefore suggesting the presence of the multidrug efflux system. After healing, only E. coli 1 showed sensibility model, however no alteration occurred in MIC(s) before and after adding the uncoupler. Those data inform the possible presence of a PMF dependent resistance mechanism that is totally or partly codified in plasmid.

Detection of a chloramphenicol efflux system in Escherichia coli isolated from poultry carcass.

An active chloramphenicol efflux system was demonstrated in a multiresistant E. coli isolated from poultry carcass. The effect of different concentrations of chloramphenicol on the original strain and on the plasmid-cured strain was determined in the presence and in the absence of CCCP, an uncoupler of the proton-motive force. Minimal inhibitory concentration (MIC) was lower in the presence of CCCP in the original strain. The plasmid-cured strain displayed lower resistance for chloramphenicol than the wild type, but the MIC was not affected by CCCP. The combined results indicate a plasmid encoded energy dependent resistance mechanism. 3H-chloramphenicol accumulation within the cells was measured by scintillation counting. The uptake or the efflux of 3H-chloramphenicol was influenced by CCCP in the original strain, but not in the plasmid-cured strain. More than one chloramphenicol resistance mechanism may exist in this strain. E. coli is an important commensal or pathogen that inhabits the gastrointestinal tracts of humans and animals, so a plasmid encoded active drug resistance mechanism can be a potential source of horizontal transfer of resistance.