A resistência bacteriana é responsável perante o fracasso no tratamento de infecções com agentes quimioterápicos. São necessárias novas alternativas para controlar estes micro-organismos. Entre essas alternativas estão plantas utilizadas tradicionalmente na medicina popular. O presente trabalho avaliou a eficiência dos extratos etanólicos e acetonicos de folhas de Cecropia pachystachya e Curatella americana com atividade antibacteriana in vitro em cepas de Staphylococcus aureus isolado de caso de mastite bovina. Os extratos de C. pachystachya e C. americana apresentam MIC de 2,5 mg.mL-1. O composto isolado de C. pachystachya denominado EEB2 apresentou actividade bactericida para uma concentração de 1,25 mg.mL-1. Estes resultados demonstram que os extratos de C. pachystachya e C. americana contém compostos anti-bacterianos. / Bacterial drug resistance is responsible for the failure of the treatment of infection using chemotherapeutic agents. Thus, new approaches are necessary for the control of these microorganisms. Included among these alternatives are several plants that are traditionally used in folk medicine. Therefore, the aim of this study was to evaluate the antibacterial activity of the ethanolic and acetone extracts of leaves of Cecropia pachystachya and Curatela americana in vitro against S. aureus bacteria isolates from cases of bovine mastitis. The extracts from C. pachystachya and Curatela americana had a MIC of 2.5 mg.mL-1. The compound isolated from C. pachystachya called EEB2 showed bactericidal activity at a concentration of 1.25 mg.mL-1. These results demonstrate that the extracts of C. pachystachya and C. americana contains antibacterial compounds.

O diagnóstico rápido e eficiente da mastite subclínica é importante para reduzir a persistência da doença e os prejuízos decorrentes. O objetivo do estudo foi avaliar a técnica de espectrometria de massas (MS) por ionização e dessorção a laser assistida por matriz - tempo-de-vôo (MALDI-TOF) para identificação de bactérias causadoras de mastite subclínica bovina por dois métodos: 1) a partir de bactérias isoladas por cultivo icrobiológico; 2) a partir da recuperação de bactérias diretamente do leite, visando eliminar completamente a necessidade de cultivo microbiológico para identificação dos patógenos. O estudo foi composto por dois experimentos (1 e 2), no experimento 1 foram utilizadas 33 amostras de leite provenientes de animais das raças Gir e Holandesa de quatro fazendas leiteiras para a identificação icrobiológica e MALDI-TOF MS. As amostras com resultados conflitantes foram confirmadas por sequenciamento do gene 16S rRNA. Os resultados de cultura microbiológica foram Staphylococcus aureus (n = 13), Streptococcus agalactiae (n = 10) e Estafilococos coagulase negativo (ECN) (n = 10). Para todas as amostras de Streptococcus agalactiae, resultados similares foram observados para a identificação microbiológica e por MALDI-TOF MS. De 13 isolados de Staphylococcus aureus, 11 foram igualmente identificados por MALDITOF, 1 isolado foi identificado como Staphylococcus haemolyticus por sequenciamento do gene 16S rRNA, e o outro isolado isolado com resultado conflitante foi caracterizado como cultura mista de Staphylococcus aureus e Enterococcus faecalis. Em relação às amostras de ECN, todas as amostras do grupo foram identificadas por MALDI-TOF em nível de gênero (S. simulans, S. epidermidis, S. Haemolyticus, S. Chromogens e S. aureus coagulase negativa). No experimento 2 foi avaliado o método de recuperação de bactérias presentes em leite e sua identificação por MALDI-TOF MS utilizando o método de contaminação experimental de leite com Escherichia coli, Enterococcus faecalis e Staphylococcus aureus. A identificação de patógenos recuperados diretamente do leite foi possível quando a concentração de E. faecalis e S. aureus foi de 106 ufc/mL, e de 107 ufc/mL para E.coli. Concluímos que o uso de MALDI-TOF MS pode acelerar a identificação de patógenos causadores de mastite subclínica bovina podendo contribuir para a adoção de medidas de controle e tratamento mais adequado. / Rapid and efficient diagnosis of subclinical mastitis is important to reduce the persistence of the disease and its losses. This study aimed to evaluate the technique of mass spectrometry (MS) and desorption ionization by matrix assisted laser time-offlight (MALDI-TOF) for identification of bacteria causing bovine subclinical mastitis by two methods: 1) from bacteria isolated by microbiological culture, 2) from bacteria recovered directly from milk, to eliminate completely the need for microbiological culture for identification of pathogens. The study consisted of two experiments (1 and 2).In experiment 1, 33 milk samples from Gir and Holstein animals collected in four dairy farms were used for microbiological identification and MALDI-TOF MS. The samples with different results were confirmed by sequencing the 16S rRNA. These samples were identified by microbiological culture as Staphylococcus aureus (n = 13), Streptococcus agalactiae (n = 10) and Staphylococcus coagulase negative (SCN) (n = 10). For all strains of Streptococcus agalactiae, similar results were observed for microbiological identification and MALDI-TOF MS. From 13 isolates of Staphylococcus aureus, 11 were also identified by MALDI-TOF, one isolate was identified as Staphylococcus haemolyticus by 16S rRNA sequencing , and the other discrepant sample was charactezided a mixed culture of Staphylococcus aureus and Enterococcus faecalis. Regarding the SCN samples, all samples of this group were identified by MALDI-TOF at gender level (S. simulans, S. epidermidis, S. haemolyticus, S. Chromogens and S. aureus coagulase negative). In experiment 2, we evaluated the method of recovery of bacteria present in milk and their identification by MALDI-TOF MS using experimental method of contamination of milk with Escherichia coli, Enterococcus faecalis and Staphylococcus aureus. The identification of pathogens recovered directly from milk was possible when the concentration of E. faecalis and S. aureus was 106 ufc/mL and 107 ufc/mL for E.coli. We conclude that the use of MALDI-TOF MS can accelerate the identification of pathogens causing bovine subclinical mastitis may contribute to the adoption of control measures and appropriate treatment.

O objetivo do presente trabalho foi verificar se as perdas na produção de leite devidas ao aumento do número de células somáticas do leite (CCS) são proporcionais à produção (dependentes dela), ou absolutas (independentes dela). Além disso, pesquisou-se a partir de que valor de CCS as vacas começam a diminuir a produção de leite. Para concretizar o objetivo deste trabalho foram realizados dois estudos. No primeiro foram usadas 7.756 observações, colhidas mensalmente de um único rebanho, durante o período de setembro de 2000 a junho de 2002, enquanto que no segundo utilizaram-se 13.725 observações, colhidas mensalmente de 6 rebanhos, durante o período de janeiro de 2001 a junho de 2002. Em ambos os casos o modelo utilizado para curva de lactação foi baseado na função gama incompleta; no primeiro estudo foram considerados os efeitos de ordem de lactação, época do parto, ocorrência de doenças no periparto e escore de condição corporal ao parto, e no segundo, os efeitos de ordem de lactação, época do parto e rebanho. A CCS foi incluída nesse modelo de duas formas: como fator multiplicativo (representando perdas relativas) e como fator aditivo (representando perdas absolutas). A escolha do melhor modelo foi baseada no critério de informação de Schwarz (BIC). Nos dois estudos concluiu-se que as perdas na produção de leite são absolutas e variam apenas com a ordem de lactação (vacas primíparas e multíparas). No primeiro estudo os dados mostraram que as perdas iniciam a partir de 14.270 células/mL, e para cada aumento unitário na escala do logaritmo natural a partir desse valor estimam -se perdas de 184 e 869 g/dia para vacas primíparas e multíparas, respectivamente. No segundo, estimou-se que as perdas começam a ocorrer a partir de 17.000 células/mL e são de 238 e 868 g/dia para vacas primíparas e multíparas, respectivamente, para cada aumento unitário na escala do logaritmo natural a partir desse valor. / The objective of the present study was to evaluate whether milk yield losses due to increasing in milk somatic cells count (SCC) are proportional to milk yield (dependent of it), or absolute (independent of it). Also, it was investigated starting from which value of SCC the losses become evident. To achieve the objective of this research, two studies were done. In the first one, 7,756 observations were used, collected monthly from a single herd, from September/2000 up to June/2002, while in the second one, 13,725 observations were used, collected monthly from 6 herds, from January/2001 up to June/2002. In both cases, the analysis model for lactation curve was based upon the incomplete gamma function; in the first study the effects of lactation order, calving season, peripartum disorder incidence and body condition score at calving were considered, and in the second one, the effects of lactation order, calving season and herd were added. Somatic cells count entered in that model in two ways: as multiplicative factor (representing relative losses) and as additive factor (representing absolute losses). The best model was chosen based on the information criteria of Schwarz (BIC). In both studies, it was concluded that losses are absolute and just vary with the lactation order (primiparous and multiparous cows). In the first case, losses become evident starting from 14,270 cells/mL, and for each unitary increase in the natural logarithm scale from that value, there are losses of 184 and 869 g/day for primiparous and multiparous cows, respectively. In the second study, it was estimated that losses start from 17,000 cells/mL and are of 238 and 868 g/day for primiparous and multiparous cows, respectively, for each unitary increase in the natural logarithm scale from that value.

Staphylococcus spp. é uma das principais causas da mastite bovina, onde o uso de antimicrobianos tem sido comprometido por mecanismos de resistência bacteriana e geração de resíduos que alteram a qualidade e segurança alimentar do leite e derivados. Foi avaliada a atividade in vitro da nisina (NS), do brometo de dioctadecildimetilamônio (DDA) e do complexo NS/DDA contra Staphylococcus spp. resistentes isolados de mastite bovina. A CBM50 da nisina e DDA foi 50 e 4μg/mL, respectivamente, enquanto que a CBM50 do complexo NS/DDA foi 3/2μg/mL, com efeito parcialmente sinérgico. O estudo de time-kill revelou redução de 3 log10 UFC/mL após uma hora de interação entre NS/DDA e a bactéria. A microscopia de fluorescência confirmou uma perda da viabilidade após 6 horas de interação. A interação NS/DDA resultou na formação de nanopartículas (148,5 nm) catiônicas (+8,84 mV) cuja interação com a superfície bacteriana negativa (-27,32 mV) resultou em ação bactericida. NS/DDA pode ser uma alternativa promissora contra Staphylococcus spp. resistentes isolados de mastite bovina. / Staphylococcus spp. is a major cause of bovine mastitis, where the use of antimicrobials has been compromised by bacterial resistance mechanisms and waste generation that change the food quality and safety of milk and dairy products. The in vitro activity antibacterial was evaluated of the nisin (NS), of the dioctadecyldimethylammonium bromide (DDA) and of the NS/DDA complex against drug-resistant Staphylococcus spp. isolated from bovine mastitis. The CBM50 of nisin and DDA were 50 and 4μg/ml, respectively, while the CBM50 NS/DDA complex was 3/2mg/mL, with partially synergistic effect. The time-kill study showed reduction of 3log10 CFU/mL after an hour of interaction between NS/DDA and bacteria. Fluorescence microscopy confirmed a loss of viability after 6h of interaction. NS/DDA interaction resulted in formation of nanoparticles (148.5 nm) cationic (+8.84 mV) which interact with negative bacterial surface (-27.32 mV) resulted in bactericidal activity. NS/DDA may be a promising alternative against drug-resistant Staphylococcus spp. isolated from bovine mastitis.

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-02-09T15:48:50Z No. of bitstreams: 1 Milena da Silva Albuquerque.pdf: 915379 bytes, checksum: dd414d9f832cb823db0efc59b06ce1fb (MD5) / Made available in DSpace on 2017-02-09T15:48:50Z (GMT). No. of bitstreams: 1 Milena da Silva Albuquerque.pdf: 915379 bytes, checksum: dd414d9f832cb823db0efc59b06ce1fb (MD5) Previous issue date: 2014-12-17 / The objective of this work was to investigate the occurrence of encoding staphylococcal enterotoxin (sea, seb, sec and seg) genes and the toxin gene 1 of Toxic Shock Syndrome (tst) from Staphylococcus aureus, coming from mastitis cases bovine in the micro region of Garanhuns, State of Pernambuco. 93 isolates from Staphylococcus aureus were analyzed, which were obtained from milk samples from cows with clinical mastitis and subclinical from 17 properties in 11 municipalities of the Micro Region of Garanhuns, State of Pernambuco. For the molecular characterization of the species Staphylococcus aureus, one of the Polymerase Chain Reaction (PCR) was performed in order to identify the presence of the nuc gene, and to the molecular characterization of enterotoxins, and Staphylococcal toxin Toxic Shock Syndrome. Specific genes were identified: sea, seb, sec, seg and tst. 93 genes were analyzed and we observed the presence of enterotoxigenic gene in 20 (21.6%) samples, of which 11 (55.0%) were positive for tst gene, seven (35.0%) for the sec gene two (10.0%) for the seg gene. 20 isolates amplified segments to the presence of the sec, seg and tst genes. 16 of these (80.0%) were positive for only one gene, and four (20.0%) were positive for both genes (tst and sec). 17 properties were studied, of which seven (41.2%) had positive cultures for at least one of the genes sec, seg and tst. This was the first hit record of encoding gene of the toxin of toxic shock syndrome in mastitis cows milk samples in the state of Pernambuco. Since there was a variation in the distribution of sec, seg and tst genes in strains from different property, it can be inferred that there is genotypic variation in S. aureus strains that cause bovine mastitis. / Objetivou-se com este trabalho pesquisar a ocorrência de genes codificadores de enterotoxinas estafilocócicas (sea, seb, sec e seg) e do gene da toxina 1 da síndrome do choque tóxico (tst) a partir de isolados de Staphylococcus aureus procedentes de casos de mastite bovina na microrregião Garanhuns, estado de Pernambuco. Foram analisados 93 isolados de Staphylococcus aureus obtidos a partir de amostras de leite de vacas com mastite clínica e subclínica, provenientes de 17 propriedades localizadas em 11 municípios da microrregião Garanhuns, no estado de Pernambuco. Para a caracterização molecular da espécie Staphylococcus aureus, foi realizada uma Reação em Cadeia da Polimerase (PCR) visando a identificação pela presença do gene nuc, assim como para a caracterização molecular das Enterotoxinas Estafilocócicas e da toxina da Síndrome do Choque Tóxico. Foram identificados os genes específicos sea, seb, sec, seg e tst. Dos 93 isolados analisados, observou-se a presença de genes enterotoxigênicos em 20 (21,6%) amostras, das quais 11 (55,0%) foram positivas para o gene tst, sete (35,0%) para o gene sec, duas (10,0%) para o gene seg. Dentre os 20 isolados que amplificaram segmentos para a presença dos genes sec, seg e tst, 16 (80,0%) foram positivos apenas para um gene e quatro (20,0%) foram positivos para dois genes (sec e tst). Das 17 propriedades estudadas, sete (41,2%) apresentaram amostras positivas para pelo menos um dos genes sec, seg e tst. Este foi o primeiro registro de ocorrência do gene codificador da toxina da síndrome do choque tóxico em amostras de leite de vacas com mastite no estado de Pernambuco. Como houve uma variação na distribuição dos genes sec, seg e tst nas cepas procedentes de diferentes propriedades, pode-se inferir que há uma variação genotípica nas cepas de S. aureus que causam mastite bovina.

Made available in DSpace on 2016-04-28T20:16:18Z (GMT). No. of bitstreams: 1 Lidiane de Castro Soares.pdf: 3621583 bytes, checksum: ed47c1ba184a35cfda362913c8e922a3 (MD5) Previous issue date: 2010-02-22 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Coagulase-negative staphylococci (SCN) take part of the normal microbiota. Although this bacteria has been considered saprophytic, nowadays there is a concern about its pathogenic potential. Nevertheless, the improvement in SCN identification assays, it continues to be neglected in laboratorial routine of infectious diseases because of the wide range of species. In spite of this, the appropriated identification of the species is necessary in order to differentiate the potential pathogenic agents and to determine its antimicrobial susceptibility pattern. The present study was performed to characterize phenotypically and genotypically the antibiotic resistance profile and virulence factors of coagulase-negative Staphylococcus spp. isolated from milk samples of cows with subclinical mastitis. Staphylococcus xylosus, S. haemolyicus, S. cohnii and S. hominis were the identified species. Antimicrobial susceptibility test yielded a high level of resistance to penicillin and ampicillin. A total of 100 isolates were studied, mecA gene was detected in 4% of isolates, also mecR1 positive. The mecI gene was detected in 47% of isolates. All mec genes (mecA-mecI-mecR1) were detected in only 2 isolates. The production of betalactamases and blaZ gene were detected in 16% of the isolates. From this, only 3 isolates showed all bla genes (blaZ, blaI e blaRI). All bla positive isolates were penicillin and ampicillin resistant and positive to nitrocefin test. The femA gene was not detected in any of the isolates. Concerning to the virulence factors, microplate technique and the congo red agar presented production of slime in 46% and 77% of the isolates, respectively. The icaA and icaD genes were detected in 9% and 10% of isolates, respectively. Hemolysis was detected in 13% of the isolates, 15,4% of total hemolysis and 84,6% partial hemolysis. The hla and hlb genes were not detected in any isolate. The hemolytic synergism was positive in 15 isolates, of these 14 showed no hemolysins. Unable to establish a correlation between phenotypic and genotypic resistance to beta-lactam antibiotics in isolates. / Os estafilococos coagulase-negativos (ECN) fazem parte da microbiota normal da pele e apesar de terem sido considerados sapr?fitas por muito tempo, o seu significado cl?nico como agente etiol?gico tem aumentado com o passar dos anos. No entanto, apesar de todo avan?o nas t?cnicas de identifica??o dos ECN e do conhecimento destes como agentes etiol?gicos em diversos processos infecciosos, estes microrganismos muitas vezes s?o negligenciados na rotina laboratorial. A identifica??o das esp?cies de ECN, embora de dif?cil realiza??o para a maioria dos laborat?rios cl?nicos, ? necess?ria para diferenciar o potencial patog?nico e o perfil de resist?ncia de cada isolado. O presente estudo foi conduzido para caracterizar fenot?pica e genotipicamente o perfil de resist?ncia aos antibi?ticos, especialmente ? oxacilina, e fatores de virul?ncia de isolados de Staphylococcus spp. coagulase-negativos provenientes de amostras de leite de vacas com mastite subcl?nica. Foram identificadas as esp?cies Staphylococcus xylosus, S. haemolyicus, S. cohnii e S. hominis. O teste de suscetibilidade antimicrobiana revelou elevada resist?ncia ? penicilina e ampicilina. Do total de 100 isolados estudados, o gene mecA foi detectado em apenas 4 (4%), os quais tamb?m foram mecR1 positivos. O gene mecI foi detectado em 47% dos isolados. Em 2 isolados foram detectados todos os genes do sistema mec (mecA-mecI-mecR1). A produ??o de beta-lactamases e do gene blaZ foi detectada em 16% dos isolados. Em 3 isolados foram detectados todos os genes do sistema bla (blaZ, blaI e blaRI). Todos os isolados bla positivos foram resistentes a penicilina e ampicilina e positivos no teste do nitrocefin. O gene femA n?o foi detectado em nenhum dos isolados avaliados. Em rela??o aos fatores de virul?ncia, a t?cnica da microplaca e o ?gar contendo vermelho congo revelaram 46% e 77% de isolados produtores de slime , respectivamente. Os genes icaA e icaD foram detectados em 9% e 10% dos isolados, respectivamente. A hem?lise foi detectada em 13% dos isolados, destes 15,4% apresentaram hem?lise total e 84,6% a hem?lise parcial. Os genes hla e hlb n?o foram detectados em nenhum isolado. O sinergismo hemol?tico foi positivo em 15 isolados sendo que destes, 14 n?o apresentaram hemolisinas. N?o foi poss?vel estabelecer uma correla??o entre os testes fenot?picos e genot?picos de resist?ncia aos antibi?ticos beta-lact?micos nos isolados avaliados.

Made available in DSpace on 2016-04-28T20:16:26Z (GMT). No. of bitstreams: 1 2008- Bruno Gomes de Castro01.pdf: 514321 bytes, checksum: bd4fc9254117e81edaedf3e2ba5305e2 (MD5) Previous issue date: 2008-04-01 / Funda??o Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / This study had the objective of evaluating the capacity of Stomoxys calcitrans fly to vehicular bacterial agents causing bovine mastitis. Just as evaluate the enterobacterian microbiota on the external surface, mouth parts and abdominal digestive tract of the stable fly. And also realize a survey of the bacterial microbiota of the cases of bovine mastitis on the visited properties. There were taken mastitis milk samples and 20 specimens of stable flies on 10 visited properties in the municipatility of Barra Mansa and Resende, Rio de Janeiro State. The collected milk was submitted to bacterial isolation in the laboratory of Bacteriology UFRRJ. Samples were subcultures on MacConkey (MC) agar, Brain-Heart Infusion agar (BHI) and Manitol-Phenol Red agar. The flies were individually washed in BHI broth, sterilized and had its mouth parts and abdominal intestinal content dissected under stereoscope microscope. These structures were macerated in BHI broth and, after 24 hours at 37 ?C, subcultured at MC agar, Agar Eosin Methilen Blue (EMB) and enriched in Sodium Tetrathionate Broth after incubation, and were subcultured in Salmonella-Shigella agar. After this stage, each isolated colony was observed for differences in morphology as size and pigment production characteristics to be identified through biochemical specific tests. It was also realized an antimicrobial resistance test to verify to which antibiotics the isolated bacteria were sensible. When were coincidences between the enterobacterian species in the milk and on the flies evaluated segments, was realized a Random Amplified Polymorphic DNA (RAPD-PCR) as an instrument of evaluating the genetic diversity and the eletrophoretic profile similarity the bacterial subpopulations. In agreement with the obtained results, was verified that the mastitis milk had as etiological agents specially the species Staphylococcus aureus, coagulasenegative staphylococci and Escherichia coli. The antimicrobials with less resistance rate were Amoxilin-Clavulanic Acid and Norfloxacin. Regarding to the enterobacterian microbiota verified on S. calcitrans segments, Escherichia coli, Enterobacter agglomerans, Enterobacter cloacae e Salmonella spp were the most prevalent bacteria. The segment with the higher number of isolations was the external surface, with 73 (45.91%) distinct colonies isolated. The mouth parts had 46 (28.93%) colonies and the abdominal digestive tract 40 (25.16%) isolated colonies. The eletrophoretic profile similarity happened just on E. coli subpopulations on milk samples and S. calcitrans segments of the same property. This way, was possible to observe that the flies do not act as a vehicle of any bovine mastitis causative E. coli. Regarding to the identified E. coli, was verified that 13.79% were Shiga Toxin-Producing, and the genes stx1, stx2 and eae were identified. On the present study, was verified that this fly has the capacity to act as vehicle to enterobacterian, on its external surface as well as inside its body. The stable fly control may contribute to the improvement on animal productivity and sanity. / Este estudo teve o objetivo de avaliar a capacidade da mosca Stomoxys calcitrans em veicular agentes bacterianos causadores de mastite bacteriana bovina, bem como avaliar a microbiota enterobacteriana na superf?cie externa, aparelho bucal e trato digest?rio abdominal da mosca dos est?bulos; e fazer um levantamento da microbiota bacteriana nos casos de mastite bovina das propriedades visitadas. Foram coletadas amostras de leite com mastite e 20 esp?cimes da mosca dos est?bulos em 10 propriedades visitadas nos munic?pios de Barra Mansa e Resende, Estado do Rio de Janeiro. No Laborat?rio de Bacteriologia da UFRRJ foi realizado o isolamento bacteriano do leite coletado em Agar MacConkey (MC), Agar Infuso C?rebro Cora??o (BHI) e Manitol Vermelho de Fenol. As moscas foram lavadas individualmente em Caldo BHI, esterilizadas e, sob a luz de um microsc?pio estereosc?pio, tinham seu aparelho bucal e conte?do intestinal abdominal dissecado. Estas estruturas foram maceradas em Caldo BHI e ap?s 24 horas a 37 ?C repicados em Agar MC, Agar Eosina Azul de Metileno (EMB) e enriquecidos em Caldo Tetrationato de S?dio que ap?s incuba??o, era repicado em Agar Salmonella-Shigella. Ap?s esta etapa, cada col?nia isolada era avaliada quanto ?s suas caracter?sticas morfo-tintoriais para serem identificadas atrav?s de testes bioqu?micos espec?ficos. Tamb?m foi realizado o teste de resist?ncia antimicrobiana, com a finalidade de se verificar, as quais antibi?ticos as bact?rias isoladas foram sens?veis. Quando havia coincid?ncia de esp?cies enterobacterianas no leite e nos segmentos avaliados das moscas, se realizava a t?cnica de an?lise do polimorfismo dos segmentos de DNA obtidos por amplifica??o rand?mica (RAPD-PCR) como instrumento de avalia??o da diversidade gen?tica e das rela??es de clonalidade entre estas subpopula??es bacterianas. De acordo com os resultados obtidos, foi verificado que o leite com mastite, teve como principais agentes etiol?gicos as esp?cies Staphylococcus aureus, Estafilococos Coagulase Negativa (ECN) e Escherichia coli. Os antimicrobianos com menor taxa de resist?ncia foram Amoxicilina/?cido Clavul?nico e Norfloxacina. Com rela??o ? microbiota enterobacteriana verificada nos segmentos de S. calcitrans, foi observado que Escherichia coli, Enterobacter agglomerans, Enterobacter cloacae e Salmonella spp foram as bact?rias mais prevalentes. O segmento com o maior n?mero de isolados foi a superf?cie externa, onde foram isoladas 73 col?nias distintas (45,91%), seguida pelo aparelho bucal com 46 col?nias (28,93%), e pelo trato digest?rio abdominal com 40 col?nias isoladas (25,16%). Houve clonalidade apenas de sub-popula??es de E. coli entre as amostras de leite e de segmentos de S. calcitrans de uma mesma propriedade. Desta forma, foi poss?vel observar que as moscas n?o veiculavam nenhuma E. coli causadora de mastite bovina. No que se refere ?s E. coli identificadas, foi verificado que 13,79% eram Shiga-Toxig?nicos, sendo identificados os genes stx1, stx2 e eaeA. No presente estudo, verificou-se que esta mosca tem a capacidade de veicular enterobact?rias, tanto em sua superf?cie externa como no interior de seu corpo. O controle da mosca dos est?bulos poder? contribuir para melhora na sanidade e produtividade animal.

Os objetivos deste trabalho foram determinar a sensibilidade e especificidade do limiar de contagem de células somáticas (CCS) de vacas Gir para o diagnóstico de mastite subclínica causada por patógenos primários e secundários e avaliar os efeitos de rebanho, vaca, mês de coleta, quarto mamário, presença de infecção intramamária, tipo de microrganismo e suas interações sobre o logCCS e composição do leite. Avaliou-se a hipótese que o limiar da CCS para detecção de mastite subclínica é igual entre vacas Gir e Holandesas. Foram utilizadas 221 vacas Gir em lactação, provenientes de três fazendas comerciais. Foram coletadas amostras de leite individuais por quarto mamário e compostas uma vez por mês, durante um ano. Foram realizadas análises de CCS, composição do leite e cultura microbiológica. O quarto mamário e a vaca foram considerados unidades experimentais. Para determinar a sensibilidade, especificidade e odds ratio (OR) dos limiares da CCS para identificação de quartos infectados, foram utilizados quatro valores de CCS: 100, 200, 300 e 400 (x 103 células/mL), assim como a correlação entre a CCS e composição do leite. Não houve efeito do rebanho sobre o logCCS para amostras individuais de quartos mamários e compostas, mas vaca dentro de rebanho foi o principal fator responsável pela variação do logCCS. Houve efeito do rebanho sobre a composição do leite, assim como o mês de coleta apresentou efeito tanto sobre o logCCS quanto para a composição do leite, considerando as duas unidades experimentais. A presença de infecção intramamária afetou negativamente a composição do leite, exceto sobre o teor de gordura; sendo que os maiores teores de lactose, proteína e ESD foram encontrados em amostras sem isolamento bacteriano. Os maiores logCCS foram obtidos em amostras infectadas. O limiar da CCS de 100 x 103 células/mL apresentou, em ambas as unidades experimentais, maiores valores de sensibilidade e valor preditivo negativo. O limiar de 200 x 103 células/mL apresentou maior chance da ocorrência de mastite do que o limiar de 100 x 103 células/mL. Foi observada correlação negativa entre CCS com lactose e extrato seco desengordurado (ESD), mas a correlação foi positiva entre CCS com gordura e proteína, tanto em nível de quarto mamário quanto de vaca. Portanto, a composição do leite foi influenciada pela CCS, os teores de lactose e ESD diminuíram em altas CCS, enquanto que as concentrações de gordura e proteína aumentaram. / The aim of this study was to determine the sensibility and specificity of somatic cell count (SCC) threshold in Gyr cows to diagnosis the subclinical mastitis caused by primary and secondary pathogens, and to evaluate the effect of herd, cows, month, mammary quarter, intramammary infection, type of microorganism and their interactions on logSCC and milk composition. The hypothesis to be tested was that the SCC threshold to detection of subclinical mastitis is the same for Holstein and Gir cows. A total of 221 lactation Gir cows from three commercial dairy farms was selected. Composed and quarter individual milk samples were collected once a month, during one year for SCC, milk composition and bacteriological analysis. The mammary quarter and the cow were considered experimental units. To determine the sensibility, specificity and odds ratio (OR) from SCC threshold to identify the infected quarters four values of SCC: 100, 200, 300 and 400 (x 103 cells/mL) were used. It was also evaluated the correlation between SCC and milk composition. There was no effect of herd on logSCC in individuals and composed samples, but cow nested within herd was major factor responsible for the logSCC variation. The month of sampling presented significant effect on logSCC and milk composition in both experimental units. The intramammary infection presence affected negatively the milk composition, except of fat concentration. Higher lactose, protein and non-fat solids (NFS) percentages were found in negative samples and higher logSCC were observed in infected samples. The SCC threshold of 100 x 103 cells/mL presented the major sensibility and negative predictive value for subclinical mastitis detection. The threshold of 200 x 103 cells/mL had higher chance to have mastitis than the threshold of 100 x 103 cells/mL. It was observed a negative correlation between SCC with lactose and NFS; but the correlation was positive between SCC with fat and protein in mammary quarters and cow level. Milk composition was influenced by SCC, once the lactose and NFS percentages decreased in samples with high SCC and the protein and fat concentration increased.

Diseases that affect dairy cattle have serious economic and animal welfare implications. The inclusion of immune response (IR) traits in breeding indices has been suggested to improve inherent animal health, and decrease the use of antimicrobials. The objectives of this research were to (1) evaluate cell-mediated (CMIR) and antibody-mediated immune responses (AMIR) on 680 Holstein cows from 58 herds across Canada, (2) estimate genetic parameters of these traits (3) examine the associations with routinely evaluated traits as well as the incidence of mastitis, (4) evaluate the correlation of natural and specific antibody and (5) perform a genome-wide association study (GWAS) to determine genetic markers associated with high or low IR. In collaboration with the Canadian Bovine Mastitis Research Network cows were immunized with both a type 1 and type 2 test antigen to stimulate CMIR and AMIR, respectively. A cutaneous delayed-type hypersensitivity test to the type 1 test antigen was used as an indicator of CMIR, and serum antibody (IgG1 and IgG2) to the type 2 test antigen as an indicator of AMIR. IR phenotypes varied significantly by cow, herd and region in Canada. Heritability estimates were moderate, 0.19 for CMIR and 0.16-0.43 for AMIR depending on time in the immunization protocol and antibody isotype. Beneficial associations between AMIR and some reproductive traits were found. Using estimated breeding values, cows were classified as high, average or low responders. High AMIR cows had significantly lower incidence rates of clinical mastitis compared to average and low cows. No difference was found when cows were classified based on CMIR. Natural antibody was not genetically correlated with specific antibody nor was it associated with mastitis. The GWAS found 198 genetic markers significantly associated with AMIR, with the majority on chromosome 23 where the major histocompatability complex is located. Other significant genes involved in IR include those associated with the complement system, interleukin 17 and tumor necrosis factor. This research confirms the benefit of identifying high IR cows and gives a glimpse of current IR profiles in Canadian Holsteins. This was the first GWAS for IR traits in dairy cattle and suggests it may be possible to include IR traits in genomic selection indices. / This research was financed by grants to B.A. Mallard from National Sciences and Engineering Research Council of Canada, Alberta Milk, Dairy Farmers of New Brunswick, Nova Scotia, Ontario and Prince Edward Island, Novalait Inc., Dairy Farmers of Canada, DairyGen council of Canadian Dairy Network, Agriculture and Agri-Food Canada, Public Health Agency of Canada, Technology PEI Inc., Université de Montréal and University of Prince Edward Island through the Canadian Bovine Mastitis Research Network. Kathleen Adele Thompson Crispi was funded by the Dairy Farmers of Ontario Doctoral Research Assistantship.

Peu d’études ont exploré l’utilisation du traitement prolongé pour les mammites cliniques et aucune ne portait sur l’utilisation du traitement prolongé à base de ceftiofur pour le traitement des mammites cliniques légères à modérées. L’objectif de cette étude était d’évaluer l’efficacité du traitement intra-mammaire prolongé à base de ceftiofur pour les mammites cliniques légères à modérées, en considérant toutes les bactéries responsables ou seulement Staphylococcus aureus (S. aureus) et les streptocoques. Des vaches laitières (n = 241) de 22 élevages du Québec et de l’Ontario ont été inclues. L’étude a été conçue comme un essai clinique à allocation aléatoire. Pour chaque cas de mammite clinique légère à modérée, 125 mg d’hydrochloride de ceftiofur (Spectramast® LC) a été administré par voie intra-mammaire une fois par jour pour 2 ou 8 jours. Le pourcentage de guérison clinique 21 jours après la fin du traitement était de 89% (n = 98/110 pour chaque groupe, p = 0,95). Les pourcentages de guérison bactériologique 21 jours après la fin du traitement pour les groupes 2 jours et 8 jours étaient 32% (n = 15/47) et 61% (n = 25/41) respectivement pour toutes les bactéries (p < 0,01); 64% (n = 9/14) et 82% (n = 9/11) respectivement pour les streptocoques (p = 0,50); et 0% (n = 0/20) et 47% (n = 9/19) pour S. aureus (p < 0,01). Il n'y avait pas de différence entre les 2 groupes pour les nouvelles infections intra-mammaires (p = 0,30). Le traitement prolongé à base de ceftiofur est un choix raisonnable pour le traitement des mammites cliniques légères à modérées, en particulier causées par S. aureus. / Little research has focused on extended therapy in lactating cows with clinical mastitis and none were with ceftiofur. The objective of the present study was to evaluate the efficacy of an intramammary extended ceftiofur treatment for mild to moderate clinical mastitis, and to determine whether it would increase cure rates when considering any bacteria or more particularly Staphylococcus aureus (S. aureus) and streptococci. Holstein dairy cows (n = 241) from 22 herds located in Quebec and Ontario, Canada, were included. The study was designed as a randomized clinical trial. For each case of mild to moderate clinical mastitis diagnosed, 125 mg of ceftiofur hydrochloride (Spectramast® LC) was administered intramammary once a day for 2 or 8 consecutive days. Clinical cure rate 21 days after the last treatment was identical in each group (89%; n = 98/110; p = 0.95). Bacteriological cure rates 21 days after the last treatment for the 2- and 8-day regimens were 32% (n = 15/47) and 61% (n = 25/41), respectively, for all bacteria (p < 0.01); 64% (n = 9/14) and 82% (n = 9/11), respectively, for streptococci (p = 0.50); and 0% (n = 0/20) and 47% (n = 9/19), respectively, for S. aureus (p < 0.01). There were no statistical differences between experimental groups for new intramammary infections. Extended therapy appears to be a reasonable choice for the treatment of mild to moderate clinical mastitis, particularly in cases where S. aureus is the pathogen involved.