RESUMO: A mastite bovina é a doença mais impactante na indústria de laticínios e é caracterizada por uma complexidade de agentes causais, os quais têm revelado uma variação geográfica entre regiões e países. Os patógenos relacionados à mastite têm sido tradicionalmente classificados como contagiosos ou ambientais, com base na fonte de infecção ou origem e nas vias de transmissão. Além disso, a gravidade das infecções mamárias tem sido associada à virulência dos patógenos, e ao estado imunológico e nutricional dos animais. Considerando esse cenário, investigamos os agentes etiológicos, os escores de gravidade clínica e os dados de dias em lactação (DEL) em 4.273 casos clínicos de mastite bovina em dez fazendas de grande porte, quatro localizadas no sudoeste do estado de São Paulo e seis no sul do Estado de Minas Gerais, Brasil. Streptococcus dysgalactiae (283/4.273 = 6,6%), Escherichia coli (190/4.273 = 4,4%), Prototheca spp. (112/4.273 = 2,6%) e Streptococcus uberis (95/4.273 = 2,2%) foram os patógenos predominantes isolados. Entre os 4.273 casos clínicos, a pontuação de gravidade clínica estava disponível em 43,8% (1.871/4.273) dos animais. Desses, 69,8% (1.306/1.871), 27,3% (510/1.871) e 2,9% (55/1.871) foram classificados como leves, moderados e graves, respectivamente. Os patógenos foram observados principalmente em amostras obtidas durante os primeiros 100 dias em lactação, e a gravidade clínica foi principalmente classificada como leve. Nossos resultados contribuem para a identificação etiológica, a pontuação de gravidade clínica e os aspectos de ordenha de vacas com mastite em fazendas leiteiras com histórico de infecções mamárias clínicas. RESUMO causais países ambientais transmissão disso animais cenário etiológicos DEL (DEL 4273 4 273 4.27 porte Gerais Brasil 283/4.273 2834273 283 (283/4.27 6,6%, 66 6,6% , 6 6,6%) 190/4.273 1904273 190 (190/4.27 4,4%, 44 4,4% 4,4%) spp 112/4.273 1124273 112 (112/4.27 2,6% 26 2 95/4.273 954273 95 (95/4.27 2,2% 22 isolados 438 43 8 43,8 1.871/4.273 18714273 1 871 (1.871/4.273 Desses 698 69 69,8 1.306/1.871, 13061871 1.306/1.871 306 (1.306/1.871) 27 3 27,3 510/1.871 5101871 510 (510/1.871 29 9 2,9 55/1.871 551871 55 (55/1.871 leves graves respectivamente 10 leve etiológica clínicas 427 4.2 283/4.27 283427 28 (283/4.2 6,6 190/4.27 190427 19 (190/4.2 4,4 112/4.27 112427 11 (112/4.2 2,6 95/4.27 95427 (95/4.2 2,2 43, 1.871/4.27 1871427 87 (1.871/4.27 69, 1306187 1.306/1.87 30 (1.306/1.871 27, 510/1.87 510187 51 (510/1.87 2, 55/1.87 55187 5 (55/1.87 42 4. 283/4.2 28342 (283/4. 6, 190/4.2 19042 (190/4. 4, 112/4.2 11242 (112/4. 95/4.2 9542 (95/4. 1.871/4.2 187142 (1.871/4.2 130618 1.306/1.8 (1.306/1.87 510/1.8 51018 (510/1.8 55/1.8 5518 (55/1.8 283/4. 2834 (283/4 190/4. 1904 (190/4 112/4. 1124 (112/4 95/4. 954 (95/4 1.871/4. 18714 (1.871/4. 13061 1.306/1. (1.306/1.8 510/1. 5101 (510/1. 55/1. 551 (55/1. 283/4 (283/ 190/4 (190/ 112/4 (112/ 95/4 (95/ 1.871/4 1871 (1.871/4 1306 1.306/1 (1.306/1. 510/1 (510/1 55/1 (55/1 283/ (283 190/ (190 112/ (112 95/ (95 1.871/ 187 (1.871/ 130 1.306/ (1.306/1 510/ (510/ 55/ (55/ (28 (19 (11 (9 1.871 18 (1.871 13 1.306 (1.306/ (510 (55 (2 (1 ( 1.87 (1.87 1.30 (1.306 (51 (5 1.8 (1.8 1.3 (1.30 1. (1. (1.3

ABSTRACT: Bovine mastitis is the most impacting disease of dairy industry, and it is characterized by a complexity of causal agents, which have revealed a geographical variation among regions and countries. The mastitis-related pathogens have been traditionally classified as contagious or environmental, based on habits of the microorganisms and transmission routes. Besides, the severity of mammary infections has been associated with the virulence of the pathogens, and immune and nutritional status of the hosts. Considering this scenario, we investigated the etiological pattern, clinical severity scores, and days in milk (DIM) data in 4,273 clinical cases of bovine mastitis among ten large-dairy farms located in Brazil. Streptococcus dysgalactiae (283/4,273 = 6.6%), Escherichia coli (190/4,273 = 4.4%), Prototheca spp. (112/4,273 = 2.6%), and Streptococcus uberis (95/4,273 = 2.2%) were the predominant pathogens isolated. Among 4,273 clinical cases, clinical severity score was available in 43.8% (1,871/4,273) animals. From these, 69.8% (1,306/1,871), 27.3% (510/1,871), and 2.9% (55/1,871) were scored as mild, moderate, and severe, respectively. Pathogens were observed mainly in samples obtained during the first 100 days in milk, and clinical severity scored mainly as mild. Our results contributed to the etiological identification, clinical severity scoring, and milking aspects of mastitic cows in dairy farms with a history of clinical mammary infections. ABSTRACT industry agents countries mastitisrelated related environmental routes Besides hosts scenario pattern scores DIM (DIM 4273 4 273 4,27 largedairy large Brazil 283/4,273 2834273 283 (283/4,27 6.6%, 66 6.6% , 6 6.6%) 190/4,273 1904273 190 (190/4,27 4.4%, 44 4.4% 4.4%) spp 112/4,273 1124273 112 (112/4,27 2.6%, 26 2.6% 2 2.6%) 95/4,273 954273 95 (95/4,27 2.2% 22 isolated 438 43 8 43.8 1,871/4,273 18714273 1 871 (1,871/4,273 animals these 698 69 69.8 1,306/1,871, 13061871 1,306/1,871 306 (1,306/1,871) 27 3 27.3 510/1,871, 5101871 510/1,871 510 (510/1,871) 29 9 2.9 55/1,871 551871 55 (55/1,871 mild moderate severe respectively 10 identification scoring 427 4,2 283/4,27 283427 28 (283/4,2 6.6 190/4,27 190427 19 (190/4,2 4.4 112/4,27 112427 11 (112/4,2 2.6 95/4,27 95427 (95/4,2 2.2 43. 1,871/4,27 1871427 87 (1,871/4,27 69. 1306187 1,306/1,87 30 (1,306/1,871 27. 510187 510/1,87 51 (510/1,871 2. 55/1,87 55187 5 (55/1,87 42 4, 283/4,2 28342 (283/4, 6. 190/4,2 19042 (190/4, 4. 112/4,2 11242 (112/4, 95/4,2 9542 (95/4, 1,871/4,2 187142 (1,871/4,2 130618 1,306/1,8 (1,306/1,87 51018 510/1,8 (510/1,87 55/1,8 5518 (55/1,8 283/4, 2834 (283/4 190/4, 1904 (190/4 112/4, 1124 (112/4 95/4, 954 (95/4 1,871/4, 18714 (1,871/4, 13061 1,306/1, (1,306/1,8 5101 510/1, (510/1,8 55/1, 551 (55/1, 283/4 (283/ 190/4 (190/ 112/4 (112/ 95/4 (95/ 1,871/4 1871 (1,871/4 1306 1,306/1 (1,306/1, 510/1 (510/1, 55/1 (55/1 283/ (283 190/ (190 112/ (112 95/ (95 1,871/ 187 (1,871/ 130 1,306/ (1,306/1 510/ (510/1 55/ (55/ (28 (19 (11 (9 1,871 18 (1,871 13 1,306 (1,306/ (510/ (55 (2 (1 ( 1,87 (1,87 1,30 (1,306 (510 (5 1,8 (1,8 1,3 (1,30 (51 1, (1, (1,3

A identificação da Escherichia coli requer conhecimento sobre os sorotipos e fatores de virulência prevalentes permitindo a classificação em patogênico/não patogênico. No entanto, algumas destas bactérias não expressam o antígeno flagelar in vitro. Neste caso, o PCR-restriction fragment length polymorphism (RFLP-PCR) e o sequenciamento do gene fliC podem ser adequados para a identificação desses antígenos, substituindo a sorologia tradicional. Nesta pesquisa foram estudadas 17 amostras de E. coli isoladas de animais e que apresentavam antígeno H não tipável (HNT). Os antígenos H foram caracterizados por PCR-RFLP e sequenciamento do gene fliC. Três novos genes da flagelina foram identificados, para os quais anti-soros específicos foram obtidos. A técnica PCR-RFLP mostrou-se mais rápida que a sorotipagem do antígeno H em E. coli, fornecendo informações sobre algumas características desses antígenos e indicou a presença de novos genes fliC.

Identification of Escherichia coli requires knowledge regarding the prevalent serotypes and virulence factors profiles allows the classification in pathogenic/non-pathogenic. However, some of these bacteria do not express flagellar antigen invitro. In this case the PCR-restriction fragment length polymorphism (RFLP-PCR) and sequencing of the fliC may be suitable for the identification of antigens by replacing the traditional serology. We studied 17 samples of E. coli isolated from animals and presenting antigen H nontypeable (HNT). The H antigens were characterized by PCR-RFLP and sequencing of fliC gene. Three new flagellin genes were identified, for which specific antisera were obtained. The PCR-RFLP was shown to be faster than the serotyping H antigen in E. coli, provided information on some characteristics of these antigens and indicated the presence of new genes fliC.

Escherichia coli has been isolated frequently, showing flagellar antigens that are not recognized by any of the 53 antisera, provided by the most important reference center of E. coli, The International Escherichia and Klebsiella Center (WHO) of the Statens Serum Institute, Copenhagen, Denmark. The objective of this study was to characterize flagellar antigens of E. coli that express non-typeable H antigens. The methods used were serology, PCR-RFLP and DNA sequencing. This characterization was performed by gene amplification of the fliC (flagellin protein) by polymerase chain reaction in all 53 standards E.coli strains for the H antigens and 20 E. coli strains for which the H antigen was untypeable. The amplicons were digested by restriction enzymes, and different restriction enzyme profiles were observed. Anti-sera were produced in rabbits, for the non-typeable strains, and agglutination tests were carried out. In conclusion,the results showed that although non-typeable and typable H antigens strains had similar flagellar antigens, the two types of strains were distinct in terms of nucleotide sequence, and did not phenotypically react with the standard antiserum, as expected. Thirteen strains had been characterized as likely putative new H antigen using PCR-RFLP techniques, DNA sequencing and/or serology.

Este trabalho teve como objetivo verificar a presença de genes de virulência em linhagens de Escherichia coli isoladas de queijo ralado comercializado em feiras livres de Cuiabá-MT. Quarenta amostras desse alimento foram submetidas a provas microbiológicas, sendo 22 (55%) positivas para E. coli. Em seguida, 64 isolados de E. coli provenientes das amostras positivas foram testados, por meio da reação da polimerase em cadeia (PCR), para a presença dos genes que codificam os seguintes fatores de virulência: stx1e stx2 (verotoxina tipo 1 e 2), eae (intimina), lt1 (toxina termolábil tipo 1), st1 (toxina termoestável tipo 1), cnf1 e cnf2 (fator de necrose citotóxico tipo 1 e 2) e cdtB (toxina citoletal distensora). Todos os isolados analisados foram negativos para os genes vt1, vt2, eae, lt1, st1, cnf1, cdtB e cinco (7,81%) foram positivos para o gene cnf2. Observou-se uma baixa prevalência de E. coli portadora de fatores de virulência associados à patogênese da diarréia. Entretanto, a presença de linhagens produtoras de CNF-2 e a possibilidade de ocorrência e dispersão de outros fatores de virulência não pesquisados neste estudo apontam para o risco vinculado ao consumo de queijo ralado de origem informal.

This research aimed to verify the presence of virulence genes in strains of Escherichia coli isolated from grated cheese sold in farmers' markets of Cuiabá-MT, Brazil. Forty samples of this food were submitted for microbiological analysis and 22 (55%) tested positive for E. coli. Next, 64 strains of E. coli were isolated from the positive samples and screened by the polymerase chain reaction (PCR) for the presence of the genes encoding the following virulence factors: stx1 and stx2 (verotoxin types 1 and 2), eae (intimin), lt1 (heat-labile toxin type 1), st1 (heat-stable toxin type 1), cnf1 and cnf2 (cytotoxic necrozing factor types 1 and 2), and cdtB (cytolethal distending toxin). All the isolates were negative for the genes stx1, stx2, eae, lt1, st1, cnf1, and cdtB, and five strains (7.81%) were positive for cnf2. A low prevalence of E. coli positive for virulence factors associated with the pathogenesis of diarrhoea was observed in this study. However, the presence of CNF-2 producing strains and the possibility of occurrence and scattering of other virulence factors that were not surveyed in the work indicate the risk related to the consumption of grated cheese from farmers' markets.

Amostras de Escherichia coli, isoladas de pacientes do sexo feminino com quadro clínico de cistite, foram caracterizadas quanto à presença de fatores de virulência associados à formação de biofilme e ao agrupamento filogenético. Os resultados da reação em cadeia da polimerase demonstraram que todas as amostras foram positivas para o gene fimH (fímbria do tipo1), 91 amostras foram positivas para o gene fliC (flagelina) 50 amostras positivas para o gene papC (fímbria P), 44 amostras positivas para o gene kpsMTII (cápsula) e 36 amostras positivas para o gene flu (antígeno 43). Os resultados dos ensaios de quantificação da formação de biofilme demonstraram que 44 amostras formaram biofilme em microplacas de poliestireno e 56 amostras apresentaram resultado ausente/fraco. Também confirmamos a incidência das amostras de Escherichia coli no grupo filogenético B2 e D.

Escherichia coli samples isolated from female patients with cystitis were characterized with regard to the presence of virulence factors associated with biofilm formation and phylogenetic groupings. Polymerase chain reaction results demonstrated that all the samples were positive for the gene fimH (type 1 fimbriae), 91 for fliC (flagellins), 50 for papC (P fimbriae), 44 for kpsMTII (capsules) and 36 for flu (antigen 43). The results from assays to quantify the biofilm formation demonstrated that 44 samples produced biofilm on polystyrene microplates and 56 samples produced weak or no biofilm. We also confirmed that Escherichia coli samples were present in phylogenetic groups B2 and D.

Adesinas (Fímbria P, fímbria S, fímbria do tipo 1 e a adesina afimbrial), toxinas (α-hemolisina e o fator necrosante citotóxico do tipo 1), sistemas de captação de ferro (aerobactina), e mecanismos de defesa do hospedeiro (cápsula ou lipopolissacarídeo) são prevalentes em amostras de Escherichia coli associadas a infecções do trato urinário. O objetivo deste trabalho foi caracterizar genotipicamente 162 amostras de Escherichia coli uropatogênica (UPEC) de pacientes com cistite através do ensaio da reação em cadeia da polimerase. Foram realizados três ensaios de PCR multiplex para os seguintes fatores de virulência: papC, papE/F, alelos de papG, fimH, sfa/foc, afaE, hly, cnf-1, usp, cdtB, iucD, e kpsMTII. Os resultados da PCR identificaram, 158 amostras fimH (97,5%), 86 amostras kpsMTII (53,1%), 53 amostras papC/papEF/papG (32,7%), 45 amostras sfa (27,8%), 42 amostras iucD (25,9%), 41 amostras hly (25,3%), 36 amostras usp (22,2%), 30 amostras cnf-1 (18,5%) e 10 amostras afa (6,2%). Nenhuma amostra foi positiva para o gene cdtB. Neste trabalho, demonstramos que podemos encontrar múltiplas adesinas em uma única amostra e que diferentes genes de fatores de virulência podem ser encontrados em associação.

Adhesins (P-fimbriae, S-fimbriae, type 1 fimbriae and afimbrial adhesin), toxins (α-hemolysin and cytotoxic necrotizing factor type 1), iron acquisition systems (aerobactin) and host defense avoidance mechanisms (capsule or lipopolysaccharide) have been shown to be prevalent in Escherichia coli strains associated with urinary tract infections. In this work, 162 Uropathogenic Escherichia coli (UPEC) strains from patients with cystitis were genotypically characterized by polymerase chain reaction (PCR) assay. We developed three multiplex PCR assays for virulence-related genes papC, papE/F, papG alleles, fimH, sfa/foc, afaE, hly, cnf-1, usp, cdtB, iucD, and kpsMTII, all of them previously identified in UPEC strains. The PCR assay results identified 158 fimH (97.5%), 86 kpsMTII (53.1%), 53 papC/papEF/papG (32.7%), 45 sfa (27.8%), 42 iucD (25.9%), 41 hly (25.3%), 36 usp (22.2%), 30 cnf-1(18.5%) and 10 afa (6.2%) strains. No strain was positive for cdtB. In this work, we also demonstrated that adhesins may be multiple within a single strain and that several virulence genes can occur combined in association.

A total of 120 strains of Pseudomonas aeruginosa, isolated from cystic fibrosis (CF) patients (n = 80) and from patients having extra-pulmonary infections (n = 40) were studied regarding the presence of some virulence factors (hemolysin, gelatinase and elastase production) and presence of the algD and algU genes as detected by polymerase chain reaction-PCR. There was not a significant difference for the production of gelatinase and hemolysin between non-mucoid strains from CF patients and other isolates from extra-pulmonary infections and mucoid strains. The production of elastase was found to be significant among these strains. The algD gene was detected by PCR in all studied strains but the algU gene was detected only in 25% of the mucoid strains. Conclusion withdrawn from the results were: (i) hemolysin and gelatinase production although present in many strains of P aeruginosa should not be considered as general virulence factors for the mucoid phenotype but could help in the pathogenic process; (ii) elastase production could be a necessary virulence factor for the initial pathogenesis process; (iii) mucoid and non-mucoid phenotypes could also be expressed according to the host's tissues or environment, and finally, (iv) more than one regulator system for alginate production is probably present in each strain.

Duzentas e cinco amostras de Escherichia coli isoladas de bezerros com diarréia da região centro oeste do Brazil foram examinados quanto a presença de fatores de virulência associados à colibacilose bovina. Cento e duas amostras (49,8%) de E. coli produziram toxinas: toxina de Shiga do tipo 1 (9,7%) e 2 (6,3%), a-hemolisina (9,7%), enterohemolisina (6,8%), Fatores Citotóxicos Necrotisantes tipo 1 (0,5%) e 2 (4,4%), enterotoxinas LT-II (8,3%), e STa (3,9%). Nenhuma amostra produziu enterotoxina LT-I. Adesinas fimbriais F5 e F17 foram produzidas por 7,3% e 4,8% das cepas, respectivamente, e nenhuma expressou F41. Sete das amostras (3,4%) apresentaram o gene eae e pertenceram aos sorotipos O26:H-; O111:H- e O118:H16. Estes resultados sugerem que bezerros no Brasil podem ser uma importante fonte de E. coli patogênica para animais e humanos.

Two hundred and five Escherichia coli strains isolated from calves with diarrhea from mid-western Brazil were screened for the presence of virulence factors associated with bovine colibacillosis. One hundred and two (49.8%) of the E. coli strains produced toxins: Shiga toxins 1 (9.7%) and 2 (6.3%), a-hemolysin (9.7%), enterohemolysin(6.8%), Cytotoxic Necrotizing Factors type 1 (0.5%), and type 2 (4.4%), enterotoxins LT-II (8.3%) and STa (3.9%). No strain produced enterotoxin LT-I. Fimbrial adhesins F5 and F17 were produced by 7.3% and 4.8% of the strains, respectivly, and none expressed F41. Seven strains (3.4%) possessed the gene eae and belonged to serotypes O26:H-; O111:H- and O118:H16. These results suggest that calves in Brazil may be an important source of pathogenic E. coli for animals and humans.

Foram avaliados semanalmente, por um período de seis semanas, os exames parasitológicos de amostras fecais, diarréicas e não-diarréicas, de 106 bezerros búfalos, com 3 a 45 dias de idade, provenientes do Vale do Ribeira-SP. Para os exames bacteriológicos e virológicos, foram colhidas amostras fecais de todos os búfalos diarréicos, e igual amostragem de animais sem diarréia. Amostras de sangue foram colhidas dos búfalos neonatos, entre o terceiro e décimo dias de idade, para determinação dos níveis de imunoglobulina G (IgG). Nos exames parasitológicos, verificou-se a maior ocorrência de Eimeria spp, Strongyloides papillosus e Toxocara vitulorum. Dentre os agentes bacterianos, observou-se maior freqüência da Escherichia coli (E. coli), Enterobacter cloacae e Klebsiella pneumoniae (isolados ou em associação). Detectou-se a enterotoxina STa em 5 das 34 amostras de E. coli isoladas de búfalos com diarréia. A norfloxacina, cloranfenicol e gentamicina foram os antimicrobianos mais efetivos frente aos microrganismos isolados. Nenhuma das amostras apresentou bandeamento característico para rotavírus, a partir da técnica de eletroforese em gel de poliacrilamida. Não foi constatada nos animais examinados a associação entre a ocorrência de diarréia e baixos níveis de IgG sérica.

Faecal samples of 106 buffalo calves aged 3 to 45 days were collected sequentially, once a week, during six weeks for parasitologic examination. For bacteriologic and virologic exams, faecal samples were collected from all diarrhoeic and the same amount on non-diarrhoeic calves. Blood samples from calves were collected at 3 to 10 days of age for serum IgG determination. Eimeria spp, Strongyloides papillosus and Toxocara vitulorum were the most common parasites. E. coli, Enterobacter cloacae and Klebsiella pneumoniae were the enteropathogens most frequently found (alone or in association with other microrganisms). Heat-stable (STa) enterotoxin was detected in 5 out of 34 samples of E. coli isolated from diarrhoeic calves. Among the antimicrobials tested susceptibilities to norphloxacin, chloramphenicol and gentamycin were the most common. None of the samples had characteristic rotavirus RNA bands in PAGE-electrophoresis. No association was observed between low levels of IgG and diarrhoea.