Staphylococcal toxin genes in strains isolated from cows with subclinical mastitis

The present study was carried out in 11 dairy herds in four municipal districts of the rural area of the State of Pernambuco, Brazil. Out of 984 quarter milk (246 cows), 10 (1.0%) were positive for clinical mastitis, 562 (57.1%) for subclinical mastitis and 412 (41.9%) were negative. A total of 81 Staphylococcus spp. isolates were obtained from milk samples from the cows diagnosed with subclinical mastitis. From these, 53 (65.0%) were S. aureus, 16 (20.0%) coagulase-positive staphylococci (CPS) and 12 (15.0%) coagulase-negative staphylococci (CNS). The isolates were further investigated for the presence of toxin genes by multiplex and uniplex PCR. The main gene observed was seg followed by seh, sei and sej. The distribution of these observed genes among the isolates obtained from different areas showed a regional pattern for the SEs. The presence of toxin genes in the strains isolated from bovine milk demonstrates a potential problem for public health.

Avaliação da superexpressão da proteína p53 e das mutações no éxon 8 do gene TP53 em carcinomas mamários caninos e glândulas normais

Este estudo foi realizado com o objetivo de avaliar a expressão da proteína p53, pela técnica de imuno-histoquímica, em neoplasmas mamários malignos em cadelas, além de investigar mutações no éxon 8 do gene supressor Tp53 por meio do padrão de bandas obtidas por PCR-RFLP. Dezenove mamas de cadelas saudáveis foram usadas como controle (Grupo 1). Amostras de 18 casos de tumores malignos (Grupo 2) e suas glândulas mamárias contralaterais (Grupo 3) foram obtidas na rotina do Hospital Veterinário da UFRPE. Os tumores foram identificados histologicamente e classificados em graus de malignidade. O método da estreptoavidina-biotina peroxidase foi utilizado para a análise da expressão de p53 por imuno-histoquímica, de acordo com a localização e intensidade da coloração. A expressão da proteína p53 não foi observada nas amostras do Grupo 1, mas foi encontrada em todas as amostras de tumores malignos (Grupo 2) seja só no núcleo, ou também no citoplasma. No Grupo 3, a expressão foi observada em quatro amostras normais e em duas que apresentavam tumor. Para a análise molecular, o DNA genômico foi extraído e submetido à PCR-RFLP com as seguintes endonucleases: AluI, BsoBI, DdeI e SmaI. O padrão de bandas foi polimórfico entre os grupos, mas não entre as variantes tumorais. Esse polimorfismo detectou mutações no fragmento estudado - éxon 8 do gene Tp53 - que podem resultar em alterações nos nucleotídeos, localizados nos sítios de restrição das enzimas. Esses achados levam a conclusão de que a imunoexpressão da p53 não tem relação com o subtipo histológico ou grau de malignidade do tumor, mas sim com a presença dos tumores no tecido mamário de cadelas. A PCR-RFLP pode ser usada como importante ferramenta para o estudo da carcinogênese mamária na cadela, possibilitando gerar diagnósticos precoces através do polimorfismo obtido com endonucleases de restrição pré-selecionadas.

Serogroups and virulence genes of Escherichia coli isolated from psittacine birds

Escherichia coli isolates from 24 sick psittacine birds were serogrouped and investigated for the presence of genes encoding the following virulence factors: attaching and effacing (eae), enteropathogenic E. coli EAF plasmid (EAF), pili associated with pyelonephritis (pap), S fimbriae (sfa), afimbrial adhesin (afa), capsule K1 (neu), curli (crl, csgA), temperature-sensitive hemagglutinin (tsh), enteroaggregative heat-stable enterotoxin-1 (astA), heat-stable enterotoxin -1 heat labile (LT) and heat stable (STa and STb) enterotoxins, Shiga-like toxins (stx1 and stx2), cytotoxic necrotizing factor 1 (cnf1), haemolysin (hly), aerobactin production (iuc) and serum resistance (iss). The results showed that the isolates belonged to 12 serogroups: O7; O15; O21; O23; O54; O64; O76; O84; O88; O128; O152 and O166. The virulence genes found were: crl in all isolates, pap in 10 isolates, iss in seven isolates, csgA in five isolates, iuc and tsh in three isolates and eae in two isolates. The combination of virulence genes revealed 11 different genotypic patterns. All strains were negative for genes encoding for EAF, EAEC, K1, sfa, afa, hly, cnf, LT, STa, STb, stx1 and stx2. Our findings showed that some E. coli isolated from psittacine birds present the same virulence factors as avian pathogenic E. coli (APEC), uropathogenic E. coli (UPEC) and Enteropathogenic E. coli (EPEC) pathotypes.

Antimicrobial resistance and detection of mecA and blaZ genes in coagulase-negative Staphylococcus isolated from bovine mastitis

The present study evaluated the pheno- and genotypical antimicrobial resistance profile of coagulase-negative Staphylococcus (CNS) species isolated from dairy cows milk, specially concerning to oxacillin. Of 100 CNS isolates, the S. xylosus was the prevalent species, followed by S. cohnii, S. hominis, S. capitis and S. haemolyticus. Only 6% were phenotypically susceptible to the antimicrobial agents tested in disk diffusion assay. Penicillin and ampicillin resistance rates were significantly higher than others antimicrobials. Four isolates were positive to mecA gene (4%), all represented by the S. xylosus species. The blaZ gene was detected in 16% of the isolates (16/100). It was noticed that all mecA + were also positive to this gene and the presence of both genes was correlated to phenotypic beta-lactamic resistance. We conclude that CNS species from bovine milk presented significantly distinct antimicrobial resistance profiles, evaluated by phenotypic and genotypic tests, which has implications for treatment and management decisions.

Detection of virulence-associated genes of Pasteurella multocida isolated from cases of fowl cholera by multiplex-PCR

The current systems of breeding poultry, based on high population density, increase the risk of spreading pathogens, especially those causing respiratory diseases and those that have more than one host. Fowl Cholera (FC) is one such pathogen, and even though it represents one of several avian diseases that should be considered in the differential diagnosis of notifiable diseases that present with sudden death, the pathogenesis and virulence factors involved in FC are still poorly understood. The objective of this study was to investigate twelve genes related to virulence in 25 samples of Pasteurella multocida isolated from FC cases in the southern region of Brazil through the development of multiplex PCR protocols. The protocols developed were capable of detecting all of the proposed genes. The ompH, oma87, sodC, hgbA, hgbB, exBD-tonB and nanB genes were present in 100% of the samples (25/25), the sodA and nanH genes were present in 96% (24/25), ptfA was present in 92% (23/25), and pfhA was present in 60% (15/25). Gene toxA was not identified in any of the samples studied (0/25). Five different genetic profiles were obtained, of which P1 (negative to toxA) was the most common. We concluded that the multiplex-PCR protocols could be useful tools for rapid and simultaneous detection of virulence genes. Despite the high frequency of the analyzed genes and the fact that all samples belonged to the same subspecies of P. multocida, five genetic profiles were observed, which should be confirmed in a study with a larger number of samples.

Identification of new flagellin-encoding fliC genes in Escherichia coli isolated from domestic animals using RFLP-PCR and sequencing methods

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.

Some virulence genes of Escherichia coli isolated from cloacal swabs of healthy Alagoas Curassows (Pauxi mitu) in Brazil

Birds of the Cracidae family (curassows, guans, and chachalacas) are endemic of the Neotropics and 50 species are currently classified. Brazil has 22 species, seven of which are considered threatened. The Alagoas Curassow (Pauxi mitu) species is considered extinct in the wild; but about 120 birds are alive in captivity. Conservation of this species depends entirely on correct management. Health reports of both wildlife and captive curassows are rare. In this study the presence of Escherichia coli was evaluated in 23 healthy Alagoas Curassows from two private breeding centres. E. coli was isolated from cloacal swabs, and the presence of genes encoding cytotoxic necrotising factor 1 (cnf1), alpha-haemolysin (hly), aerobactin (iuc), serum resistance (iss) and the following adhesions: S fimbriae (sfa), pili associated with pyelonephritis (pap) and temperature-sensitive haemagglutinin (tsh) were investigated. E. coli was isolated from 78.3% (18/23) of the birds, and the percentage of curassows colonized by E. coli was similar between the two facilities. From the 22 E. coli isolates, 15 (68.2%) were positive for at least one virulence factor by PCR, and the most frequently found gene was iss (50%). No curassows had clinical signs of disease. Nevertheless, the presence of some E. coli strains may be a concern to the wildlife in captivity. Additional health surveillance studies are essential to guarantee successful conservation programmes for threatened cracids in Brazil.

Detection of virulence-associated genes in Salmonella Enteritidis isolates from chicken in South of Brazil

Salmonella spp. are considered the main agents of foodborne disease and Salmonella Enteritidis is one of the most frequently isolated serovars worldwide. The virulence of Salmonella spp. and their interaction with the host are complex processes involving virulence factors to overcome host defenses. The purpose of this study was to detect virulence genes in S. Enteritidis isolates from poultry in the South of Brazil. PCR-based assays were developed in order to detect nine genes (lpfA, agfA, sefA, invA, hilA, avrA, sopE, sivH and spvC) associated with the virulence in eighty-four isolates of S. Enteritidis isolated from poultry. The invA, hilA, sivH, sefA and avrA genes were present in 100% of the isolates; lpfA and sopE were present in 99%; agfA was present in 96%; and the spvC gene was present in 92%. It was possible to characterize the isolates with four different genetic profiles (P1, P2, P3 and P4), as it follows: P1, positive for all genes; P2, negative only for spvC; P3, negative for agfA; and P4, negative for lpfA, spvC and sopE. The most prevalent profile was P1, which was present in 88% of the isolates. Although all isolates belong to the same serovar, it was possible to observe variations in the presence of these virulence-associated genes between different isolates. The characterization of the mechanisms of virulence circulating in the population of Salmonella Enteritidis is important for a better understanding of its biology and pathogenicity. The frequency of these genes and the establishment of genetic profiles can be used to determine patterns of virulence. These patterns, associated with in vivo studies, may help develop tools to predict the ability of virulence of different strains.

Expression of P-glycoprotein, multidrug resistance-associated protein, glutathione-S-transferase pi and p53 in canine transmissible venereal tumor

The overexpression of proteins P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP1), mutant p53, and the enzyme glutathione-S-transferase (GSTpi) are related to resistance to chemotherapy in neoplasms. This study evaluated the expression of these markers by immunohistochemistry in two groups of canine TVT, without history of prior chemotherapy (TVT1, n=9) and in TVTs presented unsatisfactory clinical response to vincristine sulfate (TVT2, n=5). The percentage of specimens positively stained for P-gp, MRP1, GSTpi and p53 were, respectively 88.8%, 0%, 44.5% and 22.2% in TVT1 and 80%, 0%, 80% and 0% in TVT2. In TVT1, one specimen presented positive expression for three markers and four specimens for two markers. In TVT2, three specimens expressed P-gp and GSTpi. In conclusion, the canine TVTs studied expressed the four markers evaluated, but just P-gp and GSTpi were significantly expressed, mainly at cytoplasm and cytoplasm and nuclei, respectively, either before chemotherapy as after vincristine sulfate exposure. Future studies are needed to demonstrate the function of these two markers in conferring multidrug resistance (MDR) or predict the response to chemotherapy in canine TVT.

Seleção, caracterização e clonagem dos genes fljB e groEL agonistas dos receptores de reconhecimento de padrão do sistema imune inato das aves

A produção recombinante de agonistas dos receptores do reconhecimento de padrão do sistema imune inato tem fornecido uma nova ferramenta para a produção de imunoestimulantes para animais. O padrão molecular associado ao patógeno (PAMP), flagelina, codificado pelo gene fljB de Salmonella Typhimurium e o padrão molecular associado ao dano (DAMP) HSP60, codificado pelo gene groEL da S. Typhimurium e S. Enteritidis, são reconhecidos por receptores de reconhecimento de padrões (RRPs) do sistema imune inato das aves. No presente estudo, foi feita a clonagem de fragmentos genéticos dos genes fljB de S. Typhimurium e groEL de S. Typhimurium e S. Enteritidis inseridos no vetor de expressão pET100/D-TOPO e transformados em células de E. coli TOP10. Os clones foram avaliados pela PCR de colônia, PCR de DNA plasmidial e sequenciamento genômico para a confirmação da presença desses genes. Na PCR de colônia, foram identificadas em 80%, 60% e 80% das colônias transformadas, a presença dos genes groEL (S. Enteritidis), groEL (S. Typhimurium) e fljB (S. Typhimurium) respectivamente. O sistema de clonagem adotado possibilitou a produção de clones dos fragmentos genéticos da HSP60 e flagelina das cepas de Salmonella, permitindo a utilização posterior desses clones em ensaios de expressão gênica, com potencial futuro de serem utilizados como imunoestimulante inespecífico das aves.

Occurrence of enterotoxin-encoding genes in Staphylococcus aureus causing mastitis in lactating goats

Staphylococcal enterotoxins are the leading cause of human food poisoning worldwide. Staphylococcus spp. are the main mastitis-causing agents in goats and frequently found in high counts in goat milk. This study aimed to investigate the occurrence of enterotoxin-encoding genes in Staphylococcus aureus associated with mastitis in lactating goats in Paraiba State, Brazil. Milk samples (n=2024) were collected from 393 farms. Staphylococcus aureus was isolated in 55 milk samples. Classical (sea, seb, sec, sed, see) and novel (seg, seh, sei) enterotoxin-encoding genes were investigated by means of polymerase chain reaction (PCR). From thirty-six tested isolates, enterotoxin-encoding genes were detected in 7 (19.5%) S. aureus. The gene encoding enterotoxin C (seC) was identified in six isolates, while seiwas observed in only one isolate. The genes sea, seb, sed, see, seg and seh were not observed amongst the S. aureus investigated in this study. In summary, S. aureus causing mastitis in goats can harbor enterotoxin-encoding genes and seC was the most frequent gene observed amongst the investigated isolates. This finding is important for surveillance purposes, since enterotoxin C should be investigated in human staphylococcal food poisoning outbreaks caused by consumption of goat milk and dairy products.

Ocorrência de genes tad associados à formação de biofilme em isolados de Pasteurella multocida de pulmões de suínos com pneumonia

Os atuais sistemas de criação intensiva de suínos aumentam a pressão de seleção microbiana propiciando a disseminação de doenças respiratórias. A bactéria Pasteurella multocida é associada a diversas patologias respiratórias em animais submetidos a esse tipo de criação, causando grandes perdas econômicas. A formação de biofilme foi descrita in vitro em P. multocida e fatores analisados indicaram a facilitação na colonização dos tecidos, aumentando a resistência às defesas do hospedeiro e aos antibióticos. Os objetivos deste trabalho foram analisar a ocorrência de P. multocida em pneumonias de suínos e na microbiota de pulmões sem lesão e a ocorrência dos genes do lócus tad nestes isolados. Foram analisados 70 isolados de P. multocida de pulmões, sendo sessenta e sete com lesão e três sem lesão. Isolados do sorotipo A ocorreram principalmente em pulmões com lesões (85,71%), enquanto em pulmões sem lesão observou-se somente o sorotipo D. Os genes tadA, tadB, tadC, tadD, tadE tadF e tadG estavam presentes em 89,55% dos isolados de pulmões com lesões. Os genes tadA, tadB e tadC estavam presentes em todos os isolados de pulmões sem lesão, porém os genes tadD, tadE, tadF e tadG estavam presentes em 0%, 33,3%, 33,3% e 66,6%, dos isolados sem lesão, respectivamente. Neste trabalho observou-se a associação da ocorrência dos genes tadD, tadE e tadF em isolados de P. multocida e a presença de lesões em pulmões.

Classification of antimicrobial resistance using artificial neural networks and the relationship of 38 genes associated with the virulence of Escherichia coli isolates from broilers

Avian pathogenic Escherichia coli (APEC) is responsible for various pathological processes in birds and is considered as one of the principal causes of morbidity and mortality, associated with economic losses to the poultry industry. The objective of this study was to demonstrate that it is possible to predict antimicrobial resistance of 256 samples (APEC) using 38 different genes responsible for virulence factors, through a computer program of artificial neural networks (ANNs). A second target was to find the relationship between (PI) pathogenicity index and resistance to 14 antibiotics by statistical analysis. The results showed that the RNAs were able to make the correct classification of the behavior of APEC samples with a range from 74.22 to 98.44%, and make it possible to predict antimicrobial resistance. The statistical analysis to assess the relationship between the pathogenic index (PI) and resistance against 14 antibiotics showed that these variables are independent, i.e. peaks in PI can happen without changing the antimicrobial resistance, or the opposite, changing the antimicrobial resistance without a change in PI.

Detecção dos genes codificantes da toxina CDT, e pesquisa de fatores que influenciam na produção de hemolisinas em amostras de Campylobacter jejuni de origem avícola

Resumo: Membros termofílicos do gênero Campylobacter são reconhecidos como importantes enteropatógenos para o ser humano e animais. A grande diversidade ecológica destes micro-organismos em diferentes habitats tais como água, animais e alimentos predispõem ao aparecimento de novos fatores de virulência. Este trabalho teve por objetivo detectar os genes codificantes da Toxina Distensiva Citoletal (CDT) por meio da técnica de PCR, pesquisar a atividade de hemolisinas e a influência de soluções quelantes e de íons nesta atividade. Foram utilizadas 45 amostras de Campylobacter jejuni de origem avícola para pesquisa de atividade hemolítica, cultivadas em Caldo Triptona de Soja (TSB). Após o crescimento bacteriano, as amostras foram semeadas em Ágar tríptico de soja (TSA) contendo 5% de sangue de ovino. Para verificar a influência de agentes quelantes e solução de íons na atividade hemolítica, as amostras de C. jejuni foram cultivadas em TSB contendo separadamente os quelantes EDTA, ácido acético, soluções de íons CaCl2, MgCl2 e FeCl3, em atmosfera de microaerofilia. Quanto à atividade de hemolisina de C. jejuni em placas de TSA - sangue ovino foi possível observar que houve hemólise em 40% das amostras analisadas apenas com caldo TSB. Somente o ácido acético apresentou ação quelante sobre a atividade de hemolisinas em amostras de C. jejuni semeadas em placas de TSA - sangue ovino. Para detecção dos genes cdtA, cdtB e cdtC através da técnica da Reação em Cadeia da Polimerase (PCR) foram utilizadas 119 amostras de C. jejuni de origem avícola. Foi possível observar que 37,8% possuíam o perfil de genes cdtABC. Os resultados demonstraram em amostras avícolas a presença de cepas de C. jejuni com potencial virulento, devido à presença dos genes da toxina CDT e potencial hemolítico, que apresentou ação reduzida in vitro com ácido acético.

Meningiomas em cães: aspectos clínicos, histopatológicos e imuno-histoquímicos

Resumo: As neoplasias no sistema nervoso central (SNC) de animais de companhia são frequentemente diagnosticadas, no entanto dados sobre prevalência são escassos. O objetivo deste estudo foi avaliar retrospectivamente a ocorrência de neoplasias primárias de SNC em cães atendidos em um Hospital-Escola Veterinário e descrever aspectos clínicos, histopatológicos e imuno-histoquímicos dos tumores mais frequentes. Quatorze casos (prevalência de 0,27%) de neoplasias primárias de SNC foram identificados no período de 1998 a 2013 e destes, 11 tiveram o diagnóstico de meningiomas. A idade média dos animais com meningioma foi 10 anos, sendo machos (7/11) e a raça Boxer (3/11) os mais afetados. Sete meningiomas eram espinhais e quatro intracranianos, sendo os principais sinais clínicos alteração na locomoção e convulsões, respectivamente. Metástase pulmonar ocorreu em dois casos. Em seis animais com meningioma espinhal foi realizada a mielografia, sendo que em um também foi realizada a tomografia. Em todos os casos os exames foram efetivos na visualização de desvio ou interrupção da coluna de contraste, com alterações sugestivas da presença de massa. Em cinco animais realizou-se cirurgia exploratória visando a confirmação da suspeita clínica ou retirada da massa, sendo que a sobrevida variou de 85 a 960 dias. Na avaliação histopatológica, os meningiomas foram classificados em transicional (4/11), meningotelial (2/11), papilar (2/11), angiomatoso (1/11), microcístico (1/11) e anaplásico (1/11). Destes, oito (8/11) apresentaram marcação positiva para tricrômio de Masson e um para vermelho congo nas técnicas histoquímicas. No painel imuno-histoquímico, todos os casos apresentaram imunomarcação positiva para vimentina, mas imunomarcação negativa para fator VIII e p53. A imunomarcação para S100 (6/11), GFAP (5/11) e pancitoqueratina (3/11) foi de intensidade variável. Na graduação histológica, dez meningiomas eram grau I e um grau III. O índice médio de proliferação celular foi de 3,2 figuras de mitose e 3,4% avaliando a expressão de Ki-67. Os resultados confirmaram que os meningiomas são a neoplasia primária de SNC mais prevalente em cães, com variação nos subtipos histológicos. A caracterização histoquímica e imuno-histoquímica contribuiu com a determinação do diagnóstico, no entanto, estudos envolvendo a expressão de outros genes são necessários para auxiliar no prognóstico dessas neoplasias.