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1.
J Dairy Sci ; 102(9): 8405-8409, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31301842

RESUMO

Different mycobacterial species are encountered in bovine medicine. The fastidiously growing mycobacteria (Mycobacterium bovis as the cause of bovine tuberculosis, and Mycobacterium avium ssp. paratuberculosis, MAP, as the cause of paratuberculosis) are well known and targeted in eradication/control or monitoring programs in different countries, whereas the rapidly growing species is only rarely identified from bovine disease. The latter have occasionally been reported as the cause of bovine clinical mastitis, but recent reports are scarce. In this study, Mycolicibacterium smegmatis (basonym Mycobacterium smegmatis) was identified as cause of granulomatous, relapsing clinical mastitis in 2 cows from one Belgian dairy herd. Milk, blood, and fecal samples were collected, as well as tissue samples after the cows were culled. Serological analysis conducted on milk and serum samples resulted in positive reactions for MAP, but negative for Mycobacterium bovis. Production of IFN-γ showed sensitization with mycobacteria or similar organisms, other than M. bovis, in one cow. Detection of MAP by bacteriological culture and IS900-based quantitative PCR on milk and feces remained negative. In conclusion, this paper describes M. smegmatis as a cause of bovine clinical mastitis in Belgium and suggests cross-reactivity of the intramammary M. smegmatis infection with routinely used serological tests for MAP.


Assuntos
Doenças dos Bovinos/microbiologia , Mastite Bovina/microbiologia , Mycobacterium smegmatis , Paratuberculose/diagnóstico , Animais , Bélgica , Bovinos , Doenças dos Bovinos/diagnóstico , Reações Cruzadas , Fezes/microbiologia , Feminino , Leite/microbiologia , Mycobacterium avium subsp. paratuberculosis/imunologia , Mycobacterium bovis/imunologia , Mycobacterium smegmatis/imunologia , Paratuberculose/microbiologia , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Tuberculose Bovina
2.
Vet Microbiol ; 235: 71-79, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31282381

RESUMO

Streptococcus agalactiae (Group B streptococcus, GBS) is a commensal of the human intestinal tract and vagina and is also an opportunistic pathogen causing serious, potentially lethal, infections preferentially in newborns and in the elderly. In cattle, it is considered an udder-specific pathogen and a common cause of mastitis. Here we investigated the host specificity of GBS by examining their colonization at various anatomical sites in both cattle and humans, as well as the possible cross-species transmission in closed barn environments. We collected more than 800 swab samples from dairy cows and herdspersons at eight dairy farms in Denmark. GBS was isolated from 12% of the samples. The GBS strains (N = 105) were characterized by biochemical test, serology, and Pulsed-Field Gel Electrophoresis (PFGE). Based on the PFGE patterns, 25 strains were selected for whole genome sequencing followed by phylogenetic analyses. The genomes were compared to each other and to a collection of publicly available GBS genomes. The study revealed that GBS clones were shared by cows and herdspersons. In phylogenetic analyses, these shared clones clustered with GBS strains from persons with no relation to farming. Horizontal cross-species transmission of the contagion in both directions was found to be highly likely within the same environment; thus, some cases of bovine mastitis are probably antrophonotic.


Assuntos
Bovinos/microbiologia , Fazendeiros , Especificidade de Hospedeiro , Infecções Estreptocócicas/microbiologia , Streptococcus agalactiae/genética , Animais , Indústria de Laticínios , Dinamarca , Reservatórios de Doenças/microbiologia , Feminino , Humanos , Masculino , Mastite Bovina/microbiologia , Leite/microbiologia , Faringe/microbiologia , Filogenia , Reto/microbiologia , Infecções Estreptocócicas/transmissão , Streptococcus agalactiae/isolamento & purificação , Vagina/microbiologia
3.
J Photochem Photobiol B ; 197: 111554, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31326843

RESUMO

Bovine mastitis is an endemic disease of dairy cattle that is considered to be one of the most frequent and costly diseases in veterinary medicine. An increase in the incidence of disease results in the increased use of antibiotics, which in turn increases the potential of bacterial resistance. This study aimed to investigate the effectiveness of antimicrobial photodynamic therapy (aPDT) in the treatment of bovine mastitis, as an alternative to systemic antibiotics. To identify the key factors affecting photoinactivation efficacy, realistic experiments in view of the end-use were conducted in milk samples using two different photosensitizers: methylene blue (MB) and silicon (IV) phthalocyanine derivative (SiPc). We explored the effects of divalent ions and fat content on the aPDT outcome and determined influence of different proteins on aPDT efficacy. Levels of bacterial sensitivity to PSs varied depending on the type of bacteria (Gram-positive vs. Gram-negative) and light exposure time. Critical interrelated factors affecting aPDT in milk were identified and an efficient combination of treatment conditions that can lead to a full photodynamic inactivation of bacteria was determined.


Assuntos
Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Leite/microbiologia , Fármacos Fotossensibilizantes/farmacologia , Animais , Bovinos , Feminino , Indóis/química , Indóis/farmacologia , Indóis/uso terapêutico , Luz , Mastite Bovina/tratamento farmacológico , Mastite Bovina/microbiologia , Mastite Bovina/patologia , Azul de Metileno/farmacologia , Azul de Metileno/uso terapêutico , Proteínas do Leite/química , Fotoquimioterapia , Fármacos Fotossensibilizantes/uso terapêutico , Oxigênio Singlete/metabolismo
4.
Animal ; 13(S1): s82-s85, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31280743

RESUMO

The use of a proteomic approach to investigate changes in the milk proteome is growing and has parralleled the increasing technological developments in proteomics moving from early investigation using a gel-based two-dimensional separation approach to more quantitative method of current focus applying chromatography and mass spectrometry. Proteomic approaches to investigate lactational performance have made substantial findings especially in the alterations in lactation during mastitis. An experimental model of Streptococcus uberis infection of the mammary gland has been used as a means to determine change not only in the milk proteome, but also in the peptidome and in the metabolome caused by the infection. Examination of the peptidome, that is the peptides of less than 25 kDa in molecular weight, demonstrated an increase in small peptides most of which were casein degradation products but also included small bioactive peptides such as mammary-associated serum amyloid A3 (MSAA3). The peptidome has also been shown to differ depending on the causative bacteria of naturally occuring mastitis. The use of a non-gel-based relative quantitative proteomic methodology has revealed major changes in the protein component of milk in mastitis. The S. uberis infection lead to increases in the concentrations of proteins such as cathelicidins, haptoglobin, MSAA3 and decreases milk content of proteins such as xanthine oxidase, butyrophilin and ß-1,4-galactosyltransferase. Analysis of all protein change data identified the acute phase, coagulation and complement pathways as well as proteins related to bile acid metabolism as being most modified. Examination of the small molecular weight organic molecules of milk using a metabolomic approach identified an increase in the content in milk during mastitis of bile acids such as taurochenodeoxycholic acid. Notable changes were also found in metabolites responding to infection of the mammary gland. Carbohydrate and nucleic acid metabolites were reduced, whereas lipid and nitrogen containing metabolites were increased. The latter included increases in amino acids along with di and tri peptides, likely to be the result of casein degradation. The use of proteomics and other omic technology is in its infancy in investigation of lactational parameters, but can already provide additional insight into the changes involved in disease and will have further value in physiological and nutritional investigation of lactation.


Assuntos
Lactação , Mastite Bovina/metabolismo , Metabolômica , Leite/metabolismo , Proteômica , Streptococcus/fisiologia , Animais , Bovinos , Feminino , Glândulas Mamárias Animais/microbiologia , Glândulas Mamárias Animais/fisiopatologia , Mastite Bovina/microbiologia , Metaboloma , Proteoma
5.
Onderstepoort J Vet Res ; 86(1): e1-e9, 2019 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-31291733

RESUMO

South Africa is a large country of approximately 1.22 million km2, made up of nine provinces with three climatic zones. Farming in the country is mostly defined by regional differences. Of the different organisms isolated from milk samples of dairy cows, Staphylococcus aureus poses a challenge to maintain udder health and wholesome dairy products for human consumption. Antibiotic resistant bacteria are therefore a potential health hazard. The objective of this study was to investigate the seasonal and regional relationships of antibiotic resistance of S. aureus, of which little is known. This study was undertaken to evaluate a data set of 3410 S. aureus isolates, taken from milk samples with a somatic cell count of > 400 000 cells/mL from commercial dairy herds. These isolates were tested for antimicrobial susceptibility using the Kirby Bauer method for ampicillin, cloxacillin, penicillin G, clindamycin, oxy-tetracycline, cephalexin, cefuroxime and tylosin. The samples were from 830 dairy herds, out of the estimated 2000 commercial dairy herds in South Africa. All the antibiotics tested, except for cephalosporins, showed a predicted prevalence of resistance of above 50% in most provinces, which is a concern. The lowest prevalence of resistance to the majority of the categories of antibiotics tested was present in KwaZulu-Natal during spring. The cephalosporins had the lowest levels of prevalence of bacterial resistance in Gauteng during winter. Resistance patterns of S. aureus to the eight antibiotics varied in the different seasons and provinces, possibly because of different weather conditions, and the action and spectrum of antibiotics.


Assuntos
Antibacterianos/farmacologia , Indústria de Laticínios , Mastite Bovina/epidemiologia , Leite/microbiologia , Infecções Estafilocócicas/veterinária , Staphylococcus aureus/efeitos dos fármacos , Animais , Bovinos , Clima , Demografia , Farmacorresistência Bacteriana , Feminino , Mastite Bovina/microbiologia , Testes de Sensibilidade Microbiana/veterinária , Prevalência , África do Sul/epidemiologia , Infecções Estafilocócicas/epidemiologia
6.
J Appl Microbiol ; 127(3): 683-692, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31216600

RESUMO

AIMS: The objective of this study was to compare qualitatively and quantitatively the results of identification of the bacteria present in milk samples from cows with subclinical mastitis using multiplex qPCR assay and matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF MS® ) after bacteriological growth. METHODS AND RESULTS: A total of 182 samples were aseptically collected from 119 cows with high somatic cell counts (>2·105 SCC per ml) on 11 farms in Belgium in 2014. The mutiplex qPCR assay was carried out on 350 µl of milk with the PathoProof® Complete-16kit. Ten microlitre of milk was streaked on Columbia blood agar and three selective agar plates. Growing colonies were identified by MALDI-TOF MS. Of the 182 samples, 90 gave positive results with either or both tests for one or two bacterial species/genera. Total qualitative agreement of the bacteria identified was observed in 41 mono- or bi-bacterial samples (46%) and partial agreement in 19 bi-bacterial samples at both or either tests (21%). The results of both tests on those mono- and bi-bacterial samples were not significantly different (McNemar test; P = 0·395) with a fair agreement (Cohen's kappa test; k = 0·375; P = 0·055). Moreover, quantitative correlation between the qPCR intensity and the numbers of growing colonies was observed in half of the 60 samples with qualitative matching results. CONCLUSIONS: Both methods give identical qualitative and quantitative results with approximately a half and a quarter of the mono- and bi-bacterial samples respectively. Several reasons can explain the differences. The multiplex qPCR assay only targets the most important mammary gland pathogens and can detect DNA of bacteria both alive and dead. Conversely, bacteria only grow when alive and the MALDI-TOF MS databases do not include all bovine milk-associated bacterial species yet. SIGNIFICANCE AND IMPACT OF THE STUDY: This study further highlights the limitations and complementarity of the genetic and phenotypic tests for the identification of bacteria present in milk samples.


Assuntos
Bactérias/isolamento & purificação , Mastite Bovina/microbiologia , Leite/microbiologia , Reação em Cadeia da Polimerase em Tempo Real , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Animais , Bactérias/genética , Bovinos , Feminino
7.
Vet Res ; 50(1): 44, 2019 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-31171032

RESUMO

The aim of this study was to analyze bacterial profiles of bovine mastitic milk samples and samples from healthy quarters using Next Generation Sequencing of amplicons from 16S rRNA genes and to compare results with microbiological results by PCR assays of the same samples. A total of 49 samples were collected from one single dairy herd during the same day. The samples were divided in two sample sets, which were used in this study. The DNA extraction as well as the library preparation and sequencing of these two sets were performed separately, and results of the two datasets were then compared. The vast majority of genera detected appeared with low read numbers and/or in only a few samples. Results of PCR and microbiome analyses of samples infected with major pathogens Staphylococcus aureus or Streptococcus uberis were consistent as these genera also covered the majority of reads detected in the microbiome analysis. Analysis of alpha diversity revealed a much higher species richness in set 1 than in set 2. The dominating bacterial genera with the highest read numbers clearly differed between datasets, especially in PCR negative samples and samples positive for minor pathogens. In addition to this, linear discriminant analysis (LDA) was conducted between the two sets to identify significantly different genera/family level microbes. The genus Methylobacterium was much more common in set 2 compared to set 1, and genus Streptococcus more common in set 1. Our results indicate amplification of contaminating bacteria in excess in samples with no or minor amounts of pathogen DNA in dataset 2. There is a need for critical assessment of results of milk microbiome analyses.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Microbiota , Leite/microbiologia , Reação em Cadeia da Polimerase/métodos , Animais , Bovinos , Feminino , Mastite Bovina/microbiologia , RNA Bacteriano/análise , RNA Ribossômico 16S/análise
8.
Vet Res ; 50(1): 49, 2019 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-31221210

RESUMO

An ethanolic extract from Rhodomyrtus tomentosa leaves (RTL) was studied as a natural alternative to control Staphylococcus aureus, which is an important pathogen responsible for bovine mastitis. The minimal inhibitory concentrations (MICs) of the RTL extract and of rhodomyrtone, a pure compound isolated from the plant, were determined by a microdilution method. Rhodomyrtone and the RTL extract exhibited antibacterial activity against S. aureus, including its persistent phenotype (SCV: small-colony variant) and a biofilm hyperproducer strain, with MICs of 0.25-0.5 and 8-16 µg/mL, respectively. Time-kill kinetics showed a strong bactericidal activity for both the RTL extract- and rhodomyrtone-treated bacteria at 2 × MIC as early as 4 h post-exposure. An additive effect of the extract at 0.5 × MIC was observed in a combination with oxytetracycline or pirlimycin against S. aureus by showing a 64- to 128-fold reduction in antibiotic MICs. Moreover, the RTL extract significantly decreased the number of intracellular SCVs inside bovine mammary epithelial cells. However, the extract or its combination with pirlimycin only slightly improved the activity of pirlimycin against the bacterial colonization of mouse mammary glands. In vitro MICs determined in the presence of casein indicated that the limited activity of the RTL extract in the murine model of mastitis could be linked to neutralization of active components by milk proteins. While the RTL extract showed interesting antibacterial properties in vitro, to be considered as an alternative to antibiotics in dairy farms, formulation studies are needed to cope with the observed reduction of activity in vivo.


Assuntos
Antibacterianos/farmacologia , Mastite Bovina/tratamento farmacológico , Myrtaceae/química , Extratos Vegetais/farmacologia , Infecções Estafilocócicas/veterinária , Staphylococcus aureus/efeitos dos fármacos , Animais , Bovinos , Modelos Animais de Doenças , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Feminino , Mastite Bovina/microbiologia , Camundongos , Testes de Sensibilidade Microbiana , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Xantonas/farmacologia
9.
J Dairy Sci ; 102(8): 6920-6922, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31178194

RESUMO

The present study investigated the effects of N-acetylcysteine (NAC) on ß-lactam antibacterial activity against 20 methicillin-resistant Staphylococcus aureus (MRSA) isolates from bovine mastitis. Minimum inhibitory concentrations (MIC) were determined by the E-test method. The presence of 10 mM NAC reduced the MIC of penicillin, ampicillin, oxacillin, cefoxitin, ceftazidime, and cefotaxime to MRSA. Importantly, the MIC of cefoxitin in MRSA in the presence of NAC was lower than the susceptible breakpoint of cefoxitin. The results provide a new way to use current ß-lactam antibiotics combined with NAC against MRSA.


Assuntos
Acetilcisteína/farmacologia , Antibacterianos/farmacologia , Mastite Bovina/microbiologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , beta-Lactamas/farmacologia , Acetilcisteína/administração & dosagem , Ampicilina/farmacologia , Animais , Bovinos , Cefoxitina/farmacologia , Feminino , Testes de Sensibilidade Microbiana , Oxacilina/farmacologia , Penicilina G/farmacologia
10.
Int J Syst Evol Microbiol ; 69(8): 2208-2213, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31225789

RESUMO

Strain S04009T, a Gram-stain-positive, coagulase-negative staphylococcus, was isolated from bovine mastitis in France. 16S rRNA gene analysis revealed it to be closely related to the coagulase-negative species Staphylococcusxylosus, Staphylococcussaprophyticus, Staphylococcuscaeli and Staphylococcus edaphicus. At the whole-genome level, strain S04009T had an average nucleotide identity value <95 % and an inferred DNA-DNA hybridization value <70 % when compared to these species. Furthermore, phenotypic characteristics distinguished S04009T from those species. From these related species only strain S04009T and S. xylosus are able to ferment xylose and these two can be distinguished by the inability of strain S04009T to express urease activity. Based on the genotypic and phenotypic results, it is proposed that this isolate is a novel species, with the name Staphylococcus pseudoxylosus sp. nov. The type strain is S04009T (=DSM 107950T=CCUG 72763T=NCTC 14184T).


Assuntos
Bovinos/microbiologia , Mastite Bovina/microbiologia , Filogenia , Staphylococcus/classificação , Animais , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/química , Feminino , França , Hibridização de Ácido Nucleico , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Staphylococcus/isolamento & purificação
11.
J Dairy Sci ; 102(8): 7476-7482, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31178197

RESUMO

The concept that ruminant mammary gland quarters are anatomically and physiologically unrelated has been recently challenged by immunological evidence. How this interdependence reflects on individual quarter milk microbiota is unknown. The aim of the present study was to cover this gap by investigating the interdependence of quarters among the same mammary gland at the milk microbiota level using next-generation sequencing of the V4-16S rRNA gene. A total of 52 samples were included in this study and classified as healthy or affected by subclinical mastitis. Extraction of DNA, amplification of the V4-16S rRNA gene, and sequencing using Ion Torrent Personal Genome Machine (Thermo Fisher Scientific, Waltham, MA) were carried out. We found that the intra-individual variability was lower than the inter-individual one. The present findings further support at milk microbiota level the hypothesis of the interdependence of quarters, as previously demonstrated following immunological studies, suggesting that individual factors (e.g., immunity, genetics) may have a role in modulating milk microbiota.


Assuntos
Glândulas Mamárias Animais/microbiologia , Mastite Bovina/microbiologia , Microbiota , Animais , Búfalos/genética , Bovinos , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Glândulas Mamárias Animais/imunologia , Mastite Bovina/imunologia , Leite , RNA Ribossômico 16S
12.
Vet Immunol Immunopathol ; 212: 1-8, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31213246

RESUMO

Bovine mastitis caused by Staphylococcus aureus is a serious problem in dairy production and effective immunoprophylaxis is an unmet goal so far. The objective of this work was to assess the humoral immune response of heifer calves against two recombinant S. aureus antigens: Clumping factor A (ClfA) and Fibronectin Binding Protein A (FnBPA), formulated with a novel adjuvant based on cationic liposomes (Lip) and CpG oligodeoxynucleotides (CpG-ODN). Six groups of 6-8 months old heifer calves received three doses biweekly of antigens, formulated with Al(OH)3, liposomes, CpG-ODN or Lip + CpG-ODN. Animals also received a fourth dose after a year (day 410) and a booster before calving. The administration of Al(OH)3+FnBPA/ClfA and Lip + FnBPA/ClfA + CpG-ODN induced the highest specific IgG levels, after the first 3 doses and induced a fast increase of antibodies after the fourth dose. All the formulations stimulated the production of specific IgG1, after the third and fourth dose. Specific IgG2 for both proteins was only stimulated after the fourth dose by Lip + FnBPA/ClfA + CpG-ODN. Pre-calving immunisation with Lip + FnBPA/ClfA + CpG-ODN led to the highest IgG levels during the calving period and to the production of the IgG2 subclass. The formulation was also able to stimulate the highest antibody levels in milk, 30 and 45 days after pre-calving booster. The combination of liposomes and CpG-ODN as adjuvant for a subunit vaccine, together with the immunisation schedule described, induced a strong humoral immune response with production of specific IgG2. The formulation demonstrated to induce immune memory allowing the application of a single pre-calving booster to maintain high antibody levels throughout the period of increased susceptibility to intramammary infections.


Assuntos
Antígenos de Bactérias/imunologia , Imunidade Humoral , Mastite Bovina/prevenção & controle , Oligodesoxirribonucleotídeos/farmacologia , Adjuvantes Imunológicos/administração & dosagem , Fatores Etários , Animais , Anticorpos Antibacterianos/sangue , Bovinos , Imunoglobulina G/sangue , Memória Imunológica , Lipossomos/farmacologia , Mastite Bovina/imunologia , Mastite Bovina/microbiologia , Staphylococcus aureus , Vacinação , Soro do Leite/imunologia
13.
Comp Immunol Microbiol Infect Dis ; 64: 117-124, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31174686

RESUMO

INTRODUCTION: Biofilms, an assemblage of microbial cells irreversibly associated with a surface and enclosed in a matrix of polysaccharide material pose serious health challenges, resulting in high economic losses. The emergence of methicillin-resistant S. aureus (MRSA) infections and ability to form biofilms in dairy animals is of emerging concern for livestock and public health owing to their association with serious infections. The present study was undertaken to examine the presence of methicillin resistance genes among the biofilm forming Staphylococcus aureus strains isolated from cases of acute and subacute bovine mastitis. A total of 150 mastitic milk samples referred to Veterinary Clinical Complex, Shuhama (Aulesteng) SKUAST-K were screened in present study. The methicillin resistant Staphylococcus aureus isolates were also screened for in vitro biofilm forming ability. RESULTS: A total of 80 (53.33%) S. aureus isolates were recovered from cases of bovine mastitis of which 20 (25%) were methicillin (mecA) gene positive. Of the 20 mecA positive isolates, 20% were positive for SCCmec I, 35% for SCCmec IV and 45% for SCCmec V subtypes. In vitro antibiotic sensitivity testing of MRSA revealed complete resistance towards methicillin and other pencillin group of antibiotics. CONCLUSION: A significant correlation was observed between in vitro biofilm formation and presence of methicillin resistance gene in S aureus isolates recovered from acute and subacute mastitis. The Staphylococcus aureus isolates positive for methicillin resistance gene (mecA) were either strong or moderate biofilm formers.


Assuntos
Biofilmes/crescimento & desenvolvimento , Mastite Bovina/microbiologia , Staphylococcus aureus Resistente à Meticilina/genética , Leite/microbiologia , Infecções Estafilocócicas/veterinária , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Bovinos , Feminino , Índia , Meticilina/farmacologia , Testes de Sensibilidade Microbiana , Proteínas de Ligação às Penicilinas/genética
14.
Microb Pathog ; 132: 355-361, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31096003

RESUMO

It is important to evaluate the antimicrobial resistance pattern by genotypic and phenotypic methods in epidemiological studies in order to control antimicrobial resistance and to improve the outcome of the treatments. Four-hundred and thirty clinical mastitis samples were collected from 14 dairy herds in five different cities. The antimicrobial susceptibility test was performed using agar disk diffusion for 70 identified Escherichia coli isolates. The antimicrobial resistance genes including strA, strB, aadA, sulI, sulII, sulIII, ampC were detected by PCR method. Phylogenic groups were determined by Clermont's multiplex PCR method, and RAPD typing was performed on all isolates. Most isolates were resistant to lincomicin and streptomycin, whereas sulfa-trimethoprim has the lowest resistance rate. Moreover, ampC, aadA and sul2 genes had the highest frequency (92.85%, 38.57%, and 32.85% respectively). 20% of all the isolates carried strA and strB genes, and 11.42% of the isolates had sul1 gene and 10% of the isolates had the less frequent sul3 gene. Of the total of 70 E. coli isolates, 26 (37.14%), 20 (28.5%), 17 (24.2%), 8 (11.4%) isolates belonged to B1, A, B2 and D phylogenic groups respectively. strA, strB, sul2and aadA resistance genes had the highest percentage in A phylogenic groups. Based on RAPD-PCR method, E. coli isolates were classified in four clusters. The result showed a high phenotypic and genotypic E. coli resistance to the current antimicrobials with a similar pattern in different cities; also the majority of E. coli isolates belonged to B1 group which mainly contains the commensal E. coli isolates.


Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Genótipo , Mastite Bovina/microbiologia , Animais , Antibacterianos/farmacologia , Bovinos , Escherichia coli/classificação , Escherichia coli/efeitos dos fármacos , Infecções por Escherichia coli/veterinária , Proteínas de Escherichia coli/genética , Feminino , Testes de Sensibilidade Microbiana/veterinária , Filogenia , Reação em Cadeia da Polimerase , Técnica de Amplificação ao Acaso de DNA Polimórfico , Estreptomicina/farmacologia
15.
J Appl Microbiol ; 127(2): 406-417, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31077513

RESUMO

AIM: To primarily estimate the sensitivity (Se) and specificity (Sp) of the commercially available Mastit4 quantitative PCR (qPCR) assay and bacterial culture (BC) for diagnosis of intramammary infections (IMI) and teat apex colonization (TAC) with coagulase-negative staphylococci (CNS) at different cut-offs for qPCR cycle threshold values using Bayesian latent class analysis. A secondary objective was to evaluate two cut-offs of BC for diagnosis of IMI and TAC with CNS. METHODS AND RESULTS: We randomly selected 13-20 cows with subclinical mastitis from eight dairy herds. Teat skin samples and aseptically collected foremilk samples were collected from the right hindquarters (n = 149) for BC and qPCR analysis. The Se of qPCR was always higher than BCSe in diagnosis of IMI, however; the Sp of BC was higher than qPCRSp . BCSe and BCSp showed no substantial difference between the tested BC cut-offs. In contrast to IMI, estimates of BC and qPCR in diagnosing TAC were different. BCSe was higher than qPCRSe at all tested cut-offs, however; qPCRSp was higher than BCSp . CONCLUSION: The overall performance of qPCR is higher than BC in the diagnosis of IMI; however, the performance of BC is better than qPCR in diagnosis of TAC. The qPCR and BC are valid diagnostics for bovine IMI with CNS. However, for TAC, both techniques require further investigation to reduce the uncertainty of the true status of the quarter and teat skin. SIGNIFICANCE AND IMPACT OF THE STUDY: We reported, for the first time, the diagnostic performance of new mastitis technology (Mastit4 PCR) and culture for detection of CNS in milk and nonmilk samples in dairy herds with automatic milking systems. Our findings will improve the interpretation of the test results of culture and qPCR assay and subsequently, will strengthen the control of IMI with CNS in dairy cows.


Assuntos
Mastite Bovina/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real , Infecções Estafilocócicas/veterinária , Animais , Teorema de Bayes , Bovinos , Feminino , Glândulas Mamárias Animais/microbiologia , Mastite Bovina/microbiologia , Leite/microbiologia , Sensibilidade e Especificidade , Infecções Estafilocócicas/diagnóstico , Infecções Estafilocócicas/microbiologia , Staphylococcus/isolamento & purificação
16.
Pol J Microbiol ; 68(1): 93-104, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31050257

RESUMO

Staphylococcus aureus is a common human and livestock opportunistic pathogen, and there is evidence of animal to human transmission. This paper aimed to recognize properties of the isolates from collections of human and livestock S. aureus strains and to estimate compatibility of results based on phenotypic tests, microarrays and the spa typing methods. The second goal was to study differences between human and animal isolates in terms of specificity of their hosts and the strain transmission among various hosts. Most strains showed multi-susceptible profiles and produced enzymes on a high level, and they were phenotypically and genetically similar. However, in contrast to the Polish bovine mastitis strains, the Slovakian strains were multi-resistant. In this research, the strains showed significant differences in terms of their phenotypic manifestations and the presence of hemolysins genes; however, other enzyme-encoding genes correlated to a higher extent with the microarrays results. Interestingly, there was a lack of enterotoxin genes in human Poultry-like protein A+ strains in comparison to other human strains. Our study showed that differences between virulence profiles of the human and animal strains correlated with their origin rather than their hosts, and any trait allowed clearly distinguishing between them based on the microarray results.Staphylococcus aureus is a common human and livestock opportunistic pathogen, and there is evidence of animal to human transmission. This paper aimed to recognize properties of the isolates from collections of human and livestock S. aureus strains and to estimate compatibility of results based on phenotypic tests, microarrays and the spa typing methods. The second goal was to study differences between human and animal isolates in terms of specificity of their hosts and the strain transmission among various hosts. Most strains showed multi-susceptible profiles and produced enzymes on a high level, and they were phenotypically and genetically similar. However, in contrast to the Polish bovine mastitis strains, the Slovakian strains were multi-resistant. In this research, the strains showed significant differences in terms of their phenotypic manifestations and the presence of hemolysins genes; however, other enzyme-encoding genes correlated to a higher extent with the microarrays results. Interestingly, there was a lack of enterotoxin genes in human Poultry-like protein A+ strains in comparison to other human strains. Our study showed that differences between virulence profiles of the human and animal strains correlated with their origin rather than their hosts, and any trait allowed clearly distinguishing between them based on the microarray results.


Assuntos
Reservatórios de Doenças/microbiologia , Mastite Bovina/microbiologia , Staphylococcus aureus Resistente à Meticilina/classificação , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Infecções Estafilocócicas/microbiologia , Animais , Antibacterianos/farmacologia , Bovinos , Farmacorresistência Bacteriana Múltipla/genética , Proteínas Hemolisinas/genética , Humanos , Gado/microbiologia , Staphylococcus aureus Resistente à Meticilina/genética , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Infecções Oportunistas/microbiologia
17.
J Vet Diagn Invest ; 31(4): 523-530, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31006359

RESUMO

We compared the effectiveness of various methods for the identification of Staphylococcus spp. other than S. aureus isolated from intramammary infections of cows on 3 dairy farms in Lower Silesia, Poland. A total of 131 isolates belonging to 18 Staphylococcus species were identified by sequence analysis of the 16S rRNA and dnaJ genes, as well using a commercial identification system (ID 32 STAPH; bioMérieux) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS; Bruker Daltonics). Sequencing of the 16S rRNA gene was found to have low discriminatory value because only 43% of isolates were recognized unequivocally. Much better results were obtained with the dnaJ gene (all isolates were correctly identified at the species level). However, some of these isolates achieved a low similarity level (<97%) and required a confirmatory test (sequencing of the rpoB gene). The performance of ID 32 STAPH was poor. Regardless of the probability level used (80% or 90%), the commercial system obtained identification rates <40%. Using MALDI-TOF MS and the commercial Bruker database, 67% of isolates were identified correctly with scores ≥2.0 (acceptable species-level identification) but this number increased to 97% after the database was expanded. The definitive identification of Staphylococcus spp. other than S. aureus causing intramammary infections in cattle often requires a combination of different procedures, and the existing databases should be updated.


Assuntos
Mastite Bovina/microbiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/veterinária , Infecções Estafilocócicas/veterinária , Staphylococcus/classificação , Staphylococcus/genética , Animais , Bovinos , Feminino , Genótipo , Humanos , Mastite Bovina/epidemiologia , Polônia/epidemiologia , RNA Ribossômico 16S/genética , Infecções Estafilocócicas/epidemiologia , Infecções Estafilocócicas/microbiologia
18.
J Microbiol ; 57(7): 587-596, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30982119

RESUMO

Staphylococcus aureus is the major pathogen leading to bovine mastitis globally while livestock-associated methicillin resistant S. aureus (LA-MRSA) has become a potential threat to public health. MRSA from bovine mastitis is not common but a methicillin susceptible S. aureus (MSSA) genotype, rpoB sequence type (RST)10-2 (RST10-2), is prevalent in Korea. To date, many genomic sequences from S. aureus have been elucidated, but the complete genome sequences of RST10-2 MSSA from bovine mastitis has never been reported. In this study, we determined the complete genome sequence of two RST10-2 MSSA that differ from each other in staphylococcal protein A and molecular prophage types [PMB64-1 (t2489/ mPPT0) and PMB81-4 (t127/mPPT1-2-3)] and conducted a comparative genomics study. The genomic sequences of PMB64-1 and PMB81-4 were more homologous to the representative human RST10-2 strains (MSSA476, MW2 etc.) compared to other RSTs. Most of them shared five common pseudogenes, along with high amino acid identity of four variable virulence genes that were identified in this study. However, PMB64-1 and PMB81-4 acquired different strainspecific pseudogenes and mobile genetic elements than the human strains. The unique pseudogene profile and high identity of the virulence genes were verified in RST10-2 field strains from bovine mastitis. Thus, bovine mastitic RST10-2 MSSA may have an evolutionary relationship with the human RST10-2 community-associated (CA) MSSA and CA-MRSA strains but may have adapted to cows.


Assuntos
RNA Polimerases Dirigidas por DNA/genética , Mastite Bovina/microbiologia , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/veterinária , Staphylococcus aureus/genética , Animais , Bovinos , DNA Bacteriano/genética , Feminino , Sequências Repetitivas Dispersas , Pseudogenes , República da Coreia , Fatores de Virulência/genética , Sequenciamento Completo do Genoma
19.
N Z Vet J ; 67(4): 203-209, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31023170

RESUMO

Aims: To assess the use of different cut-points based on individual cow somatic cell counts (SCC) to define cows with intramammary infection (IMI) at drying-of, in a herd with a high mean bulk tank SCC. Methods: Results for SCC from four herd tests during lactation and bacterial culture of milk samples collected before drying-off were obtained for 139 cows from a herd with an average bulk milk SCC of >300,000 cells/mL over the final 4 months of the 2006/07 lactation. Based on culture results, cows were defined as being infected with a major (Staphylococcus aureus, Streptococcus uberis or Nocardia spp.) or any pathogen. Receiver-operator characteristics (ROC) curves were used to determine optimum cut-points for maximum, average and last herd test SCC, for predicting IMI. Multivariable logistic regression models were used to determine which variables were associated with IMI, and the sensitivity (Se), specificity (Sp) and positive predictive value (PPV) were determined for different cut-points. Results: At the cow level, 75/139 (54.0%) cows had IMI with a major pathogen and 123/139 (88.5%) with any pathogen. A SCC ≥150,000 cells/mL at ≥2 herd tests and a SCC ≥299,000 cells/mL at the last herd test, for cows aged ≥4 years, were associated with IMI with a major pathogen at drying-off (p<0.05). A SCC ≥150,000 cells/mL at ≥2 herd tests was associated with IMI with any pathogen at drying-off (p<0.001). A cut-point of ≥150,000 cells/mL at any herd test had the highest Se (0.97 and 0.94), but the lowest Sp (0.19 and 0.44) and PPV (0.58 and 0.93) for infection with major and any pathogens, respectively. A cut-point of ≥150,000 cells/mL at ≥2 herd tests doubled the Sp and increased the PPV without large decreases in test Se for infection with either a major or any pathogen. Conclusions and clinical relevance: In this herd with a high bulk milk SCC, use of a cut-point of a SCC ≥150,000 cells/mL at any herd test to define IMI would be appropriate, where the goal at drying-off is to ensure that cows infected with any pathogen receive antimicrobial treatment. Where the goal is to reduce the use of antimicrobial dry cow therapy in uninfected cows while limiting the number of infected cows not being treated, use of a cut-point of SCC ≥150,000 cells/mL at ≥2 herd tests to define IMI may be more appropriate.


Assuntos
Contagem de Células/métodos , Infecções por Bactérias Gram-Positivas/veterinária , Mastite Bovina/diagnóstico , Mastite Bovina/microbiologia , Leite/microbiologia , Animais , Bovinos , Indústria de Laticínios , Feminino , Bactérias Gram-Positivas/isolamento & purificação , Infecções por Bactérias Gram-Positivas/diagnóstico , Infecções por Bactérias Gram-Positivas/epidemiologia , Modelos Logísticos , Glândulas Mamárias Animais/microbiologia , Mastite Bovina/epidemiologia , Nova Zelândia/epidemiologia , Curva ROC , Estudos Retrospectivos
20.
J Dairy Sci ; 102(7): 6442-6453, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31030918

RESUMO

A longitudinal study was conducted to assess to what extent intramammary infection (IMI) with non-aureus staphylococci (NAS) within the first 4 d after calving in dairy heifers affects quarter milk yield (qMY) and quarter milk somatic cell count (qSCC) during the first 4 mo of lactation. In total, 324 quarters from 82 Holstein Friesian heifers from 3 commercial dairy herds equipped with an automatic milking system were included and followed from calving up to 4 mo in lactation. The automatic milking system allowed us to precisely determine the daily qMY. A milk sample from each quarter was collected in early lactation (between 1 and 4 d in milk) for bacteriological culturing and measurement of the qSCC. Subsequently, milk samples were taken on a biweekly basis for measurement of the qSCC. The milk prolactin level in early lactation was measured, and the relation with NAS IMI was determined. Overall, NAS IMI in early lactation caused only a slight but significant increase in qSCC compared with milk from noninfected quarters during the first 4 mo in lactation, whereas no significant difference in daily qMY was present between NAS-infected and noninfected quarters. The milk prolactin level in early lactation did not differ between NAS-infected and noninfected quarters either. Our data suggest that IMI with NAS (as a group) present shortly after calving do not have an adverse effect on later production. The milk prolactin concentrations were not dissimilar between NAS-infected and noninfected quarters and thus cannot explain why NAS-infected quarters do not produce less than noninfected quarters.


Assuntos
Glândulas Mamárias Animais/fisiopatologia , Mastite Bovina/metabolismo , Leite/metabolismo , Infecções Estafilocócicas/veterinária , Staphylococcus aureus/fisiologia , Animais , Bovinos , Contagem de Células/veterinária , Feminino , Lactação , Estudos Longitudinais , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/microbiologia , Mastite Bovina/microbiologia , Mastite Bovina/fisiopatologia , Leite/microbiologia , Prolactina/metabolismo , Infecções Estafilocócicas/metabolismo , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/fisiopatologia
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