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1.
Vet Res ; 52(1): 137, 2021 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-34711282

RESUMO

Although extensive research has been performed on bovine non-aureus staphylococci (NAS), several aspects such as bacteria-host interaction remain largely unstudied. Moreover, only a few mastitis pathogen challenge studies in cows have been conducted in the dry period, an important period that allows intramammary infection (IMI) to cure and new IMI to occur. We challenged 16 quarters of 4 Holstein Friesian cows at dry off with 100; 100 000 or 10 000 000 CFU of the udder-adapted S. chromogenes IM strain. Four quarters from one cow served as negative controls. Internally sealed quarters remained untouched, whereas non-sealed quarters were sampled 3 times during the dry period. After parturition, colostrum and daily milk samples were taken during the first week of lactation of all quarters. In total, 8 quarters appeared to be colonized, since S. chromogenes IM was recovered at least once during the experiment, as substantiated using Multilocus Sequence Typing. S. chromogenes IM shedding was highest in dry quarters inoculated with 10 000 000 CFU. Colonized quarters had the highest quarter somatic cell count (qSCC) in early lactation. Inoculated quarters (both colonized and non-colonized) had lower IL-6 and IL-10 concentrations in the dry period, whilst IFN-γ levels tended to be higher in colonized quarters compared to non-inoculated quarters. Also, IgG2 levels were higher in inoculated compared to non-inoculated quarters and the IgG2/IgG1 ratio was on average above 1. To conclude, we showed that dry quarters can be colonized with S. chromogenes IM, resulting in a shift towards a Th1 response in late gestation and early lactation characterised by an increased IgG2 concentration. However, further research is needed to confirm our findings.


Assuntos
Imunidade Inata , Mastite Bovina/microbiologia , Infecções Estafilocócicas/veterinária , Staphylococcus/fisiologia , Animais , Bovinos , Contagem de Células/veterinária , Feminino , Lactação , Mastite Bovina/imunologia , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia
2.
BMC Vet Res ; 17(1): 321, 2021 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-34620161

RESUMO

BACKGROUND: Bovine mastitis is an important cause of economic loss in dairy farms. Streptococcus uberis is among the most frequently isolated bacterial species isolated from cows with mastitis. The aim of this study was to perform an in-depth genetic assessment of S. uberis strains isolated from bovine clinical mastitis (CM) and to perform a phylogenetic analysis to represent the evolutionary relationship among S. uberis sequences. RESULTS: A total of 159 isolates was genetically characterized using whole genome sequencing. According to the virulence determinants, all strains harbored the hasC, leuS, perR, purH, and purN virulence genes. Thirty-four resistance genes were identified in at least one strain. In terms of acquired genes, we observed that 152 (95.6 %) strains had a resistance gene to lincosamine (lnuD), 48 (30.2 %) to tetracycline (tetM), 4 (2.51 %) to tobramicine (ant6), and 1 to lincosamide (lsa(E)). MLST detected the Sequence Type (ST)797 (n = 23), while 85.5 % of the strains did not match to known STs. CONCLUSIONS: Then, eleven distinct ST were identified after we submitted the new alleles to assign new STs. The other prevalent STs observed were ST1215 (n = 58), ST1219 (n = 35), and ST1213 (n = 15). And it was not possible to identify the MLST of four strains. Phylogenetic lineages indicated a high genomic diversity of S. uberis in our collection, confirming that most strains isolated from bovine mastitis have different reservoirs, typical of environmental pathogens.


Assuntos
Variação Genética , Mastite Bovina/microbiologia , Streptococcus/genética , Animais , Bovinos , Farmacorresistência Bacteriana/genética , Feminino , Tipagem de Sequências Multilocus , Filogenia , Infecções Estreptocócicas/veterinária , Virulência/genética , Sequenciamento Completo do Genoma/veterinária
3.
J Dairy Res ; 88(3): 286-292, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34372953

RESUMO

This study aimed to obtain a better understanding of the regulatory genes and molecules involved in the development of mastitis. For this purpose, the transcription factors (TF) and MicroRNAs (miRNA) related to differentially expressed genes previously found in extracorporeal udders infected with Streptococcus agalactiae were investigated. The Gene-TF network highlighted LOC515333, SAA3, CD14, NFKBIA, APOC2 and LOC100335608 and genes that encode the most representative transcription factors STAT3, PPARG, EGR1 and NFKB1 for infected udders. In addition, it was possible to highlight, through the analysis of the gene-miRNA network, genes that could be post-transcriptionally regulated by miRNAs, such as the relationship between the CCL5 gene and the miRNA bta-miR-363. Overall, our data demonstrated genes and regulatory elements (TF and miRNA) that can play an important role in mastitis resistance. The results provide new insights into the first functional pathways and the network of genes that orchestrate the innate immune responses to infection by Streptococcus agalactiae. Our results will increase the general knowledge about the gene networks, transcription factors and miRNAs involved in fighting intramammary infection and maintaining tissue during infection and thus enable a better understanding of the pathophysiology of mastitis.


Assuntos
Simulação por Computador , Regulação da Expressão Gênica , Mastite Bovina/genética , RNA-Seq/veterinária , Animais , Bovinos , Feminino , Perfilação da Expressão Gênica/métodos , Perfilação da Expressão Gênica/veterinária , Predisposição Genética para Doença , Glândulas Mamárias Animais/metabolismo , Mastite Bovina/imunologia , Mastite Bovina/microbiologia , MicroRNAs/genética , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/veterinária , Streptococcus agalactiae , Fatores de Transcrição/genética
4.
J Dairy Res ; 88(3): 302-306, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34372963

RESUMO

In this Research Communication we evaluate the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify 380 bacteria isolated from cases of bovine mastitis in Brazil. MALDI-TOF MS identifications were compared to previous identifications by biochemical tests and 16S rRNA sequencing. MALDI-TOF MS achieved a typeability of 95.5%. The accuracy of MALDI-TOF MS for the identification of Staphylococcus isolates was 93.2%. The agreement between MALDI-TOF MS and biochemical identification of Streptococcus agalactiae was 96%, however, the agreement between these techniques for identifying other catalase-negative, Gram-positive cocci was lower. Agreement in identifying Gram-negative bacteria at the genus level was 90.5%. Our findings corroborate that MALDI-TOF MS is an accurate, rapid and simple technique for identifying bovine mastitis pathogens. The availability of this methodology in some research institutions would represent a significant step toward increasing the diagnosis and epidemiological studies of bovine mastitis and other animal infectious diseases in Brazil.


Assuntos
Mastite Bovina/microbiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/veterinária , Animais , Brasil , Bovinos , Feminino , Bactérias Gram-Negativas/classificação , Bactérias Gram-Negativas/genética , Bactérias Gram-Positivas/classificação , Bactérias Gram-Positivas/genética , Leite/microbiologia , RNA Ribossômico 16S/genética , Sensibilidade e Especificidade , Análise de Sequência de RNA , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Staphylococcus/genética , Streptococcus agalactiae/genética
5.
J Dairy Res ; 88(3): 321-325, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34382532

RESUMO

Variations in the levels of acute phase proteins and lactoferrin in serum and milk for diagnosis of subclinical mastitis in dairy cows are described in this research paper. Milking animals from two organized dairy farms in Kerala, India, were screened by California Mastitis Test (CMT), Electrical Conductivity test (EC) and Somatic Cell Count (SCC) test to identify animals affected with sub clinical mastitis (SCM). The concentrations of acute phase proteins (APP) Haptoglobin (Hp), C- reactive protein (CRP), Albumin, Lactoferrin (Lf) and α- 1 acid glycoprotein (AGP) in milk and Hp, Albumin, Serum Amyloid A (SAA) and CRP in the serum of 40 normal cows and 40 cows affected with sub clinical mastitis were assessed. Solid phase ELISA was employed for assessment of all parameters except the albumin levels, for which spectrophotometry was used. The values of Hp in milk; and SAA, AGP and Lf in serum, were significantly elevated in the group with sub clinical mastitis. Such variations were found to be independent of the specific bacterial organism causing the disease. These results show that significant variations exist in the levels of acute phase proteins Hp, AGP and Lf in milk, and SAA in serum of animals affected with subclinical bovine mastitis that are not affected by specific bacterial etiology.


Assuntos
Proteínas de Fase Aguda/análise , Lactoferrina/análise , Lactoferrina/sangue , Mastite Bovina/diagnóstico , Leite/química , Animais , Biomarcadores/análise , Biomarcadores/sangue , Proteína C-Reativa/análise , Bovinos , Contagem de Células/veterinária , Feminino , Haptoglobinas/análise , Índia , Programas de Rastreamento/métodos , Programas de Rastreamento/veterinária , Mastite Bovina/microbiologia , Leite/citologia , Proteína Amiloide A Sérica/análise
6.
J Dairy Res ; 88(3): 326-329, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34382922

RESUMO

Our objective was to evaluate relationships between milk components (acute phase proteins, enzymes, metabolic parameters and oxidative indices) and the spontaneous cure outcome of Staphylococcus aureus subclinical mastitis in dairy cows. The values of haptoglobin, serum amyloid A (SAA), malondialdehyde (MDA), total antioxidant capacity, milk urea nitrogen (MUN), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), electrolytes (Cl and K), total protein, albumin, α-lactalbumin, ß-lactoglobulin, and immunoglobulin were measured in milk samples of S. aureus subclinical mastitis cows with spontaneous cure (n = 23), S. aureus subclinical mastitis cows without spontaneous cure (n = 29) and healthy cows (n = 23). The comparison of measured parameters revealed that subclinical mastitis cows with spontaneous cure had lower ALP and haptoglobin concentrations both at diagnosis and after cure (P < 0.05). In contrast, total antioxidant capacity and MDA concentration in subclinical mastitis cows without spontaneous cure significantly increased with time (P < 0.05). We can suggest that elevated haptoglobin concentration and higher ALP activity indicative of enhanced oxidative stress could potentially serve as early diagnostic indicators of chronic disease and the persistence of S. aureus subclinical mastitis in dairy cows.


Assuntos
Mastite Bovina/diagnóstico , Leite/química , Infecções Estafilocócicas/veterinária , Staphylococcus aureus , Fosfatase Alcalina/análise , Animais , Antioxidantes/análise , Bovinos , Doença Crônica/veterinária , Feminino , Haptoglobinas/análise , Malondialdeído/análise , Mastite Bovina/metabolismo , Mastite Bovina/microbiologia , Proteínas do Leite/análise , Estresse Oxidativo , Remissão Espontânea , Proteína Amiloide A Sérica/análise
7.
J Dairy Res ; 88(3): 293-301, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34425921

RESUMO

Staphylococcus aureus is a common pathogen of bovine mastitis which can induce autophagy and inhibit autophagy flux, resulting in intracellular survival and persistent infection. The aim of the current study was to investigate the role of p38α in the autophagy induced by intracellular S. aureus in bovine mammary epithelial cells. An intracellular infection model of MAC-T cells was constructed, and activation of p38α was examined after S. aureus invasion. Through activating/inhibiting p38α by anisomycin/SB203580, the autophagosomes, LC3 and p62 level were analyzed by immunofluorescence and western blot. To further study the detailed mechanism of p38α, phosphorylation of ULK1ser757 was also detected. The results showed that intracellular S. aureus activated p38α, and the activation developed in a time-dependent manner. Inhibition of p38α promoted intracellular S. aureus-induced autophagy flow, up-regulated the ratio of LC3 II/I, reduced the level of p62 and inhibited the phosphorylation of ULK1ser757, whereas the above results were reversed after activation of p38α. The current study indicated that intracellular S. aureus can inhibit autophagy flow by activating p38α in bovine mammary epithelial cells.


Assuntos
Autofagia/fisiologia , Células Epiteliais/microbiologia , Mastite Bovina/microbiologia , Infecções Estafilocócicas/veterinária , Staphylococcus aureus/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia , Animais , Bovinos , Linhagem Celular , Ativação Enzimática , Células Epiteliais/fisiologia , Feminino , Glândulas Mamárias Animais/citologia , Mastite Bovina/fisiopatologia
8.
Int J Mol Sci ; 22(16)2021 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-34445550

RESUMO

Within-host adaptation is a typical feature of chronic, persistent Staphylococcus aureus infections. Research projects addressing adaptive changes due to bacterial in-host evolution increase our understanding of the pathogen's strategies to survive and persist for a long time in various hosts such as human and bovine. In this study, we investigated the adaptive processes of S. aureus during chronic, persistent bovine mastitis using a previously isolated isogenic strain pair from a dairy cow with chronic, subclinical mastitis, in which the last variant (host-adapted, Sigma factor SigB-deficient) quickly replaced the initial, dominant variant. The strain pair was cultivated under specific in vitro infection-relevant growth-limiting conditions (iron-depleted RPMI under oxygen limitation). We used a combinatory approach of surfaceomics, molecular spectroscopic fingerprinting and in vitro phenotypic assays. Cellular cytotoxicity assays using red blood cells and bovine mammary epithelial cells (MAC-T) revealed changes towards a more cytotoxic phenotype in the host-adapted isolate with an increased alpha-hemolysin (α-toxin) secretion, suggesting an improved capacity to penetrate and disseminate the udder tissue. Our results foster the hypothesis that within-host evolved SigB-deficiency favours extracellular persistence in S. aureus infections. Here, we provide new insights into one possible adaptive strategy employed by S. aureus during chronic, bovine mastitis, and we emphasise the need to analyse genotype-phenotype associations under different infection-relevant growth conditions.


Assuntos
Adaptação Fisiológica , Hemólise , Adaptação ao Hospedeiro , Glândulas Mamárias Animais/patologia , Mastite Bovina/patologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/patogenicidade , Animais , Apoptose , Bovinos , Feminino , Glândulas Mamárias Animais/microbiologia , Mastite Bovina/microbiologia , Fenótipo
9.
Sci Rep ; 11(1): 17392, 2021 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-34462461

RESUMO

Mastitis is a common and costly disease on dairy farms, commonly caused by Staphylococcus spp. though the various species are associated with different clinical outcomes. In the current study, we performed genomic analyses to determine the prevalence of adhesion, biofilm, and related regulatory genes in 478 staphylococcal species isolated from clinical and subclinical mastitis cases deposited in public databases. The most prevalent adhesin genes (ebpS, atl, pls, sasH and sasF) were found in both clinical and subclinical isolates. However, the ebpS gene was absent in subclinical isolates of Staphylococcus arlettae, S. succinus, S. sciuri, S. equorun, S. galinarum, and S. saprophyticus. In contrast, the coa, eap, emp, efb, and vWbp genes were present more frequently in clinical (vs. subclincal) mastitis isolates and were highly correlated with the presence of the biofim operon (icaABCD) and its transcriptional regulator, icaR. Co-phylogenetic analyses suggested that many of these adhesins, biofilm, and associated regulatory genes could have been horizontally disseminated between clinical and subclinical isolates. Our results further suggest that several adhesins, biofilm, and related regulatory genes, which have been overlooked in previous studies, may be of use for virulence profiling of mastitis-related Staphylococcus strains or as potential targets for vaccine development.


Assuntos
Adesinas Bacterianas/genética , Biofilmes/crescimento & desenvolvimento , Mastite Bovina/patologia , Infecções Estafilocócicas/patologia , Staphylococcus/genética , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bovinos , Feminino , Mastite Bovina/microbiologia , Filogenia , Infecções Estafilocócicas/microbiologia , Staphylococcus/classificação , Staphylococcus/isolamento & purificação , Staphylococcus/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Virulência/genética
10.
BMC Vet Res ; 17(1): 282, 2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34433467

RESUMO

BACKGROUNDS: The present study explored the viability of bovine milk macrophages, their intracellular production of reactive oxygen and nitrogen species (RONS), and their phagocytosis of Staphylococcus aureus, as well as the profile of lymphocytes, from healthy udder quarters and udder quarters infected by Corynebacterium bovis. The study included 28 healthy udder quarters from 12 dairy cows and 20 udder quarters infected by C. bovis from 10 dairy cows. The percentages of macrophages and lymphocytes were identified by flow cytometry using monoclonal antibodies. Macrophage viability, RONS production, and S. aureus phagocytosis were evaluated by flow cytometry. RESULTS: Milk samples from quarters infected with C. bovis showed a lower percentage of macrophages but an increased number of milk macrophages per mL and a higher percentage of macrophages that produced intracellular RONS and phagocytosed S. aureus. No effect of C. bovis infection on macrophage viability was found. Udder quarters infected by C. bovis showed a higher percentage of T cells and CD4+ T lymphocytes, but no effect was found on the percentage of CD8+ CD4- T, CD8- CD4- T, or B lymphocytes. CONCLUSIONS: Thus, our results corroborate, at least in part, the finding that intramammary infections by C. bovis may offer protection against intramammary infections by major pathogens.


Assuntos
Macrófagos/fisiologia , Mastite Bovina/microbiologia , Leite/citologia , Animais , Bovinos , Corynebacterium , Feminino , Linfócitos , Mastite Bovina/patologia , Fagocitose , Espécies Reativas de Nitrogênio , Espécies Reativas de Oxigênio , Staphylococcus aureus
11.
Vet Ital ; 57(1): 41-47, 2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-34313097

RESUMO

Streptococcal species are known to be responsible for bovine mastitis. The aim of the present study was to determine antimicrobial drug resistance patterns of hemolytic streptococci distributed according to Lancefield serogrouping. Streptococcus sp. strains were isolated from 124 bovine milk samples from 31 cows with subclinical or clinical mastitis submitted to Mehmet Akif Ersoy University Faculty of Veterinary Medicine, Department of Microbiology Laboratory in Burdur province, Turkey from January 2015 to January 2017. A total of 63 Streptococcus sp. were isolated and the most frequently obtained isolates were classified as Lancefield's serogroup B (84.13%), the remaining isolates as serogroup F (15.87%). Out of 63 isolates, 53 (84.13%) showed beta­hemolytic activity whereas 10 (15.87%) alpha­hemolytic activity. Antimicrobial resistance was assessed by disk diffusion test against the most common antibiotics used in the field. Among the 63 Streptococcus sp. tested, the highest antimicrobial resistance patterns were observed for neomycin (95.24%), trimethoprim sulphamethoxazole (87.30%) and gentamicin (69.84%). None of the isolates showed resistance to amoxicillin­clavulanic acid, except for one serogroup F isolate. The resistance rates for the other antimicrobials ranged from 1.59% to 38.04%. A total of 50 isolates exibited multi­drug resistance to ≥ 3 antimicrobial agents tested. Overall, our results suggested that there is an urgent need to enhance awareness among the dairy farmers in choosing the appropriate drug for treating mastitis.


Assuntos
Antibacterianos/uso terapêutico , Gestão de Antimicrobianos , Farmacorresistência Bacteriana , Mastite Bovina/tratamento farmacológico , Streptococcus/isolamento & purificação , Animais , Antibacterianos/farmacologia , Bovinos , Indústria de Laticínios , Feminino , Mastite Bovina/microbiologia , Streptococcus/efeitos dos fármacos , Turquia/epidemiologia
12.
Int J Mol Sci ; 22(12)2021 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-34200743

RESUMO

Mastitis is a common disease in dairy cows that is mostly caused by E. coli, and it brings massive losses to the dairy industry. N6-Methyladenosine (m6A), a methylation at the N6 position of RNA adenine, is a type of modification strongly associated with many diseases. However, the role of m6A in mastitis has not been investigated. In this study, we used MeRIP-seq to sequence the RNA of bovine mammary epithelial cells treated with inactivated E. coli for 24 h. In this in vitro infection model, there were 16,691 m6A peaks within 7066 mRNA transcripts in the Con group and 10,029 peaks within 4891 transcripts in the E. coli group. Compared with the Con group, 474 mRNAs were hypermethylated and 2101 mRNAs were hypomethylated in the E. coli group. Biological function analyses revealed differential m6A-modified genes mainly enriched in the MAPK, NF-κB, and TGF-ß signaling pathways. In order to explore the relationship between m6A and mRNA expression, combined MeRIP-seq and mRNA-seq analyses revealed 212 genes with concomitant changes in the mRNA expression and m6A modification. This study is the first to present a map of RNA m6A modification in mastitis treated with E. coli, providing a basis for future research.


Assuntos
Adenosina/análogos & derivados , Metilação de DNA , Células Epiteliais/metabolismo , Infecções por Escherichia coli/veterinária , Regulação da Expressão Gênica , Glândulas Mamárias Animais/metabolismo , Mastite Bovina/genética , Adenosina/química , Animais , Bovinos , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/imunologia , Feminino , Perfilação da Expressão Gênica , Glândulas Mamárias Animais/imunologia , Glândulas Mamárias Animais/microbiologia , Mastite Bovina/imunologia , Mastite Bovina/microbiologia
13.
J Dairy Res ; 88(3): 334-336, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34233770

RESUMO

This Research Communication describes the relation between somatic cells and microbial content in milk from Jersey cattle. Milk samples were classified in groups: healthy, dirty and mastitic (from Staphylococcus spp., Escherichia coli, Coliforms). The somatic cells in each of those groups were analysed by two methods - flow cytometric and automatic fluorescent cell counting. Those methods were compared. Total somatic cell count (SCC), neutrophil count, and lymphocytes with cluster of differentiation 4 (CD4+cells) were determined. There was a positive relationship between microbes and somatic cells. It was noticed that the neutrophil count was generally increased together with SCC, whilst the CD4+ cell count was higher in healthy milk samples (about 8%) compared to mastitic ones (about 3%). Lower number of CD4+ cells (from 1 to 4%) was determined in samples positive for Staphylococcus spp. but with lower SCC (from 2.7 to 4.0 × 105 cells/ml). Also, the number of CD4+ cells in Staphylococcus spp.-positive samples increased (to 4.8%) together with higher SCC, something that was not observed in the other mastitic samples. Knowledge of those relations could be useful for veterinary medical tests in the initial phase of inflammation.


Assuntos
Contagem de Linfócito CD4/veterinária , Contagem de Células/veterinária , Mastite Bovina/patologia , Leite/citologia , Neutrófilos , Animais , Carga Bacteriana/veterinária , Bovinos , Enterobacteriaceae/isolamento & purificação , Escherichia coli/isolamento & purificação , Feminino , Citometria de Fluxo/veterinária , Contagem de Leucócitos/veterinária , Mastite Bovina/microbiologia , Leite/microbiologia , Staphylococcus aureus/isolamento & purificação
14.
Sci Rep ; 11(1): 15228, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34315981

RESUMO

Staphylococcus aureus is a major etiological agent of clinical and subclinical bovine mastitis. Owing to the mostly backyard dairy practices, we hypothesized that genetic diversity among mastitis-associated S. aureus from India would be high, and investigated 166 isolates obtained mostly from the Southern State of Karnataka, but also from a few other states. The results revealed (a) 8 to 13 fragments in pulsed-field gel electrophoresis (PFGE), forming 31 distinct patterns, and (b) 34 spa types, of which three (t17680, t18314, and t18320) were newly identified. Multi-locus sequencing typing (MLST) identified 39 sequence types (STs), with ST2454 (34.4%) and ST2459 (24%) being the most commonly represented, which clustered to clonal complexes (CC) CC9 and CC97, respectively; 12 STs were newly identified. Thirty-four (20.5%) of the 166 isolates displayed oxacillin resistance. On the other hand, whereas none were mecC+, 44 (26.5%) isolates were mecA+, with a predominance of SCCmecIVb (26/32 isolates, others being untypeable); 24 isolates (14.46%) were oxacillin-susceptible methicillin-resistant S. aureus (OS-MRSA; mecA+ but OS). Integrated analysis revealed that CC9-ST2454- and CC97-ST2459-SCCmecIVb were the predominant MRSA, although the distribution of CC9 and CC97 was similar between methicillin-resistant and -susceptible isolates. By PCR, 56.25%, 28.75% and 47.5% of the 166 isolates were positive for hlg, tsst and pvl genes, respectively. Our results, for the first time describe the application of a combination of various molecular methods to bovine mastitis-associated S. aureus isolates from India, corroborate the worldwide distribution of CC97 and CC9, and suggest pathogenic potential of the isolates.


Assuntos
Mastite Bovina/microbiologia , Infecções Estafilocócicas/veterinária , Staphylococcus aureus/genética , Animais , Bovinos , Eletroforese em Gel de Campo Pulsado , Feminino , Genes Bacterianos , Índia , Staphylococcus aureus Resistente à Meticilina/genética , Tipagem de Sequências Multilocus , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/isolamento & purificação
15.
BMC Vet Res ; 17(1): 239, 2021 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-34233667

RESUMO

BACKGROUND: The threat of methicillin-resistant Staphylococcus aureus (MRSA) exists globally and has been listed as a priority pathogen by the World Health Organization. One of the sources of MRSA emergence is livestock and its products, often raised in poor husbandry conditions. There are limited studies in Nepal to understand the prevalence of MRSA in dairy animals and its antimicrobial resistance (AMR) profile. A cross-sectional study was conducted in Chitwan, one of the major milk-producing districts of Nepal, from February 2018 to September 2019 to estimate the prevalence of MRSA in milk samples and its AMR profile. The collected milk samples (n = 460) were screened using the California Mastitis Test (CMT) and positive samples were subjected to microbiological analysis to isolate and identify S. aureus. Polymerase Chain Reaction (PCR) was used to identify the presence of the mecA gene and screen for MRSA. RESULTS: In total, 41.5% (191/460) of milk samples were positive in the CMT test. Out of 191 CMT positive milk samples, the biochemical tests showed that the prevalence of S. aureus was 15.2% (29/191). Among the 29 S. aureus isolates, 6.9% (2/29) were identified as MRSA based on the detection of a mecA gene. This indicates that that 1.05% (2/191) of mastitis milk samples had MRSA. The antibiotic sensitivity test showed that 75.9% (22/29) and 48.3% (14/29) S. aureus isolates were found to be sensitive to Cefazolin and Tetracycline respectively (48.3%), whereas 100% of the isolates were resistant to Ampicillin. In total 96.6% (28/29) of S. aureus isolates were multidrug-resistant (MDR). CONCLUSIONS: This study revealed a high prevalence of S. aureus-mediated subclinical mastitis in dairy herds in Chitwan, Nepal, with a small proportion of it being MRSA carrying a mecA gene. This S. aureus, CoNS, and MRSA contaminated milk poses a public health risk due to the presence of a phenotype that is resistant to very commonly used antibiotics. It is suggested that dairy herds be screened for subclinical mastitis and treatments for the animals be based on antibiotic susceptibility tests to reduce the prevalence of AMR. Furthermore, future studies should focus on the Staphylococcus spp. to explore the antibiotic resistance genes in addition to the mecA gene to ensure public health.


Assuntos
Mastite Bovina/microbiologia , Staphylococcus aureus Resistente à Meticilina/genética , Leite/microbiologia , Infecções Estafilocócicas/veterinária , Animais , Antibacterianos/farmacologia , Bovinos , Estudos Transversais , Farmacorresistência Bacteriana , Feminino , Genes Bacterianos , Mastite Bovina/epidemiologia , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Testes de Sensibilidade Microbiana/veterinária , Nepal/epidemiologia , Prevalência , Infecções Estafilocócicas/epidemiologia , Infecções Estafilocócicas/microbiologia
16.
Pol J Vet Sci ; 24(2): 303-305, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34250786

RESUMO

In a dairy cow herd consisted of herd of 200 lactating Holstein-Friesian cows and heifers, clinical signs of mastitis in 40 out of 170 animals were observed. Treatments with antibiotics were proved ineffective. Milk bacterial cultures from 15 affected animals revealed Pseudomonas aeruginosa. An autogenous vaccine was administered subcutaneously, twice in a month period, to all adults. Cases of clinical mastitis declined significantly (p⟨0.0001) during next 3 months.


Assuntos
Surtos de Doenças/veterinária , Mastite Bovina/microbiologia , Infecções por Pseudomonas/terapia , Infecções por Pseudomonas/veterinária , Vacinas contra Pseudomonas/imunologia , Animais , Bovinos , Feminino , Mastite Bovina/prevenção & controle , Mastite Bovina/terapia , Infecções por Pseudomonas/epidemiologia , Infecções por Pseudomonas/prevenção & controle , Vacinas contra Pseudomonas/administração & dosagem , Pseudomonas aeruginosa
17.
Vet Immunol Immunopathol ; 238: 110290, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34217108

RESUMO

Staphylococcus aureus is a leading cause of bovine intramammary infections (IMI). Standard antibiotic treatments are not very effective and currently available vaccines lack tangible efficacy. Developing a vaccine formulation for S. aureus mastitis is challenging and selection of target antigens is critical. The gene products of six S. aureus genes that are highly expressed during IMI were selected as antigens for this study. The vaccine contained six recombinant proteins formulated with Emulsigen®-D, a CpG oligodeoxynucleotide and indolicidin. Nine cows in mid-lactation received the vaccine while ten received saline (placebo). Two immunizations were performed 10 weeks apart. All the antigens induced an immune response. A balanced immune response (IgG2/IgG1 ratio of 1) was observed for antigen SACOL0442 while a predominant Th2 response was observed for the other antigens (IgG2/IgG1 ratio <1). Immunizations induced CD4+ cell proliferation in response to SACOL0442, SACOL0029, SACOL0720 and SACOL1912 while a CD8+ cell proliferation was induced by SACOL0720. Four weeks after the second immunization, three quarters per animal were experimentally infused with ∼60 CFU of S. aureus. Although no difference in S. aureus counts was observed between the two groups after this robust infectious challenge, a sustained reduction in milk somatic cells counts (SCC) was observed in vaccinated cows. A correlation between SCC and S. aureus counts in milk was also observed. Altogether, this indicates that the collective immune responses induced by the antigens certainly contribute to the observed benefits of the whole vaccine. More work is needed to understand how different antigens stimulate a different response using the same adjuvant.


Assuntos
Proteínas de Bactérias/imunologia , Mastite Bovina/prevenção & controle , Vacinas Antiestafilocócicas/imunologia , Staphylococcus/classificação , Adjuvantes Imunológicos , Animais , Anticorpos Antibacterianos/sangue , Bovinos , Feminino , Imunidade Celular , Imunidade Humoral , Imunoglobulina G/sangue , Mastite Bovina/microbiologia , Vacinação
18.
PLoS Comput Biol ; 17(6): e1009108, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34115749

RESUMO

Staphylococcus aureus is a serious human and animal pathogen threat exhibiting extraordinary capacity for acquiring new antibiotic resistance traits in the pathogen population worldwide. The development of fast, affordable and effective diagnostic solutions capable of discriminating between antibiotic-resistant and susceptible S. aureus strains would be of huge benefit for effective disease detection and treatment. Here we develop a diagnostics solution that uses Matrix-Assisted Laser Desorption/Ionisation-Time of Flight Mass Spectrometry (MALDI-TOF) and machine learning, to identify signature profiles of antibiotic resistance to either multidrug or benzylpenicillin in S. aureus isolates. Using ten different supervised learning techniques, we have analysed a set of 82 S. aureus isolates collected from 67 cows diagnosed with bovine mastitis across 24 farms. For the multidrug phenotyping analysis, LDA, linear SVM, RBF SVM, logistic regression, naïve Bayes, MLP neural network and QDA had Cohen's kappa values over 85.00%. For the benzylpenicillin phenotyping analysis, RBF SVM, MLP neural network, naïve Bayes, logistic regression, linear SVM, QDA, LDA, and random forests had Cohen's kappa values over 85.00%. For the benzylpenicillin the diagnostic systems achieved up to (mean result ± standard deviation over 30 runs on the test set): accuracy = 97.54% ± 1.91%, sensitivity = 99.93% ± 0.25%, specificity = 95.04% ± 3.83%, and Cohen's kappa = 95.04% ± 3.83%. Moreover, the diagnostic platform complemented by a protein-protein network and 3D structural protein information framework allowed the identification of five molecular determinants underlying the susceptible and resistant profiles. Four proteins were able to classify multidrug-resistant and susceptible strains with 96.81% ± 0.43% accuracy. Five proteins, including the previous four, were able to classify benzylpenicillin resistant and susceptible strains with 97.54% ± 1.91% accuracy. Our approach may open up new avenues for the development of a fast, affordable and effective day-to-day diagnostic solution, which would offer new opportunities for targeting resistant bacteria.


Assuntos
Diagnóstico por Computador/veterinária , Mastite Bovina/diagnóstico , Penicilina G/farmacologia , Infecções Estafilocócicas/veterinária , Staphylococcus aureus , Animais , Proteínas de Bactérias/química , Bovinos , Biologia Computacional , Diagnóstico por Computador/métodos , Diagnóstico por Computador/estatística & dados numéricos , Farmacorresistência Bacteriana Múltipla , Feminino , Humanos , Mastite Bovina/tratamento farmacológico , Mastite Bovina/microbiologia , Staphylococcus aureus Resistente à Meticilina/química , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Testes de Sensibilidade Microbiana , Modelos Moleculares , Mapas de Interação de Proteínas , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Infecções Estafilocócicas/diagnóstico , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/química , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/isolamento & purificação , Aprendizado de Máquina Supervisionado , Reino Unido
19.
J Dairy Res ; 88(2): 170-175, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33958019

RESUMO

To limit the use of antimicrobials in dairy cattle, farmers are increasingly encouraged to adopt targeted treatment decisions based on knowledge of the pathogens causing clinical mastitis (CM), whereby treatment of non-severe CM is generally recommended for gram-positive mastitis but not for gram-negative or culture-negative mastitis. The objectives of this study were to conduct a laboratory-based evaluation of the performance of a simplified slide test as a tool to differentiate gram-positive CM from other cases of CM, and to compare its performance against a commercially available on-farm test that is commonly used in our area (VétoRapid). Test outcomes after 24-48 h incubation were compared to results from bacteriological culture and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS). Milk samples (n = 156) were obtained from cases of severe and non-severe CM on seven farms and collected by farm personnel. After removal of small numbers of contaminated samples and organisms with unknown species identity, the simplified slide test showed high sensitivity and accuracy (>80%), similar to the comparator test. For most outcomes of interest (culture positive, Escherichia coli, or gram-positive growth), the specificity of the slide test was higher than the specificity of the comparator test. When considering non-severe cases of CM only, and interpreting detection of gram-positive organisms as indicative of the need for antimicrobial treatment, the simplified test had higher specificity (77.4% v. 60.4%) and higher positive predictive value (79.7% v. 70.0%) than the comparator test and similar sensitivity (83.9% v. 87.5%). The proportion of sampled CM cases, contaminated samples and gram-positive mastitis cases - which affects the positive and negative predictive value, the economic value of diagnostic testing and its potential to reduce antimicrobial use - differed between farms. The simplicity and accuracy of the slide test could make it an attractive tool for farmers to target antimicrobial treatment of non-severe clinical mastitis.


Assuntos
Laboratórios , Mastite Bovina/microbiologia , Mastite Bovina/terapia , Testes Imediatos , Animais , Anti-Infecciosos/uso terapêutico , Técnicas Bacteriológicas/métodos , Bovinos , Fazendas , Feminino , Bactérias Gram-Negativas/isolamento & purificação , Bactérias Gram-Positivas/isolamento & purificação , Leite/microbiologia , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/veterinária
20.
J Dairy Res ; 88(2): 179-184, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33993898

RESUMO

Biofilm formation is a central feature to guarantee staphylococcal persistence in hosts and is associated with several diseases that are difficult to treat. In this research paper, biofilm formation and antimicrobial susceptibility were investigated in staphylococcal strains belonging to several species. These strains were isolated from the milk of cows with subclinical mastitis and most of them were coagulase-negative, with the prevalence of Staphylococcus chromogenes. High genetic diversity was observed among the strains by pulsed field gel electrophoresis. Antimicrobial resistance was assessed by disk diffusion and more than 50% of the strains were resistant to ampicillin and penicillin G, with multi-resistance profiles (13.6%) also being observed. Most strains (65.9%) formed biofilms when cultivated in BHI supplemented with 1% glucose. Most strains (72.7%) carried the intercellular adhesion gene (icaA), while less than half (36.3%) carried the biofilm-associated protein gene (bap). Concentrations of up to 10xMIC of erythromycin and tetracycline were not sufficient to suppress cell viability in preformed biofilms. Our results revealed that a genetically diverse group of biofilm-forming Staphylococcus species can be involved in subclinical mastitis. Since high antimicrobial concentrations cannot eradicate biofilm cells in vitro, their use in dairy animals may be ineffective in controlling infections, while supporting selection of resistant microorganisms. These data reinforce the need for alternative therapies aiming at disrupting biofilms for effective disease control.


Assuntos
Biofilmes/crescimento & desenvolvimento , Farmacorresistência Bacteriana/fisiologia , Mastite Bovina/microbiologia , Infecções Estafilocócicas/veterinária , Staphylococcus/efeitos dos fármacos , Staphylococcus/fisiologia , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Bovinos , Coagulase/análise , Farmacorresistência Bacteriana/genética , Feminino , Variação Genética , Mastite Bovina/tratamento farmacológico , Testes de Sensibilidade Microbiana/veterinária , Infecções Estafilocócicas/microbiologia , Staphylococcus/genética
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