No abstract

The present study aimed to compare two MALDI-TOF identification methods [(a) direct sample identification after pre-incubation; or (b) use of bacteria isolated on pre-culture)] to standard, traditional bench microbiology. A total of 120 quarter milk samples from 40 Holstein lactating cows were screened based on culture-positive results obtained by microbiological culture (reference method) with the following numbers of quarters positive per cow: 4 cows with 1, 8 cows with 2, 12 cows with 3 and 16 cows with 4 infected quarters per cow. For direct identification method, quarter milk samples (n = 120) were skimmed by centrifugation (10,000 × g/10 min) and pre-incubated at 37 °C for 12 h. After pre-incubation, quarter milk samples were submitted to total bacterial count by flow cytometry and for a preparation protocol for bacterial ribosomal protein extraction followed by MALDI-TOF MS analysis. The direct MALDI-TOF MS identification method compared to microbiological culture correctly identified isolates of coagulase-negative Staphylococci (27.2%), Streptococcus agalactiae (21.8%), Staphylococcus aureus (14.2%), and Streptococcus uberis (5.2%). The pre-incubation protocol of milk samples, associated to the direct identification method by MALDI-TOF MS, did not increase the identification at species level (score >2.0) of pathogens causing subclinical mastitis in comparison to the method without previous incubation.

The emergence of antimicrobial resistance to commonly used antibiotics has necessitated the development of new antimicrobial products. Crude extracts produced by actinomycetes contain antimicrobial metabolites that can inhibit bacterial growth. The objective of our study was to evaluate the antimicrobial activity of crude extracts (Caat1-54 and CaatP5-8) produced by actinomycetes against isolates of Staphylococcus aureus, Staphylococcus chromogenes, Streptococcus dysgalactiae, and Streptococcus uberis, which were obtained from the milk of cows affected by mastitis in 23 dairy herds. Twenty isolates of each bacterial species were used to define minimum inhibitory concentrations (MIC) of both crude extracts and ceftiofur (positive control). The MIC and MIC were defined at the concentration required to inhibit the growth of 50 and 90% of bacterial isolates tested, respectively. The MIC results were evaluated by survival analysis. Staphylococcus aureus isolates presented MIC of Caat 1-54 ≥6.25 µg/mL, ceftiofur ≥12.5 µg/mL, and Caat P5-8 ≥100 µg/mL. Streptococcus uberis presented MIC of ceftiofur ≥0.39 µg/mL, Caat 1-54 ≥50 µg/mL, and Caat P5-8 ≥100 µg/mL. Staphylococcus chromogenes isolated from subclinical mastitis presented MIC of Caat 1-54 ≥0.78 µg/mL and ceftiofur and Caat P5-8 of ≥6.25 and ≥100 µg/mL, respectively. Streptococcus dysgalactiae isolated from clinical mastitis presented similar MIC values between antimicrobials tested (ceftiofur, Caat 1-54, and Caat P-58), but these values (≥100 µg/mL) were higher than the values obtained from other pathogens evaluated in the present study. Our results indicate that Caat 1-54 and Caat P5-8 crude extracts present in vitro antimicrobial activity against isolates of Staph. aureus, Staph. chromogenes, Strep. dysgalactiae, and Strep. uberis isolated from clinical and subclinical mastitis.

A contagem de células somáticas (CCS) é um teste de triagem para avaliação de infecção intramamária, porém, as alterações na composição do colostro observadas durante a colostrogênese podem promover o aumento fisiológico da CCS e inviabilizar o uso desta prova para diagnóstico da mastite. Assim, o objetivo desta pesquisa foi avaliar a CCS do colostro como parâmetro indicativo de infecção mamária em vacas Holandesas periparturientes. Para tanto, foram colhidas 80 amostras de colostro de primeira ordenha, provenientes de 20 vacas, para a CCS manual e exame bacteriológico (EB). Observou-se crescimento bacteriano em 36,62% dos cultivos, com predomínio de Staphylococcus coagulase-negativa (SCN) (76,92%). O valor mediano da CCS de vacas infectadas (1,8 x106 células/mL) foi maior do que o obtido para as vacas sadias (0,9 x106 células/mL) (P=0,0451). A sensibilidade (100 a 15%), especificidade (100 a 2,2%) e falso-positivo (100 a 2,2%) diminuíram gradativamente quando os limiares de 0,2 a 10,0 x106 células/mL foram adotados. Em contrapartida, a proporção de falso-negativo (0 a 84,6%) apresentou perfil inverso. O limiar de maior concordância entre a CCS e EB foi de 10,0 x106 células/mL, porém os índices de sensibilidade (15,4%), especificidade (2,2%) e falso-positivo (2,2%) foram muito baixos. Com base nos resultados encontrados, conclui-se que a CCS apresentou elevação diante dos processos infecciosos da glândula mamária (GM), causados especialmente por bactérias do grupo SCN. No entanto, as alterações fisiológicas decorrentes da colostrogênese resultaram em baixa concordância entre a CCS e o exame bacteriológico do colostro.

RESUMO: Este trabalho avaliou a influência da infecção bacteriana da glândula mamária (GM) sobre a transferência de imunidade passiva (TIP) em bezerros recém-nascidos. Vacas holandesas (n=13) foram observadas no momento da parição e ordenhadas para a obtenção de forma asséptica das amostras de colostro (n=52) para os testes microbiológicos. Os recém-nascidos receberam 6 litros de colostro de uma ordenha nas primeiras 12 horas de vida, proveniente de suas respectivas mães. Amostras de sangue foram colhidas antes (D0) e após (D2) o manejo do colostro. A TIP foi avaliada por meio de testes bioquímicos, eletroforese e leucograma. Os bezerros foram distribuídos conforme a ausência (IB-) ou presença (IB+) de infecção mamária em pelo menos uma GM de suas respectivas mães. Todas as amostras de colostro (n=52) foram negativas ao cultivo fúngico. Das 13 fêmeas, 8 (61%) apresentaram crescimento bacteriano em ≥1 quartos mamários. Considerando-se os quartos mamários, foi obtido isolamento bacteriano em 21,15% (11/52), observando-se predomínio de espécies bacterianas do grupo Staphylococcus coagulase negativa. Não foram encontradas diferenças entre os parâmetros de acordo com os grupos experimentais. Em relação aos momentos, foi possível verificar aumento nos valores de proteína total, globulinas, atividade sérica da gama glutamiltransferase e frações eletroforéticas beta e gamaglobulina após a ingestão do colostro materno. A mastite subclínica não influencia a transferência de imunidade passiva em bezerros recém-nascidos da raça holandesa, avaliados por teste bioquímicos, eletroforese e leucograma.

Polyhexamethylene biguanide (PHMB) nanoparticles (NP) developed for this study presented antimicrobial activity against mastitis-causing Staphylococcus aureus at lower concentrations than PHMB alone, chlorhexidine digluconate, povidone-iodine, and sodium dichloroisocyanurate. Thus, PHMB NP present potential for the development of new dipping solutions. Highlights• The PHMB NP were developed by layer-by-layer assembly.• Mastitis-causing Staph. aureus were inhibited by PHMB NP at low concentrations relative to commonly used teat-dip ingredients. • The NP formulation potentiated the antimicrobial activity of PHMB.

The objective of this study was to evaluate the efficacy of the antibiotic therapy associated with vaccination on the microbiological cure rate of subclinical mastitis caused by Staphylococcus aureus in lactating dairy cows. A total of five herds, from which 72 cows (120 mammary quarters - MQ) were diagnosed with S. aureus subclinical mastitis, were included in this study. Cows were randomly allocated to one of three treatment groups: a) Control (no treatment); b) ATB (antibiotic therapy); and c) ATB+VAC (antibiotic therapy plus vaccination against S. aureus). Intramammary treatment consisted of twice-daily infusion of ampicillin 75mg + cloxacillin 200mg, for 5 days. Parenteral treatment was done by injection of a single dose (7.5mg/kg) of enrofloxacin, on the first day of the treatment protocol. Vaccinated cows received three doses of a commercial vaccine 14 days before treatment (d-14), on the first day of treatment protocol (d1), and 14 days after the treatment protocol (d+14). Non-treated cows had a lower cure rate (0.06) than cows treated with ATB (0.84) and ATB+VAC (0.85). No difference in cure rate was observed between cows treated with ATB and ATB+VAC. On the other hand, vaccinated cows had lower somatic cell count (SCC) after 28 days of the treatment protocols (4.76 log10) than non-treated cows (5.37 log10). In conclusion, treatment with intramammary ampicillin and cloxacillin, associated with intramuscular enrofloxacin presented a high cure rate for SCM caused by S. aureus during lactation. The use of vaccination against S. aureus in association with antibiotic therapy did not increase the cure rate of MQ during lactation, but it was effective in reducing the SCC when compared to non-treated MQ. Although to ensure that the decrease of the SCC in ATB+VAC group was associated with the vaccination, the study should have included an additional group of only vaccinated cows, without antimicrobial therapy, with was not done in the present study, and therefore is one of the limitations of the experimental protocol used.