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ABSTRACT: Prototheca spp. is a frequent cause of bovine mastitis and is highly resistant to commonly used disinfectants. This study aimed to: (1) evaluate the antimicrobial activity of polyhexamethylene biguanide (PHMB) against mastitis-causing Prototheca spp., and (2) evaluate the biofilm production ability of Prototheca spp. A total of 85 Prototheca bovis and 2 Prototheca blaschkeae isolates from bovine mastitis cases were submitted to biofilm production assays and antimicrobial susceptibility tests against PHMB and disinfectants commonly used in dairy herds (chlorhexidine digluconate, povidone-iodine, sodium dichloroisocyanurate, and sodium hypochlorite). The minimal inhibitory concentration (MIC) and minimal algicidal concentration (MAC) were determined by microdilution assays. We observed that PHMB (MIC90: ≥2 µg/mL and MAC90: ≥4 µg/mL) and chlorhexidine gluconate (MIC90 and MAC90: ≥2 µg/mL) presented the highest antimicrobial activity against P. bovis isolates, followed by sodium dichloroisocyanurate (MIC90 and MAC90: ≥1,400 µg/mL), sodium hypochlorite (MIC90 and MAC90: ≥2,800 µg/mL), and povidone-iodine (MIC90 and MAC90: ≥3,200 µg/mL). Concerning P. blaschkeae isolates, PHMB (MIC and MAC ≥1 µg/mL) and chlorhexidine gluconate (MIC and MAC ≥1 µg/mL) were the disinfectants that presented the lowest concentration values required to inhibit the isolates. Regarding biofilms formation, 63.5% (n = 54/85) of the P. bovis isolates were classified as strong, 28.3% (n = 24/85) moderate, and 8.2% (n = 7/85) weak biofilm producers. In contrast, the P. blaschkeae isolates were classified as weak and moderate biofilm producers. These findings suggest that PHMB has the potential to be used for teat and milking-equipment disinfection for the prevention of mastitis-causing Prototheca spp. in dairy herds.

The in vitro algaecide activity of quaternary ammonium (QA) against Prototheca isolated from bovine clinical mastitis was investigated, in which the clinical severity was scored, milk samples were subjected to microbiological culture, and algal species were identified by molecular typing. A total of 4275 milk clinical samples of different cows from ten large dairy farms were used. Forty-four (1%) samples of cows from three dairy farms yielded growth of Prototheca, of which 88.6% (39/44) were identified as Prototheca bovis and 11.3% (5/44) as Prototheca sp. by MALDI-TOF MS, whereas 100% of the isolates were identified as P. bovis using PCR sequencing of the cytb gene. Among cows for which clinical severity scoring was available, 78.8% (26/33) and 21.2% (7/33) had mild and moderate infections, respectively, whereas no animal showed severe clinical signs. The algaecide activity of QA in Prototheca was observed in low concentrations among all isolates, in 20.4% (9/44) at 35 ppm, 36.4% (16/44) at 17 ppm, and 43.2% (19/44) at an 8 ppm, in addition to activity on three reference Prototheca strains. Overall, the study highlights the predominance of P. bovis as the causative agent of algal mastitis in bovines. Prototheca induced abnormalities preponderantly in the milk and mammary gland tissue of cows, and to our knowledge, our study is the first to apply clinical severity scoring in protothecal mastitis. In addition, the study underlines the activity of QA in low concentrations against Prototheca, indicating its potential use as an antiseptic/disinfectant in milking facilities and dairy environments.

The primary objective of this study was to investigate the association between the bulk tank somatic cell count (SCC) and dairy farmers’ knowledge about milk quality, mastitis control, and their socioeconomic characteristics. Additionally, we estimated the association between the bulk tank SCC and bulk tank milk hygienic quality in dairy herds. Bulk tank milk samples from 120 dairy herds enrolled in the milk quality payment program were collected for the determination of the SCC, the total bacterial count (TBC), the preliminary incubation count (PIC), the laboratory pasteurization count (PC), and the coliform count (CC). Based on the bulk tank SCC results, 63 herds were selected and categorized into three groups: (a) low SCC: ≤250,000 cells/mL (n = 16); (b) medium SCC: >250,000 ≤ 400,000 cells/mL (n = 24); and (c) high SCC: >400,000 cells/mL (n = 23). Socioeconomic profiles, attitudes, and knowledge of somatic cell count and milk quality were assessed using previously tested questionnaires, which were used to interview the herd managers of the 63 selected dairy herds. Among the findings, 87.0% of the dairy herds had an SCC < 400 × 103 cells/mL, and presented milk production as the main economic activity of the farm, whereas only 47.0% of dairy herds with an SCC > 400 × 103 cells/mL had milk production as the main economic activity of the farm (p < 0.031). In a total of 95% of the selected herds (n = 60), milking machines were used, with pipeline milking machines being more predominant, accounting for 70.8% in herds with a medium somatic cell count (SCC) and 78.3% in herds with a high SCC, as opposed to herds with a low SCC at 50% (p < 0.031). The frequency of dairy producers’ awareness of the maximum legal requirements for the bulk tank SCC and TBC was higher in herds with a higher SCC than in herds with a medium SCC. In conclusion, the results of this study indicate a significant association between the bulk tank somatic cell count (SCC) and dairy farmers’ knowledge about milk quality, mastitis control, and socioeconomic characteristics. These findings emphasize the importance of knowledge and the socioeconomic profiles of dairy farms in maintaining and enhancing milk quality in dairy herds.

Enterococcus spp., including E. faecalis and E. faecium, pose risks to dairy farms as opportunistic pathogens. The study evaluates antimicrobial resistance (AMR) and virulence characteristics of Enterococcus spp. isolated from bovine milk. Bile esculin agar was used to assess 1471 milk samples, followed by colony identification, gram staining, catalase tests, and 45 °C incubation. PCR analysis targeted E. faecalis and E. faecium in characteristic Enterococcus spp. colonies, with MALDI-TOF used for negative samples. Multiple tests, including disk diffusion, chromogenic VRE agar for vancomycin resistance, Vancomycin Etest® for MIC determination, and PCR for virulence factors (cylA, esp, efaA, ace, asa1, gelE, and hyl genes), were performed. Out of 100 identified strains, E. durans (30.66%), E. faecium (26.28%), and E. faecalis (18.25%) were predominant. AMR in Enterococcus spp. varied, with the highest rates against rifampicin (27%), tetracycline (20%), and erythromycin (18%). Linezolid (5%), vancomycin, ciprofloxacin, and teicoplanin (3% each) had lower prevalence. E. faecium and E. faecalis showed high AMR to rifampicin, erythromycin, and tetracycline. Thirty-two strains (18.98%) grew on VRE Chromoselect agar, while 4 (2 E. faecalis and 2 E. faecium) showed vancomycin resistance by MIC values. E. faecalis carried gelE (45.5%) and asa1 (36%), and E. gallinarum had 9.1% with the asa1 gene. Detecting resistant Enterococcus in bovine milk supports control strategies for enterococci on dairy farms, highlighting AMR concerns in the food chain.

The present study aimed to evaluate the diagnostic performance specificity (Sp), sensitivity (Se), positive predictive value (PPV), negative predictive value (NPV), and accuracy (Acc) of two chromogenic culture media for rapid identification of Gram-positive bacteria causing subclinical mastitis (SCM) in dairy cows. For this, the performance of chromogenic culture media Gram-positive (GP) and Staphylococcus (Staph) (CHROMagar ™, Paris—France) was evaluated in milk samples collected from: (1) lactating cows with SCM (n = 504), and (2) cows in the post-partum period (PP) (7 ± 3 days post-partum; n = 536). Rapid identification of Gram-positive bacteria in chromogenic media was performed by visual inspection of colony colors after 24 h of incubation at 37°C. Bacterial identification by MALDI-TOF mass spectrometry was considered the reference methodology for calculating: Acc, Se, Sp, PPV, NPV, and Cohen's Kappa coefficient of agreement (k). The chromogenic media GP showed high Acc for Strep. agalactiae/dysgalactiae identification in both samples of SCM (Se: 89.1%; Sp: 96.3% and Acc: 95.6%) and of cows in PP (Se: 100%; Sp: 99.0% and Acc: 99.1%). Similar results were observed for Strep. uberis/Enterococcus spp. identification (Se: 90.5%; Sp: 92.5% and Acc: 92.3%) in SCM samples and Se: 100%; Sp: 99.6% and Acc: 99.6% in samples of PP cows using the GP media. However, the GP chromogenic media showed low Se (25.0% in SCM samples and 50.0% in samples of cows in PP) for Staph. aureus identification, despite Sp and Acc were high (Sp: 98.3% and Acc: 95.4% in SCM and Sp samples: 99.4% and Acc: 98.9% in PP cow samples). Staph culture media showed high Acc for Staph. aureus identification (Se: 80.0%; Sp: 98.8% and Acc: 98.0% in SCM samples and Se: 66.7%; Sp: 100% and Acc: 99.6% in PP cow samples), although the low prevalence of Staph. epidermidis and Staph. saprophyticus limit inferences about the performance of identifying these pathogens in Staph media. In conclusion, despite the limitation of the GP media for identification of Staph. aureus, GP, and Staph chromogenic media obtained satisfactory diagnostic performance results for the rapid identification of the main Gram-positive pathogens associated with SCM.