This study aimed to evaluate the diagnostic performance (specificity, Sp; sensitivity, Se; accuracy; positive predictive value; negative predictive value; and Cohen's kappa coefficient, κ, of agreement) of chromogenic culture media for rapid identification of microorganisms isolated from cows with clinical (CM) and subclinical mastitis (SCM). For this, 2 experiments were carried out: evaluation of (1) biplate, and (2) triplate of chromogenic culture media for rapid identification of mastitis-causing microorganisms. For the evaluation of diagnostic performance, identification of microorganisms by MALDI-TOF mass spectrometry was considered the standard methodology. In experiment 1, 476 milk samples collected from cows with CM and 660 from cows with SCM were evaluated by inoculation in 2 selective chromogenic culture media (CHROMagar) for gram-positive bacteria and another for gram-negative bacteria. In experiment 2, 476 milk samples from cows with CM and 500 from cows with SCM were evaluated by inoculation in triplate chromogenic culture media (Smartcolor2, Onfarm), selective for Streptococcus and Strep-like organisms, Staphylococcus, and gram-negative bacteria. In experiment 1 for the CM samples, the use of biplates with gram-positive and gram-negative culture media showed Se that ranged from 0.56 (0.32-0.81; Staphylococcus aureus) to 0.90 (0.80-0.99 Streptococcus uberis), Sp varied from 0.94 (0.92-0.96; Strep. uberis) to 1.00 (Prototheca spp. or yeast), and κ ranged from 0.47 (0.26-0.67; Staph. aureus) to 0.84 (0.78-0.9; Escherichia coli). The Se of biplates for SCM samples ranged from 0.50 (0.15-0.85; E. coli) to 0.94 (0.87-1.00; Staph. aureus), Sp varied from 0.95 (0.93-0.97; Strep. uberis) to 0.99 (0.98-1.00; Staph. aureus and Strep. Agalactiae or dysgalactiae), and κ ranged from 0.18 (0.00-0.40; Escherichia coli) to 0.88 (0.80-0.95; Staph. aureus). In experiment 2, the Se of the triplate chromogenic media in CM samples ranged from 0.09 (0.00-0.26; Serratia spp.) to 0.94 (0.85-1.00; Klebsiella spp. and Enterobacter spp.), Sp varied from 0.94 (0.92-0.96; Strep. agalactiae and Strep. dysgalactiae) to 1.00 (Serratia spp.) and κ ranged from 0.07 (0.00-0.24; Serratia spp.) to 0.85 (0.75-0.94; Klebsiella spp. and Enterobacter spp.). For SCM samples, the use of the triplate with the chromogenic culture media showed Se that varied from 0.25 (0.10-0.40; Lactococcus spp.) to 1.00 (Strep. Agalactiae or dysgalactiae), Sp ranged from 0.92 (0.90-0.94; Strep. Agalactiae and Strep. dysgalactiae) to 0.99 (0.98-1.00; Klebsiella spp. and Enterobacter spp.), and κ varied from 0.28 (0.00-0.72; E. coli) to 0.72 (0.60-0.82; Staph. aureus). Our results suggest that the diagnostic accuracy of the biplate and triplate of chromogenic culture media varies according to pathogen, and the results of chromogenic culture media may be useful for rapid decision-making on mastitis treatment protocols of the main mastitis-causing microorganisms, but their use for implementation of mastitis control measures will depend on each farm specific needs.

We evaluated the use of MALDI-TOF MS for the identification of 3 major, dairy-associated Prototheca species, namely, Prototheca bovis (formerly P. zopfii genotype 2), P. blaschkeae, and P. ciferrii (formerly P. zopfii genotype 1). The MALDI-TOF MS spectra established for those species were introduced into the reference spectra library of the Bruker Biotyper MALDI-TOF MS analysis software. Next, 31 Prototheca isolates from Holstein cows with mastitis, from herds located in the midwestern area of São Paulo State, Brazil, were subjected to MALDI-TOF MS profiling. MALDI-TOF MS allowed identification of 22 of 27 P. bovis and 3 of 4 P. blaschkeae isolates with scores >2.0, with 5 of 27 P. bovis and 1 of 4 P. blaschkeae isolates identified only to the genus level. With our extended algae database, MALDI-TOF MS can contribute to quick and effective speciation of Prototheca from mastitis cases.

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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.

We aimed to evaluate the impact of selective dry cow therapy (SDCT) (protocol 1: antimicrobial combined with internal teat sealant (ITS); vs. protocol 2: ITS alone) on bacterial diversity and the abundance of quarter milk. Eighty high production cows (parity ≤ 3 and an average milk yield of 36.5 kg/cow/day) from the largest Brazilian dairy herd available were randomly selected; milk quarter samples were collected for microbiological culture (MC) on the day of drying-off (n = 313) and on day 7 post-calving (n = 313). Based on the results of the MC before and after calving, 240 quarters out of 313 were considered healthy, 38 were cured, 29 showed new infections and 6 had persistent infections. Mammary quarters were randomly selected based on intramammary information status and SDCT protocols for bacterial diversity analyses. The bacterial diversity was similar when comparing both healthy and cured quarters submitted to both drying-off protocols. Despite healthy cows that were treated at dry-off using only teat sealant showing no alteration in the alpha and beta bacterial diversity, they did show a higher abundance of bacterial groups that may be beneficial to or commensals of the mammary gland, which implies that antibiotic therapy should be reserved for mammary quarters with a history of mastitis.