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This study aimed to evaluate the effects of chronic subclinical mastitis (CSM) on milk production and component yields in dairy cows. A total of six herds located in the Midwest area of São Paulo State, Brazil were selected. Herds were visited once every 2 weeks to measure milk yield and to collect milk samples from lactating Holstein cows. Milk samples were collected at two stages (1 and 2), and each stage comprised three milk samplings. In stage 1, a total of 117 of 647 cows were diagnosed with CSM based on at least two of three repeated somatic cell counts (SCC) > 2000,000 cells/mL and positive bacterial milk culture results (BC). Cows with CSM were selected for the second stage. In stage 2, selected cows had quarter sampling aseptically collected for BC analyses prior to milking, and quarter milk yield was measured. Milk components (total protein, fat, lactose, and total solids) were measured using mid-infrared spectroscopy. Mammary quarters were considered healthy if all three repeated SCC results were ≤ 200,000 cells/mL and no bacterial growth was detected on BC. All quarters with positive bacterial growth were classified as having (non-chronic) subclinical mastitis when only one of three SCC results were > 200,000 cells/mL, and CSM when at least two of three SCC results were > 200,000 cells/mL. The effects of CSM by type of pathogen on milk and components yield were assessed using a linear mixed model. Mammary quarters with CSM caused by major pathogens had milk loss of 1.1 kg/quarter milking in comparison to healthy quarters. Milk losses were 0.8 and 1.3 kg/quarter milking when CSM was caused by Staphylococcus aureus or environmental streptococci, respectively. In addition, healthy quarters produced more milk components than quarters with CSM caused by major pathogens. Minor pathogens causing CSM (non-aureus staphylococci and Corynebacterium spp.) had no effect on milk yield. Quarters with CSM had lower milk and component yields when compared with healthy quarters. Milk losses varied according to the type of pathogen and were higher when associated with major pathogens such as S. aureus and environmental streptococci compared with healthy quarters.

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The objective was to compare standard versus on-plate sample preparation protocols for identification of mastitis bacteria by MALDI-TOF MS. A total of 186 bacterial isolates from cows with subclinical mastitis were identified by MALDI-TOF MS after preparation using two extraction protocols. On-plate protocol was performed by applying the bacterial colony directly from the culture plate onto the plate spot. For the standard protocol, lysis of bacterial colonies using reagents was performed in a cryotube, and the resulting extract was applied onto the plate spot for analysis. The on-plate protocol showed a similar bacteria identification rate (91.4%, n = 170/186) in comparison to the standard (94.6%, n = 176/186). Identification was higher for both protocols when scores used for species-level identification (≥ 2.0) was reduced to genus-level (≥ 1.7); genus-level identification score rate increased from 94.6 to 100% when using the standard protocol, and from 91.4 to 94.6% when using the on-plate protocol. However, when compared standard (as gold standard) versus on-plate protocol, genus-level identification score rate ranged from 87.1 to 89.8%. Therefore, when the on-plate protocol fails to identify any specie, the standard extraction may be more suitable as a reference protocol for use. Strategy for increasing identification with the on-plate protocol may include upgrading the reference database library. Choice of protocol for preparation may be influenced by the bacterial type to be identified. Standard and on-plate extraction protocols of bacterial ribosomal proteins associated with MALDI-TOF MS might be alternatives to conventional microbiology methods for identification of subclinical mastitis pathogens.

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Determination of antimicrobial susceptibility (AMS) of Escherichia coli causing clinical mastitis (CM) according to the phylogenetic groups and its association with descriptors at the cow and herd level may help improve specific strategies for treatment and control of this pathogen in dairy herds. The aims of the present study were to (a) determine the frequency of phylogenetic groups of E. coli isolated from CM in dairy cows, and its association with cow-level descriptors (parity, lactation stage, CM severity, and affected quarter position), housing system, and season; and (b) determine and compare AMS among E. coli phylogenetic groups. A quadruplex PCR method was used to classify E. coli isolates into 1 of the 7 phylogenetic groups. Minimal inhibitory concentrations were determined for 10 antimicrobials, and survival analysis was performed to evaluate the AMS differences among E. coli phylogroups. Most E. coli isolates belonged to phylogroups A (52%) and B1 (38%). None of the cow- and herd-level descriptors were associated with the E. coli phylogenetic groups. Overall, E. coli isolates were mostly susceptible to ceftiofur (96.8%), sulfadimethoxine (75.5%), and cephalothin (74.5%). Based on the survival analysis, differences in AMS between phylogenetic groups of E. coli was observed only for cephalothin, in which strains of phylogroup A were inhibited at lower minimum inhibitory concentration than strains of phylogroup B1. Results of this study indicated low susceptibility of E. coli isolates identified from CM to most antimicrobials. In addition, differences in AMS can occur among E. coli phylogenetic groups, although they may be uncommon as they were limited to only one antimicrobial (i.e., cephalothin).

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The present study aimed to evaluate the effect of dietary cation-anion difference (DCAD) on ruminal fermentation, total apparent digestibility, blood and renal metabolism of lactating dairy cows. Sixteen Holstein cows were distributed in four contemporary 4×4 Latin Square designs, which consisted of four periods of 21 days and four treatments according to DCAD: +290; +192; +98 and -71 milliequivalent (mEq)/kg dry matter (DM). Ruminal pH and concentrations of acetic and butyric acid increased linearly according to the increase of DCAD. Similarly, NDF total apparent digestibility linearly increased by 6.38% when DCAD increased from -71 to 290 mEq/kg DM [Y=65.90 (SE=2.37)+0.0167 (SE=0.0068)×DCAD (mEq/kg DM)]. Blood pH was also increased according to DCAD, which resulted in reduction of serum concentrations of Na, K and ionic calcium (iCa). To maintain the blood acid-base homeostasis, renal metabolism played an important role in controlling serum concentrations of Na and K, since the Na and K urinary excretion increased linearly by 89.69% and 46.06%, respectively, from -71 to 290 mEq/kg DM. Changes in acid-base balance of biological fluids may directly affect the mineral composition of milk, as milk concentrations of Na, K, iCa and chlorides were reduced according to blood pH increased. Thus, it can be concluded that the increase of DCAD raises the pH of ruminal fluid, NDF total apparent digestibility, and blood pH, and decreases the milk concentration of cationic minerals, as well as the efficiency of Na utilization to milk production.

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The aim of this study was to evaluate the effect of subclinical intramammary infection (IMI) caused by coagulase-negative staphylococci (CNS) as a group and by specific CNS species on milk yield and composition and somatic cell count (SCC) of dairy cows. Selection of cows with IMI caused by CNS was performed by microbiological cultures of composite samples collected from 1,242 dairy cows distributed in 21 dairy herds. After selection of cows, milk yield was measured and milk samples were collected at the mammary quarter level (i.e., 1,140 mammary samples collected from 285 cows) for analysis of milk composition and SCC. In total, 108 isolates of CNS were identified at the species level by PCR-RFLP analysis. Forty-one pairs of contralateral mammary quarters, with and without IMI, were used to evaluate the effect of CNS on milk yield and composition. Mammary quarters infected with CNS had higher geometric mean SCC (306,106 cells/mL) than noninfected contralateral mammary quarters (62,807 cells/mL). Intramammary infection caused by CNS had no effect on milk yield or on contents of fat, crude protein, casein, lactose, total solids, and solids-not-fat. Staphylococcus chromogenes was the most prevalent CNS species in this study and the only species that allowed within-cow evaluation. The IMI caused by S. chromogenes increased SCC but had no effect on milk yield and composition at the quarter level. In conclusion, subclinical mastitis caused by CNS increased the SCC but had no effect on milk yield and composition of dairy cows.

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This study aimed to investigate the in silico biofilm production ability of Staphylococcus aureus strains isolated from milking parlor environments on dairy farms from São Paulo, Brazil. The Staph. aureus isolates were obtained from 849 samples collected on dairy farms, as follows: milk from individual cows with subclinical mastitis or history of the disease (n=220); milk from bulk tank (n=120); surfaces of milking machines and utensils (n=389); and milk handlers (n=120). Thirty-one Staph. aureus isolates were obtained and categorized as pulsotypes by pulsed-field gel electrophoresis and submitted to assays for biofilm formation on polystyrene, stainless steel, rubber, and silicone surfaces. Fourteen (45.2%) pulsotypes were considered producers of biofilm on the polystyrene microplate assay, whereas 13 (41.9%) and 12 (38.7%) pulsotypes were biofilm producers on stainless steel and rubber, respectively. None of the pulsotypes evaluated produced biofilms on silicone. Approximately 45% of Staph. aureus pulsotypes isolated from different sources on dairy farms showed the ability to produce biofilms in at least one assay, indicating possible persistence of this pathogen in the milking environment. The potential involvement of Staph. aureus in subclinical mastitis cases and its occurrence in milk for human consumption emphasize the need to improve hygiene practices to prevent biofilm formation on the farms studied.

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Background. Food allergies have been shown to reduce serum triacylglycerol, glucose, cholesterol, and free fatty acid levels in mice. In turn, dyslipidemias, especially dyslipidemias presenting with low levels of HDL cholesterol, are important risk factors for the development of atherosclerosis. However, the consequences of food allergies on dyslipidemia and atherosclerosis have not been fully investigated. Methods. Food allergy was induced using an egg white solution (EWS) in ovalbumin- (OVA-) sensitized C57BL/6 and low-density lipoprotein receptor knockout mice (LDLr(-/-)) for 5 weeks and was confirmed by the high production of anti-OVA IgE and IgG1 antibodies in both mouse strains. Results. The allergic C57BL/6 mice exhibited EWS aversion that was associated with less visceral fat and high levels of anti-Ova IgE antibodies after 5 weeks of EWS intake compared to controls. However, LDLr(-/-) allergic mice showed reduced anti-Ova IgE levels that were similar to the nonsensitized group. The LDLr(-/-) allergic mice also demonstrated a reversal of food aversion and sustained visceral fat after 5 weeks of allergy. Although HDL cholesterol levels were reduced in both sensitized mouse strains, lipid deposition in thoracic and abdominal aorta as well as area and composition of atherosclerotic plaques as unaffected by chronic ingestion of EWS. Conclusion. LDLr(-/-) mice develop an attenuated food allergy, as they showed a reversal of food aversion and lower IgE production after 5 weeks of induced allergy. The development of atherosclerosis, in turn, was not accelerated in the allergic LDLr(-/-) group despite the more atherogenic lipid profile.

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This research aimed to evaluate the occurrence of Staphylococcus aureus isolates in milk and in the milking environment of 10 small-scale farms (<400 L/d) located in the regions of Franca and Ribeirão Preto, state of São Paulo, Brazil. Two-hundred twenty samples of milk were collected from individual cows, along with 120 samples from bulk tank milk, 389 samples from milking equipment and utensils (teat cups, buckets, and sieves), and 120 samples from milkers' hands. Fifty-six Staph. aureus strains were isolated from 849 analyzed samples (6.6%): 12 (5.5%) from milk samples of individual cows, 26 (21.7%) from samples of bulk tank milk, 14 (3.6%) from samples collected from equipment and utensils, and 4 (3.3%) from samples from milkers' hands. Pulsed-field gel electrophoresis typing of the 56 Staph. aureus isolates by SmaI restriction enzyme resulted in 31 profiles (pulsotypes) arranged in 12 major clusters. Results of this study indicate a low incidence, but wide distribution of Staph. aureus strains isolated from raw milk collected from individual cows and surfaces of milkers' hands and milking equipment in the small-scale dairy farms evaluated. However, the high percentage of bulk milk samples found with Staph. aureus is of public health concern because raw, unprocessed milk is regularly consumed by the Brazilian population.

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Elevated blood cholesterol is an important risk factor associated with atherosclerosis and coronary heart disease. Several studies have reported a decrease in serum cholesterol during the consumption of large doses of fermented dairy products or lactobacillus strains. The proposed mechanism for this effect is the removal or assimilation of intestinal cholesterol by the bacteria, reducing cholesterol absorption. Although this effect was demonstrated in vitro, its relevance in vivo is still controversial. Furthermore, few studies have investigated the role of lactobacilli in atherogenesis. The aim of the present study was to determine the effect of Lactobacillus delbrueckii on cholesterol metabolism in germ-free mice and the possible hypocholesterolemic and antiatherogenic action of these bacteria using atherosclerosis-prone apolipoprotein E (apo E) knock-out (KO) mice. For this purpose, Swiss/NIH germ-free mice were monoassociated with L. delbrueckii and fed a hypercholesterolemic diet for four weeks. In addition, apo E KO mice were fed a normal chow diet and treated with L. delbrueckii for 6 weeks. There was a reduction in cholesterol excretion in germ-free mice, which was not associated with changes in blood or liver cholesterol concentration. In apo E KO mice, no effect of L. delbrueckii was detected in blood, liver or fecal cholesterol. The atherosclerotic lesion in the aorta was also similar in mice receiving or not these bacteria. In conclusion, these results suggest that, although L. delbrueckii treatment was able to reduce cholesterol excretion in germ-free mice, no hypocholesterolemic or antiatherogenic effect was observed in apo E KO mice.

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