Identification of bacterial species in milk by MALDI-TOF and assessment of some oxidant-antioxidant parameters in blood and milk from cows with different health status of the udder.

This study aimed to identify bacterial pathogens in milk samples from dairy cows with subclinical and clinical mastitis as well as to assess the concentrations of oxidant-antioxidant parameters [malondialdehyde (MDA), reduced glutathione (GSH), and total GSH levels] in both blood and milk samples. From a total of 200 dairy cows in 8 farms, 800 quarter milk samples obtained from each udder were tested in the laboratory for the presence of udder pathogens. Cultivated bacteria causing intramammary infection from milk samples were identified by Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF). In addition, from tested animals 60 cows were selected including 20 healthy cows that were CMT negative, 20 cows with subclinical mastitis (SM), and 20 cows with clinical mastitis (CM) for detection of MDA, GSH, and total GSH levels in blood and milk samples. Three hundred and eighty (47.5%; 380/800), 300 (37.5%; 300/800), and 120 (15%; 120/800) of milk samples, respectively were CMT positive or SM and CM, and those positives were cows from different farms. We observed that 87.4% (332/380), 25.3% (76/300), and 34.2% (41/120) of cows with CMT positive, CMT negative, and CM had bacterial growth. The most predominantly identified bacteria were Staphylococcus chromogenes (18.7%) obtained mainly from SM and Staphylococcus aureus (16.7%) as the most frequent cause of CM. According to our results, dairy cows with CM had the highest MDA levels, the lowest GSH, and total GSH levels in both blood and milk samples however, high MDA levels and low GSH levels in milk samples with SM were observed. Based on our results, lipid oxidant MDA and antioxidant GSH could be excellent biomarkers of cow's milk for developing inflammation of the mammary gland. In addition, there was no link between nutrition and MDA and GSH levels.

Assessment of the main pathogens associated with clinical and subclinical endometritis in cows by culture and MALDI-TOF mass spectrometry identification.

Clinical endometritis (CE) and subclinical endometritis (SCE) are diseases that affect dairy cows during the puerperium, causing negative effects on the animals' milk production and fertility. The objective of this study was to assess the main bacteria related to cases of CE and SCE from uterine samples of dairy cows in Brazilian herds. Selective and differential media were used for isolation of aerobic and anaerobic bacteria and further MALDI-TOF mass spectrometry (MS) identification. A total of 279 lactating dairy cows with 28 to 33 d in milk from 6 commercial farms were evaluated. Initially, cows were classified in 3 groups: cytologic healthy cows (n = 161), cows with CE (n = 83), and cows with SCE (n = 35). Healthy animals presented 97 species, followed by the CE group with 53 identified species, and SCE cows presented only 21 bacterial species. We found a significantly higher isolation rate of Trueperella pyogenes in CE (26.5%) cows compared with healthy and SCE cows. Some anaerobic species were exclusively isolated from the CE group, even though they presented lower frequency. Interestingly, 18.1% of samples from CE cows and 40% of SCE cows were negative to bacterial isolation. Despite the use of culture-dependent methods instead of molecular methods, the present study enabled the identification of a complex community of 127 different species from 48 genera, composed of aerobic and anaerobic bacterial species among the 3 different animal groups. The method of sample collection, culture, and identification by MALDI-TOF MS were essential for the success of the analyses.

Assessment of the potential use of MALDI-TOF MS for the identification of bacteria associated with chilled vacuum-packaged lamb meat.

The focus of this study was to compare the effectiveness of MALDI-TOF MS and partial 16S rRNA gene sequencing for the identification of bacteria isolated from VP lamb meat stored chilled at 5 °C for 21 days, at the same time gaining insights into bacterial changes over time. The identity of bacterial isolates on non-selective and selective agars was determined by both methods and results compared. Results showed that total bacterial numbers increased over the 21 days (as expected) with Staphylococcus and Pseudomonas (day 0) being replaced by Carnobacterium, Brochothrix and members of the Enterobacteriaceae family by day 21. A high level of agreement (86-100%) for bacterial isolates' identity at genus level was observed between MALDI-TOF MS and partial 16S rRNA gene-based sequencing for isolates where identification was possible. With its cheaper cost and faster turnaround time, once optimized, MALDI-TOF MS could become a useful alternative to 16S rRNA gene-sequencing for the rapid identification of red meat bacterial isolates.

Isolation of Atlantic halibut pituitary hormones by continuous-elution electrophoresis followed by fingerprint identification, and assessment of growth hormone content during larval development.

Continuous-elution electrophoresis (CEE) has been applied to separate putative hormones from adult Atlantic halibut pituitaries. Soluble proteins were separated by size and charge on Model 491 Prep Cell (Bio-Rad), where the homogenate runs through a cylindrical gel, and protein fractions are collected as they elute from the matrix. Protein fractions were assessed by SDS-PAGE and found to contain purified proteins of molecular size from 10 to 33 kDa. Fractions containing proteins with molecular weights of approximately 21, 24, 28 and 32 kDa, were identified as putative growth hormone (GH), prolactin, somatolactin and gonadotropins, respectively. These were analyzed further by mass spectrometry and identified with peptide mass protein fingerprinting. The CEE technique was used successfully for purification of halibut GH with a 5% yield, and appears generally well suited to purify species-specific proteins often needed for research in comparative endocrinology, including immunoassay work. Thus, the GH obtained was subsequently used as standards and iodination label in a homologous radioimmunoassay, applied to analyze GH content through larval development in normally and abnormally metamorphosing larvae. As GH is mainly found in the pituitary, GH contents were analyzed in tissue extracts from the heads only. The pituitary GH content increases proportionally to increased larval weight from first feeding to metamorphic climax. No difference in relative GH content was found between normal and abnormal larvae and it still remains to be established if GH has a direct role in metamorphosis.

Quantification of circulating peptides and assessment of peptide uptake across the gastrointestinal tract of sheep.

Gastrointestinal absorption of peptides was examined in sheep fed a forage-based diet. Peptide concentrations were determined in arterial, portal, and mesenteric blood and plasma by quantification of amino acid concentrations before and after acid hydrolysis of samples that had been first deproteinized then subjected to Sephadex G-15 gel-filtration to remove residual protein. In contrast to other studies of ruminants, peptide concentrations for individual amino acids were lower than for the corresponding free amino acids with peptide (expressed as a proportion of total nonprotein amino acid) representing not more than .25 to .3 of total amino acid. Peptide concentrations in arterial, mesenteric, and portal blood and plasma were similar, indicating that on this diet there was no net uptake of peptides from the small intestine (mesenteric-drained viscera, MDV) or the whole tract (portal-drained viscera, PDV). Increasing the intake of alfalfa pellets from 800 to 1,200 g/d, while increasing the absorption and net flux across the MDV and PDV of free amino acids, had no effect on peptide absorption. Preparation of blood and plasma samples for peptide analysis with methods used in studies in which substantial peptide absorption has been reported indicated no net MDV or PDV flux of peptide. Such conflicting data on the extent of gastrointestinal peptide flux are discussed in the context of methodological differences and the importance of diet and physiological state of the animal.