The present study investigates the effect of adaptive resistance to ciprofloxacin (Cip) and benzalkonium chloride (BC) on biofilm formation potential (BFP), efflux pump activity (EPA) and haemolysin activity of Escherichia coli isolates of dairy origin. All the isolates, irrespective of antimicrobial susceptibility, developed significant adaptive resistance (P < 0·05). All the resistant phenotypes (antibiotic resistant: AR; & biocide resistant: BR) were stronger biofilm former and post-adaptation, an insignificant change was observed in their BFP. Whereas, post-adaptation, non-resistant isolates (antibiotic non-resistant: ANR; biocide non-resistant: BNR) transformed from poor or moderate to strong biofilm formers. Post-adaptive percentage increase in EPA was highly significant in non-resistant categories (P < 0·01) and significant at P < 0·05 in BR category. Interestingly, post-adaptive increase in EPA in BR isolates was more than that in AR yet, the latter exhibited greater adaptive resistance than the former. These findings indicated prevalence of some other specific resistance mechanism/s responsible for adaptive resistance against Cip. Strain specific variations were observed for stability of adaptive resistance and haemolysin activity for all the categories. Our findings especially in reference to post-adaptation upgradation of BFP status of non-resistant isolates seems to be providing an insight into the process of conversion of non-resistant isolate into resistant ones with enhanced BFP. These observations emphasize the serious implications of sub-lethal residual levels of antimicrobials in food environments and suggest a role of food chain in emergence of antimicrobial resistances.

In this work the effects of addition of different amounts of sodium chloride, during cheese making, on shelf life of mozzarella cheese were evaluated. The mozzarella cheese quality decay was assessed during storage at 9 °C by monitoring microbiological, sensory and physico-chemical changes in the product. Results showed that Pseudomonas spp. growth was responsible for cheese unacceptability, whereas the sensory quality did not limit cheese shelf life. In particular, the highest shelf life values were obtained for mozzarella without salt and with the lowest salt concentration (0·23 g NaCl), and amounted to about 5 and 4 d, respectively. On the contrary, high salt concentrations affected product shelf life, probably as a consequence of progressive solubilisation of cheese casein, due to the phenomenon of ‘salting in’.

In order to optimise the pre-ripening of pasta filata cheeses, two traditional Italian cheeses, Caciocavallo and Scamorza, were dried for different times at 15 °C, 50% relative humidity and airflow rate of 1000 m3/h, by using a pre-ripening plant. During ripening, microbiological, chemical-physical and sensorial analyses were applied to the products in order to evaluate the impact of the innovative pre-ripening technique used on the cheese characteristics. The used plant led to commercial time saving and to the standardisation of the process, making it unaffected by the effects of temperature and humidity variations, common in small industries. The final products showed good chemical-physical and sensorial qualities and resulted in a higher microbiological safety, preserving their traditional characteristics.

In this study the fate of enterotoxigenic Staphylococcus aureus and staphylococcal enterotoxins in Feta and Galotyri cheeses were studied. Initially, the enterotoxigenic abilities of four Staph. aureus LHA, LHB, LHC and LHD strains isolated from raw ovine milk were examined in both BHI broth and ovine milk. In BHI broth, the Staph. aureus LHA, LHB, LHC and LHD strains were found toxigenic at 37 °C producing the staphylococcal enterotoxins (SEs) serotypes SEA, SEB, SEC and SED, respectively, whereas in ovine milk at 37 °C, Staph. aureus LHD was found to produce only SED, while no SE production was observed for the other examined strains. Thus, the fate of only Staph. aureus LHD and SED were examined in Feta and Galotyri cheeses. The cheeses were made from raw ovine toxic milk with preformed SED or raw ovine milk contaminated with high (ca 6 log cfu/ml) and low inocula (ca 3 log cfu/ml) of Staph. aureus LHD. Results showed that the pathogen was eliminated at slower rate in Galotyri cheese than in Feta cheese, for the high (5 d vs. 16 d) or the low (1 d vs. 12 d) inoculum trials. In both cheeses produced from the toxic milk, SED was detected during manufacturing and storage. SED was also detected in the curd (2 h), when Staph. aureus LHD populations had reached ca 7 log cfu/g, and up to the end of storage for the high inoculum trials of both cheeses. No SED was observed for the low inoculum trials of either cheese.

The susceptibility of total casein and the individual caseins in reconstituted skim milk to transglutaminase (TGase)-induced cross-linking was studied as a function of incubation temperature (5–40 °C), pH (5·0–7·0) and mineral addition. Within the ranges studied, the level of total casein cross-linked increased with increasing temperature, pH and concentration of added trisodium citrate, whereas adding calcium chloride had the opposite effect. These effects can be largely related to the effects of these parameters on TGase activity. In addition, the parameters were also found to influence the susceptibility of κ-casein, and to a lesser extent β-casein, to cross-linking, whereas the susceptibility of αs1-casein was not affected. The susceptibility of κ-casein to cross-linking increased with increasing temperature and calcium chloride addition, but decreased with increasing pH and citrate content, whereas the susceptibility of β-casein to TGase-induced cross-linking decreased with increasing temperature, but was not affected by other parameters. These findings highlight the fact that selection of environmental conditions during cross-linking can be applied to tailor the surface, and hence possibly colloidal stability, of casein micelles in TGase-treated milk.

Periparturient hypocalcaemia (milk fever) is a disorder of Ca metabolism in dairy cattle primarily affecting multiparous cows. The major reasons for the rapid decrease of blood Ca concentration after calving are the prompt increase of Ca secretion into the colostrum and the delayed activation of Ca regulation mechanisms including calcitriol, a metabolite of vitamin D. In man, vitamin D receptor (VDR) gene polymorphisms are reported to be associated with disturbances of Ca metabolism, whereas data confirming the same in dairy cows are still missing. Moreover, polymorphisms that only affect non-coding regions are sometimes difficult to ascribe to a specific disorder as pathways and unequivocal links remain elusive. Therefore, the idea of the present study was to investigate in a small group of dairy cows with documented clinical records whether polymorphisms in the coding regions of the VDR gene existed and whether these potentially found variations were correlated with the incidence of periparturient hypocalcaemia. For this purpose, blood DNA was isolated from 26 dairy cows in their 4th to 6th lactation, out of which 17 had experienced hypocalcaemia at least once, whereas 9 cows had never undergone periparturient hypocalcaemia in their lifetime. The 10 VDR exons and small parts of adjacent introns were sequenced and compared with the Bos taurus VDR sequence published on NCBI based on the DNA of one Hereford cow. In total, 8 sequence alterations were detected in the fragments, which were primarily heterozygous. However, only 4 of them were really located on exons thereby potentially causing changes of the encoded amino acid of the VDR protein, but were not correlated with the incidence of periparturient hypocalcaemia. Certainly, this lack of statistical correlation could be due to the small number of animals included; anyhow, it was not encouraging enough to initiate a larger study with hundreds of cows and document blood Ca levels post partum for at least four lactations.

An association study between the milk yield trait and the stearoyl-CoA desaturase (SCD) polymorphism (g.133A > C) in Italian Mediterranean river buffalo was carried out. A full characterization of the river buffalo SCD promoter region was presented. Genotyping information was provided and a quick method for allelic discrimination was developed. The frequency of the C allele was 0·16. Test-day (TD) records (43 510) of milk production belonging to 226 lactations of 169 buffalo cows were analysed with a mixed linear model in order to estimate the effect of g.133A > C genotype, as well as the effect of parity and calving season. The SCD genotype was significantly associated with milk yield (P = 0·02). The genotype AC showed an over-dominance effect with an average daily milk yield approximately 2 kg/d higher than CC buffaloes. Such a difference represents about 28% more milk/d. The effect of the genotype was constant across lactation stages. The contribution of SCD genotype (r2SCD) to the total phenotypic variance in milk yield was equal to 0·12. This report is among the first indications of genetic association between a trait of economic importance in river buffalo. Although such results need to be confirmed with large-scale studies in the same and other buffalo populations, they might offer useful indications for the application of MAS programmes in river buffalo and in the future they might be of great economic interest for the river buffalo dairy industry.

The overall objective of this study was to investigate how relevant research publications address the validity of diagnostic methods for acute puerperal metritis (APM) in dairy cows, a disease commonly treated with antibiotic drugs. Therefore, a literature search was conducted in Journal of Dairy Science, Theriogenology, Animal Reproduction Science and The Veterinary Journal utilizing the ScienceDirect database. The search revealed 259 articles addressing APM. After applying exclusion criteria, a total of 48 trials remained. It was determined whether the author gave a clear definition of APM, the time of diagnosis relative to calving, and the person who performed the diagnosis. Studies were checked for the presence of definitions of possible findings, thresholds, and test characteristics of the methods used. Overall 9 different diagnostic methods were employed. On average 2·5 ± 1·75 diagnostic methods were used in a study. References to support the use of the diagnostic methods were provided in 10 of 48 articles (20·8%). Vaginal discharge, transrectal palpation and rectal temperature were examined in 39, 22, and 21 of the studies, respectively. Thresholds for diagnostic tests and test characteristics were mentioned in 6 and 3 of the 48 articles, respectively. Based on this systematic review of 48 research papers the evidence supporting the use of the diagnostic methods to identify cows with APM has either been not reported or is weak. In conclusion, the reporting of the diagnostic methods to identify cows with APM needs to be improved and further high-quality research is necessary to improve diagnostic performance of the methods employed.

The prevalence of bovine leukaemia virus (BLV) was determined in 113 Michigan dairy herds by ELISA testing for anti-BLV antibodies in milk. Additionally, an interview regarding management practices with cooperating herd managers identified farm-level variables thought to be associated with prevalence of BLV. Twenty-three risk factors (P ⩽ 0·1) were identified on one-way ANOVA or simple linear regression. Multivariate analysis identified several management practices whose predictive value for increased prevalence of BLV may relate to transmission among herd mates, e.g. reuse of hypodermic needles, lack of fly control, gouge dehorning and increased use of injections in dry cows. Additionally, exclusive breeding of heifers with artificial insemination was associated with decreased BLV prevalence, as compared with at least some use of natural service by a bull. Although intervention studies are needed before causal relationships can be concluded, and unaccounted variables related to transmission exist among dairy herds, these findings suggest management practices that may help dairy producers reduce the transmission of BLV within their herds.

Fractionation of bovine milk was performed using chymosin-induced separation, isoelectric precipitation or ultracentrifugation as separation techniques prior to gel-based proteomic analysis. This approach allowed for comparative display and identification of proteins partitioned into casein and whey, respectively. Initially, three different staining methods (silver staining, colloidal Coomassie Blue G-250 or fluorescent Flamingo Pink staining) for two-dimensional gel electrophoresis (2-DGE) analysis were compared for their suitability as staining agent, especially in relation to their suitability to reveal differences in the casein fractions. Fluorescent staining proved to be the most appropriate for this purpose, giving a high sensitivity, and using this staining method, characteristic 2-DGE fingerprints were obtained for each casein and whey fraction from each separation method. A number of protein spots in both casein and whey fractions varied with separation method and these spots were subsequently identified using tandem mass spectrometry (MS). In rennet casein, proteolytic fragmentation of caseins (αs1-, αs2,-, β- and κ-) was identified as a result of chymosin hydrolysis, whereas the 2-DGE profile of acid and ultracentrifuged casein was dominated by the presence of multiple isoforms of κ-caseins. Furthermore, casein remnants were identified in milk serum after ultracentrifugation. This study shows that gel-based proteomic analysis is suitable for characterisation of subtle variations in protein composition of milk fractions that occur as a consequence of different milk fractionation strategies.

The solubility of milk protein concentrate (MPC) powders was influenced by the method used for preparing the concentrate, drying conditions, and the type of dryer used. Increasing total solids of the ultrafiltered concentrates (23% total solids, TS) by diafiltration to 25% TS or evaporation to 31% TS decreased the solubility of MPC powders (80–83% protein, w/w dry basis), with ultrafiltration followed by evaporation to higher total solids having the greater detrimental effect on solubility. High shear treatment (homogenisation at 350/100 bar, microfluidisation at 800 bar or ultrasonication at 24 kHz, 600 watts) of ultrafiltered and diafiltered milk protein concentrates prior to spray drying increased the nitrogen solubility of MPC powders (82% protein, w/w dry basis). Of the treatments applied, microfluidisation was the most effective for increasing nitrogen solubility of MPC powders after manufacture and during storage. Manufacture of MPC powders (91% protein, w/w dry basis) prepared on two different pilot-scale dryers (single stage or two stage) from milk protein concentrates (20% TS) resulted in powders with different nitrogen solubility and an altered response to the effects of microfluidisation. Microfluidisation (400, 800 and 1200 bar) of the concentrate prior to drying resulted in increased long term solubility of MPC powders that were prepared on a single stage dryer but not those produced on a two stage spray dryer. This work demonstrates that microfluidisation can be used as a physical intervention for improving MPC powder solubility. Interactions between the method of preparation and treatment of concentrate prior to drying, the drying conditions and dryer type all influence MPC solubility characteristics.

An appropriate composition of milk fatty acids (FA) improves the nutritional value of milk and milk products, and improves milk processing. Polish dairy farms in the mountainous region are rather small, animal nutrition there is based on locally produced forages and this, together with the transitional climate zone brings about seasonal changes in FA composition of milk. The aim of the study was to evaluate the composition of FA in bovine milk fat in relation to fat intake in forages and their FA profiles. The study involved 5 herds reared in low-input mountain farms located at an altitude of 670–780 m above sea level (Beskid Mountains). The cows were fed forages produced locally. FAs in forages and milk samples were subjected to gas chromatography. Highest fat intake observed in grazing season (4·2–4·7%) and high amounts of polyunsaturated FA in forages from that period (51·8–64·1 g/100 g FA) resulted in a markedly high content of valuable FAs: t-11 C18:1 (3·22 g/100 g FA), c-9, t-11 C18:2 (CLA; 1·20 g/100 g FA) in milk. Lower fat intake of forages containing high amount of SFA (32·42–38·83 g/100 g FA) in the indoor period resulted in changes in milk composition. The content of total short-chain saturated FA (SCFA) was highest in winter and early spring samples (14·10 and 13·44 g/100 g FA, respectively), like the amounts of myristic C14:0 and palmitic C16:0 acids (11·80 and 37·92 g/100 g FA). Total odd- and branched-chain fatty acids (OBCFA; 6·58 g/100 g FA) content was highest at the beginning of the grazing period. Fresh grass consumed by cows promoted the activity of Δ9-desaturase in mammary gland as evidenced by higher C14:1 : C14:0 (0·054) and C16:1 : C16:0 (0·026) ratios in grazing than in the indoor periods.

The objective of this field study was to compare the udder health status as well as the clinical mastitis rate during the first 100 d of lactation in cows that received long-acting dry cow antibiotic alone (group AB) or in combination with an internal teat sealant (group AB + OS). The study was conducted during a 9-month period and included 136 Holstein cows from 12 dairy farms in Hessia, Germany. Between days 1 and 5 after calving a California mastitis test (CMT) was performed. Milk-samples were collected for bacteriological culture before drying off, between days 6 and 14 and days 35 and 56 of lactation. Additionally the cows were monitored for the occurrence of clinical mastitis events until 100 d post partum. Within the 12 herds cow-pairs were formed on the basis of age, milk yield and SCC. A cow-pair consisted of one cow from group AB and one cow from group AB + OS. For statistical analysis within every cow-pair one quarter that has been dried off with internal teat sealant and dry cow antibiotic (group AB + OS) was compared with one quarter that has been dried off with dry cow antibiotic (group AB) alone. As criterion for the matching process of udder quarters the cytobacteriological udder health status before drying off was used. A total of 544 quarters (136 cows) were used in this analysis. In the first 5 d after calving, group AB had significantly more quarters with a positive CMT reaction than group AB + OS (85 vs. 57; P <0·001), and in the first 100 d of lactation, group AB had more quarters with clinical mastitis than group AB + OS (25 vs. 15; P = 0·03). In the time periods 6–14 and 35–56 d of lactation, there were fewer quarters in group AB + OS populated with Corynebacterium spp. (days 6–14, P = 0·05; days 35–56, P = 0·02) and aesculin-positive streptococci (days 35–56, P = 0·02). The internal teat sealant was a promising tool for the prevention of new intramammary infections (IMI) of dry cows with environmental udder pathogens as expressed during early lactation.

The objective of the present work was to study the differences in the fatty acid (FA) composition of raw sheep milk fat under commercial milk production conditions throughout lactation, in two consecutive years. Particular attention was placed on the C18:2cis-9,trans-11 isomer, C18:1trans-11 acid, and unsaturated FA as the feeding regimen of 10 commercial flocks of latxa dairy sheep changed from indoor feeding to part-time grazing conditions (from early spring) as traditionally practiced in the Basque Country (Northern Spain). Farms located at an altitude of between 600 and 700 m, in two different geographical areas with different rainfall were selected. Milk samples were collected monthly from late January (indoor feeding) until mid-, or end of, June (outdoor feeding), during two consecutive years. In spite of some interannual variability (most likely due to large differences in rainfall), the evolution of individual FA throughout lactation was comparable between years, indicating that it was reproducible under commercial milk production conditions. The average concentrations of C18:2cis-9,trans-11 isomer and C18:1trans-11 acid in milk from the commercial flocks increased about 200% during the transition period (end of March or early April until May), from indoor feeding (late January or early February until the end of March) to the outdoor period (early May to mid-June), remaining constant during the outdoor period (27·53 ± 9·32 μmol/g fat and 71·58 ± 20·53 μmol/g fat, respectively). Non-atherogenic FA comprised approximately 50% of all saturated FA at any time during lactation, whereas the milk atherogenicity index decreased significantly during the outdoor period. The Trolox-equivalent antioxidant capacity of the water-soluble milk fraction did not appear to be influenced by feeding management. The FA composition of cheeses made during the second year with milk from the indoor or outdoor periods reflected those of the corresponding milks. A principal components analysis clearly showed that differences in the milk FA composition were primarily due to outdoor grazing, with very little contribution from the geographical zone or the year.

Sialic acid, being a biologically active compound, is recognised as an important component of milk and milk products. Almost all the sialic acid estimation protocols in milk require prior hydrolysis step to release the bound sialic acid followed by its estimation. The objective of this work was to estimate sialic acid in milk and milk products by fluorimetric assay which does not require a prior hydrolysis step thus decreasing the estimation time. The recovery of added sialic acid in milk was 91·6 to 95·8%. Sialic acid in milk was found to be dependent on cattle breed and was in the range of 1·68–3·93 g/kg (dry matter basis). The assay was further extended to detect adulteration of milk with sweet whey which is based on the detection of glycomacropeptide (GMP) bound sialic acid in adulterated milk. GMP is the C-terminal part of κ-casein which is released into the whey during cheese making. For detection of adulteration, selective precipitation of GMP was done using trichloroacetic acid (TCA). TCA concentration in milk was first raised to 5% to precipitate milk proteins, especially κ-casein, followed by raising the TCA concentration to 14% to precipitate out GMP. In the precipitates GMP bound sialic acid was estimated using fluorimetric method and the fluorescence intensity was found to be directly proportional to the level of sweet whey in adulterated milk samples. The method was found to detect the presence of 5% sweet whey in milk.