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In 2 epidemiological studies, we evaluated the effect of mastitis induced by gram-positive Streptococcus and gram-negative Escherichia coli on impaired reproductive performance in lactating Holstein cows. In the first study, 52,202 cows from 178 dairy farms throughout Israel were divided into groups based on infection before first artificial insemination (AI) with Streptococcus or E. coli, 3 groups with elevated somatic cell count (SCC) without infection by those pathogens [low SCC (200-400) × 103 cell/mL; medium SCC (401-1,000) × 103 cell/mL; high SCC, >1,000 × 103 cell/mL], and uninfected controls. Pregnancy per first AI (P/1stAI) and pregnancy rate at 300 d in milk (PREG 300) were analyzed by the GLIMMIX procedure (SAS); number of AI per pregnancy (AI/P), days open, and rest days (calving to first AI) were analyzed by the MIXED procedure (SAS Institute Inc., Cary, NC). Values of P/1stAI were similarly low for Streptococcus and E. coli (27-28%) versus 42% in controls; PREG 300 was lower for Streptococcus (76%) than for E. coli (79%) versus 88% for uninfected controls and a mean 83% for the elevated SCC groups. Days open and number of AI/P were higher than in controls and similar in Streptococcus and E. coli groups. The second study included 778 cows on 6 dairy farms; the cows were infected before first AI by Streptococcus or E. coli or uninfected. Resumption of cyclicity was determined by an automated activity-monitoring system, and data were sorted by time of infection before or after cyclicity resumed. The Streptococcus group had lower P/1stAI before and after cyclicity (26 and 27%, respectively) than the E. coli group (31 and 34%, respectively) and uninfected controls (42%). Notably, PREG 300 in the Streptococcus group before (73%) and after (67%) cyclicity was much lower than for the E. coli group (85 and 93%, respectively) and the controls (95%). A marked rise in day of cyclicity resumption (∼80 d) was observed in cows that were infected early on. Number of AI/P was higher in the mastitic groups than in uninfected controls. Uterine disease postpartum, although more prevalent among Streptococcus cows, did not substantially alter the larger reduction in P/1stAI and PREG 300 in Streptococcus versus E. coli cows. Thus, long-term Streptococcus-induced mastitis disrupted fertility more than short-term acute E. coli-induced mastitis, resulting in a much higher percentage of Streptococcus cows in late lactation that did not conceive due to reproduction failure.

The effects of subclinical intramammary infection (SCIMI) in sheep are ignored in most cases by both the farmers and the industry. Due to the high prevalence of coagulase negative staphylococci (CNS) and the lack of feasibility to treat the infection during lactation, prominent actions must concentrate on its prevention. On the sheep level, SCIMI caused by CNS clearly reduces milk yield and changes milk composition that directly influence the product’s properties. High proportion of milk from infected glands in the milk tank is associated with higher losses of fat and protein in the whey during the coagulation process. Moreover, extended storage of milk, irrespective of its initial quality, deteriorates its final quality for cheese making and extended storage of low-quality milk before processing, further increases this deterioration. Studies related to the quality of cheese and yogurt indicated that milk composition has been changed owing to the presence of different bacteria. The review aims to draw attention to the effects of SCIMI in sheep caused by CNS on milk production losses along with changes in its composition that influences milk coagulation properties and chasse yield.

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Differentiation of cells by flow cytometry provides informative somatic cell counts (SCCs) that allow analyzing leukocyte population patterns in udder infections of different etiologies. Postulating that this approach also enhances the statistical power to detect genetic variants linked to cell levels in milk of healthy mammary glands, we used monoclonal antibodies anti-CD18, anti-CD4, anti--CD14, and anti-PMN to count cells presenting these surface antigens, and performed a genome-wide association study of these counts in 125 Israeli Holsteins genotyped using SNP BeadChips. We identified an informative haplotype of 15 SNPs in the centromeric end of BTA3 that was strongly associated with CD18 cells (p &lt, 2.3 &times, 10&minus, 9). Within this region, examination of the network of genes interacting with ITGB2 (CD18) indicated an Fc-&gamma, receptor gene cluster, including FCGR2A (CD32). Sanger-sequence analysis of FCGR2s-linked exon 3 variation to CD18 counts. Meta-analysis of RNA-Seq data revealed a significant negative correlation (R = &minus, 0.51) between expression of CD32 and CD18 in milk. Assembly of DNA-Seq reads uncovered FCGR copy-number variation and a variant, designated V7, was abundant in dairy cattle, probably reflecting adaptation to selection pressure for low SCC in Holstein milk.

The mammary immune and physiological responses to distinct mammary-pathogenic E. coli (MPEC) strains were studied. One gland in each of ten cows were challenged intra-mammary and milk composition (lactose, fat, total protein, casein), biochemical (glucose, glucose-6-phosphate (Glu6P), oxalate, malate, lactate, pyruvate and citrate, malate and lactate dehydrogenases, lactate dehydrogenase (LDH), nitrite, lactic peroxidase, catalase, albumin, lactoferrin, immunoglobulin) and clotting parameters were followed for 35 days post-challenge. Challenge lead to clinical acute mastitis, with peak bacterial counts in milk at 16–24 h post-challenge. Biochemical and clotting parameters in milk reported were partially in accord with lipopolysaccharide-induced mastitis, but increased Glu6P and LDH activity and prolonged lactate dehydrogenase and Glu6P/Glu alterations were found. Some alterations measured in milk resolved within days after challenge, while others endured for above one month, regardless of bacterial clearance, and some reflected physiological responses to mastitis such as the balance between aerobic and anaerobic metabolism (citrate to lactate ratios). The results suggest that E. coli mastitis can be divided into two stages: an acute, clinical phase, as an immediate response to bacterial infection in the mammary gland, and a chronic phase, independent of bacteria clearance, in response to tissue damage caused during the acute phase.

Colostrum samples of first, second, and third milking were collected from 72 cows. Immunoglobulins were determined by colostrometer, Brix refractometer, online Afilab spectrometer, and ELISA. Corre...

We studied the mammary immune response to different mammary pathogenicEscherichia coli(MPEC) strains in cows, hypothesising that the dynamics of response would differ.E. coliis a major aetiologic agent of acute clinical bovine mastitis of various degrees of severity with specific strains being associated with persistent infections. We compared challenge with three distinct pathogenic MPEC strains (VL2874, VL2732 and P4), isolated from different forms of mastitis (per-acute, persistent and acute, respectively). A secondary objective was to verify the lack of mammary pathogenicity of an environmental isolate (K71) that is used for comparison against MPEC in genomic and phenotypic studies. Twelve cows were challenged by intra-mammary infusion with one of the strains. Cellular and chemokine responses and bacterial culture follow-up were performed for 35 d. All cows challenged by any of the MPEC strains developed clinical mastitis. Differences were found in the intensity and duration of response, in somatic cell count, secreted cytokines (TNF-α, IL-6 and IL-17) and levels of milk leucocyte membrane Toll-like receptor 4 (TLR4). A sharp decrease of TLR4 on leucocytes was observed concomitantly to peak bacterial counts in milk. Intra-mammary infusion of strain K71 did not elicit inflammation and bacteria were not recovered from milk. Results suggest some differences in the mammary immune response to distinct MPEC strains that could be correlated to their previously observed pathogenic traits. This is also the first report of anE. colistrain that is non-pathogenic to the bovine mammary gland.

Upon entry into the mammary gland, mammary pathogenic strains of Escherichia coli rapidly grow using milk as a nutrient source, in a manner highly dependent on the expression of the ferric-dicitrate system by these bacteria. Intra-mammary challenge with distinct mammary pathogenic E. coli (MPEC) strains result in development of clinical acute mastitis, with peak bacterial counts in milk at 16-24 h post-challenged and profound immune changes found in the milk. The main biochemical changes measured in milk were partially in accord with lipopolysaccharide-induced mastitis, with increased glucose-6-phosphate and lactate dehydrogenase activity or prolonged lactate dehydrogenase, and Glu6P/Glu alterations. Changes also reflect physiological responses to inflammation in the mammary gland, as in the balance between aerobic and anaerobic metabolism (citrate to lactate ratios). Some alterations measured in milk resolved with days after challenge but other remained for above one month, regardless of bacterial clearance. The results suggest that E. coli mastitits can be divided into two stages: an acute, clinical phase, as an immediate response to bacterial infection in the mammary gland and a chronic phase, independent of bacteria clearance, and in response to the tissue damage caused in the first phase.

The current study measured the influence of milk of subclinically infected glands by different bacteria species on the cow’s milk and suggests different parameters for milk payment. The effects of bacterial infection or inflammation on gland milk yield were related to the bacteria species that caused the infection. The volume of milk of the inflamed gland from the cow’s milk yield was significantly lower (P) for the glands previously infected byEscherichia coli(PIEc) and those infected withStreptococcus dysgalactiae.Coagulation properties, rennet clotting time (RCT) and curd firmness (CF) also depended on the bacteria causing the infection. RCT values of all the inflamed glands were significantly longer (P) and CF values were significantly lower than that of the healthy ones. Moreover, in the whole milk, CF was also significantly lower and not proportional to the volume of the milk from the inflamed gland of the cow’s milk. Calculating the predicted 40% dry matter curd weight (PCW) on the cow level, including the healthy and inflamed glands or the healthy glands alone, found that for 9 of 13 PIEc cows, the presence of the affected gland’s milk in the whole cow milk resulted in a negative PCW value. Likewise, 5 of 20 cows infected byS. dysgalactiaehad negative delta values. Unlike the latter bacteria, PCW from milk of glands infected with CNS increased, although in a lower magnitude than in the healthy glands. No correlation was found between logSCC in the whole cow milk (healthy and inflamed glands) and PCW.

This study focused on the need for bulk milk tank somatic cell count (BMTSCC) thresholds and cut-off levels indicating a decrease in milk quality that consequently influences product quantity and quality. First, 226 ewes and 231 goat bulk tank milk samples were collected from different Israeli herds and coagulation properties were determined. Second, soft cheese was produced. No correlation of coagulation properties was found with BMTSCC for sheep milk up to 3264 × 103 and goat milk up to 6452 × 103 cells mL−1. Coagulation properties of goat milk with cell count higher than the latter resulted in a significant decrease in curd firmness. For breeds and management system in Israel, 2500 × 103 cells mL−1 is suggested as the cut-off level for sheep and 3500 × 103 cell mL−1 for goats. The cell count cut-off level and milk price according to BMTSCC should be tested and then determined for every breed and management and final dairy product.

Mastitis, particularly in its subclinical form, is a widely spread disease that reduces the fertility of lactating cows. A major cause of poor conception risk has been associated with delayed ovulation of a large subgroup of subclinical mastitic cows. This study examined 2 approaches to improve fertility in this subgroup. Subclinical mastitic cows were defined by somatic cell count elevated above a threshold of 150,000 cells/mL of milk determined in all monthly test day samples collected before AI. Uninfected (control) cows were defined by somatic cell count below threshold. In experiment 1, we examined a hormonal approach aimed to correct the timing of ovulation in mastitic cows in which it would otherwise be delayed. The probability of conception of mastitic and uninfected groups following Ovsynch (OVS) and timed AI versus AI following detected estrus (E) was examined (n=1,553 AI) and analyzed by a multivariable, logistic model statement using the GLIMMIX procedure of SAS. The OVS protocol significantly elevated the probability of conception of mastitic cows to a level similar to that of their uninfected counterparts. Actual mean conception risks for uninfected-E, subclinical-E, uninfected-OVS, and subclinical-OVS groups were 41.8, 26.4, 39.3, and 40.5%, respectively. The OVS protocol did not improve probability of conception in cows diagnosed with uterine disease postpartum. In experiment 2, a management approach aimed to better synchronize timing of ovulation with timing of AI in subclinical mastitic cows was examined. A second AI was added 24h after the first (routine) AI, following detection of natural estrus. Probability of conception did not differ between subclinical mastitic cows inseminated once or twice. Lack of improvement in conception risk might be related to low preovulatory LH surge in mastitic cows, which is likely to induce not only delayed ovulation but also disruption of oocyte maturation. Thus the OVS protocol can improve fertility of subclinical mastitic cows, probably due to "corrected" timing of ovulation in cows in which it would otherwise be delayed.

Clinical and subclinical mastitis affects 30% of cows and is regarded as the most significant economic burden on the dairy farm reducing milk yield and quality and increasing culling rate. A proprietary Acoustic Pulse Therapy (APT) device was developed specifically for treating dairy cows. The APT device was designed to produce deep penetrating acoustic pulses that are distributed over a large treated area at a therapeutic level. This paper presents findings from a clinical assessment of this technology for the treatment of dairy cows with subclinical and clinical mastitis. In subclinical mastitis, a group of 116 cows from 3 herds were identified with subclinical intramammary infection and enrolled in the study; 78 cows were assigned to the treatment group and 38 cows to the control group. Significant differences (P

The concentrations of glucose and glucose-derived carbons in milk reflect their concentrations in the mammary epithelial cell cytosol. We hypothesized that the sharp reduction in milk secretion observed during acute inflammation in the mammary gland is associated with conversion of the gland's metabolism to aerobic glycolysis and reduced extraction of glucose from the blood, in support to the innate immune system. Acute inflammation was induced by challenging one mammary gland in 5 cows with a single dose of 10 μg bacterial lipopolysaccharide. The glandular response was followed up to 96 h post-challenge. The challenge induced increases in polymorphonuclear leukocytes, milk malondialdehyde concentration and casein degradation. The response peaked at 24 h post-challenge and the inflammation began to decrease after 48 h, but at 96 h post-treatment, values had not yet returned to pre-treatment levels. Milk secretion, and milk lactose, glucose and citrate concentrations decreased sharply, reaching minimal levels at 24 h post-treatment. The correlations between these parameters and inflammation parameters were negative and statistically significant. The reduction of ∼50% in milk yield and lactose concentration in the treated gland indicated that extraction of glucose from the blood was reduced from a rate of ∼740 g d−1 to 190 g d−1 (i.e., by 550 g d−1) at the peak of response. The concentrations of glucose-6-phosphate, malate, oxaloacetate, lactate and pyruvate and the activities of the enzymes glucose-6-phosphate dehydrogenase, malate dehydrogenase and lactate dehydrogenase increased and, in general, were positively and significantly correlated to inflammation parameters. It was concluded that inflammation shifts the passage of glucose-derived carbons to the pentose phosphate pathway and shifts cell metabolism to glycolysis at the expense of mitochondrial activity.

Loss of milk quality caused by subclinical infection in dairy sheep has a negative effect on cheese manufacture. As milk from each single animal is not systematically evaluated for somatic cell count, milk from animals with undetected subclinical mastitis often reaches the refrigeration tanks, mixing with normal milk and reducing its technological suitability for cheese manufacture. This study was undertaken to investigate the effect of subclinical mastitis in the coagulation properties of ewe milk using a light backscatter fiber optic sensor. Manchego-type cheese was manufactured using milk from Lacaune and Manchega sheep. Milk from infected and non-infected udders was coagulated and monitored at laboratory scale using both a NIR fiber optic light backscatter sensor and a rheometer. Simultaneously, clotting and cutting time were visually evaluated by an experienced cheesemaker. Optical parameters t max , t 2max , and t 2min were highly correlated (0.914 r P Staphylococcus had longer coagulation and cutting time. Prediction models using light backscatter parameters alone or in combination with protein/solids concentration were successfully obtained for visually determined clotting and cutting time, rheologically derived gelation and cutting times and for tan δ at cutting with R 2 values ranging from 0.799 to 0.999. Our results suggest that early detection of subclinical mastitis and milk coagulation monitoring using light scatter can diminish the negative impact of mixing milk of infected animals, when milk is used for cheese manufacture.

Humans learned to exploit ruminants as a source of milk about 10,000 years ago. Since then, the use of domesticated ruminants as a source of milk and dairy products has expanded until today when the dairy industry has become one of the largest sectors in the modern food industry, including the spread at the present time to countries such as China and Japan. This review analyzes the reasons for this expansion and flourishing. As reviewed in detail, milk has numerous nutritional advantages, most important being almost an irreplaceable source of dietary calcium, hence justifying the effort required to increase its consumption. On the other hand, widespread lactose intolerance among the adult population is a considerable drawback to dairy-based foods consumption. Over the centuries, three factors allowed humans to overcome limitations imposed by lactose intolerance: (i) mutations, which occurred in particular populations, most notably in the north European Celtic societies and African nomads, in which carriers of the lactose intolerance gene converted from being lactose intolerant to lactose tolerant; (ii) the ability to develop low-lactose products such as cheese and yogurt; and (iii) colon microbiome adaptation, which allow lactose intolerant individuals to overcome its intolerance. However, in a few examples in the last decade, modern dairy products, such as the popular and widespread bio-cultured yogurts, were suspected to be unsuitable for lactose intolerant peoples. In addition, the use of lactose and milk-derived products containing lactose in non-dairy products has become widespread. For these reasons, it is concluded that it might be important and helpful to label food that may contain lactose because such information will allow lactose intolerant groups to control lactose intake within the physiological limitations of ~12 g per a single meal.

Manchego is a high-fat, long ripened hard cheese, which is produced by enzymatic coagulation of sheep milk. Manchego produced in the La Mancha region in Spain and has Protected Designation of Origin (PDO) status. The objective of the study was to evaluate the effects of intramammary infection (IMI) in Manchega and Lacaune dairy sheep with various coagulase negative staphylococci bacteria (CNS) on milk coagulation properties and cheese whey syneresis. The effect of the length of milk storage before processing on cheese yield and its quality was also evaluated. The study included 20 Manchega and 16 Lacaune dairy ewes. The half-udder model was applied: each of the selected animals had one gland free of infection, while the contra-lateral gland was infected with various CNS species. Pasteurized milk (10 L) from uninfected glands, and 50:50 and 75:25 milk blends from uninfected:CNS-infected glands was used for cheese production. Overall, 23 batches of cheese were made and ripened for 63 d. Cheeses were analyzed for organic components, physical and mechanical structure. The taste and sensual qualities of the cheese were evaluated by experts and consumers in two separate panels. Milk yield in the glands infected with CNS was significantly lower than in control glands. The milk of infected glands had higher somatic cell count and poorer coagulation properties in comparison to controls. Cheese made from infected milk had a significant effect on % fat, total fat, fat loss, total protein, protein loss and dry matter in both breeds. However, cheeses made of blends of 25% or 50% milk from infected glands had only minor losses of fat and protein content, which were not associated with noticeable changes in the final weight of the cheese; though, as discussed herein, further research in this line is warrant. The cheeses made of milk stored under refrigerated conditions were clearly perceived as different from the other cheeses because it had more intense attributes related with tangy taste (i.e., bitter, sharp, intense taste, acid) and soft texture (i.e., soft, pasty, not tough).

A hypothesis that the source of milk-serum derived vesicles (MSDVs) is the Golgi apparatus (GA) was examined. Using dynamic light scattering and electron microscopy, it was shown that MSDVs are composed of globular structures with hydrodynamic sizes of 70 ± 15 nm. More than 60% of the total protein content of MSDVs was associated with the MSDV lumen and 30% was associated with the MSDV membrane. Casein was the major protein found in the MSDV lumen. The conclusive markers of the GA, lactose synthase components (α-lactalbumin and galactosyltransferase) and activity (synthesis of lactose from glucose and UTP-galactose), the presence of casein in micellar form in the MSDV lumen, and a high luminal content of citric acid, were demonstrated in the lumen of MSDVs. Though MSDVs composed only 0.7% of the milk mass, they accounted for a high proportion of the total milk content of reactive (15% Cu and 18% Fe) and toxic minerals (60% Cd and 65% Pb), which strongly suggests that MSDVs serve as an avenue to protect mammary epithelial cells from the toxic effects of these minerals by storing them intraluminally and secreting them into milk. The presence of micellar casein in the MSDV lumen, along with the presence of metal transporters in their membranes, is responsible for this impressive capacity for storing reactive and toxic minerals. Exposing a single mammary gland to lipopolysaccharide challenge induced changes in regulatory proteins stored in the lumen of MSDVs (tissue plasminogen activators, plasminogen and plasmin) and in the activity of xanthine oxidase and alkaline phosphatase attached to the outer membrane of MSDVs. Thus, we have demonstrated that MSDVs are under the regulation of the nucleus and respond to extracellular signals.

This study establishes an experimental model for subclinical mastitis induced by Gram-positive (Gþ) exosecretions of Staphylococcus aureus origin or Gram-negative (G ) endo-toxin of Escherichia coli origin to examine its effects on follicular growth and steroidconcentrations in Holstein dairy cows. Cows were synchronized with the Ovsynch protocolfollowed by a series of follicular cycles that included GnRH and PGF 2 a doses administeredevery 8 days. Cows received small intramammary doses of either Gþ (10 m g, n ¼ 10) or G (0.5 m g, n ¼ 6) toxin, or saline (n ¼ 6; uninfected control) every 48 hours for 20 days.Follicular fluids were aspirated from preovulatory follicles before (aspiration one: control),at the end of (aspiration two: immediate effect), and 16 days after the end of (aspirationthree: carryover effect) toxin exposure. During the 3 weeks of subclinical mastitis inducedby Gþ or G , no local inflammatory signs were detected in the mammary gland and nosystemic symptoms were noted: body temperatures of the treated cows did not differ fromcontrols; plasma cortisol and haptoglobin concentrations were not elevated and did notdiffer among groups. Somatic cell count was higher in the treated groups than in controls,and higher in the G versus Gþ group. For analysis of reproductive responses, cows werefurther classified as nonaffected or affected based on an more than 20% decline in follicularandrostenedione concentration in aspiration two or three relative to the first, controlaspiration. Most G (5/6) and 40% of Gþ (4/10) cows were defined as affected by inducedmastitis. An immediate decrease in the number of medium-size follicles was recorded onDay 4 of the induced cycle, toward the end of the 20-day mastitis induction, in the affectedGþ compared with uninfected control group (1.0 0.5 vs. 3.0 0.4 follicles; P < 0.05); theaffected G and nonaffected Gþ subgroups exhibited a similar numerical decline in thenumber of follicles. A carryover (but not immediate) decrease to 51% and 62% in follicularestradiol concentrations in G affected group and Gþ affected group was detected relativeto controls (P < 0.05). The nonaffected Gþ subgroup did not differ from its controlcounterparts. Based on the current experimental model, subclinical IMI induced by Gþ orG toxin disrupts follicular functions, and it seems that the ovarian pool of early antralfollicles is susceptible to subclinical mastitis. 2014 Elsevier Inc. All rights reserved.

Bulk milk is the mixture of all the milked udders in a given herd. Nowadays, about 15–40% of the udders in most herds are intramammary infected by different bacteria species, mainly coagulase negative staphylococci. The presences of bacteria in the lumen of the mammary gland induce impairment of milk quality and increase the number of somatic cells. A positive relationship between % casein (casein/total protein) and curd firmness (CF) and negative relationship between lactose, or somatic cell count (SCC) and CF are associated with bacterial infection and with late lactation milk, and therefore with reduction in cheese yield and quality. On the other hand, in milk of goats with intramammary infection, the correlation between the levels of fat, protein, casein and curd yield is minor compared to milk of uninfected animals. Thus, gross milk composition is an insufficient predictor of milk quality for cheese production, since a high percent of the bulk milk originates from subclinically infected glands. Research carried out in the past few years highlighted the effectiveness of lactose as a predictor of milk quality. The correlation between lactose and CF was higher than that for % casein and SCC. Lactose concentration of ≤4% is associated with non-coagulating milk and therefore, such milk is unsuitable for making cheese, but still meets the criterion for consumption as pasteurized milk. A model that describes the simultaneous and close association between reduction in lactose concentration and milk yield on the one hand and reductions in lactose concentration and milk quality on the other hand is presented. The physiological and biochemical basis for deterioration of milk quality in subclinically infected and in late lactation animals is reviewed and suggestions to improve the quality of milk produced by farmers and acquired by dairies are presented.

Use of DNA-based methods, such as real-time PCR, has increased the sensitivity and shortened the time for bacterial identification, compared with traditional bacteriology; however, results should be interpreted carefully because a positive PCR result does not necessarily mean that an infection exists. One hundred eight lactating dairy ewes (56 Manchega and 52 Lacaune) and 24 Murciano-Granadina dairy goats were used for identifying the main bacteria causing intramammary infections (IMI) using traditional bacterial culturing and real-time PCR and their effects on milk performance. Udder-half milk samples were taken for bacterial culturing and somatic cell count (SCC) 3 times throughout lactation. Intramammary infections were assessed based on bacteria isolated in ≥2 samplings accompanied by increased SCC. Prevalence of subclinical IMI was 42.9% in Manchega and 50.0% in Lacaune ewes and 41.7% in goats, with the estimated milk yield loss being 13.1, 17.9, and 18.0%, respectively. According to bacteriology results, 87% of the identified single bacteria species (with more than 3 colonies/plate) or culture-negative growth were identical throughout samplings, which agreed 98.9% with the PCR results. Nevertheless, the study emphasized that 1 sampling may not be sufficient to determine IMI and, therefore, other inflammatory responses such as increased SCC should be monitored to identify true infections. Moreover, when PCR methodology is used, aseptic and precise milk sampling procedures are key for avoiding false-positive amplifications. In conclusion, both PCR and bacterial culture methods proved to have similar accuracy for identifying infective bacteria in sheep and goats. The final choice will depend on their response time and cost analysis, according to the requirements and farm management strategy.

The assumption, that metabolites derived from the activity of the mammary gland epithelial cells reflect changes in milk secretion and its coagulation properties, was tested in dairy cows. The experiment included cows with uninfected udders and cows with one of the glands infected by different bacteria specie. Analysis were carried at the cow level (including all four glands), or at the gland level. High and significant correlations among the concentrations of lactose, glucose, glucose-6-posphate, milk related respiratory index (the ratio between the concentrations of citrate/lactate+malate in milk) and milk-derived glycolytic index (the ratio between glucose-6-phosphate and glucose in milk) and milk clotting parameters were found. The physiological basis for these relations and their ability to predict the deterioration in milk quality in subclinically infected glands and in glands previously clinically infected with Escherichia coli are discussed.

Escherichia coli is one of the most frequently diagnosed causes of bovine mastitis, and is typically associated with acute, clinical mastitis. The objective of the present study was to evaluate the long term effects of intramammary infections by E. coli on milk yield and quality, especially milk coagulation. Twenty-four Israeli Holstein cows diagnosed with clinical mastitis due to intramammary infection by E. coli were used in this study. Mean lactation number, days in milk (DIM) and daily milk yield (DMY) at the time of infection was 3.3 ± 1.3, 131.7 days ± 78.6 and 45.7 L ± 8.4, respectively. DMY, milk constituents, somatic cells count (SCC), differential leukocytes count and coagulation parameters were subsequently assessed. Two patterns of inflammation were identified: 'short inflammation', characterized by15% decrease in DMY and30 days until return to normal (n = 5), and 'long inflammation', characterized by15% decrease in DMY and30 days to reach a new maximum DMY (n = 19). The estimated mean loss of marketable milk during the study was 200 L/cow for 'short inflammation' cases, and 1,500 L/cow for 'long inflammation' ones. Significant differences between 'short' and 'long inflammation' effects were found in almost all parameters studied. Long-term detrimental effects on milk quality were found regardless of clinical or bacteriological cure of affected glands.

The aim of this study was to verify the existence of a nitric oxide (NO) cycle in goat milk and to study how changes in it affect milk composition during subclinical mastitis. Fifteen lactating dairy goats in which one udder-half was free from bacterial infection and the contra-lateral one was naturally infected with various species of coagulase-negative staphylococci were used. In comparison to uninfected glands, subclinical mastitis was associated with a decrease in milk yield, lactose concentration, and curd yield and an increase in nitrite and nitrate concentrations and with measurements reflecting increased formation of NO-derived free-radical nitrogen dioxide. The occurrence of NO cycling in goat milk was largely confirmed. The increase in the NO-derived stress during subclinical infection was not associated with significant increase in oxidatively modified substances, 3-nitrotyrosine, and carbonyls on proteins, but with increased levels of peroxides on fat. However, the relatively modest nitrosative stress in subclinically infected glands was associated with significant reduction in total antioxidant capacity and vitamin C levels in milk. We concluded that subclinical mastitis in goats caused by coagulase-negative staphylococci imposes negative changes in milk yield, milk quality for cheese production, and negatively affects the nutritional value of milk as food. Thus, subclinical mastitis in goats should be considered as a serious economic burden both by farmers and by the dairy industry.

This review focuses on recent evidence showing that various types of udder inflammation (mastitis) are associated with increased concentration of NO˙-derived metabolites, nitrite and nitrate, and oxidatively modified organic components under commercial farming and experimental conditions. In milk, NO˙ constantly cycles through: (i) auto oxidation to nitrite; (ii) hydrogen peroxide-dependent conversion of nitrite into NO2˙ by lactoperoxidase; (iii) interaction of NO2˙ with thiyl (RS˙) radicals on proteins formed by NO˙ to generate S-nitrosothiols; and (iv) disintegration of NO˙ from S-nitrosothiols, which completes the cycle. The main mechanism which restrains this cycle is conversion of nitrite to nitrate by catalase in a hydrogen peroxide dependent manner. The main source of hydrogen peroxide in milk derives from the oxidation of secreted hypoxanthine and xanthine by xanthine oxidoreductase. Formation of NO2˙ has an important role in the glandular innate defense system because it has bactericidal effects towards major pathogens that infect the mammary gland. However, increased formation of NO2˙ that occurs during mastitis and extended storage of milk for more than three days, even when kept in cold, dark conditions, induce nitrosative stress on milk organic components. Nitrosative stress in milk is reflected by a marked increase in the concentration of 3-nitrotyrosine, carbonyl and lipid peroxides. Thus, it is possible that current criteria for dairy plants acceptance of milk overlook important information on milk safety for consumption by humans. The literature regarding the presence of nitrite and nitrate in milk under experimental, farm and marketed milk is reviewed and the potential implications discussed. Relevant conclusions to improve safety of milk for human consumption are derived, and the particular importance in applying such recommendations for milk designated for the manufacturing of infant formulas is outlined.

The recent burst of duplication and divergence of the bovine PLA2G2D genes is considered typical of immune response genes, and it was recently shown that PLA2G2D is abundantly expressed in mouse leukocytes and acts as an immunosuppressive phospholipase. Analysis of 1,143 Holstein bulls indicated that the four common haplotypes spanning PLA2G2D display copy number variation ranging from 1 to 4 per haploid genome. Association of the fourth haplotype with negative total merit remained significant (P

Previously it was shown that a low molecular mass fraction isolated from the proteoseepeptone preparation of milk, fraction E, inhibited milk coagulation. Here, the composition and molecular mass of fraction E, and its effect on milk clotting parameters, was investigated to better understand its mechanism of action. Fraction E comprised casein-derived peptides of 1e3 kDa rich in phosphorus residues. Fraction E content increased substantially in milk from glands infected with Escherichia coli and Streptococcus dysgalactiae, and during storage of the milk. However, the specific activity of fraction E on milk clotting parameters was the same whether sampled from healthy, infected or stored milk. The inhibitory effect was reversible on adding 0.75 mM CaCl2, suggesting that chelation of Ca by fraction E was involved in the inhibitory mechanism. However, only partial recovery was achieved and an excess of Ca was required, suggesting the involvement of additional pathways in the process.

Escherichia coli is a major agent of bovine mastitis worldwide. However, specific E. coli virulence factors associated to pathogenicity during intra-mammary infections are yet unknown and this pathotype remains uncharacterized. The objectives of the present work were to assess the presence of a wide range of known virulence factors in a large set of E. coli strains isolated from bovine mastitis (mastitis set) and to study the genotypic distribution of strains in the mastitis set in comparison to a set of strains isolated from cows’ environment in dairy farms (environmental set). Virulence factors were assessed by DNA hybridization microarray. The three most prevalent virulence factors found in the mastitis set were lpfA (long polar fimbriae), iss (increased serum resistance) and astA (enteroaggregative E. coli heat-stable enterotoxin 1). None, however, characterized the majority of these strains. Genotyping was assessed by ECOR phylogenetic grouping, multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Strains in the mastitis and environmental sets were differentially distributed into ECOR phylogenetic groups; groups A and B1 being the most prevalent ones. Multiple MLST strain types were found in the two sets of strains, but only a few were common to both, and diversity was higher in the environmental set. A variety of PFGE patterns were found in the mastitis and environmental sets. Two clusters comprising mostly highly similar mastitis strains were identified. The results confirm that mastitis E. coli strains mostly lack known E. coli virulence factors. In addition, it is shown that the genotypic diversity of mastitis strains does not reflect the diversity found in the environmental E. coli population.

Passive vaccination is used to treat a wide range of infections and cancer. However, this approach has some limitations. An immune complex termed Y-complex was developed to intensify the effect of the passive vaccine. The complex is composed of a microbead that carries specific antibodies and an inducer. It enables targeting of pathogen or abnormal cells, and stimulation of a desired response by innate immune cells, depending on the inducer. The production and efficacy of Y-complex as a passive immune prophylaxis is demonstrated in this study by its use in treating cow mastitis. In an in vitro assay, Y-complex inhibited propagation and induced phagocytosis of bacteria. In challenge experiments, cows were inoculated through the udder with Escherichia coli or Streptococcus dysgalactiae. Following treatment with Y-complex, no bacteria were isolated in the milk and N-acetyl-ß-D-glucosaminidase activity had returned to normal levels. Thus the Y-complex approach can be used as an effective treatment for mastitis. Due to its modularity, this approach may serve as a treatment for a variety of disease agents.