Gabriel Leitner

Effect of subclinical intramammary infection on somatic cell counts, NAGase activity and gross composition of goats' milk

The study was aimed at identifying the pathogens causing subclinical udder infections in representative Israeli dairy goat herds and determining their effect on milk quality. Five hundred goats in ten flocks of various breeds and crossbreeds were surveyed. Of the 500 goats, 13.4% were in their first lactation, 36.4% were in their second lactation and 50.2% were in their third or higher lactation. Percentages of udder halves with subclinical intramammary infection in the flocks ranged from 35 to 71%. The effect of the bacteriological infection on somatic cells count (SCC) was significant (P<0.001). Various species of coagulase-negative staphylococci (CNS), mainly Staphylococcus caprae and Staphylococcus epidermidis, were the main pathogens in infected udder halves. Lactation number did not significantly influence either infection rate of udder halves or SCC, although the percentage of udder halves with no bacteriological findings was higher at the first lactation than at the third lactation. Milk composition (fat, protein and lactose) varied among flocks, with lower mean total protein in uninfected halves than in infected ones and higher lactose in uninfected than infected halves.

Sex-related differences in immune response and survival rate of broiler chickens

In a comparison between male and female broiler chicks, the mortality rate of males was found to be significantly higher than that of females, starting from the second week of age until marketing at 7 or 8 weeks of age. The main causes of death during this period were various infectious diseases. This observation was explained by differences in the activity of humoral and cell-mediated immune responses between the sexes. In tests of antibody responses of young chicks to a variety of antigens (bacterial-E. coli, viral-Newcastle disease virus, and protein antigen-bovine serum albumin), females responded 24-72 h earlier than males and with higher peak antibody titers. In-vitro proliferation of T-lymphocytes to purified protein derivative and E. coli showed an earlier and greater response in females. The correlation between immune responsiveness and survival, as tested by challenging vaccinated chicks with pathogenic E. coli, showed a significantly higher mortality rate in vaccinated males, that was correlated with their lower antibody titer. We concluded, therefore, that sex-related differences in mortality rates of broiler chicks may result from a less efficient immune response in males.

Association of conception rate with pattern and level of somatic cell count elevation relative to time of insemination in dairy cows

The aim was to evaluate the effects of mastitis, determined by the pattern and level of somatic cell count (SCC) around first artificial insemination (AI), on conception rate (CR). Data from 287,192 first AI and milk records covering a 7-yr period were obtained from the Israeli Herd Book. Analyses examined the association of probability of conception with SCC elevation relative to timing of AI, using generalized linear mixed models. A SCC threshold of 150,000 cells/mL of milk was set to distinguish between uninfected cows and cows with mastitis. Accordingly, cows with high SCC before and low SCC after AI were designated cured, those with low SCC before and high SCC after AI were designated newly infected, and cows with high SCC before and after AI were designated chronic (likely subclinical) mastitic cows. Compared with uninfected cows, the cured, newly infected, and chronic subgroups showed reduced CR (39.4±0.1, 36.6±0.2, 32.9±0.3, and 31.5±0.2, respectively). In the chronic, subclinical group, probability of conception was lowered by 14.5% in the mild and moderately elevated SCC subgroups and by 20.5% in cows with high SCC elevation compared with the uninfected group (CR of 29.7 vs. 39.4%, respectively). A single high elevation of SCC (>10(6) cells/mL on only 1 milk test day) lowered the probability of conception by 23.6% when it occurred during the 10 d immediately before AI, but not when it occurred earlier. For 30 d after AI, probability of conception was lowered by about 23%, as reflected in a CR of about 27% compared with the uninfected group. Probability of conception was lowered in cows with uterine and foot health problems (33.9%), in multiparous cows (34.1%), and in cows in the summer (29.1%), but no interactions with mastitis were detected. Results indicate that SCC elevation around AI, typical for subclinical mastitis, was associated with a significant reduction in probability of conception, and that even mild SCC elevation reduced CR. Severe elevation of SCC before AI, typical for clinical intramammary infection, reduced the probability of conception.

Vaccine development for the prevention of staphylococcal mastitis in dairy cows.

Staphylococci are the most common and costly mammary disease of dairy cattle worldwide. Target of RNAIII Activating Protein (TRAP), a membrane associated 167AA protein, is highly conserved among staphylococci and was shown in Staphylococcus aureus to be involved in bacterial stress response. The aims of this study were to test the safety and efficacy of recombinant TRAP (rTRAP) vaccine in dairy animals. The vaccine was safe as 2-3 subcutaneous injections of rTRAP (54-100 μg) with adjuvant ISA 206 to cows and goats did not lead to any abnormal symptoms of sensitivity to the vaccine. The rTRAP vaccine was immunogenic and caused the induction of a humoral immune response that remained high for at least 160 d post second immunization. The rTRAP vaccine was efficacious; at parturition only 13.5% (5/37) heifers in the immunized group were infected with Staphylococcus chromogenes as compared to 42.9% (18/42) in the non-immunized group. Additionally, when cows were immunized in mid-lactation, the difference between somatic cell count (SCC) in immunized and control animals was profound (45 ± 7 vs. 470 ± 194, respectively). At the same time, the difference in milk yield was also evident (48.3 ± 1.4 L d(-1)vs. 44.3 ± 0.9 L d(-1), respectively). Put together, these studies indicate the value of the rTRAP vaccine in preventing new udder infections by staphylococci, which significantly lead to lowered SCC and some increase in milk yield. TRAP is conserved among all strains and species and is constitutively expressed in any strain of S. aureus or CNS tested so far, including those isolated from cows. TRAP may thus serve as a universal anti-staphylococcus vaccine.

Immediate and carryover effects of Gram-negative and Gram-positive toxin-induced mastitis on follicular function in dairy cows

This study compared immediate and carryover effects of mastitis induced by Gram-negative endotoxin (E. coli LPS) and Gram-positive exosecretions (Staph. aureus ex.) on preovulatory follicle function. Synchronized, uninfected cyclic lactating Holstein cows were treated with PGF(2α) on day 6 of the cycle and 36 h later, a dose of either E. coli LPS (n = 8), S. aureus ex. (n = 10), or saline (n = 9) was administered into the mammary gland. Follicular fluids and granulosa cells were aspirated 6 h later from the preovulatory follicles and cows were treated with GnRH. This (cycle 1; immediate effect) was repeated three times (excluding the mammary injections) to induce three 7 d cycles (cycles 2, 3, and 4; carryover effect). E. coli LPS increased body temperature, plasma cortisol concentration, and somatic cell count (SCC), whereas S. aureus ex. induced a minor, subclinical elevation of SCC and slight rise (NS) in body temperature and cortisol concentration. Follicular estradiol, androstenedione, and progesterone concentrations in the E. coli LPS group decreased (P < 0.05) in cycle 1 to about 40%, 13%, and 35%, respectively, of control levels, whereas in the S. aureus ex. group, only estradiol decreased (P < 0.05), to 56% of control concentrations. In cycles 3 and 4, follicular steroids in the E. coli LPS group returned to control concentrations, whereas in the S. aureus ex. group, follicular concentrations of estradiol and androstenedione were lower (P < 0.10) than in controls. In the control group, the concentrations of all follicular and circulating steroids remained stable (P > 0.05) throughout the study. Follicle size was similar in all groups, but the S. aureus ex. treatment caused a decrease (P < 0.02) in the number of follicles developed in cycles 3 and 4. The mRNA expression of steroidogenic genes and LHCGR in the granulosa cells was not affected (P > 0.05) by either treatment during the study, except for a tendency toward lower (P < 0.1) expression in cycle 1 and lower (P < 0.05) expression in cycle 4 of the latter in the S. aureus ex. group. Strain levels, such as SCC and body temperature, following toxin injection correlated well with the magnitude of the immediate decline in follicular steroids. As is typical for Gram-negative clinical events, E. coli LPS-induced acute mastitis caused immediate, short-term, but not long-term impairment of follicular responses, whereas the Gram-positive S. aureus ex.-induced subclinical mastitis exhibited both immediate and carryover disruptive effects on preovulatory follicle function.

The Interrelationships between Lactose Intolerance and the Modern Dairy Industry: Global Perspectives in Evolutional and Historical Backgrounds

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.

Outbreak of subclinical mastitis in a flock of dairy goats associated with atypical Staphylococcus haemolyticus

Staphylococcus haemolyticus is a pathogen frequently isolated from dairy cows and small ruminants. However, it always appears in only a few animals and not as a major pathogen. Recently, in a dairy goat herd of approximately 250 milking animals, 25.6% (46/180 goats) had milk cultures with atypical highly mucoid colonies accompanied by elevated somatic cell counts. The isolates were identified as Staph. haemolyticus. The present study describes the steps used in an attempt to identify the bacterium and to compare it with other coagulase-negative staphylococci (CNS) including Staph. haemolyticus. Species identification performed with the API STAPH-IDENT 32 kit showed >99.4% identity confirmed by 16S rDNA sequencing tests. Microscopically the atypical Staph. haemolyticus strains showed unique cuboidal tetrad clusters reminiscent of those of the genus Sarcina. The outbreak caused by an atypical CNS underlines the need for accurate biochemical and genetic methods for ultimate identification of CNS to the species level.

Genomic Comparative Study of Bovine Mastitis Escherichia coli.

Escherichia coli, one of the main causative agents of bovine mastitis, is responsible for significant losses on dairy farms. In order to better understand the pathogenicity of E. coli mastitis, an accurate characterization of E. coli strains isolated from mastitis cases is required. By using phylogenetic analyses and whole genome comparison of 5 currently available mastitis E. coli genome sequences, we searched for genotypic traits specific for mastitis isolates. Our data confirm that there is a bias in the distribution of mastitis isolates in the different phylogenetic groups of the E. coli species, with the majority of strains belonging to phylogenetic groups A and B1. An interesting feature is that clustering of strains based on their accessory genome is very similar to that obtained using the core genome. This finding illustrates the fact that phenotypic properties of strains from different phylogroups are likely to be different. As a consequence, it is possible that different strategies could be used by mastitis isolates of different phylogroups to trigger mastitis. Our results indicate that mastitis E. coli isolates analyzed in this study carry very few of the virulence genes described in other pathogenic E. coli strains. A more detailed analysis of the presence/absence of genes involved in LPS synthesis, iron acquisition and type 6 secretion systems did not uncover specific properties of mastitis isolates. Altogether, these results indicate that mastitis E. coli isolates are rather characterized by a lack of bona fide currently described virulence genes.

Milk metabolites as indicators of mammary gland functions and milk quality

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.

Lipopolysaccharide challenge of the mammary gland in bovine induced a transient glandular shift to anaerobic metabolism

Support of milk production in modern dairy cows demands a large proportion of its own metabolic resources, such as glucose, which might be required under stressful situations. The aim of the experiment was to test the hypothesis that acute immune stress shifts oxidative metabolism to glycolysis. Two mammary quarters in 6 Holstein cows were infused with lipopolysaccharide (LPS), whereas the 2 counter quarters served as controls to the treatment. An additional 6 cows were infused with saline and served as running controls. The LPS challenge induced dramatic transient increases in milk lactate (75-fold) and malate (11-fold) concentrations (both markers of glycolysis) at 24h posttreatment. No significant changes in lactate and malate concentrations were recorded in control quarters and control animals, indicating that the effect of LPS was restricted to the treated gland. The LPS challenge induced a dramatic transient decrease in milk yield, and lactose and citrate (a marker of mitochondrial metabolism) secretion at 24h posttreatment. The kinetics were inversely proportional to those of lactate and malate concentrations. Thus, our data suggest that LPS challenge induces acute conversion of epithelial cell metabolism from principally mitochondrial-oxidative to principally cytosolic (glycolytic), which allows the diversion of metabolic resources normally used to synthesize milk to support the immune system. An in vitro bacterial growth test showed that concentrations of lactate, malate, and lactose equivalent to those found in the in vivo experiment delayed and reduced the growth of a pathogenic Escherichia coli strain, suggesting that they play a role in diminution of bacterial multiplication in the mammary gland.