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We studied the mammary immune response to different mammary pathogenic Escherichia coli (MPEC) strains in cows, hypothesising that the dynamics of response would differ. E. coli is 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 an E. coli strain that is non-pathogenic to the bovine mammary gland.
Intramammary infection (IMI), comprises a group of costly diseases affecting dairy animals worldwide. Many dairy parlours are equipped with on-line computerised data acquisition systems designed to detect IMI. However, the data collected is related to the cow level, therefore the contribution of infected glands to the recorded parameters may be over estimated. The present study aimed at evaluating the influence of single gland IMI by different bacteria specieson the cow's overall milk quality. A total of 130 cows were tested 239 times; 79 cows were tested once and the others were examined 2–8 times. All of the analysed data refer to the number of tests performed, taking into account the repeated testing of the same cows. Of the cows tested ~50% were free of infection in all 4 glands and the others were infected in one gland with different coagulase negative staphylococci (CNS), Streptococcus dysgalactiae, or were post infected with Escherichia coli (PIEc), i.e., free of bacterial infection at the time of sampling but 1–2 months after clinical infection by E. coli. Overall, infection with bacteria had significant effects on somatic cell count (SCC) and lactose concentration. Examining each bacterium reveals that the major influence on those parameters was the sharp decrease in lactose in the PIEc and curd firmness in PIEc and Strep. Individual gland milk production decreased ~20% in Strep. dysgalactiae- and ~50% in PIEc-infected glands with respect to glands with no bacterial findings. Significant differences were found in lactose, SCC, rennet clotting time and curd firmness in the milk of infected glands and among those, these parameters were significantly higher in Strep. dysgalactiae and PIEc than in CNS infected cows. The current results using quarter-milking reinforces the importance of accurate IMI detection in relation to economic and welfare factors, and moreover, emphasises the need for technical sensing and constant reporting to the farmer about changes in the milk quality of every animal.
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.
Real-time analysis of milk coagulation properties as performed by the AfiLab™ milk spectrometer introduces new opportunities for the dairy industry. The study evaluated the performance of the AfiLab™ in a milking parlor of a commercial farm to provide real-time analysis of milk-clotting parameters –Afi-CF for cheese manufacture and determine its repeatability in time for individual cows. The AfiLab™ in a parlor, equipped with two parallel milk lines, enables to divert the milk on-line into two bulk milk tanks (A and B). Three commercial dairy herds of 220 to 320 Israeli Holstein cows producing ∼11 500 l during 305 days were selected for the study. The Afi-CF repeatability during time was found significant (P < 0.001) for cows. The statistic model succeeded in explaining 83.5% of the variance between Afi-CF and cows, and no significant variance was found between the mean weekly repeated recordings. Days in milk and log somatic cell count (SCC) had no significant effect. Fat, protein and lactose significantly affected Afi-CF and the empirical van Slyke equation. Real-time simulations were performed for different cutoff levels of coagulation properties where the milk of high Afi-CF cutoff value was channeled to tank A and the lower into tank B. The simulations showed that milk coagulation properties of an individual cow are not uniform, as most cows contributed milk to both tanks. Proportions of the individual cow's milk in each tank depended on the selected Afi-CF cutoff. The assessment of the major causative factors of a cow producing low-quality milk for cheese production was evaluated for the group that produced the low 10% quality milk. The largest number of cows in those groups at the three farms was found to be cows with post-intramammary infection with Escherichia coli and subclinical infections with streptococci or coagulase-negative staphylococci (∼30%), although the SCC of these cows was not significantly different. Early time in lactation together with high milk yield >50 l/day, and late in lactation together with low milk yield<15 l/day and estrous (0 to 5 days) were also important influencing factors for low-quality milk. However, ∼50% of the tested variables did not explain any of the factors responsible for the cow producing milk in the low – 10% Afi-CF.
The aims of this study were to test the assumption that tissue-type plasminogen activator (t-PA) and plasminogen (PG) are closely associated with the casein micelle and form a functional complex that rules casein degradation. This assumption was essentially verified for bovine milk under conditions wherein the plasmin system was activated by treatment with casein hydrolysate. It was also shown that urokinase-type PA (u-PA), the second type of plasminogen activator present in milk, was not involved in casein degradation. In agreement with previous studies, we show that treatment with casein hydrolysate precipitously reduced mammary secretion, disrupted the tight junction integrity (increase in Na+ and decrease in K+ concentrations), induced hydrolysis of casein, and activated various elements of the innate and acquired immune system. In the present study, we have identified t-PA as the principal PA, which is responsible for the conversion of PG to plasmin. It was found that t-PA and plasminogen are present in freshly secreted milk (less than 10 min from its secretion), suggesting that they are secreted as a complex by the mammary gland epithelial cells. Further research is needed to provide the direct evidence to verify this concept.
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.
During 1998–2002 outbreaks of Pseudomonas sp. mastitis among more than 15 Israeli sheep and goat dairy herds were observed. The animals presented a wide spectrum of clinical signs ranging from subclinical to gangrenous udder. Ninety-five isolates of Pseudomonas sp. were isolated from clinical and subclinical mastitis of 47 sheep, 17 goats and 31 cows from 34 different farms. Biochemical and genetic analyses revealed that the all-causative organism was Ps. aeruginosa. Selections of isolates were further analysed on the bases of colony morphology, biochemical traits and capacity to form biofilm. All the strains displayed a wide heterogeneity in all the tested traits. No association between bacterial isolates, farm of origin and type of animal was found. Pulsed-field gel electrophoresis and cluster analysis showed no clonality among the tested strains. The present study revealed that a large variety of Ps. aeruginosa strains may cause mastitis outbreaks in sheep, goat and cattle in Israel.
Although there has been little study of the origin of intramammary infection (IMI) in goats, a common view is that most bacterial infection in goats occurs during milking. In the present study, the dynamics of occurrence of udder infection during and between lactations in three Anglo-Nubian goat farms in Israel was monitored. Coagulase-negative staphylococci were the predominant bacteria in the IMIs. We found that about 15% of the yearling does were already infected with bacteria when they joined the flock, whereas about 8% of the goats that dried-off returned with new IMIs. Moreover, virtually none of the goats acquired infection during lactation. Thus, our study showed that the aetiology of IMI in goats is very similar to that in dairy cows. A preventive treatment during the dry period should, therefore, be considered as an effective means of reducing the current rate of bacterial infections in goats.
The involvement of Staphylococcus aureus exosecretions in bovine udder infection (Younis et al. 2003) suggests that four different monomer protein bands appearing between 36 and 31 kDa, are associated with the severity of the cow's infection response. Three out of these four bands have been identified by means of protein sequencing. Band B, with a MW of 35 kDa was identified as Panton-Valentaine leucocidin LukF'-PV chain- Staph. aureus; band C, with a MW of 32 kDa was identified as leucocidin chain LukM precursor- Staph. aureus; and band D was found to be similar, but not identical, to phosphatidylinositol-specific phospholipase-C-X. Bands B and C were purified by gel filtration using FPLC. The ability of these proteins to induce udder inflammation in vivo, and proliferation response in vitro and cytokine secretion were tested for both the crude exosecretions and purified bands. Three cows were inoculated intracisternally, with three quarters receiving either 0·007–0·008 mg (as total proteins) of Staph. aureus FR2449/1 bacterial exosecretion, pooled fraction 39–41 (bands B and C), or culture broth medium. The fourth quarter was left free as a control. Quarters that received fraction 39–41 of Staph. aureus FR2449/1, exhibited induced inflammation, which was indicated by increased somatic cell count and enhanced NAGase activity that was significantly higher than that of the original Staph. aureus FR2449/1 bacterial exosecretion. Proliferation tests of bovine blood lymphocytes in vitro showed that the pooled fraction 39–41 stimulated bovine proliferation of mononuclear cells much more than the original Staph. aureus FR2449/1 bacterial exosecretion. Secretion of TNF-α, IL-1β, IL-6 and IL-8 was in accordance with the contents of LukF'-PV and LukM precursor in the exosecretions. The results suggest that LukM/LukF' induce inflammation into the udder by a mechanism similar to that of LPS or by a unique mechanism(s) which requires further investigation.
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.
The progress of Staphylococcus aureus infection from inoculation to the
early chronic stage was examined in 12 Israeli-Holstein cows (four primiparous and
eight multiparous) for up to 48 d after inoculation. Before inoculation, the
primiparous cows were free from any infection and the multiparous cows were
infected by coagulase-negative staphylococci. Two quarters in each cow were
inoculated intracisternally following milking with 2000 cfu of a local prevailing
Staph. aureus strain, VL-8407. Infection was established in 21 out of 24 quarters. The
control quarters remained free from infection during the study, with no significant
change in function. No statistically significant differences were found between
primiparous and multiparous cows in the responses examined. Somatic cell count
(SCC) increased within 24 h of inoculation and remained high for the duration of the
study. In the infected quarters mean ln (SCC) increased within 24 h from 9·9±0·5
before inoculation to 13·0±0·2 after inoculation; most of the cells were neutrophils.
N-acetyl-β-glucosaminidase activity, expressed as ln (nnmol/min per l), was
increased from 0·9±0·6 to 2·4±0·2 by inoculation, and was highly correlated with
SCC. The Staph. aureus count fluctuated with no particular relationship with SCC.
The phagocytic activity of neutrophils was significantly lower in the inoculated than
in the control quarters and this difference increased with time after inoculation.
CD8+ T lymphocytes were the main subpopulation of lymphocytes found in
inoculated quarters. After inoculation, maximum but not minimum electrical
conductivity (EC) recorded during milking increased significantly. The rises in
maximum EC varied significantly among cows. The rises in SCC were associated with
a persistent increase in EC in only one of the eight cows examined. No clinical signs
were observed, and milk yield and composition were not affected during the study
period. The results suggest that some strains of Staph. aureus may induce a relatively
mild response in mammary glands of cows in mid lactation, and that the concomitant
development of such chronic Staph. aureus infections in two quarters may not be
detected by changes in the EC of composite milk and in the yield of the cow.
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