G. Leitner

Development of a Staphylococcus aureus vaccine against mastitis in dairy cows. I. Challenge trials.

A vaccine composed of three field isolates of Staphylococcus aureus (S. aureus) derived from cases of mastitis in cows was developed. The vaccine was administered to nine uninfected cows while 10 other cows were used as controls. All cows were challenged with a highly virulent S. aureus strain administered into two quarters of each cow. Quarters were tested for clinical signs, secretion of S. aureus, and somatic cell count (SCC). No systemic effects were observed in any of the cows, vaccinated or control. Vaccinated cows had 70% protection from infection compared with fewer than 10% in the controls. Moreover, all quarters challenged in the vaccinated cows, regardless of whether they were successfully infected or not with S. aureus, exhibited very mild inflammatory reactions, identified by their low SCCs (<100,000).

Udder disease etiology, milk somatic cell counts and NAGase activity in Israeli Assaf sheep throughout lactation.

Bacterial pathogens causing udder infections in Israeli Assaf dairy sheep were identified and changes occurring throughout lactation were monitored to study the correlation between the contaminant and the severity of the infection, as measured by somatic cell count (SCC) and NAGase tests. A total of 159 Israeli Assaf dairy sheep on one farm, in their first (69), second (13) or third and more (77) lactations were included in this study. Udder halves were tested for bacterial condition, SCC and NAGase activity 2-3 weeks post lambing and every 4 weeks after until drying-off. At first sampling, in 60.7% (193/318 quarters) of the halves no bacterial growth (NBG) was detected. Different species of coagulase negative staphylococci (CNS) were the main pathogen group in infected udders. Streptococci were isolated from 14 halves, most of them in the two udder halves. The percent of udder infection in sheep in their third or further lactations was 2.8 greater (P<0.05) than in that of sheep in their first lactation. During the lactation, 90.6% of the halves did not change their classification status, suggesting that most infections occur before lambing and/or during the following first few days. The arithmetic mean of SCC and NAGase of total half udder milk and samplings (during the lactation) were 1144+/-48x10(3)cells/ml and 49.4+/-2.5, respectively. The average SCC in the milk of halves classified as NBG was 321+/-35x10(3)cells/ml and was not significantly changed during the lactation period. In halves infected with CNS, average SCC was 1371+/-150x10(3)cells/ml at the first testing and increased to 2129+/-347x10(3)cells/ml at drying-off. No significant differences were found in SCC and NAGase activity between the different species of the CNS. The mean SCC over the types of bacteria isolated, lactation number and days in lactation was significantly different (P<0.0001). In 4% of the halves, from all samples, SCC was above 5000x10(3)cells/ml although no bacteria were detected in their milk. The higher SCC in the CNS infected halves contrasted with the more moderate SCC found in dairy cows similarly infected, suggesting that the sheep udder has a lower resistance and an augmented immunological response against this group of bacteria. Thus, this should be considered accordingly in schemes for sheeps milk quality payment.

Udder infection and milk somatic cell count, NAGase activity and milk composition—fat, protein and lactose—in Israeli-Assaf and Awassi sheep

The present study aimed to identify the pathogens that cause subclinical udder infections in Israeli dairy sheep and evaluate their influence on milk yield and composition. Eight hundred and fifteen Israeli-Assaf and Israeli-Awassi dairy sheep were surveyed. More than half of the sheep were in their third or higher lactation (513/815 sheep) and in 14 out of 20 flocks; the sheep were in their peak (second to third month) of lactation. The percentage of bacteriological infected udders in the flocks ranged from 8.6 to 64.3%. The effect of the bacteriological infection on somatic cells count (SCC) was significant ( P> 0.001). Various species of coagulase-negative staphylococci (CNS) mainly S. chromogenesand S. epidermidis, formed the main pathogen group in infected udders. Lactation number did not significantly influence either the infection rate of udder halves or the milk SCC, although the percentage of udder halves with no bacteriological findings found was higher at first lactation than at the second and third lactation. Milk yield was significantly higher in uninfected than in infected halves. Milk composition—fat, proteins and lactose—varied among flocks, with mean total protein lower in uninfected halves than in infected ones and lactose higher in uninfected than in infected halves.

Identification of a bovine mastitis Escherichia coli subset.

Eleven Escherichia coli isolates from clinical bovine mastitis cases (mastitic strains) and 11 from the cowshed environment (environmental strains) were compared, to determine if the former were a subset of the latter. The mastitic and environmental strains could not be distinguished according to O antigen and antibiotic sensitivity. All mastitic isolates showed significantly (P<0.0001) faster growth in milk and faster lactose fermentation than most (approximately 64%) environmental strains, but growth rates in nutrient broth did not differ. The rates of lactose fermentation and growth in milk were positively correlated. Adhesion and phagocytosis of mastitic strains by bovine PMN were significantly (P<0.0001) lower than those of environmental strains, and correlated negatively with growth in milk and lactose fermentation. The average percentages of killing by bovine leukocytes in the two sources were not statistically different. All mastitic strains were serum sensitive, whereas most ( approximately 72%) environmental ones were resistant. Finally, pulse-field gel electrophoresis revealed two main pulse type clusters, sharing a similarity coefficient of 79%. Cluster 1 comprised only environmental strains, whereas cluster 2 comprised mostly mastitic strains and only three environmental ones. Four mastitic strains shared a similarity coefficient of less than 74% with the other strains and were not included in the clusters. Our results suggest that clinical bovine mastitis E. coli isolates may form a subset of the general environmental E. coli population; they seem better able to multiply in the udder medium and to evade the host cellular innate immune response, and are genetically distinct from most environmental strains.

Mammary infection with Staphylococcus aureus in cows: progress from inoculation to chronic infection and its detection.

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-beta-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.

Staphylococcus aureus vaccine against mastitis in dairy cows, composition and evaluation of its immunogenicity in a mouse model

Bovine mastitis caused by Staphylococcus aureus (S. aureus) is a most important infection disease that affects both the quality and the quantity of milk production. Antibiotic therapies formulated for intramammary use are generally unsuccessful in eliminating existing S. aureus infections. Vaccination is a logical approach to the control of S. aureus udder infections. However, to date commercially available S. aureus vaccine have shown limited efficacy under field conditions, mainly due to the paucity of information regarding relevant antigens which will induce a broad spectrum immunization. In the present paper the attempt to develop a new vaccine designated MASTIVAC I is described. MASTIVAC I is composed of three strains of S. aureus namely: VLVL8407; ZO3984 and BS449 which were isolated from clinical and sub-clinical cases of bovine mastitis. A mouse model was used to evaluate the S. aureus specific antibody production and protection of mice against virulent S. aureus strains. The results obtained showed that this vaccine exhibits a broad spectrum of antigenic and immunogenic properties that protects mice from homologues and hetrologous S. aureus challenge.

Efficacy of dry-off treatment in sheep

The efficacy of dry-off treatment of sheep in curing chronic intramammary infections and preventing new ones during the dry-off period and during the following lactation was studied in the present work. A total of 85 Israeli-Assaf dairy sheep on one farm in their first, second, third or more lactations were divided into pairs according to bacteriological condition, somatic cell count (SCC), NAGase activity and lactation number, and were then randomly allocated to one of two groups: one group to receive drying-off treatment, the second as an untreated control. At drying off, sheep in the treatment group received intramammary treatment with a commercial cattle dry-off treatment, which is a combination of penicillin, nafcillin and dihydrostreptomycin. n nAt the first sampling (I), 15–20 days after lambing, cure rates (CR) of 64.9 and 6.5% for the treated and control groups, respectively were significantly different (P<0.001). At the second (II) sampling, 35–50 days after lambing, no change was found in the CR of the control sheep while that of the treated group decreased to 46.4%; this difference was also found to be significant (P<0.005). New infection rates (NIRs) in the treated and control groups were 15.6 and 28.6%, respectively, at sampling I and 27.3 and 42.9%, respectively, at sampling II. Although the NIRs were lower in the treated group, the difference from the control group was not significant. The new infections were mainly due to different coagulase-negative staphylococci. Overall, in both samplings SCCs and NAGase activities were significantly higher (P<0.05) in the infected than in the non-infected halves. No significant differences in these parameters were found between treated and control halves that exhibited no infection nor between those found to be infected. However, at sampling II, the mean SCC and NAGase activity in the infected halves were higher in the treated than in the control ones. Antibiotic residues were detected in five of the 25 sheep sampled on the first day, in three out of 25 on the second day and in only in two sheep on the third and fourth days after lambing. On the fifth day no antibiotic residues were detected. n nThese data suggested that dry-off treatment could reduce intramammary infection and somatic cell count of dairy sheep, but other methods are required to both prevent new infection and maintain healthy udders in the herd.

Genotyping and virulence factors assessment of bovine mastitis Escherichia coli.

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.

Feedback-Based, System-Level Properties of Vertebrate-Microbial Interactions

Background Improved characterization of infectious disease dynamics is required. To that end, three-dimensional (3D) data analysis of feedback-like processes may be considered. Methods To detect infectious disease data patterns, a systems biology (SB) and evolutionary biology (EB) approach was evaluated, which utilizes leukocyte data structures designed to diminish data variability and enhance discrimination. Using data collected from one avian and two mammalian (human and bovine) species infected with viral, parasite, or bacterial agents (both sensitive and resistant to antimicrobials), four data structures were explored: (i) counts or percentages of a single leukocyte type, such as lymphocytes, neutrophils, or macrophages (the classic approach), and three levels of the SB/EB approach, which assessed (ii) 2D, (iii) 3D, and (iv) multi-dimensional (rotating 3D) host-microbial interactions. Results In all studies, no classic data structure discriminated disease-positive (D+, or observations in which a microbe was isolated) from disease-negative (D–, or microbial-negative) groups: D+ and D– data distributions overlapped. In contrast, multi-dimensional analysis of indicators designed to possess desirable features, such as a single line of observations, displayed a continuous, circular data structure, whose abrupt inflections facilitated partitioning into subsets statistically significantly different from one another. In all studies, the 3D, SB/EB approach distinguished three (steady, positive, and negative) feedback phases, in which D– data characterized the steady state phase, and D+ data were found in the positive and negative phases. In humans, spatial patterns revealed false-negative observations and three malaria-positive data classes. In both humans and bovines, methicillin-resistant Staphylococcus aureus (MRSA) infections were discriminated from non-MRSA infections. Conclusions More information can be extracted, from the same data, provided that data are structured, their 3D relationships are considered, and well-conserved (feedback-like) functions are estimated. Patterns emerging from such structures may distinguish well-conserved from recently developed host-microbial interactions. Applications include diagnosis, error detection, and modeling.

Long term effects of Escherichia coli mastitis

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 by <15% decrease in DMY and <30 days until return to normal (nu2009=u20095), and long inflammation, characterized by >15% decrease in DMY and >30 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.