Naturally occurring mastitis disrupts developmental competence of bovine oocytes.

We examined the effects of naturally occurring mastitis on bovine oocyte developmental competence in vitro. Specifically, we investigated the effects of intramammary infection on the ovarian pool of oocytes (i.e., follicle-enclosed oocytes) and their ability to undergo in vitro maturation, fertilization, and further development to the blastocyst stage. Culled Holstein cows (n=50) from 9 commercial dairy farms in Israel were allotted to 3 groups according to somatic cell count (SCC) records of the last 3 monthly milk tests as well as of quarter samples collected before slaughter: (1) low SCC (n=7), (2) medium SCC (n=16), or (3) high SCC (n=27). Means of SCC values differed among low-, medium-, and high-SCC groups: 148,000, 311,000 and 1,813,000 cell/mL milk, respectively. Milk yield and days in milk did not differ among the 3 groups. Bacterial isolates included coagulase-negative staphylococci, Escherichia coli, Streptococcus dysgalactiae, or no bacteria found. Ovaries were collected at the abattoir and brought to the laboratory. Cumulus oocyte complexes were recovered separately from each cow and subjected individually to in vitro maturation and fertilization, followed by 8d in culture. The number of aspirated oocytes did not differ among groups, with a range of 17 to 21 oocytes per cow. The proportion of oocytes that cleaved into 2- to 4-cell-stage embryos (86.1 ± 3.4%) did not differ among groups. In contrast, mean percentages of embryos developed to the blastocyst stage on d 7 and 8 after fertilization were less in both medium- and-high SCC groups than in the low-SCC group (5.6 ± 2.3 and 4.1 ± 1.8 vs. 18.1 ± 4.6%, respectively). Additional analysis indicated that cleavage and blastocyst-formation rates did not differ among the bacterial types in the low-, medium-, and high-SCC groups. These are the first results to demonstrate that naturally occurring mastitis disrupts the developmental competence of the ovarian pool of oocytes, (i.e., oocytes at the germinal vesicle stage). The disruption was associated with elevation of SCC rather than bacterial type. The results may provide a partial explanation for the low fertility of cows that have contracted mastitic pathogens before insemination.

The effect of caprine arthritis encephalitis virus infection on production in goats.

Three consecutive years of monitoring 248 goats in the same flock, found that the first lactation milk yield was significantly higher in seronegative (578L) than in seropositive (447L) animals but this difference disappeared in the subsequent second to fourth lactations. No significant differences were found in the proportions of seronegative and seropositive does in the flock, the percentage of animals culled, the number of offspring, or in the number of cases of udder bacterial infection, irrespective of age. Removal of kids from their dams before suckling and the feeding of pasteurised colostrum resulted in reduced numbers of seropositive animals. Nevertheless, by approximately 24 months of age, 76.9% of these initially seronegative animals were seropositive, a factor that significantly contributed to flock seropositivity. This finding could be attributed to lateral virus transmission from seropositive to seronegative kids because of lack of segregation within the flock.

Concentrations of ganglioside type M1 and immunoglobulin G in colostrum are inversely related to bacterial infection at early lactation in cows.

The levels of IgG and ganglioside type M1 in the colostrum of cows and heifers were analyzed to examine their utility in predicting acquisition of intramammary infection (IMI) during the first weeks postpartum. In general, high levels of IgG and ganglioside type M1 in cows were associated with lower new incidence of IMI, and linear discriminate analysis based on these 2 variables yielded 69.4% successful classification into cows that did or did not acquire new IMI. This analysis was less successful in heifers because a high proportion of them joined the herd when already infected with bacteria in their udders. It is suggested that application of a wider range of measures that reflect the immune status would enable the identification of most cows prone to new IMI.

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.

Pseudomonas aeruginosa mastitis outbreaks in sheep and goat flocks: antibody production and vaccination in a mouse model.

Pseudomonas aeruginosa is most often associated with sporadic clinical mastitis. Outbreaks among more than 15 Israeli dairy sheep and goat herds presented clinical signs, including gangrenous infections. Characterization of the P. aeruginosa isolates revealed the presence of multiple environmental strains in the various farms, and it was hypothesized that the infected herds were temporarily immunocompromised. In spite of the variability of the isolates, because of the economic impact on the small-ruminant farms, we decided to search for common antigens that might induce an immune response that would protect the animals from the various P. aeruginosa strains present in the herds. Responses to selected isolates, representative of various animals and farms, were further studied in mouse models for capability to induce antibodies and to protect from homologous and heterologous challenge after immunization with an experimental primary vaccine. 1-D-SDS-PAGE tests revealed two patterns of protein bands among the various P. aeruginosa isolates, whereas the immunoblot and ELISA tests of sera of mice immunized with either MA-58, GE-61 monovalent, or (MA-58 + GE-61) bivalent bacterial vaccine preparations revealed two subgroups with low mutual cross-reactivity. Immunization with a vaccine prepared from either of the subgroups protected mice only from the homologues but not from the heterologues, whereas the bivalent vaccine protected mice from both subgroups.

Effect of bovine lactoferrin on a transmissible AIDS-like disease in mice.

The effect of bovine lactoferrin (bLF) was examined on an AIDS-like disease (ALD) in mice. Induction of disease was achieved by inoculation with infected cell-free plasma from diseased mice to uninfected ones. The effect of treatment with bLF was investigated when administered simultaneously with the virus, 20 days prior to infection, or 20 days after infection. Animals underwent clinical surveillance and enumeration of white blood cells (WBC) and lymphocytes, as well as fluorescent staining of CD4 and CD8 bearing cells. Simultaneous administration of bLF and virus did not affect the pattern of ALD progress along the course of the experiment. Pretreatment with bLF prior to virus inoculation abolished on day 21 the detrimental effect of viral infection that lasted for two months. An opposite outcome was observed when bLF was administered 20 days after the virus. It seems that bLF had played a preventive role for a restricted period of time. However, an adverse response was elicited when bLF was administered 20 days after viral infection.

Staphylococcus aureus exosecretions and bovine mastitis.

The role of Staphylococcus aureus and coagulase-negative staphylococcal exosecretions in bovine udder infection was tested by monitoring the cows' response to in vivo inoculation of bacterial exosecretions into udder quarters. Twenty Israeli-Holstein dairy cows were included in the study; two or three of the udder quarters of each cow were intracisternally inoculated with 0.04-0.05 mg/quarter (total proteins) of the various sterile bacterial exosecretions in a sterile pyrogen-free saline. Each udder was inoculated with two or three different bacterial exosecretions or placebo (Columbia Broth). Cows were monitored for 96 h post-inoculation for rectal temperature, heart and respiratory rates, alimentary tract activity (rumen contraction), udder temperature, pain, oedema and udder size. Milk samples were examined bacteriologically and for somatic cell count, N-acetyl-D-glucosaminidase (NAGase) activity and somatic cell differentiation. No enterotoxins (beta-G) or toxic shock syndrome toxin-1 were detected in response to any of the bacteria tested. Control quarters or those inoculated with Columbia Broth, showed similar NAGase and somatic cell count values throughout the experiment. Twelve of the 18 strains tested, induced inflammation in the inoculated quarters while six did not. Of the 12 strains causing local inflammation, only six were found significantly different from the control and were considered as high response (group 1). The other six that caused a local inflammation did not differ significantly from the control, and were considered to be moderate response (group 2). The six S. aureus isolates that did not cause an inflammatory response were considered to have low response (group 3). In all quarters inoculated with S. aureus bacterial exosecretions belonging to groups 1 and 2, the polymorphonuclear cells and macrophages were proportionally increased while CD4+ and CD8+ T-lymphocyte populations decreased. One-dimensional NuPAGE (7%) Tris-acetate gel electrophoresisof the bacterial exosecretions revealed four different bands appearing between 36 and 31 kDa, marked from top to bottom as A, B, C and D. An association was found between the combinations of expressed bands and the cow responses: the majority of the cases could be linked to the expression of bands B and C.

Immune cell differentiation in mammary gland tissues and milk of cows chronically infected with Staphylococcus aureus.

This study identifies and compares the distribution of mononuclear cells in the mammary gland tissues and milk of healthy and chronically infected with Staphylococcus aureus cows. Somatic cell counts (SCCs) during the 3 months before the study were > 1 x 10(6) cell/ml in the infected quarters and < 1 x 10(5) cell/ml in the infection-free quarters. Immediately after slaughter, samples from the tissues above the gland cistern and supra-mammary lymph node were collected. No histological differences were found between the supra-mammary lymph nodes of the healthy and infected udders, and both appeared normal. In the milk of the healthy infection-free mammary glands, SCC was < 50,000 cells/ml) while epithelial cells were the predominant type. The percentage of CD18+ was low than 45%, of which over three-quarters were polymorphonuclear (PMN), and less than one- quarter were mononuclear cells. The later comprised CD4+ or CD8+ T-lymphocytes, macrophages (Mo) but not B-cells. In the tissues, there were few CD18+ leukocytes, and most of the cells were T-lymphocytes. The number of B-lymphocytes bearing CD21+ was similar to that of CD8+ and were localized in the connective tissue as clusters of 2-5 cells, mainly in areas with no alveoli, or as single cell having a dendritic like form. The number of Mos was negligible. In the milk of the infected glands, SCC exceeded 700,000 cells/ml, of which > 95% were CD18+ positive. The distribution of the leukocytes had two patterns: one presented (> 80%) of PMN cells and a small number of mononuclear cells; the second had less than 50% PMN and many mononuclear cells. The CD8+ cells in these infected sections were observed throughout the mammary epithelial cells (MEc) around the alveoli and in the alveolar lumen (AL). The numbers and the location of CD21+ B-lymphocytes were similar to those in the infection-free mammary glands. The number of CD5+ positive cells was lower than T and B- cells combined and were located throughout the mammary epithelial cells, around the alveoli and within the connective tissue. Mo numbers were high in most of those infected quarters, and were localized around the connective tissue and within the AL.

Staphylococcus aureus strains isolated from bovine mastitis: virulence, antibody production and protection from challenge in a mouse model.

Septic arthritis in mice was used as a model to evaluate the virulence of Staphylococcus aureus and coagulase-negative staphylococci (CNS) isolated from cases of bovine mastitis. In addition, the model was used to evaluate the cross protection elicited by heterologous antibodies. Mice were intramuscularly inoculated with serial bacterial doses of different strains of S. aureus or CNS, for virulence determination; they were monitored for arthritis, gangrene or death up to 20 days. Antibody response, cross reactivity and resistance to challenge were tested by subcutaneous inoculation with a low dose of one of the S. aureus or CNS strains followed by challenge with two S. aureus strains. S. aureus alpha-hemolysin isolate was the most virulent, followed by alpha+beta-hemolysin and beta-hemolysin isolates. The least virulent isolates were the non-hemolytic S. aureus strains but even they were more virulent than the CNS strains tested. Antibodies against three different S. aureus antigens were detected by the ELISA in all mice that were inoculated with the S. aureus strains but not in any of those with the CNS strains. Immunoblot test against various S. aureus strains as antigens showed high cross-reactivity among the S. aureus strains but only a slight similarity, restricted to the bands above 36 kDa, with the CNS sera. Low-dose inoculation of alpha or alpha+beta strains before challenge with homologous and heterologous strains protected the mice, whereas the two beta strains provided only partial protection. The inoculations of non-hemolytic S. aureus or the CNS strains did not elicit any protection. Our findings demonstrate that pre-exposure of mice to a low dose of certain S. aureus strains could provide protection and that the antibodies produced could have an important protective role.

Influence of Staphylococcus aureus exosecretions isolated from bovine mastitis on leukocyte activity in vitro.

The role of Staphylococcus aureus and coagulase-negative staphylococcal exosecretions was tested for its ability to elicit in vitro proliferation of bovine blood lymphocytes, which we determined by means of the 3H-thymidine proliferation assay and by flow cytometry. Exosecretions of 32 field strains of S. aureus isolated from bovine udder infection and one of each of S. intermedius (M2), S. hyicus (M5), S. xylosus (M6) and S. chromogenes (M10) were used. Of the 32 S. aureus bacterial exosecretions, only 14 stimulated bovine mononuclear cells to proliferate. A high degree of association was found when the proliferation indexes were compared with the virulence as determined by intracisternal inoculation. All the six S. aureus strains that were categorized as highly virulent and that were tested in the proliferation assay exhibited a proliferation index > 20, whereas the five S. aureus strains that were categorized as low did not stimulate at all. Cells treated with media or Columbia broth supplemented with 0.1% D-glucose, yeast extract, and 0.5% NaCl (CBs) did not exceed 15% of the T-cells double positive with CD25+, whereas incubation with Con A activated the T-cells to display CD25+ up to 90%. Cells treated with one of the exosecretions that stimulated bovine mononuclear cells to proliferate, stimulated CD3+ and CD4+ T-cells to exhibit CD25+ receptor significantly higher (P < 0.05) than that found in media and CBs treatments, but lower than those found in Con A treatments. The exosecretions that did not stimulate mononuclear cells to proliferate also did not activate T-cells to exhibit CD25+ receptor. Con A activated 74% out of the total CD8+ to exhibit ACT2 receptor and 50% out of the total CD4+ to exhibit ACT3 receptor. A few but not all of the exosecretions that activated the CD25 receptor on T-cells also activated the ACT3 receptor on CD4+ cells.

Milk leucocyte population patterns in bovine udder infection of different aetiology.

This study compared the different leucocyte populations in milk from udders infected with different mastitic pathogens and in different stages of infection. Milk samples were collected from quarters free of intramammary infection, acutely infected with Escherichia coli or Staphylococcus aureus and chronically infected with S. aureus, coagulase-negative staphylococci (CNS) or Streptococcus dysgalactiae. Udder bacteriological status was confirmed after three consecutive bacteriological examinations from weekly quarter milk samples. At the time of the trial, milk samples were tested for somatic cell count (SCC) and differential cell count by both light microscopy (LM) and flow cytometry. Monoclonal antibody (mAb) CD11a/CD18 was used in order to differentiate between leucocytes and epithelial cells when tested by flow cytometry. Udder quarters free of intramammary infection had a mean SCC lower than 107 x 10(3) cells/ml in which the epithelial cells were the main cell type followed by polymorphonuclear cells (PMNs), while macrophages and lymphocytes had a lower concentration. Only 56% of the cells were labelled with the mAb anti-CD11a/CD18. In either acute E. coli- or S. aureus-infected quarters, SCC were significantly higher (P < 0.0001) than in samples from the time of inoculation, with over 90% of the cells labelled with the mAb anti-CD11a/CD18. The main cell type was neutrophils. In chronically infected cows, differences in SCC and in leucocyte patterns were found between infecting pathogens as well as between quarters harbouring the same pathogen. In all the chronically infected quarters, SCC was significantly higher (P < 0.05) than in uninfected ones. The distribution of the leucocyte patterns in the quarters infected with S. dysgalactiae did not differ from that in quarters with acute infection with both E. coli and S. aureus. In the cows chronically infected with S. aureus or CNS, the proportion of PMN was higher but not significantly different from quarters free of intramammary infection, while epithelial cells were significantly lower (P < 0.05). The T lymphocytes bearing CD4+ or CD8+ were significantly higher in quarters chronically infected with S. aureus than in quarters free of intramammary infection and in quarters acutely infected with either E. coli or S. aureus. In all samples B cells were negligible.

Milk leucocyte populations in heifers free of udder infection.

Improvement of udder health through a process of genetic selection is related to heritability and the role of the specific trait in the probability of an individual cow developing an infection. It was suggested that different patterns of leucocyte population of the healthy gland are a significant factor in mastitis. Thus, in order to analyse the heritability of a trait and its correlation with udder health, the present study examined the leucocyte populations of uninfected mammary glands, their variability among quarters in a particular cow, and the changes that occur during lactation. Each one of the 20 cows examined was tested on average 3.06 times during lactation. The somatic cell count (SCC)/ml ranged from 12,000 to 151,000, the coefficients of determination (R2) were higher than 0.5 for SCC. No significant differences were found in the dependent variables between the sampled times (test) nor any interaction between the slopes calculated for the cows over time. No significant differences were found among quarters within a cow for any of the dependent variables including SCC. The effect of the cow trait was found to be significant for polymorphonuclear (PMN), macrophage (MO), and T-lymphocyte-bearing CD4+. The number of lymphocytes labelled with the anti-B monoclonal antibodies was negligible. In conclusion the patterns of leucocyte populations in milk together with the variance among cows should enable an analysis of the heritability of this trait and its correlation with udder health in a future study.

Coagulase-negative staphylococci and mammary gland infections in cows.

Coagulase-negative staphylococci (CNS) are the most frequently isolated bacteria from bovine mammary gland milk samples. The objective of this study was to determine the type of inflammation evoked by CNS in the mammary gland of cows during their first lactation. Twenty-four Israeli-Holstein heifers in their first lactation were tested for bacteriological status, somatic cell count (SCC) and differential leucocyte count in milk 60-120 days postparturition and every 50-60 days after until drying off. Following the first testing, the 96 quarters of the 24 heifers were classified as follows: 69.8% as no bacterial growth (NBG), 27.1% infected with CNS and 3.1% infected with Staphylococcus aureus. During lactation, 84.5% quarters had no change in their classification, 6.2% were newly infected with other pathogens, 3.1% were classified as self-cured and in 6.2% sporadic bacteria were isolated. Among the CNS, S. intermedius, S. chromogenes and S. haemolyticus were the most frequently isolated. Milk from CNS-infected quarters had significantly higher SCC than milk from NBG quarters. An analysis of the leucocyte pattern in milk from CNS vs. NBG quarters revealed a significant increase in polymorphonuclears and a significant decrease in the percentage of total lymphocytes and lymphocytes bearing CD4+ or CD8+. The high percentage of CNS-infected quarters that remained unchanged in their bacterial status during the first lactation, indicates that those CNS have the ability to elude the immune system and persist in the mammary gland for a long time. The persisting infection, resulting to some extent from an increase of SCC by some CNS strains, suggests that in the near future control steps will have to be taken into consideration, in order to enhance the improvement of milk quality.