The survival of serum resistant Escherichia coli in the bovine mammary gland following experimental infection.

Serum resistant strains of Escherichia coli were injected into one or two quarters of the udders of eight healthy dairy cows. Animals receiving infection into two quarters showed variation in their ability to eliminate the bacteria. This variation extended from elimination from both glands to complete failure to remove the organisms from either gland. In most cases, the organisms were removed from one gland before the clinical signs of infection were observed, but persisted in the other gland for three to four days. Following a single infection most animals eliminated the organisms before the appearance of clinical signs, but one retained the bacteria for four days. The retention of bacteria within the gland for a period longer than the initial inflammatory response resulted in their survival within neutrophils; and in some of these glands spasmodic clinical signs of mastitis reappeared for up to at least 40 days after the initial infection. These signs were associated with the reappearance of the same serological strain of E coli. Bovine serum albumin levels in the milk were found not to constitute the most effective marker of the serum components involved in the bactericidal activity.

The pathogenesis of experimental Escherichia coli mastitis in newly calved dairy cows.

Experimental infections of the mammary gland of newly calved cows with 500 serum resistant Escherichia coli produced a very severe form of mastitis when compared with animals in mid-lactation. Ten hours after infection the bacteria had multiplied in the milk to very high numbers (10(6)--10(7)/ml) and the animals showed signs of pyrexia, anorexia and diarrhoea. Initially the gland and milk showed little or no clinical signs of mastitis, but later the secretion became a viscous, serous fluid with little or no casein or fat. A delay in diapedesis of neutrophils into the gland appears to be the reason for the peracute state and lack of clinical signs. This form of pathogenesis may produce a paradoxical situation where the most severe cases of E coli mastitis cannot be diagnosed at a stage early enough for the animal to respond to therapy.

The elimination of serum-resistant Escherichia coli from experimentally infected single mammary glands of healthy cows.

Neutrophils isolated from mammary glands stimulated with a staphylococcal culture filtrate efficiently killed serum-resistant strains of Escherichia coli. This study was extended and it was shown that an infusion of wide ranging numbers (5 X 10(1) to 5 X 10(6)) of the same strains of E coli into a single mammary gland resulted in bacterial growth, which was eliminated following neutrophil infiltration. This elimination occurred before the appearance of any clinical signs. Once bacterial kill had started in the gland, it continued in the milk after withdrawal from the gland. These results offer an explanation of why causative microbial agents cannot be isolated from some cases of clinical mastitis.

The requirement of specific antibody for the killing of E. coli by the alternate complement pathway in bovine serum.

Experiments using the selective divalent cation chelator, ethylene glycol tetraacetic acid (EGTA) showed that the alternate complement pathway was involved in the bactericidal action of bovine serum on two strains of E. coli. The bactericidal system was shown, in experiments involving EGTA and epsilon-amino caproic acid, to be independent of Ca++ and the C1 unit of complement. The fixation of the complement components C3-C9 by endotoxin does not require a functional classical pathway ((C1, C4 and C2). In the case of one coliform strain (W1) however, there was an almost total dependence on a factor absorbable at 0 degrees with homologous bacteria, and which appeared to be a natural antibody. These results suggest that in certain circumstances in the cow, gamma globulins may be directly involved in the activation of the alternate complement pathway.

Fertility of boar spermatozoa after freezing in the absence of seminal vesicular proteins.

PIP: A comparison of spermatozoa from intact boars and from boars without seminal vesicles, frozen in various diluents, was made to determine any differences in sperm quality after thawing and the conception rates of gilts. Sperm samples were frozen at -196 degrees C. 50 semen pellets were thawed after 1-3 weeks storage and assessed for postthaw motility at 37 degrees C. The percentage of eosinophilic spermatozoa was estimated. Random samples were assayed for L-aspartate 2-oxoglutarate aminotransferase activity. Enzyme release was measured. Gilts were inseminated 24 and 32 hours after 1st standing to a boar and gave slightly higher conception rates than those from intact animals although the differences were insignificant statistically. Overall conception rate was 35% for spermatozoa from boars without seminal vesicles and 29% for those who were intact. The diluents with 20% egg yolk and 2% casin with cholesterol and phosphatidyl serine provided significantly greater (p less than .05) cryoprotection than did that with 2% casein. In general, levels of enzyme release from the spermatozoa after thawing were 3 times those before freezing, indicating substantial membrane damage. It is suggested that the presence of seminal vesicular fluid at ejaculation has only a small effect on the fertilizing ability of spermatozoa frozen and thawed in suitable diluents.^ieng

Increased antibacterial activity against Escherichia coli in bovine serum after the induction of endotoxin tolerance.

Small amounts of endotoxin injected intramuscularly into cows induced endotoxin pyrogenic tolerance and an increase in the rate at which the serum killed a strain of Escherichia coli. Most of the difference between normal serum and serum from the endotoxin-tolerant animal was shown to be due to a bentonite-adsorbable factor other than lysozyme or beta-lysin. The antibacterial activity was not completely removed from either type of serum after bentonite adsorption. Electron microscope studies and measurement of the rate of release of radioactively labeled cytoplasmic contents showed that the bentonite-adsorbable factor was important in the final breakdown of the cell membrane and release of cellular contents. The antibacterial system was totally dependent on complement, and the importance of antibodies could not be entirely ruled out because adsorption at O C with homologous cells eliminated the killing activity.

Seminal plasma proteins and the reaction of spermatozoa from intact boars and from boars without seminal vesicles to cooling.

Spermatozoa from intact boars and from boars without seminal vesicles were resuspected in diluent and cooled at different rates to 0 degrees C. Glutamic oxaloacetic transaminase and lactate dehydrogenase activities were greater in the diluents which had contained spermatozoa from intact boars than in those which contained spermatozoa from animals without seminal vesicles. The incubation of seminal plasma from an intact boar with spermatozoa from a vesiculectomized animal before cooling also increased the enzyme activity in the diluent. The factors responsible for this effect were associated with the basic protein fractions of boar seminal plasma, in particular the proteins with haemagglutinating activity which may have been adsorbed onto the spermatozoa. Spermatozoa were exposed to colloidal Fe(OH)2+ to determine by electron microscopy the charge on the surface of the plasma membrane of washed epididymal spermatozoa and ejaculated spermatozoa from intact and vesiculectomized boars. Epididymal spermatozoa bound the positively charged particles more readily than the ejaculated spermatozoa from the intact boars, due to the absence of membrane-bound protein.

The binding of labelled basic proteins by boar spermatozoa.

Seminal plasma basic proteins were labelled with 131I. The efficiency of the labelling was studied by superimposing protein density traces on a radioactive fractionation plot. These labelled proteins were incubated with spermatozoa and shown to bind more readily to spermatozoa from boars after the removal of the vesicular glands than to spermatozoa obtained from their normal litter mates. Most of the labelled protein became bound to the membranes which were isolated by sucrose density gradient centrifugation. The membranes were separated into two bands which equilibrated at the relative densities of 1-150 and 1-165. These fractions consisted of membrane vesicles of different size; the smaller band on the gradient, which equilibrated at 1-165, consisted of denser membrane material.

Isoelectric focusing of boar spermatozoa.

The isoelectric points of washed spermatozoa from intact boars and from boars after removal of the seminal vesicles were determined using isoelectric focusing on natural pH gradients. Normal boar spermatozoa focused at a higher pH than spermatozoa from boars without seminal vesicles. The isoelectric point of the latter was increased to a value approaching normal by preincubation in normal seminal plasma. This indicates that seminal plasma alters the membrane surface charge of boar spermatozoa on ejaculation.