The effect of an experimentally induced acute mastitis on the test results of an Ostertagia ostertagi milk ELISA

Antibody levels against Ostertagia ostertagi in the milk are a promising parameter to identify dairy cows or herds with production losses due to gastrointestinal nematodes. However, milk antibody levels can be influenced by udder-related factors. In this study, the effect of an experimentally induced mastitis on the test results (ODR) of a milk O. ostertagi ELISA was investigated. Twenty-five cows that were naturally infected with gastrointestinal nematodes, were inoculated in their left udder quarters with Escherichia coli P4:O32 and quarter milk samples were collected at several intervals from 24 h before until 144 h after experimental infection. The effect of the contribution of milk from one or more infected quarters on the bulk tank milk ODR was estimated, based on a titration experiment. The mean O. ostertagi antibody levels of the infected udder quarters were significantly (P < 0.001) higher than those of the uninfected udder quarters at each sampling time post-infection. The largest difference was observed at 24 h post-infection with a mean difference of 0.251 ODR (95%CI: 0.172; 0.330). There was also a significant increase (P < 0.001) in total IgG levels with the largest difference being observed at 24 h post-infection. Highly significant (P < 0.005) correlation coefficients were observed between O. ostertagi ODR, total IgG ODR, Na+ and Cl- ion concentration and log transformed somatic cell counts at 24 h post-infection. The results demonstrate that an acute mastitis causes a flow of specific and non-specific antibodies from serum to milk with a subsequent increase in the O. ostertagi ODR values. The effect of the contribution of milk from infected quarters on the bulk tank milk O. ostertagi ODR was estimated to be minor if the relative number of infected quarters is small (< 3%).     

Thyroid hormones (TH) and 5'-monodeiodinase (5'-MD) activity in goat's milk from the early, mid- and late lactation period

The physiological significance of thyroid hormones (TH) present in colostrum and milk is still under consideration. The present study was aimed at determining milk thyroxine (T4) and triiodothyronine (T3) levels in three lactation phases (early, mid- and late) of the goat, and to measure activity of the milk 5'-deiodinase (5'-MD) enzyme responsible for the intramammary conversion of prohormone T4 to its metabolically highly active form T3. Thirty-two milk goats (Polish White breed) fed a standard diet were used for milk sampling. The highest TH levels in mammary secretion were recorded during the first 2-3 days post partum. Then the hormone levels decreased, and by about Day 7 fluctuated around the overall mean for the early-lactation phase (Days 1 to 24 of lactation), recording 0.134+/-0.059 microg T4 and 150.8+/-2.80 ng T3 in 100 ml of the milk. Such T4 concentrations appeared to be comparable to those in the rabbit and human, whereas the concentration of T3 was higher than in the cow, pig and mare's milk. Milk 5'-MD activity was higher (P < 0.01) during early and late lactation, compared to the mid-lactation phase. It coincided with low T4 and high T3 milk levels during early lactation, and with high milk T4 and low T3 concentrations during late lactation. The quantity of T4 and T3 available to newborn kids in milk suggests that TH ingested with the colostrum may have a physiological role during the early postnatal life of suckling goats.     

Changes in the bovine udder quarters naturally infected by Corynebacterium bovis

Of 272 bovine udder quarters studied for mastitis, 19 of them naturally infected with Corynebacterium bovis alone, were compared with 16 others infected by C. bovis together with other bacteria and another 36 non-infected quarters. While there was no significant difference in milk somatic cell counts between the quarters infected by C. bovis alone and those affected by C. bovis together with other bacteria (33.37 +/- 20.28 X 10(3) and 33.86 +/- 23.18 X 10(3)/ml of milk, respectively), there was a significant difference between these and the non-infected quarters (5.60 +/- 3.23 X 10(3)/ml of milk). Microscopically, quarters infected by either C. bovis alone or C. bovis in combination with other bacteria had inflammatory changes in the teat cisterns, Furstenberg's rosettes and/or mammary parenchyma. The non-infected quarters had no changes. In all 82 quarters no pathological changes could be seen in the teat canals.     

The use of polyethylene intramammary device in protection of the lactating bovine udder against experimental infection with Staphylococcus aureus or Streptococcus agalactiae.

The susceptibility of lactating bovine udder quarters fitted with a polyethylene intramammary device to infection was investigated. Following experimental challenge with Streptococcus agalactiae or Staphylococcus aureus, the incidence of infection was significantly (p less than 0.05) lower in intramammary device-fitted quarters compared to control quarters. In general, total foremilk and strippings milk somatic cell counts for intramammary device-fitted and control quarters were not significantly (p less than 0.05) different. Differential foremilk and strippings milk somatic cell counts were significantly (p less than 0.05) higher in samples from intramammary device-fitted quarters compared to control quarters.     

Effect of intramammary devices on the outcome of induced Escherichia coli infection of bovine mammary quarters

Eighteen Holstein cows, free of intramammary infection, were fitted with smooth (n = 9) or abraded (n = 9) intramammary devices (IMD) in 2 diagonally opposed quarters within 4 weeks after calving. The 2 other quarters of each cow were used as controls. Three to 6 weeks after IMD insertion, depending on when milk somatic cell counts returned to a base-line value of less than 4 X 10(5)/ml, all cows were subjected to bacterial challenge exposure in the front or rear quarters by intracisternal injection of about 30 colony-forming units of Escherichia coli/quarter. Challenge exposure was done immediately after milking. Three weeks after the initial bacterial exposure, the other quarter pairs were similarly challenge exposed. Quarter bacteriologic status, concentration of milk somatic cells, and clinical observations (rectal temperature, milk appearance, udder palpation, and general condition of the cow) were monitored. Infection developed in 14 of 16 (88%) quarters with smooth IMD vs 16 of 16 (100%) control quarters and in 7 of 17 (41%) quarters with abraded IMD vs 17 of 17 (100%) control quarters. The difference in infection frequency between quarters with smooth IMD and quarters with abraded IMD was significant (P less than 0.05). Protection against establishment of infection was associated with somatic cell counts greater than 8.0 X 10(5)/ml in milk collected immediately after milking (7 of 12 quarters) or 4 hours later (11 of 12 quarters). In 10 quarters (59%) of cows fitted with abraded IMD, secretory abnormalities appeared before bacterial challenge inoculation. Abnormal milk or visible blood was observed over periods varying from 2 weeks after insertion through the entire lactation.     

Somatic cells count in cow's bulk tank milk

The objective of this study was therefore to present factors affecting somatic cell counts in bovine bulk milk as a result of intramammary infections as well as non-infectious factors. The paper presents also the impact of on-farm management practices on the level of bulk milk somatic cell counts and presents quality indicators in bulk tank milk. At the farm level bulk milk bacterial infection takes place through three main sources: bacterial contamination from the external surface of the udder and teats, from the surface of the milking equipment, and from mastitis microorganisms within the udder. The threshold of 200,000 cells/ml identifies bacteriological negative quarters of the udder. The counts of mammary pathogens in bulk tank milk are relatively low, on average not exceeding 1,000 cfu/ml. Environmental pathogens predominate in bulk tank milk samples with somatic cells count <300 × 10(3) ml.     

An intramammary implant in the prevention of infectious mastitis in cows

A sterile polyethylene device was introduced in the milk cistern of two mammary quarters of forty dairy cows. The cows were divided into three groups according to the length of exposure of the device in the cistern (3 days, 14 days, 365 days). The somatic cell counts were studied for a year in the first 10 ml fraction of milk and an increase in the somatic cell counts was found in the quarters having the intramammary device, as compared with the control quarters (having no devices). This difference was marked during lactation and prior to the onset of drying off. For the reduction in the frequency of occurrence of new natural intramammary infections, the activity of the device against S. aureus was limited and no activity against S. agalactiae was proved. The proportions of polymorphonuclears, round-cell elements and macrophages were histologically studied and compared for the udder quarters with and without the intramammary body in correlation with the time of exposure of the device to the milk cistern milieu. The most marked differences in favour of the udder quarters with the intramammary device were recorded in the alveoli containing more cells with a significant proportion of polymorphonuclear leucocytes. Small differences were found in the interstitial and subepithelial zone of the milk cistern. The activity of acid and alkaline phosphatase and adenosine triphosphatase was histochemically determined in the tissue structures of the udder and was found not to change under the influence of the device. Leucocytes and macrophages adhering to the surface of the body were observed under scanning electron microscope.     

Effect of intramammary infusion of bacterial lipopolysaccharide on experimentally induced Staphylococcus aureus intramammary infection

Mastitis due to Staphylococcus aureus is a significant problem in the dairy industry and is refractory to antibiotic treatment and/or vaccine prevention. Relative to other mastitis-causing pathogens, S. aureus elicits a diminutive host inflammatory response during intramammary infection. To determine whether induction of a heightened inflammatory response could influence outcome of infection, the highly pro-inflammatory molecule bacterial lipopolysaccharide (LPS) was infused into udder quarters experimentally infected with S. aureus. Relative to S. aureus-infected udder quarters receiving saline, quarters infused with LPS demonstrated a heightened inflammatory response as demonstrated by the induction of TNF-alpha and higher milk somatic cell counts and albumin levels. Although there was no overall effect on bacterial clearance, a trend toward reduced bacterial numbers during the immediate pro-inflammatory response following LPS infusion was observed suggesting that this novel approach to treating S. aureus intramammary infection may warrant further investigation.     

Effect of the intramammary device on milk infection status, yield, and somatic cell count and on the morphological features of the lactiferous sinus of the bovine udder

Twenty primiparous heifers were fitted intramammarily with polyethylene coils in both quarters of one random right or left udder half at 5 days after parturition. Foremilk samples were collected and udder-half milk yields were measured at the afternoon milking on days - 1, 3, 7, and 14 and on every 14th day for 8 months after the device was inserted. Three weeks after the heifers were fitted with the intramammary device, 6 were euthanatized for gross observation of devices and tissues and cytologic evaluation of the gland cistern epithelium. There were significantly fewer bacterial isolations (P less than 0.01) and less clinical mastitis (P less than 0.05) in treated quarters than in the control quarters. Coagulase-negative staphylococci were isolated at frequencies of 0 and 15.2% for treated and control quarters. The reduction in isolation frequency for treated, compared with control, quarters was less marked with other organisms. Intramammary devices in no way interfered with the milking process. Milk yields per milking were 4.2 kg for treated udder halves compared with 4.4 kg for control halves; however, this 0.2 kg difference was not significant. Mean milk somatic cell counts, as determined by electronic counter, were 34 X 10(3) and 81 X 10(3) cells/ml for control and treated quarters (P less than 0.05). Mean bovine serum albumin values were 0.160 and 0.175 mg/ml for control and treated udder halves (P less than 0.05), indicating an increased capillary permeability due to the device. Quantitative morphologic analysis of gland cisterns showed a significant (P less than 0.05) change toward a single layer of epithelial cells in treated quarters compared with a double layer in control quarters.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bovine serum albumin and cell counts in the diagnosis of subclinical udder infection

Summary

Puncture of the milk cisterns was performed in 120 bacteriologically positive quarters of forty‐seven lactating dairy cows on three farms. This method was used to determine whether the existing infection was an infection of the teat canal or one of the udder. The results were related to the concentration of bovine serum albumin (BSA) and the cell count in the milk. Of the bacteriologically negative quarters, both BSA levels (in 91 per cent of the quarters the BSA concentration was 0.20 mg. per ml. of milk or less) and cell counts (92 per cent contained less than 500,000 cells per ml. of milk) were low. In cases of udder infection with primary pathogenic bacteria there was a marked increase in cell count (90 per cent more than 500,000 cells per ml. of milk), whereas the increase in BSA was rather small (51 per cent still contained 0.20 mg. BSA per ml. of milk or less). While the difference in cell counts of milk from quarters with udder infections and teat canal infections with primary pathogenic bacteria was significant, the difference between the BSA levels of these two groups was not. Therefore, the cell count supplies more reliable information than does the BSA level of the milk. Of all infections, 23 per cent were found to be infections of the teat canal.     

Role of leukotriene B4 in the pathogenesis of Klebsiella pneumoniae-induced bovine mastitis

Mastitis was induced in 4 lactating cows by inoculation of Klebsiella pneumoniae (10(7) organisms/ml) via the teat canal. Sterile isotonic saline solution (1 ml) was instilled into designated control quarters via the teat canal. Changes in milk leukotriene B4 and C4 (LTB4, LTC4) concentrations, milk somatic cell counts, and milk bovine serum albumin concentration were monitored over a 24-hour postinoculation period. Milk LTB4 concentration before inoculation in control quarters and quarters later to be infected was 376 +/- 45 and 326 +/- 56 pg/ml of milk, respectively. A significant (P less than 0.05) increase in milk LTB4 concentration in the infected quarters was first observed at postinoculation hour 6, and milk LTB4 concentration in infected quarters generally remained significantly high through postinoculation hour 14. Thereafter, milk LTB4 concentration in infected quarters was not significantly different from the concentration in control quarters. Measurable amounts of LTC4 were not detected in the milk of either control or infected quarters. Milk bovine serum albumin concentration in the infected quarters generally was high throughout the study, as were milk somatic cell counts. The results of this study suggested that LTB4 contributes to the pathogenesis of bovine mastitis.     

Physiological and morphological changes in bovine mammary glands following intramammary infusion of recombinant interferon-gamma.

Eleven lactating dairy cows were used to evaluate the response of bovine mammary glands to increasing doses of recombinant bovine interferon (rBoIFN)-gamma. Right front and rear quarters were intramammarily infused with eight different doses (10(2) U to 2 x 10(8) U/quarter) of rBoIFN-gamma; each dose was tested in at least two quarters. Left udder halves served as within animal controls in which quarters were injected with a saline placebo or were not infused at all. Milk secretion samples for compositional analysis were collected from each quarter prior to infusion and at 6, 24, 36 and 48 h following infusion. Animals were slaughtered immediately following the 48 h sampling period and mammary tissue was obtained for morphometric analyses. Milk composition was similar between control quarters and those quarters infused with up to 10(5) U of rBo-IFN-gamma during the entire sampling period. Quarters infused with 10(6) U and 10(7) U of rBoIFN-gamma had higher milk somatic cell counts (SCC) following treatment compared with preinfusion values. Changes in the composition of mammary secretion were most dramatic in quarters infused with greater than or equal to 10(8) U of rBoIFN-gamma as indicated by the significant increase in SCC and milk pH with a concomitant decrease in lactose concentration when compared with pre-infusion values or with control quarters. Morphometric analysis of tissue demonstrated an increase in stroma, a decrease in luminal area, and a marked increase in the number of infiltrating leukocytes in those quarters infused with the higher doses of rBoIFN-gamma.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacokinetics of polymyxin B administered via the bovine mammary gland

Polymyxin B was infused into normal, chronically inflamed, and acutely inflamed quarters of the mammary gland of lactating cows at dosages ranging between 1 and 2 million units (100–200 mg) per quarter. Samples of milk from treated and non-treated quarters, jugular venous blood, subcutaneous abdominal (mammary) venous blood, and urine were collected at intervals after treatment and were assayed using microbiological test methods for polymyxin B concentrations. The drug was not absorbed from normal and chronically inflamed quarters; more than 90% of the infused dose was recovered in milk within 24 h after treatment, and drug residues were detected up to the ninth milking. Drug concentrations in milk from acutely inflamed quarters were significantly lower than in milk from normal quarters; 55% of the infused dose was recovered in the milk within 24 h after treatment. The drug was detected in milk from non-treated quarters, in blood from the subcutaneous abdominal vein, and in the urine during 36–48 h after acutely inflamed quarters were infused with the drug. These data indicate that polymyxin B is well distributed throughout, and is absorbed to a significant degree into the systemic circulation from the acutely inflamed udder.     

Clinical pharmacokinetics of carbenicillin, carfecillin, ticarcillin and BL-P 1654 in dairy cows

The minimal inhibitory concentrations (MIC) of carbenicillin, ticarcillin and BL-P 1654 for gram-negative udder pathogens were determined using the agar plate dilution method. The MIC of the drugs for 50% and 90% of the isolates examined ranged for Escherichia coli and Aerobacter spp. from 1.56 to 25 micrograms/ml, and for Klebsiella spp. and Pseudomonas spp. from 3.12 to 50 micrograms/ml. The Serratia spp. were relatively non-susceptible for the drugs studied (MIC greater than 50 micrograms/ml). Each drug was administered intravenously at 5 g and 15 g per cow to different groups of cows with normal and inflamed quarters of the udder. Distribution and elimination kinetic parameters calculated from serum drug level data were very similar to those of other beta-lactam antibiotics. Although drug concentrations in milk from inflamed quarters were higher than in milk from normal quarters, they were considerably below the MIC for the majority of gram-negative udder pathogens. The data suggest that parenteral treatment of gram-negative udder infections with carbenicillin, carfecillin, ticarcillin and BL-P 1654 at the dose levels used in the present study is unlikely to result in a bacteriological cure and would probably be clinically ineffective.     

The contribution of mammary infections by coagulase-negative staphylococci to the herd bulk milk somatic cell count

Quarter foremilk samples were taken at 2–3 weekly intervals for several years in a experimental herd comprising about 45 cows. The samples were submitted to bacteriological analysis and somatic cell counting. The most prevalent quarter infections from 1982 to 1988 were by coagulase-negative staphylococci (15–20% of all the quarters sampled). Most of these (75.6%) persisted until drying-off Dry cow therapy eliminated 86.5% of these infections. Comparison of udder quarters within cows, involving 775 samples from pairs of non-infected quarters and quarters infected by coagulase-negative staphylococci, yielded geometric means of somatic cell counts of 210 000 and 420 000 cells/ml, respectively. The correlation (r=0.87) between the herd bulk milk somatic cell count (SCC) and its estimation from the quarter milk somatic cell count performed on the same day allowed us to evaluate the contribution of the different categories of quarters, according to their infection status, to the herd bulk milk SCC. Quarters infected by a major pathogen (8.5% of samples) gave rise to 46.6% of the total number of cells, while quarters infected by coagulase-negative staphylococci (17.8% of samples) gave rise to 18.1%. Although coagulase-negative staphylococci represented only a secondary source of somatic cells as compared to major pathogens, they were not a negligible source considering the threshold of 300 000 somatic cells advocated for herd milk of good quality.     

Somatic cell counts in bovine milk

Factors which influence somatic cell counts in bovine milk are reviewed and guidelines for their interpretation are presented. It is suggested that the thresholds of 300 000 and 250 000 cells/mL be used to identify infected quarters and cows respectively. However, it is stressed that somatic cell counts are general indicators of udder health which are subject to the influence of many factors. Therefore the evaluation of several successive counts is preferable to the interpretation of an individual count.

Relationships between somatic cell counts and both milk production and milk composition are discussed. Subclinical mastitis reduces milk quality and decreases yield although the relationship between production loss and somatic cell count requires clarification. Finally the availability of somatic cell counting programs in Canada is presented.

     

Evaluation of interleukin-2 treatment for prevention of intramammary infections in cows after calving

A low-dose treatment based on interleukin-2 (IL-2) was investigated for preventing mastitis in dairy cows. The treatment consisted of a single dose of IL-2 injected into the skin region drained by the supramammary lymph node 3-5 days after calving. The study included 45 cows (23 treated and 22 controls) from three commercial dairy herds. The results showed that the treatment had no side effects. The treatment with IL-2 induced the significant increase of several milk markers related to leukocyte and epithelial cell functions, i.e. SCC (somatic cell counts), serum amyloid A (SAA), lactoferrin and NAGase. The increased concentration of milk markers suggested also an activity of IL-2 on epithelial cells, resulting in a higher resistance to invading pathogens. Indeed, the increased efficiency of cells in the udder is supported by the higher frequency of healthy quarters observed in the treated group until day 17-19 after calving, in comparison with the control one.     

The Influences of Dietary Selenium and Vitamin E Intakes on Milk Somatic Cell Counts and Mastitis in Cows

Dietary supplements of selenium and vitamin E in greater amounts than are required for nutritional adequacy can have complementary functions in reducing somatic cell counts and both the severity and duration of clinical mastitis. Selenium inadequacy is geographically widespread and can frequently be a year-round problem. In contrast, an adequate intake of fresh grass and quality grass silage or other green, leafy material should provide adequate vitamin E. Many observations indicate that in farm situations where there is good udder hygiene and where long-acting antibiotic treatment is given at drying off, significant correlations are found between the mean bulk milk somatic cell counts and the blood selenium concentration or glutathione peroxidase activity in the blood, even where plasma vitamin E concentration is fully adequate. The accompanying reduced incidence of clinically affected quarters diminishes the need for corrective antibiotic treatment during lactation. Presentation of selenium and vitamin E within a sustained-release rumen bolus system during the dry period and into the succeeding lactation is a convenient means of supplementation to avoid over- or under-consumption by individual cows within a group. Adequate hygiene of the environment, the milking equipment and the udder are essential.     

Microbiological studies on bacterial spectra of milk samples from healthy udder quarters and from those with increased cell counts and/or conductivity values]

The germ levels of 2,182 milk samples obtained from udder quarters with subclinical mastitis were compared to milk sampled from 2,061 udder quarters with physiological cell counts or conductivity values. Three cattle herds were involved in the test programme. No germ growth was established from 9.5 per cent of all samples taken from udder quarters with increased cell counts and conductivities and from 4.1 per cent of those samples taken from intact udder quarters. Samples taken from udder quarters with subclinical mastitis exhibited the following rises in bacteria, as compared to samples from intact quarters: staphylococci by 3.1 per cent, staphylocci in germ mixtures by 3.0 per cent, CAMP-positive streptococci by 2.2 per cent, alpha-haemolytic CAMP-negative streptococci by 0.8 per cent, anhaemolytic streptococci in germ mixtures by 0.4 per cent, beta-haemolytic streptococci by 0.5 per cent, and Pseudomonas aeruginosa by 1.4 per cent. Other germ species and mixtures exhibited declining trends along with growing subclinical affection of udder quarters. All findings so far obtained in the presence of subclinical mastitis are likely to suggest that 11.4 per cent of detected bacteria were of pathogenicity to udders. However, attempts to localise those 11.4 per cent were unsuccessful, since no significant difference could be calculated by comparison of intact with affected udder quarters. Reference is made in the discussion to primary and secondary germ levels of milk samples and their relevance to the problem and its elucidation.     

Persistent Experimental Salmonella dublin Intramammary Infection in Dairy Cows

Experimental intramammary infections were induced in five post-parturient Holstein cows by inoculation of low numbers (5000 colony forming units) of virulent Salmonella dublin via the teat canal of mammary gland quarters. Rectal temperature, pulse and respiratory rates, milk yield, and milk quality as assessed by the California Mastitis Test (CMT) and somatic cell counts (SCC) were recorded every 12 hours at milking. Bacteriologic cultures of foremilk quarter samples and feces were obtained daily, as were complete blood counts. ELISA titers for IgG and IgM recognizing S. dublin lipopolysaccharide (LPS) were obtained weekly on serum and quarter milk samples. All cows excreted S. dublin intermittently from infected quarters, but no changes were detected in rectal temperature, appearance of the mammary gland or secretions, CBC, milk yield, and pulse and respiratory rates. Somatic cell counts were modestly increased in infected quarters as compared with uninfected quarters (P = .015, paired t test); however, CMT scores after infection remained low, and were not significantly different from pre-infection scores (P greater than .10, sign test). After infection, administration of dexamethasone resulted in signs of clinical mastitis and increased excretion of S. dublin from mammary quarters (P = .0004, paired t test). One cow had necrotizing mastitis and S. dublin septicemia and was euthanatized. In the four surviving cows, clinical improvement was observed after systemic gentamicin therapy and intramammary infusion with polymyxin B, but all cows continued to excrete S. dublin intermittently from one or more quarters and occasionally from feces for the remaining period of observation. All infected cows demonstrated a rise in IgG and IgM ELISA titers recognizing S. dublin LPS in serum and milk. At necropsy (13-25 weeks postinfection), S. dublin was recovered only from the mammary tissue or supramammary lymph nodes in three of four cows. In one cow, mammary gland and lymph-node samples were negative for S. dublin despite positive milk cultures. In all cows, histopathologic examination revealed multifocal areas of chronic active mastitis. These lesions were similar to histopathologic findings from mammary gland carriers with naturally acquired S. dublin infection.