Endotoxin-induced bovine mastitis: immunoglobulins, phagocytosis, and effect of flunixin meglumine

Milk whey immunoglobulins (Ig) and phagocytosis of staphylococci by milk polymorphonuclear neutrophilic leukocytes (PMN) were measured in 12 cows (allotted to 6 pairs) during acute bovine mastitis induced by intramammary inoculation of endotoxin. Six of these cows (or 1 in each pair) were treated with flunixin meglumine and were compared with the others (given only saline solution). The endotoxin inoculation comprised 10 micrograms of Escherichia coli O26:B6 lipopolysaccharide injected into one of the rear quarters (mammae). Flunixin meglumine was administered parenterally at a dosage of 1.1 mg/kg every 8 hours (total of 7 doses) beginning at 2 hours after endotoxin was injected. Milk samples were obtained, and whey samples were prepared from each quarter of each cow 3 times before inoculation and at 2, 4, 8, 12, 24, 48, 72, 96, 120, 144, 168, and 336 hours after endotoxin was inoculated. Significant increases (P less than 0.05) in milk whey IgG1, IgG2, IgM, and IgA concentrations were observed in whey samples from endotoxin-inoculated quarters. Greatest relative increase was seen for IgG2. Increased whey Ig concentrations were not observed in quarters which were not inoculated with endotoxin. Concentrations of whey IgG1 and IgM in endotoxin-inoculated quarters were significantly (P less than 0.05) decreased in flunixin meglumine-treated cows, compared with those in saline solution-treated cows. Significant increases in phagocytosis of staphylococci by milk PMN were observed in whey samples from endotoxin-inoculated quarters. Significant differences in PMN phagocytosis were not found in whey samples from cows given flunixin meglumine when compared with whey samples from cows given saline solution.     

In vitro function of bovine neutrophils against Actinomyces pyogenes

Factors that influenced the in vitro bactericidal activity of bovine neutrophils against Actinomyces pyogenes were investigated. Neutrophils and serum from 2 clinically normal donor cows were incubated with bacteria for 2 hours. To determine bactericidal activity, colony-forming units were counted after a 48-hour incubation on blood agar plates. Microscopic examination indicated that in the presence of serum, bacteria were cell associated after incubation, whereas when serum was replaced by medium, bacteria were not cell associated. Bactericidal activity of neutrophils was similar whether the sera were heat-treated at 56 C for 30 minutes or were not heated. Heating the serum at 65 C for 30 minutes significantly (P less than 0.001) reduced bactericidal activity. Bactericidal activity decreased (P less than 0.001) as serum concentration (less than 10%) decreased. More than 80% of the bacteria were killed within the 40 minutes of incubation. The opsonizing capacity of serum varied significantly (P less than 0.01) among 12 cows. Similarly, neutrophil bactericidal activity (by cow) was affected significantly (P less than 0.001). Preincubation of serum with A pyogenes significantly (P less than 0.001) reduced the opsonizing ability of the serum. Culture filtrate of A pyogenes was not chemotactic for neutrophils in vitro.     

Systemic and local immune response of cows to intramammary infection with Staphylococcus aureus

The association between Staphylococcus aureus chronic mammary gland infection and the resulting immune response expressed by the production of specific IgG and IgA antibodies in blood and milk was studied in Israeli Holstein cows. Specific antibodies of the IgG class were detected in sera of 82.6 per cent of the cows chronically infected by S aureus, while in 17.4 per cent no such antibodies could be detected. Specific IgG antibodies to S aureus were neither detected in sera of cows free of mammary infection nor in those infected with different coagulase-negative staphylococci (CNS) such as S intermedius, S chromogenes or S haemolyticus. In milk, specific IgG antibodies to S aureus were detected only in cows with positive serology. The end point dilutions in the milk were 5 to 30 per cent of that of blood from the same cow. No significant difference in IgG titres was found in the same cow if the quarter was infected with S aureus or not. Specific antibodies to S aureus of the IgA class could not be detected in the sera of any of the cows included in this study. In milk, a specific IgA antibody was detected only in the samples from the S aureus infected quarters in which S aureus was isolated at the time of the experiment. In the same cow, quarters infected by S aureus were found to have a significantly higher IgA titre (P < 0.0001) than that of the non-infected ones.     

Use of enzyme-linked immunosorbent assay to measure bovine milk and serum antibodies to alpha toxin, beta toxin, and capsular antigens of Staphylococcus aureus

An enzyme-linked immunosorbent assay (ELISA) procedure was used to quantitate milk and serum antibodies (IgG) to Staphylococcus aureus alpha and beta toxins, and S. aureus 2-8 and Smith diffuse strain capsular antigens. Milk samples were collected on two occasions. A comparison was made between levels of milk antibodies specific for the two toxins and capsular antigens for 41 cows that were infected with S. aureus on both sampling dates, and 18 cows not S. aureus-infected on either date. Staphylococcus aureus-infected cows were grouped according to somatic cell counts. All groups of infected cows, regardless of somatic cell counts, had significantly higher milk antibody levels to alpha and beta toxins than did the non-infected cows (P less than .002). Serum samples taken for 13 infected and 4 non-infected cows also indicated that significant elevations in anti-alpha toxin and anti-beta toxin IgG were present in S. aureus-infected cows, compared to non-infected cows. A similar immune response was not seen to capsular antigens, however. No significant differences were present between the two groups of cows for either milk or serum antibodies to Smith diffuse strain capsular antigens. Milk antibodies to 2-8 capsule were significantly elevated only in infected cows with somatic cell counts greater than 10(6)/ml, compared to non-infected cows; no differences were present for serum antibodies to 2-8 capsule between infected and non-infected cows. These results indicate that significant increases in milk (and possibly serum) antibodies to alpha and beta toxins are present in cows with chronic staphylococcal mastitis, apparently resulting from a systemic immune response to these toxins. There does not appear to be a similar immune response to capsular antigens.     

Milk whey induction of agglutination in ovine and bovine mastitis Staphylococcus aureus

A total of 59 mastitis staphylococcic strains were tested for growth agglutination upon supplementation of growth media with ovine and bovine milk whey and mammary secretions from dry cows. Differences were observed when comparing bacterial species or origins (ovine vs. bovine) of bacteria and whey. All of the ovine and bovine S. aureus strains tested, but only 4 among 22 other ovine mastitis staphylococcic strains, showed growth agglutination in Todd Hewitt broth (THB) supplemented with greater than or equal to 30% (v/v) ovine milk whey. None of the strains agglutinated during growth in regular THB medium. Ovine whey had an agglutination induction capacity higher than bovine whey (P less than 0.005), concerning the number of responsive ovine and bovine S. aureus strains. There were no differences between whey samples from different ewes with regard to their capacity to induce agglutination. Ovine S. aureus strains were more responsive than bovine strains of this bacterial species, concerning the number of responsive strains (P less than 0.001) to bovine whey (greater than or equal to 30% in THB), the proportion of responsive strains at low (10%) ovine whey concentration (P less than 0.001), and the strength of reaction (precipitation timing and clump size). Secretions from dry cows systematically induced agglutination in all of the bovine and ovine S. aureus strains tested.     

IgG antibody response to Corynebacterium pyogenes,Peptococcus indolicus and microaerophilic cocci in natural and experimental mastitis

A mixed inoculum of C. pyogenes, P. indolicus and a microaerophilic coccus was used to induce experimental mastitis in two heifers. During the course of the infection, the IgG titres against these bacteria were monitored by the indirect immunofluorescent antibody technique. At the same time, the course of the infection was studied bacteriologically and by determination of clinical-chemical parameters. In both heifers, high titres of IgG against C. pyogenes, P. indolicus and the microaerophilic coccus were detected. The reactivity of the IgG towards surface-associated antigens of these bacteria seemed to reside mainly in the IgG2 subclass. Sera obtained from Denmark from heifers with experimental mastitis, which had been induced similarly, were also found to have significantly raised IgG titres against C. pyogenes and P. indolicus. Sera from naturally infected heifers and dry cows, from whose udder secretions C. pyogenes and P. indolicus had been isolated, were examined comparatively with sera from healthy heifers and dry cows, respectively, for the presence of IgG against these two bacteria. Statistically significant differences in IgG titres against both bacteria were demonstrated between pregnant heifers with mastitis and healthy pregnant heifers and between dry cows with mastitis and clinically healthy controls.     

Enzyme-linked immunosorbent assay for detection of milk immunoglobulins to leukocidin toxin of Staphylococcus aureus

Leukocidin toxin from a bovine strain of Staphylococcus aureus was partially purified by ion exchange chromatography. An enzyme-linked immunosorbent assay was developed to quantitate antibodies specific for leukocidin in bovine milk. This was used to assay quarter samples from 88 cows in a S aureus-infected herd for antibody levels to the toxin. Milk samples from 65 cows with S aureus infections in at least one quarter produced a mean optical density of 1.054, whereas milk samples from 23 cows that were free of bacteria on cultural examination had a mean optical density of 0.584. There was a significant difference (P less than 0.001) in milk anti-leukocidin levels between these 2 groups. Evaluation of serum samples from 40 of these cows indicated that the milk anti-leukocidin concentrations were reflective of systemic anti-leukocidin values. The capability of 57 milk samples to neutralize the cytolytic effect of minimal amounts of leukocidin on bovine peripheral blood neutrophils was examined. Good correlation existed between the enzyme-linked immunosorbent assay antibody concentration and toxin-neutralizing capability of individual milk samples.     

Enzyme immunoassay using mouse monoclonal anti-bovine antibodies for the detection of Brucella abortus antibodies in cow milk

Individual milk samples and artificially constructed tank milk samples from cows with naturally occurring brucellosis were examined by the enzyme-linked immunosorbent assay (ELISA) using sonicated B. abortus S-99 antigen, and mouse monoclonal anti-bovine IgM, IgA, and IgG1 conjugates. ELISA results were compared with the results of the milk ring test using either 1 ml milk (MRT-1) or 8 ml milk (MRT-8). The ELISA using mouse monoclonal anti-bovine IgG1 conjugate was sensitive and specific. In testing individual milk samples and constructed tank milk samples containing milk with low titers in the MRT-1 the ELISA was superior to the MRT-1, and MRT-8. In testing other milk samples, the ELISA was as sensitive or slightly less sensitive than the MRT-8. From a total of 5,910 milk samples collected from cows free from brucellosis, only 24 (0.4%) samples tested positive in the ELISA. All 500 tank milk samples collected from farms negative for brucellosis tested negative in the ELISA. We concluded that the ELISA is a good substitute for the MRT-1 to detect antibodies against Brucella in milk from individual cows. When tank milk is tested for antibodies against Brucella, however, both the MRT-8 and the ELISA should be used.     

Immune response of pregnant cows to bovine rotavirus immunization

Fifteen pregnant Holstein cows were freely assigned to 3 experimental groups (5 cows in each group). Cows in group I were inoculated IM and intramammarily (IMm) with Ohio Agricultural Research and Development Center (OARDC) tissue culture-propagated modified-live Nebraska calf diarrhea bovine rotavirus with added adjuvant (OARDC vaccine-immunized cows). Group II cows were given IM injections of a commercial modified-live rotavirus-coronavirus vaccine (commercial vaccine-immunized cows), and the remaining 5 cows were noninoculated controls (group III). Rotavirus antibody in colostrum and milk was mainly associated with immunoglobulin (Ig)G1, and less so with IgG2, IgA, and IgM, as analyzed by the enzyme-linked immunosorbent assay (ELISA), using monospecific anti-bovine IgG1, IgG2, IgM, and IgA sera. In serum, the rotavirus antibody was distributed almost equally between IgG1 and IgG2. The same relationships appeared in both immunized and nonvaccinated cows. All OARDC vaccine-injected cows had virus-neutralization (VN) and ELISA IgG1 rotavirus antibody titers in serum and mammary secretions at significantly increased levels (at least 100-fold; P less than 0.05) compared with the titers in groups II (commercial vaccine-immunized cows) and III (controls). Serum, colostrum, and milk antibody titers from these latter 2 groups did not differ statistically. The ELISA IgG2, IgA, and IgM rotavirus antibody titers also were significantly greater in mammary secretions from OARDC vaccine-immunized cows than in groups II and III cows. There was a high correlation between ELISA IgG1 and VN rotavirus antibody titers for all samples tested (r = 0.97, P less than 0.001), but ELISA IgG1 antibody titers were consistently higher than VN titers. The ELISA IgG1 and VN antibody titers of milk samples collected from cows 30 days after parturition were higher from the OARDC vaccine-immunized cows (ELISA IgG1, geometric mean titer (GMT) = 3,511; VN GMT = 1,689) than were titers from the group II cows (ELISA IgG1 GMT = 39; VN GMT = 33) or group III cows (ELISA IgG1 GMT = 21; VN GMT = 19). These results indicate that IM plus IMm immunization of pregnant cows, using modified-live bovine rotavirus with added adjuvant, may significantly enhance serum, colostrum, and milk rotavirus antibody titers, whereas IM vaccinal inoculation of pregnant cows with a commercial modified-live rotavirus-coronavirus vaccine may not.     

Immunity to Escherichia coli in pigs: Antibody Secretion by the Mammary Gland after Intramammary or Intramuscular Vaccination with an E. coli Vaccine

Indirect hemagglutinating antibody titres in individual gland samples of colostrum and milk from 13 sows were measured. Five of the sows were vaccinated via a mammary gland and five by the intramuscular route with a live formalinised Escherichia coli vaccine and three remained as non-vaccinated controls.

Antibody titres were higher in colostral and milk whey from the vaccinated sows than from non-vaccinated groups. The inoculated gland in the group of sows given vaccine by the intramammary route secreted milk containing markedly more antibodies to the vaccine E. coli strain than did the non-vaccinated glands. Milk from the vaccinated gland did not contain higher titres to heterologous E. coli O antigens than milk from non-vaccinated glands. Serum titres were the same or higher than the titres in colostrum from non-vaccinated glands.

     

Use of ELISA for detection of immunoglobulins G and M that recognize Salmonella dublin lipopolysaccharide for prediction of carrier status in cattle

Immunoglobulin reactions to Salmonella dublin in serum and milk from 4 groups of lactating cows were measured by an indirect ELISA. The groups consisted of (1) cows that were natural carriers of S dublin in the mammary gland, (2) experimentally infected cows that did not become carriers, (3) cows inoculated with a commercial S dublin bacterin, and (4) cows used as S dublin-negative controls. Milk and serum samples were obtained at monthly intervals. Models for predicting carrier status were developed by use of stepwise logistic regression. Independent variables consisted of serum and milk IgG and IgM titers to S dublin lipopolysaccharide and a ratio of IgG to IgM. The utility of a single sample vs multiple samples obtained at 1-month or 2-month intervals was tested by comparison of goodness-of-fit chi 2 P values for 8 models predicting carrier status. Immunoglobulin reactions specific to S dublin were a significant predictor of carrier status (P less than 0.001). Serum IgG titers specific for S dublin were the most important variable for predicting carrier status. Two serum IgG titers to S dublin obtained 2 months apart was a better predictor of carrier status than measurement of the IgG:IgM ratio from a single serum sample. Immunoglobulin recognizing S dublin epitopes also were detected in milk samples. In milk, performing 2 ELISA 60 days apart to determine IgG and IgM reactions to S dublin appeared to be useful for the prediction of carrier status, but was not as accurate as models for serum immunoglobulin reactions.     

Cause, occurrence, and clinical signs of mastitis and anorexia in cows in a Wisconsin study

Microbiologic culture revealed the following cause of mastitis and anorexia in 145 cows in Wisconsin to be Escherichia coli, 66 cows; Klebsiella spp, 3; Corynebacterium pyogenes, 27; streptococci, 21; staphylococci, 20; yeasts, 1; and no bacterial growth, 7. Mastitis was detected with approximately equal frequency throughout the year. Escherichia coli was isolated throughout the year, but was more common and was the predominant organism during the summer. Corynebacterium pyogenes was isolated most often in winter and spring; streptococci in fall, winter, and spring; and staphylococci throughout the year. Corynebacterium pyogenes caused most of the mastitis in nonlactating cows. Escherichia coli, C pyogenes, streptococci, and staphylococci were isolated with about equal frequency at parturition, whereas E coli was the predominant cause of mastitis in early and late lactation. Of cases of mastitis, 27% were seen 10 days before and after parturition. Local and systemic clinical signs of infection were similar for all causes, except that C pyogenes caused more (P less than 0.01) malodorous and purulent milk than did other organisms and was isolated more commonly from quarters with injured teats. Recovery was significantly (P less than 0.01) higher in cows with E coli infections, compared with recovery in cows with gram-positive organism infections. Cows with C pyogenes infections frequently had quarters that ultimately ceased lactation. A few cows were recumbent at initiation of antimicrobial therapy and a few were not eating 24 hours later; however, 50% of these cows recovered. Criteria such as season of year, stage of lactation, appearance of milk and udder, and appetite permitted the cause (gram-negative or gram-positive organisms) of the mastitis to be predicted with 77% accuracy.     

Influence of genotype and yield and composition of milk on interval to first postpartum ovulation in milked beef and dairy cows.

Our objectives were to investigate the effects of genotype, yield and composition of milk, and changes in BW and body condition on the initiation of cyclic ovarian activity in nonsuckled beef and dairy cows milked twice daily. Nulliparous heifers were selected from three breeds, 12 Angus, 13 Simmentals, and 26 Holsteins, based on their EBV for milk yield. Cows were machine-milked twice daily, and daily milk yield was recorded; composite samples of milk were collected twice weekly for analyses of milk components. Blood was collected from all cows thrice weekly for 75 d postpartum, and concentrations of progesterone in serum measured by RIA were used to estimate day of first postpartum ovulation. Holstein cows produced more (P less than .05) milk (unadjusted or 3.5% fat-corrected) than Angus or Simmental cows during the first 30 d of lactation. Holstein cows had higher (P less than .01) peak yield of milk than Simmental and Angus cows. Days to peak milk yield were similar for Simmental and Holstein cows, and both were more (P less than .05) than those for Angus cows. Percentages of fat, protein, and total solids in milk were highest (P less than .05) for Simmental cows, whereas milk of Holstein cows had the highest (P less than .05) percentage of lactose and lowest (P less than .05) concentration of somatic cells. Average BW at ovulation differed (P less than .05) among breeds. Estimated daily changes in BW from calving to first ovulation were different (P less than .05) for Holstein and Simmental cows but were similar to those of Angus cows.(ABSTRACT TRUNCATED AT 250 WORDS)     

Milk yield increase after anthelmintic treatment of dairy cattle related to some parameters estimating helminth infection

On 81 farms blood samples were taken from adult dairy cattle, on pasture in October 1986 and after stabling in December 1986, to measure antibody titres against the nematodes Dictyocaulus viviparus, Cooperia spp. and Ostertagia spp., and the trematode Fasciola hepatica, and serum pepsinogen values. Faecal samples, collected in October, were examined to confirm the presence of parasites by means of egg counts and larval identifications. From December until the end of the stabling period, dry cows were either treated with albendazole or left untreated in alternate sequence of calving date. Treated cows produced 132.9 kg milk per cow per lactation more than untreated cows (P less than 0.01). Fat and protein percentage were not significantly influenced by anthelmintic treatment. The mean herd milk yield response to treatment varied from -889 to +1231 kg milk per cow per lactation. There was a significant between-herd variation in antibody titres against nematodes and in pepsinogen values. However no significant correlations between these parameters and the mean herd milk yield response to treatment were found.     

Capture ELISA systems for the detection of bovine coronavirus-specific IgA and IgM antibodies in milk and serum

Isotype-capture ELISAs for BCV-specific IgA and IgM were developed and tested on milk and serum samples from Swedish cattle. The capture ELISAs showed higher sensitivity than indirect ELISAs for detection of BCV-specific IgA and IgM. In the capture ELISAs the agreement between detection in milk and serum samples was 94% for IgA and 86% for IgM. The correlation between log(10) titres in milk and serum was r=0.82 (P<0.001) for IgA and 0.84 (P<0.001) for IgM. Milk seemed a better target than serum for diagnosing specific IgA at low levels. There was no variation in the isotype-specific BCV antibody titres between healthy quarters of the same udder, but subclinical mastitis was associated with higher levels of IgA antibodies and weak false IgM positive reactions in undiluted milk. Bovine IgA and IgM antibodies in milk and serum showed high stability towards freezing and thawing and storage at room temperature.The antibody responses to BCV were followed in milk and serum from six dairy cows and in serum from four calves for a period of 1 year after an outbreak of winter dysentery (WD). In this outbreak some animals became reinfected with BCV. The IgA and IgM capture ELISAs differentiated between primarily BCV infected and reinfected animals. In the primarily infected cattle, IgM antibodies were first detected in milk and serum four to nine days after the first WD symptoms observed, and were subsequently detected for at least 2-3 weeks. IgM was also detected in the reinfected cows, but mostly at lower levels and for a shorter period of time than in the primarily infected animals. In milk, however, the IgM response of the reinfected cows was detected for a longer period of time than in serum. Six months after the outbreak, IgA was still detected in both serum and milk of all six cows and also in serum of one calf. The reinfected cows showed higher and more long-lasting peak levels of IgA in milk and serum than the primarily infected cows, indicating boosting of the IgA response.     

Effect of anthelmintic treatment of dairy cattle on milk production related to some parameters estimating nematode infection

On 31 farms, blood samples were taken from adult dairy cattle in September 1985, when pastured, and in November-December 1985, when stabled, to assess serum pepsinogen levels and level of nematode antibody titres. Faecal samples taken in September were examined to establish the presence of parasites by means of egg counts and larval identification. During the stabling period, dry cows were either treated with ivermectin or with a placebo in alternate sequence of expected calving date. As a result, 285 cows were treated with ivermectin while 242 cows served as controls. Anthelmintic treatment resulted in a significant increase in the 305-day milk production of 205.1 kg (P less than 0.01). Fat and protein percentages were not significantly influenced by anthelmintic treatment. There was a significant between-herd variation in nematode antibody titres and in pepsinogen values. The mean herd milk-production response to treatment correlated positively with the mean herd Ostertagia antibody titre measured in September 1985 (r = 0.364, P less than 0.05).     

Phagocytosis, bactericidal activity, and oxidative metabolism of milk neutrophils from dairy cows fed selenium-supplemented and selenium-deficient diets

Six primiparous Holstein cows were fed a Se-deficient diet, beginning at least 90 days before their first calving, and 6 other primiparous cows were given the same diet plus a supplement of 2 mg of Se/cow/d as sodium selenite. All cows were fed their diets for the duration of the experimental period. One uninfected quarter of each cow was injected with 25 micrograms of Escherichia coli endotoxin at postpartum week 5. Leukocytes were isolated by centrifugation from milk collected at postinjection hour 16. Isolated cells were 92 +/- 3% neutrophils and were incubated with Staphylococcus aureus or E coli in a 1:300 ratio. Phagocytosis and intracellular killing by neutrophils were assessed after 0, 30, 60, and 90 minutes by a fluorochrome assay, using acridine orange. Viability of neutrophils was assessed by use of trypan blue. Superoxide anion production and hydrogen peroxide production by neutrophils also were determined. Cows fed Se-deficient diets had significantly (P less than 0.05) lower blood Se concentration and blood glutathione peroxidase activity than cows fed Se-supplemented diets. Selenium status had no effect on the phagocytic capacity of neutrophils. Neutrophils obtained from cows fed Se-supplemented diets killed a significantly (P less than 0.05) higher percentage of ingested bacteria than did neutrophils from cows fed the Se-deficient diet. Viability was significantly (P less than 0.05) reduced by incubation with S aureus in neutrophils from both groups of cows, with neutrophils from Se-deficient cows having lower viability.(ABSTRACT TRUNCATED AT 250 WORDS)     

Natural antibodies in bovine milk and blood plasma: variability among cows, repeatability within cows, and relation between milk and plasma titers

Innate immunity plays an important role in preventing (barrier function) or combating infection (effector function). An important humoral component of innate immunity is formed by natural antibodies (NAb). The objectives of this study were to determine presence, variation among cows and repeatability within cows over time of total NAb titers directed to the pathogen-associated molecular patterns lipopolysaccharide, lipoteichoic acid (LTA) and peptidoglycan, and titers of NAb directed to the glycoprotein keyhole limpet hemocyanin in milk and plasma of individual cows. Furthermore in milk the antibody isotypes IgG1, IgG2, IgM and IgA binding LTA were analyzed. Ten milk and blood samples were obtained from each of 20 clinically healthy dairy cows from first to seventh parity during a period of 3 weeks. Total NAb binding lipopolysaccharide, LTA, peptidoglycan, and keyhole limpet hemocyanin were detected in milk and plasma, with titers considerably higher in plasma than in milk. Total NAb titers showed significant variation among cows, and repeatability within cows over time (ranging from 0.60 to 0.93). Titers of NAb in milk and plasma were positively correlated (correlation ranging from 0.69 to 0.91). Natural antibodies in milk binding LTA were of all 4 isotypes tested, although IgG2 was on average only present at low titers. All 4 isotypes in milk binding LTA also showed variation among cows, and repeatability within cows over time (ranging from 0.84 to 0.92). We conclude that NAb can be measured in a consistent and repeatable manner in bovine milk and blood plasma.     

Listeriosis in sheep. Listeria monocytogenes excretion and immunological state in sheep in flocks with clinical listeriosis.

The excretion of Listeria monocytogenes (Lm) in the faeces and milk, and humoral immunity against Lm, were examined in a sheep flock with outbreaks of listeric encephalitis and in a flock with outbreaks of listeric abortion. The encephalitis flock consisted of 86 ewes and 20 hoggs, the abortion flock of 45 ewes and 3 hoggs, all of them pregnant. Faecal excretion rate in the encephalitis flock varied from about 25 % in the first part of the indoor season to nearly zero 1 month later, to about 30 % 1 month before lambing and about 15 % at lambing. About 15 % of the animals also excreted Lm in the milk. Lm 4 was the dominating serotype.In the abortion flock about 2/3 of the animals excreted Lm in the faeces and 1/3 in the milk at lambing. All the isolates belonged to serotype 1, which also was isolated from grass silage and strawbedding samples.In the encephalitis flock ewes with ≥ 3 foetuses had a higher excretion rate than the remainder, while no such differences were found in the abortion flock.Antibody titres against Lm in sera and whey in the encephalitis flock were of the same order as in the healthy flock described in an earlier publication (Grønstøl 1979), except that the highest titres were found in the hoggs. Serum titres from the abortion flock after lambing were significantly higher than in the encephalitis flock, while whey titres were of the same order.Treatment with 2-mercapto-ethanol reduced the titres substantially in sera from the abortion flock, indicating that the antibodies belonged to the IgM-fraction, while only a slight reduction was seen after similar treatment of the whey.     

Sources of variation introduced into a phagocytosis assay as a result of the isolation of neutrophils from bovine blood

A study was conducted to examine sources of variation introduced into a phagocytosis assay as a result of the isolation of neutrophils from bovine blood, including variation attributable to isolation of neutrophils from blood, variation between duplicate determinations of percentage phagocytosis, and the variation in the ability of neutrophils isolated from blood (over repeated collections from the jugular vein) to phagocytose. For the phagocytosis assay, jugular venous blood from each of 4 cows was divided into 2 equal portions. The neutrophils were isolated by lysis of red blood cells with 0.2% sodium chloride. The neutrophils (2 x 10(7)) were incubated in duplicate with 32P-labeled Staphylococcus aureus ([32P]SA; 2 x 10(8)) in skimmed milk samples (2.5% final concentration) prepared from 4 cows. This process was repeated thrice on neutrophils isolated from 4 cows at 2-week intervals. The proportions of variation in percentage of 32P-labeled S aureus phagocytosed between duplicate neutrophil isolations and between duplicate assay determinations were 0 and 1%. Differences among skimmed milk sources and among runs, using blood neutrophils taken at different times from the same donor cow, accounted for 62 and 36% of the total variation. The results indicated that variation arising from blood neutrophil isolation introduced into a phagocytosis assay within a single-day trial is of no concern. The large variation among skimmed milk sample sources indicated differences among cows in the ability of their milk to support phagocytosis. The variation in neutrophil isolations over time for any cow was considered too large to allow for evaluation of physiologic and environmental effects on phagocytosis of neutrophils isolated from blood.