Comparison of milk antitrypsin,albumin, n-acetyl-β-d-glucosaminidase,somatic cells and bacteriological analysis as indicators of bovine subclinical mastitis

Thirty-two quarters, five of which harbored subclinical mastitis, were examined daily for one month. The usefulness of milk antitrypsin, BSA (bovine serum albumin), NAGase, somatic cells and bacteriological analysis in differentiating the inflamed quarters from the healthy control quarters was analysed. Inter-quarter evaluation clearly improved each indirect mastitis parameter; NAGase and antitrypsin were better indicators of differences between infected and non-infected quarters than BSA or the somatic cell count.     

Antitrypsin and N-acetyl-beta-D-glucosaminidase as markers of mastitis in a herd of Ayshire cows

Microplate analyzers for N-acetyl-beta-D-glucosaminidase (NAGase) and antitrypsin in milk were high-capacity systems for determination of subclinical mastitis on a mammary-quarter basis in a herd of 80 Ayshire cows. Infected quarters could be better differentiated from non-infected quarters by interquarter evaluation of milk NAGase and antitrypsin, than by adaptation of threshold values based on measured NAGase and antitrypsin values. This differentiation between healthy quarters and mastitic quarters was further improved by using absolute values and interquarter evaluations in combination. Therefore, quarter-based sample collection in combination with microplate technology and computerization is proposed as the method of choice for monitoring udder health in herds.     

Effect of frequent milkings on milk NAGase, plasmin, trypsin inhibitory capacity and the quality of whey as the growth medium for mastitis pathogens

Unequal milking intervals affected milk somatic cell count and N-acetyl-beta-D-glucosaminidase (NAGase) activity. The total daily output of milk NAGase and plasmin decreased, if quarters were emptied frequently during the day. Mastitis pathogens showed stimulated growth in whey prepared from filled quarters as compared with growth in whey from quarters emptied frequently during the day. The quality of whey as growth medium for mastitis pathogens paralleled plasmin activity in respective milk samples. Adaptation of mastitis pathogens to grow in whey had an enhancing effect on bacterial growth during subsequent inoculations in whey. Bacteria probably "learn" to overcome the effect from endogenous antibacterial factors and to use nutrients present in whey.     

Effect of some vehicles used in intramammary mastitis preparations on inflammation indicators in milk

The irritant effect of some vehicles used in intramammary mastitis preparations was assessed by indirect mastitis indicators in milk. Mineral oil, soybean oil, peanut oil, a combination of aluminium monostearate, stearic acid and mineral oil as well as isotonic saline were infused intracisternally in cows during mid-lactation. These vehicles induced a selective increase in the number of somatic cells without any notable effects on the permeability marker antitrypsin or the lysosomal enzyme N-acetyl-beta-D-glucosaminidase (NAGase).     

Intramammary antibiotics with complementary acupuncture decreases milk serum N-acetyl-beta-D-glucosaminidase concentrations in dairy cattle with subclinical mastitis

The objective of this research is to determine whether intramammary antibiotics with complementary acupuncture can reduce bovine mammary inflammation due to subclinical mastitis. Lactating cows were selected based on milk with a somatic cell count (SCC) greater than 500,000 cells/ml. Pre- and post-treatment milk samples were collected to determine SCC, aerobic bacterial content, milk ion conductivity, total protein, lactate dehydrogenase (LDH) and N-acetyl-beta-D-glucosaminidase (NAGase) concentrations. Milk serum was prepared from milk samples by double centrifugation. Concentrations of LDH and NAGase were determined using commercial enzyme-linked immunosorbent assays. Cows being treated with intramammary antibiotics were separated by random assignment to the acupuncture group (n = 10) and a no-acupuncture (control) group (n = 9). Both the acupuncture and control group were restrained for 30 min in a head catch 12 hr apart for a total of four times. For front quarters affected by subclinical mastitis, the acupuncture points used were spleen (SP) 12, SP 17, SP 18, SP 21, stomach (ST) 18 and conception vessel (CV) 12. For rear quarters affected by subclinical mastitis, the acupuncture points used were bladder (BL) 30, BL 30-1, BL 49, kidney (KI) 10, conception vessel (CV) 2 and CV 3. All parameters were compared using a Student t test. Significance was defined as p < .05. Compared to control cows, complementary acupuncture treatment reduced NAGase enzymatic activity in quarters of cows with subclinical mastitis. The reduction in NAGase suggests that complementary acupuncture treatment may be associated with healing of the damaged mammary epithelial cells, which are the primary source of NAGase activity in milk serum.     

乳酸链球菌素治疗奶牛乳房炎效果观察

乳酸链球菌素（Nisin）是由乳酸链球菌产生的合有34个氨基酸的抗菌肽,对人体无害。本研究将患临床型乳房炎奶牛92头（107个乳区）和隐性乳房炎奶牛90头（90个乳区）随机分成试验组和对照组。试验组的治疗方法是在患病乳区灌注Nisin;临床型乳房炎以庆大霉素乳房内灌注作对照,隐性乳房炎的对照组不作任何处理。临床型乳房炎的Nisin治疗组细菌学治愈率和临床治愈率分别为60．8％和90．2％,而庆大霉素治疗组则分别为44.6％和91．2％,两者无统计学差异。隐性乳房炎经Nisin治疗后,高体细胞数奶牛的数量和牛奶中NAGase的活性均显著下降,细菌转阴率提高。乳酸菌发酵试验表明,经Nisin乳房内灌注治疗临床型乳房炎后,牛奶在第36小时恢复正常发酵,而庆大霉素治疗则需要72h。     

Pathogenicity of different species of staphylococci in caprine udder

Aseptic foremilk samples were collected from Finnish landrace goats. Ten different species of staphylococci, causing subclinical infections were detected. Twelve goats with persistent subclinical staphylococcal infection were followed on a monthly basis and compared with foremilk samples of nine goats suffering from clinical mastitis. Parameters of inflammation based on the activity of the California Mastitis Test (CMT). N-acetyl-beta-D-glucosaminidase (NAGase) and antitrypsin were determined from the milk. Staphylococci were further classified using the API STAPH system. On the basis of elevations of activities of CMT, NAGase and antitrypsin, Staphylococcus aureus was the most pathogenic in clinical and subclinical mastitis, S. hyicus showed only marginal pathogenicity. Subclinical infections were persistent and the infective organism was not always detected from milk by culture. The biochemical reactions of subclinical staphylococci seemed to vary within the same gland by time. Antitrypsin was most effective in differentiating between subclinical and clinical infection. A teat cistern puncture technique was found to be suitable for the goat.     

Efficacy of targeted 5-day combined parenteral and intramammary treatment of clinical mastitis caused by penicillin-susceptible or penicillin-resistant Staphylococcus aureus

Combined parenteral and intramammary treatment of mastitis caused by Staphylococcus aureus was compared to parenteral treatment only. Cows with clinical mastitis (166 mastitic quarters) caused by S. aureus treated by veterinarians of the Ambulatory Clinic of the Faculty of Veterinary Medicine during routine farm calls were included. Treatment was based on in vitro susceptibility testing of the bacterial isolate. Procaine penicillin G (86 cases due to beta-lactamase negative strains) or amoxycillin-clavulanic acid (24 cases due to beta-lactamase positive strains) was administered parenterally and intramammarily for 5 days. Efficacy of treatments was assessed 2 and 4 weeks later by physical examination, bacteriological culture, determination of CMT, somatic cell count and NAGase activity in milk. Quarters with growth of S. aureus in at least one post-treatment sample were classified as non-cured. As controls we used 41 clinical mastitis cases caused by penicillin-susceptible S. aureus isolates treated with procaine penicillin G parenterally for 5 days and 15 cases due to penicillin-resistant isolates treated with spiramycin parenterally for 5 days from the same practice area. Bacteriological cure rate after the combination treatment was 75.6% for quarters infected with penicillin-susceptible S. aureus isolates, and 29.2% for quarters infected with penicillin-resistant isolates. Cure rate for quarters treated only parenterally with procaine penicillin G was 56.1% and that for quarters treated with spiramycin 33.3%. The difference in cure rates between mastitis due to penicillin-susceptible and penicillin-resistant S. aureus was highly significant. Combined treatment was superior over systemic treatment only in the beta-lactamase negative group.     

Examination of milk samples from experimentally infected heifers by an N-acetyl-β-D-glucosaminidase test and an antigen-capture elisa

Milk samples collected from normal and experimentally infected quarters of five heifers throughout their first lactation were examined by bacterial culture, milk cell count (MCC), N-acetyl-beta-D-glucosaminidase (NAGase) test and a monoclonal antibody based antigen-capture ELISA. The results were analysed according to the presence (mastitis positive) or absence (mastitis negative) of bacteria on culture, and the numbers of false negative and false positive results of the other three tests defined in relation to this. Similar numbers of false negative results were observed with the MCC (20), NAGase (18) and ELISA (13). False positive results due to the physiological factors present in early lactation were evident in the MCC, prominent in the NAGase test and absent from the ELISA. The major difference in false positive results associated with experimental infection between the three tests was the more rapid return to negative values of the ELISA following resolution of infection, compared with MCC and NAGase.     

Phenotypic characteristics of Staphylococcus aureus isolated from bovine mastitis in Israeli dairy herds

A study of the characterization of the phenotypic patterns of Staphylococcus aureus strains isolated from bovine subclinical mastitis in Israeli dairy herds and their correlation with the severity of the disease was undertaken. A total of 400 chronically S. aureus-infected Israeli-Holstein cows, from 15 dairy herds were included in this study. Based on the results of the biochemical reactions, of the anti-biogram and phage typing, one major type of S. aureus was determined in each herd, its prevalence being between 54 and 100% of the total isolates from that same herd. The majority of the isolates were found to be non-haemolytic (62.7%). The most common phage type was 3/A,3/C,55,71, which was predominant in five herds. In two herds none of the isolates (24) were typable by this set of phages. All isolates were susceptible to methicillin, erythromycin, cephalotin, norfloxacin, trimethoprin-sulphamethoxazole and novobiocin. Most isolates were resistant to penicillin (96.6%) and 52% to oxytetracyclin. Differences in protein patterns between 50 and 36 kDa were found by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis. No correlation between any combination of the phenotypic characteristics was found when correlation was done with milk yield and somatic cell count, corresponding to the 6 months before sampling. Otherwise, a positive correlation was found between type of haemolysis and the N-acetyl-beta-glucosaminidase (NAGase) values. In milk from quarters infected with the-non-haemolytic strains, the level of NAGase was significantly lower (P < 0.05) than that from quarters infected with the haemolytic strains (69.7 and 105.9, respectively). However, the level of NAGase activity in the milk of the quarters infected with the non-haemolytic strains was significantly higher (P < 0.05) when compared to the milk of quarters infected with coagulase-negative staphylococci (43.5).     

Sequestration of N-acetyl-beta-D-glucosaminidase in somatic cells during experimental bovine mastitis induced by endotoxin, Staphylococcus aureus or Streptococcus agalactiae

Experimental mastitis was induced in cows by intramammary infusion of endotoxin, Staphylococcus aureus or Streptococcus agalactiae. The inflammatory response was monitored by somatic cell counting and N-acetyl-beta-D-glucosaminidase (NAGase). NAGase activity was analysed in fresh milk samples in parallel with samples treated by a cycle of freezing and thawing combined with detergent treatment to release the cell-bound NAGase. Before the udder reacted by inflammation, the total NAGase activity consisted of free extracellular activity. Later on when the inflammation was established, much of the milk NAGase remained sequestered intracellularly. S agalactiae was linked with a high degree of cellular NAGase sequestration indicating a blockage of the lysosomal release function from the phagocytes. S aureus delayed the inflammatory response.     

Bacterial growth in whey from mastitic and nonmastitic quarters

Growth of Escherichia coli and Staphylococcus aureus was analyzed by turbidometry in 456 selected whey samples. The samples had been graded mastitic or nonmastitic as determined by antitrypsin assay and bacteriologic examination. Whey samples from inflamed quarters (antitrypsin increased) and infected quarters significantly promoted bacterial growth as compared with whey samples from control quarters. The growth-stimulatory effect was limited to the diseased quarters, since control quarters from the same animals did not support enhanced bacterial growth. The observations indicate that in chronic mastitis, the inflammatory process leads to changes in the whey that promote bacterial growth in vitro.     

Effects of estrus and exogenous estrogen on circulating neutrophils and milk somatic cell concentration, neutrophil phagocytosis, and occurrence of clinical mastitis in cows.

In a study involving 56 quarters (14 cows), it was seen that standing estrus did not affect circulating neutrophil and milk somatic cell concentrations, neutrophil phagocytosis, milk production, or occurrence of clinical mastitis. The subcutaneous injection of 17beta-estradiol (E2) (0.05 mg/kg of body weight 2 times daily for 2 consecutive days) did not affect neutrophil phagocytosis or milk somatic cell values. However, a significant increase in circulating neutrophils and a significant decrease in milk production were observed. The injection of E2 was followed by clinical mastitis in 4 uninfected quarters, in 7 quarters infected with Corynebacterium bovis, and in 1 quarter infected with Streptococcus uberis and Escherichia coli.     

Indicators of inflammation to evaluate the recovery from acute bovine mastitis

The recovery of 39 mastitic cows was evaluated using alpha 1-antitrypsin, N-acetyl-beta-D-glucosaminidase (NAGase), plasmin and somatic cell counting for monitoring the inflammatory process. Bacteriological investigation was undertaken during the acute phase and at days 21 and 50 after treatment. In the quarters with a bacteriological cure the concentrations of all inflammatory markers in the milk returned to normal within three weeks. The quarters which harboured subclinical infection at days 21 and 50 after the onset of the disease showed higher values of the inflammatory parameters than uninfected, healed quarters. NAGase was the best predictor of the bacteriological status during the healing period (three weeks after therapy). The study suggests that NAGase determination in each quarter (with interquarter evaluation) possibly in conjunction with a bacteriological investigation three weeks after therapy, would be a good combination for assessing the outcome of treatment.     

Variations During Lactation in Total and Differential Leukocyte Counts,N-acetyl-ß-D-glucosaminidase,Antitrypsin and Serum Albumin in Foremilk and Residual Milk from Non-infected Quarters in the Bovine

Quarter samples of foremilk and residual milk were taken approximately every second week from 2 days post partum (pp) throughout lactation month 9, from 5 dairy cows in their second lactation period. Bacteriologically positive milk samples were excluded. The aim was to study the variation in total and differential leukocyte counts, N-acetyl-ß-D-glucosaminidase (NAGase), antitrypsin (ATR) and serum albumin (BSA) in milk during the lactation period and different stages of oestrous cycle. Also the between milkings variation was studied from lactation month 4 to 9. At 2 days pp, each fraction of milk contained significantly higher numbers of leukocytes and had a higher activity of NAGase and ATR than later in the lactation period. In foremilk the highest content of BSA was also recorded at 2 days pp. From lactation month 2 to 9, stage of lactation had, in general, a slight effect on the variation in the variables measured. The total leukocyte count in residual milk tended to increase as lactation proceeded. The proportion of monocyte-macrophages in foremilk was significantly decreased during the last 4 months. NAGase and BSA in both fractions and ATR in residual milk increased significantly towards the end of the lactation period. From lactation month 4 to 9 the highest recorded ranges of variation between milkings, within quarter and stage of lactation, in the total leukocyte count, proportions of neutrophils, lymphocytes, monocyte-macrophages, NAGase, ATR and BSA in foremilk were 215 × 10³ml, 42 %, 34 %, 54 %, 6.68 units, 0.36 units and 0.14 mg/ml respectively. The corresponding figures in residual milk were higher except for the variation in BSA which was slightly lower in residual milk than in foremilk. In residual milk there was a positive correlation between the proportion of neutrophils and the total leukocyte count, when calculated on data from all cows and the entire experimental period. During the oestrous periods, the proportion of neutrophils in residual milk was higher than during the dioestrous periods. Foremilk and residual milk differed in the total as well as the differential leukocyte counts in all the various stages of lactation, whereas the contents of NAGase, ATR and BSA were equal in both fractions. The exception was 2 days pp when the proportions of lymphocytes were equal in both fractions and BSA-significantly higher in foremilk than in residual milk.     

The use of Markov chain Monte Carlo for analysis of correlated binary data: patterns of somatic cells in milk and the risk of clinical mastitis in dairy cows

Two analytical approaches were used to investigate the relationship between somatic cell concentrations in monthly quarter milk samples and subsequent, naturally occurring clinical mastitis in three dairy herds. Firstly, cows with clinical mastitis were selected and a conventional matched analysis was used to compare affected and unaffected quarters of the same cow. The second analysis included all cows, and in order to overcome potential bias associated with the correlation structure, a hierarchical Bayesian generalised linear mixed model was specified. A Markov chain Monte Carlo (MCMC) approach, that is Gibbs sampling, was used to estimate parameters.

The results of both the matched analysis and the hierarchical modelling suggested that quarters with a somatic cell count (SCC) in the range 41,000–100,000 cells/ml had a lower risk of clinical mastitis during the next month than quarters <41,000 cell/ml. Quarters with an SCC >200,000 cells/ml were at the greatest risk of clinical mastitis in the next month. There was a reduced risk of clinical mastitis between 1 and 2 months later in quarters with an SCC of 81,000–150,000 cells/ml compared with quarters below this level. The hierarchical modelling analysis identified a further reduced risk of clinical mastitis between 2 and 3 months later in quarters with an SCC 61,000–150,000 cells/ml, compared to other quarters.

We conclude that low concentrations of somatic cells in milk are associated with increased risk of clinical mastitis, and that high concentrations are indicative of pre-existing immunological mobilisation against infection. The variation in risk between quarters of affected cows suggests that local quarter immunological events, rather than solely whole cow factors, have an important influence on the risk of clinical mastitis. MCMC proved a useful tool for estimating parameters in a hierarchical Bernoulli model. Model construction and an approach to assessing goodness of model fit are described.     

Inflammatory reactions in the teat and udder of the dry cow

The inflammatory reactions in the teat and udder of the dry cow were studied by total and differential somatic cell counts (SCC) and by measuring bovine serum albumin, N-acetyl-beta-D-glucosaminidase (NAGase), plasminogen and plasmin. The teat and udder cisterns were surgically separated from each other in two udder quarters of each cow. Salmonella endotoxin was infused in one teat cistern and one udder quarter, and saline was infused in one teat cistern and one udder quarter. The inflammatory response was followed by several samplings post infusion. The reactions in the dry udder quarters were mainly in line with the results of similar studies in lactating glands. The differential SCC and the NAGase results were, however, somewhat different. The teats were capable of a strong inflammatory response. Reactions different from those seen in the glands were observed with regard to permeability changes and NAGase. The experimental model used showed promising results and is suitable for further studies of the inflammatory process.     

Experimental infection of lactating bovine mammary glands with Streptococcus uberis in quarters colonized by Corynebacterium bovis

Twenty-seven quarters of 18 lactating dairy cows were inoculated intramammarily with 3.6 X 10(4) colony-forming units (CFU) of a strain of Streptococcus uberis isolated from a cow with clinical mastitis. Before quarters were inoculated, 22 were considered as naturally colonized with Corynebacterium bovis, and 5 were considered bacteriologically negative. Streptococcus uberis was isolated from all quarters within 2 days after inoculation, and all quarters developed clinical mastitis by 3 days after inoculation. Mastitis was acute, and most cows had increased rectal temperatures. The number of somatic cells increased significantly (P less than 0.05), and milk production decreased significantly. In many cows, rectal temperatures remained increased, and Str uberis was isolated from infected glands after intramammary and systemic antimicrobial treatments were given. A decreased number (110 CFU) of the same strain of Str uberis caused equally severe mastitis in 3 quarters colonized with C bovis and in 1 bacteriologically negative quarter in 2 cows. Streptococcus uberis was isolated from all inoculated quarters, and all quarters developed clinical mastitis by 2 days after inoculation. Two quarters colonized with C bovis and 2 bacteriologically negative quarters were inoculated once with 25 CFU and once with 240 CFU of a different strain of Str uberis (ATCC 27958). Streptococcus uberis was never isolated from inoculated quarters, and changes in milk yield or number of somatic cells were not observed.     

Bacterial cure and somatic cell count response of dairy cows with a positive California Mastitis Test at calving to therapy with cephapirin sodium

Cows without signs of clinical mastitis were evaluated by the California Mastitis Test at calving (Day 0). Milk samples from 117 of 184 quarters (64 cows) were positive by this test for mastitis and were submitted for bacterial culture and determination of somatic cell counts. Cows with infected quarters were randomly allocated to treatment with cephapirin sodium by intramammary infusion or to be untreated as controls. Two and 4 weeks following calving, milk was again sampled from the infected quarters and tested. By the 4-week evaluation, the quarters treated with cephapirin sodium had significantly (P < or = .05) fewer positive bacterial cultures and somatic cell counts were significantly (P < or =.05) reduced compared with untreated control quarters.     

Milk plasmin, antitrypsin, N-acetyl-beta-D-glucosaminidase and bacterial growth in lactoserum during the early post partum period

The activities of plasmin, N-acetyl-β-d-glucosaminidase (NAGase) and antitrypsin in milk were determined during 1 week post partum. The concentration of NAGase and antitrypsin in milk decreased significantly during this period. A slight decrease in plasmin activity was also seen. Replication rates of E. coli and S. aureus in lactoserum were also determined. Both test bacteria showed a tendency for increasing growth rates towards the end of the period. Growth of E. coli was significantly (p < 0.01) enhanced in day 2 samples as compared with samples collected during the first day post partum.