Milk NAGase Activity Research Articles

N-acetyl -β-D-glucosaminidase activity in cow milk as an indicator of mastitis.

Activity of lysosomal N-acetyl-β-d-glucosaminidase (NAGase) in milk has been used as an indicator of bovine mastitis. We studied NAGase activity of 808 milk samples from healthy quarters and quarters of cows with spontaneous subclinical and clinical mastitis. Associations between milk NAGase activity and milk somatic cell count (SCC), mastitis causing pathogen, quarter, parity, days in milk (DIM) and season were studied. In addition, the performance of NAGase activity in detecting clinical and subclinical mastitis and distinguishing infections caused by minor and major bacteria was investigated. Our results indicate that NAGase activity can be used to detect both subclinical and clinical mastitis with a high level of accuracy (0·85 and 0·99). Incomplete correlation between NAGase activity and SCC suggests that a substantial proportion of NAGase activity comes from damaged epithelial cells of the udder in addition to somatic cells. We therefore recommend determination of NAGase activity from quarter foremilk after at least six hours from the last milking using the method described. Samples should be frozen before analysis. NAGase activity should be interpreted according to DIM, at least during the first month of lactation. Based on the results of the present study, a reference value for normal milk NAGase activity of 0·1-1·04 pmoles 4-MU/min/μl for cows with ≥30 DIM (196 samples) could be proposed. We consider milk NAGase activity to be an accurate indicator of subclinical and clinical mastitis.

Milk haptoglobin, milk amyloid A, and N-acetyl-β-d-glucosaminidase activity in bovines with naturally occurring clinical mastitis diagnosed with a quantitative PCR test

The associations between quantitative bacteriological results from a real-time PCR test and concentrations of acute-phase proteins (APP) and N-acetyl-β-d-glucosaminidase (NAGase) activity in milk in naturally occurring clinical mastitis were investigated. Milk APP concentrations and NAGase activity in clinical mastitis caused by different udder pathogens were studied. The associations between the severity of the clinical signs and concentrations of APP and NAGase activity were estimated. Milk samples from 281 cases of clinical mastitis were collected from 3 Estonian dairy farms and analyzed by PCR to identify pathogens. Twenty-seven samples out of 281 (9.6%) were PCR negative. Milk samples containing 4 or more bacterial species (n=28) were considered possibly contaminated and excluded from all further analyses. In total, 443 bacterial identifications were made from the remaining 226 milk samples. A single bacterial species was detected in 68 samples (30.1%), 2 species were detected in 99 samples (43.8%), and 3 species were detected in 59 (26.1%) samples. To determine the inflammatory response in the udder, the concentrations of milk amyloid A (MAA) and haptoglobin (Hp) and NAGase activity in the milk were analyzed. A significant positive association was found between the severity of the clinical signs and inflammatory markers in the milk. Milk amyloid A and Hp concentrations and NAGase activity were significantly higher in samples with large quantities of bacterial DNA from Escherichia coli or Streptococcus dysgalactiae compared with milk samples not containing those species. Large quantities of bacterial DNA from Trueperella pyogenes or Streptococcus uberis in the milk were associated with elevated concentrations of Hp and high NAGase activity, but not with increased MAA concentrations. Milk samples containing Corynebacterium bovis and coagulase-negative staphylococci had significantly lower concentrations of MAA and Hp and lower NAGase activity compared with samples where these species were not detected. It can be concluded that concentrations of APP and NAGase activity in the milk were associated with the quantity of bacterial DNA in the milk samples.

Somatic cell count, lactoferrin and NAGase activity in milk of infected and non-infected udder halves of dairy goats

Based on a repeated sampling the influence of an intramammary infection on the somatic cell count (SCC), the content of lactoferrin (Lf) and the activity of N-acetyl-β- d-glucosaminidase (NAGase) in goats’ milk was investigated. 58 dairy goats (German Improved Fawn) were sampled weekly over 3 consecutive weeks and the udder halves were classified according to the results of the bacteriological analysis of the foremilk samples and the results of their parallel half into three groups: uninfected halves with an uninfected parallel half (NoInf/NoInf), uninfected halves with an infected parallel half (NoInf/Inf) and infected halves with an uninfected parallel half (Inf/NoInf). None of the goats had two infected halves, thus, this group was omitted. 15 out of the 58 goats were infected on one udder half. The bacteria detected were Staphylococcus aureus ( n = 4), coagulase-negative staphylococci ( n = 7), corynebacteria ( n = 3) and esculin-positive streptococci ( n = 1). Log 10 SCC, log 10 Lf and log 10 NAGase were strongly correlated to each other and changed over the sampling period but not uniformly, revealing a significant effect of the sampling day on the variables ( F 6,98 = 29.13, p < .001). This could not be explained by an underlying effect due to the stage of lactation or the estrus season the animals were in, and therefore needs further investigation. The infection status had a significant effect on log 10 SCC ( F 2,103 = 20.22, p < .001), log 10 Lf ( F 2,103 = 11.18, p < .001) and log 10 NAGase ( F 2,103 = 12.06, p < .001). Inf/NoInf differed significantly from NoInf/NoInf as well as NoInf/Inf for log 10 SCC ( p < .01) and log 10 Lf ( p < .001) whereas the NoInf/NoInf did not differ from NoInf/Inf indicating that the infected halves did not influence their uninfected parallel half. For log 10 NAGase this was different: infected halves differed significantly from NoInf/NoInf ( p < .01) but not from NoInf/Inf which might be caused by a dependency of the udder halves. Results support the approach to monitor mastitis in goats by means of Lf or NAGase instead of SCC. Further studies should explore the effect of other independent variables, such as estrus, on these indicators and aim for thresholds indicating an intramammary infection.

Efficacy of enrofloxacin in the treatment of naturally occurring acute clinical Escherichia coli mastitis

The efficacy of the combination of systemic enrofloxacin (5mg/kg twice with a 24-h interval, first dose i.v., second dose s.c.) and the nonsteroidal antiinflammatory agent ketoprofen (3mg/kg i.m. or 4mg/kg per os daily for 1 to 3 d) treatment was compared with antiinflammatory treatment only in dairy cows with naturally occurring acute clinical Escherichia coli mastitis. A total of 132 cows with acute clinical mastitis and with confirmed growth of E. coli in a pretreatment milk sample were randomly allocated to 1 of 2 treatment groups. Response to treatment was evaluated clinically and by bacteriological culturing and determination of N-acetyl-β-d-glucosaminidase (NAGase) activity on d 2 and 21 posttreatment. Enrofloxacin treatment did not increase bacteriological (90.5% of treated vs. 86.8% of nontreated cured) or clinical cure (46.7% of treated vs. 57.1% of nontreated cured), cow survival (95.3% of treated vs. 92.7% of nontreated), or quarter milk production assessed 21 d posttreatment (21.8 vs. 29.3% return to preinfection level for nontreated cows), nor did it decrease mammary gland tissue damage estimated using determination of milk NAGase activity (24.0±0.3 vs. 18.3±1.3 pmol of 4-methylumbelliferone per min per μL for nontreated cows). Treatment did not influence the number of study cows remaining in the herd after 6 mo (71.9% of treated vs. 80.6% of nontreated). The only significant effects of enrofloxacin were enhancing the bacteriological cure (odds ratio=3.32 for treated cows) and decreasing the clinical cure (odds ratio=0.05 for treated cows) on d 2 posttreatment. Our results did not support the use of enrofloxacin to treat acute clinical E. coli mastitis.

Host response in bovine mastitis experimentally induced with Staphylococcus chromogenes

An experimental infection model was developed to study host response to intramammary infection in cows caused by Staphylococcus chromogenes. CNS intramammary infections have become very common in modern dairy herds, and they can remain persistent in the mammary gland. More information would be needed about the pathophysiology of CNS mastitis, and an experimental mastitis model is a means for this research. Six primiparous Holstein–Friesian cows were challenged with S. chromogenes 4 weeks after calving . One udder quarter of each cow was inoculated with 2.1 × 10 6 cfu of S. chromogenes. All cows became infected and clinical signs were mild. Milk production of the challenged quarter decreased on average by 16.3% during 7 days post-challenge. Cows eliminated bacteria in a few days, except for one cow which developed persistent mastitis. Milk indicators of inflammation, SCC and N-acetyl-β- d-glucosaminidase (NAGase) returned to normal within a week. Milk NAGase activity increased moderately, which reflects minor tissue damage in the udder. Concentrations of serum amyloid A (SAA) and milk amyloid A (MAA) were both elevated at 12 h PC. MAA was affected by the milking times, and was at its highest before the morning milking. In our experimental model, systemic acute phase protein response with SAA occurred as an on–off type reaction. In conclusion, this experimental model could be used to study host response in CNS mastitis caused by the main CNS species and also for comparison of the host response in a mild intramammary infection and in more severe mastitis models.

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 β-lactamase negative strains) or amoxycillin-clavulanic acid (24 cases due to β-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 β-lactamase negative group.

Bovine clinical mastitis due to coagulase-negative staphylococci and their susceptibility to antimicrobials.

A total of 168 coagulase-negative staphylococci (CNS) strains were isolated from milk samples taken from cows with clinical mastitis. The samples were collected between January 1990 and August 1992 from cows in the veterinary surveillance area of the Ambulatory Clinic, College of Veterinary Medicine, Hautjärvi, Finland. In 100 cases the effect of antibiotic treatment was evaluated 3-4 weeks after initial sampling. Clinical symptoms of the animals were recorded, and the inflammatory status of their udders was evaluated using the CMT test and assessing milk NAGase activity. CNS mastitis was most common in young cows during early lactation. Staphylococcus hyicus, S. simulans and S. epidermidis were the most frequently isolated CNS. Clinical symptoms were most severe with S. hyicus. Cure rates for CNS induced mastitis were high.

Prediction of Mastitis Using Milk Somatic Cell Count, N-Acetyl-β-D-Glucosaminidase, and Lactose

The objectives of this work were 1) to examine the responsiveness of SCC, lactose concentration, and NAGase activity in milk to changes in bacteriological status and 2) to develop models for predicting bacteriological status of mammary glands. Data included 550 cows in 10 commercial herds. Natural logarithm NAGase and log cell count were most responsive to changes in bacterial status. The log NAGase was relatively more effective in identifying major from minor pathogen infections, whereas log SCC was better able to differentiate between infected and uninfected classes. Non-transformed NAGase, SCC, and lactose were considerably less responsive to infection status. Logistic regression of bacterial status on herd, lactation number, milk, log SCC, log NAGase, and stage of lactation was performed. The least significant variables were removed in a stepwise process. Final predictors of infection status were herd, log SCC, and log NAGase. The role of log SCC was to discriminate infection from no infection, whereas log NAGase discriminated major from minor pathogens. The log NAGase, alone or in combination with log SCC, added substantially to the detection power of the model. Chi-square goodness of fit tests found no significant differences between observed and predicted infection probabilities. Substitution of herd averages for log SCC and log NAGase for the herd variables resulted in significant differences between predicted and observed herd infection probabilities.

N-acetyl-β-d-glucosaminidase as a predictor of milk loss and recovery after clinical mastitis

Summary Milk samples were collected at onset of 508 episodes of clinical mastitis on a 1,700-cow dairy farm in Michigan. Daily milk production and disease events were recorded for all cows in the herd. Despite statistical association with severity of mastitis, this association was too weak for Nacetyl-(β-d-glucosamimdase (NAGase) activity to be of great value as a prognostic test for clinical mastitis. High milk NAGase activity was significantly (P &lt; 0.0001) associated with: increased duration of treatment; increased duration of clinical signs of mastitis; decreased daily milk production; and increased risk of the cow being culled because of mastitis. The NAGase value was combined with days in milk production, baseline milk production before mastitis onset, parity, and season of onset to predict the outcome of clinical cases as measured by the first 3 aforementioned variables. Statistical models explained little of the variability among cows in duration of treatment (R2 = 0.11), duration of clinical signs of infection (R2 = 0.11), and milk production change (R2 = 0.09).

Influence of Exogenous Estradiol on the Concentration of Antitrypsin, Albumin, N‐Acetyl‐pβ‐D‐Glucosaminidase and Somatic Cells in Milk of Cows at Various Lactation Stages

Estradiol benzoate (0.02 mg/kg of body weight) was injected (i.m.) daily to 13 ovariectomized cows in their first or third lactations during early (60-90 days), mid (140-200 days) and late (240-300 days) lactation. The majority of the udder quarters were free of bacteria throughout the experiment. Signs of clinical mastitis were observed after 9-19 days of treatment in cows at mid and late lactation. This was accompanied by a decrease in milk yield, an increase in milk somatic cell count (MSCC) and increases in milk concentration of antitrypsin and serum albumin (as indicator of increased permeability); and milk NAGase activity (as indicator of epithelial cell damage or release of the enzyme from phagocytes). NAGase activity was first to increase and on some occasions was not accompanied by an increased MSCC. The response was either absent (first lactation) or relatively mild (third lactation) in cows at the early stages of lactation. The intensity of the inflammatory response increased with advancing lactation stage. Generally, the response of cows in the third lactation was greater than that of cows in the first lactation at a corresponding stage of lactation. Three cows which received the control treatment with vehicle alone showed no changes in any of the parameters except a slight decrease in antitrypsin. All inflammatory indicators correlated negatively with quarter milk production; milk NAGase activity was superior in this respect (r = -0.75). All correlation coefficients were generally higher after, rather than before the hormone treatment.

Determination of NA-Gase activity in milk for diagnosis of subclinical caprine mastitis

Two goat herds were examined to determine the prevalence and causes of subclinical mastitis and to assess the value of some laboratory tests currently used as aids in the diagnosis of caprine mastitis. Due to large variation in mean cell count, the Whiteside test and California mastitis test were unreliable in diagnosis of caprine mastitis. N-acetyl-β-D-glucosaminidase (NA-Gase) activity in the milk was a sensitive method in detecting subclinical mastitis in goats. In 381 samples from mammary gland halves of 191 goats, the prevalence of infection in the two herds ranged from 13.0% to 20.0%. Organisms isolated were coagulase-negative staphylococci 54.2%, coagulase-positive staphylococci 20.3%, alpha-haemolytic streptococci 2.0%, Pasteurella haemolytica biotype A 5.0%, Mycoplasma agalactiae 6.0% and Escherichia coli 4.0%.

N-Acetyl-β-D-Glucosaminidase Activity in Whole Milk and Milk Fractions

Experiment 1 was conducted to determine NAGase activity in skim, fat, and cell pellet fractions of foremilk and stripping milk from infection-free quarters. Changes in milk NAGase activity during a 12h in vitro incubation were also determined. Eight cows, two quarters per cow, were used. One quarter of each cow received an intramammary infusion of oyster glycogen. N-Acetyl-β-D-glucosaminidase activity was highest in stripping milk and in milk from infused quarters. The percentages of NAGase activity in skim, fat, and cell pellet fractions were 62.6, 22.4, and 12.6. The NAGase activity of milk incubated in vitro did not significantly change over time. Experiment 2 was conducted to determine if neutrophils lost NAGase activity during extravasation into milk. Leukocytosis was induced in infection-free quarters of five cows. The NAGase activities of peripheral neutrophils and milk neutrophils were not significantly different. Results from both studies suggest that the major source of milk NAGase is the mammary epithelial cell and that milk somatic cells contribute less than 15% of the total milk NAGase activity.

The Effect of Intramammary Antibiotic Therapy at Calving on Udder Health Traits

The effect of intramammary antibiotic therapy at calving on mastitis infection prevalence, linear score milk somatic cell count, and milk NAGase activity, 30 d postpartum, and on milk production, 90 to 120 d postpartum, was tested. Cows (n=175) were split into treatment and control groups at drying off. All cows received commercial dry cow therapy. At calving, treated cows received commercial lactating cow therapy in all quarters after the first two milkings; control cows were not treated. Composite milk samples were aseptically collected from all cows at drying off, calving, and 30 d postpartum. Udder health traits: linear score milk SCC, NAGase activity, and bacterial content in milk, were determined on all samples. The first three DHI milk weights were recorded for all cows.Treatment and control cows had similar prevalences of intramammary infections during the dry and 30-d postpartum periods. Least squares means of linear score milk SCC and NAGase activities were similar at drying off and calving. Cell count scores were similar between groups; NAGase activities were higher in control cows at 30 d postpartum. Control cows tended to produce more milk postpartum. Results demonstrated no advantage of intramammary therapy at calving in improving milk production or udder health.