N-Acetyl-13-D-Glucosaminidase, Etiologic Agent, and Duration of Clinical Signs for Sequential Episodes of Chronic Clinical Mastitis in Dairy Cows D. J. WILSON, P. S. HERER, and P. M. SEARS Quality Milk Promotion Services Cornell University Ithaca, NY 14850

NAGase in mastitic quarter, NAGR = NAGase in reference quarter, days OUT = days out of the lactating herd following clinical mastitis, WMT = Wisconsin mastitis test, 4·MU = 4-methylumbelliferone.

ABSTRACT

This study examined effects of repeated episodes of clinical mastitis in chronically infected quarters on milk Nacetyl-~-D-glucosaminidase activity and duration of clinical signs. Milk samples were collected at each clinical onset from 49 chronic mastitis cases on a 1700-cow Michigan dairy farm. There were 49 first episodes of clinical mastitis, 49 second episodes, and 13 episodes of third or more. Agents isolated were Staphylococcus aureus (18.4%), Staphylococcus spp. (7.3%), no growth (20.2%), environmental pathogens (streptococci other than agalactiae, Escherichia coli, Klebsiella spp., Enterobacter spp., and Citrobacter spp.) (22.0%), other pathogens (Serratia spp., Bacillus spp., diphtheroids [Corynebacterium spp. and Actinomyces pyogenes], Pseudomonas spp., and Nocardia spp.) (11.9%), mixed pathogens (two agents isolated) (12.8%), and contaminated samples (7.3%). Etiologic agents, duration of clinical signs, and NAGase did not differ by episode number. The correlation between log of NAGase and log of time until clinical recovery was .34. The relationship between NAGase and duration of clinical signs was strongest for second episodes, and weakest for third and greater episodes of chronic mastitis. (Key words: N-acetyl-~-D-glucosamini­ dase, clinical mastitis, clinical recovery)

INTRODUCTION

Clinical mastitis has become more important as management improvements have resulted in greater control of subclinical mastitis in dairy cows. In herds with low SCC and low incidence of subclinical mastitis, increased clinical mastitis has been reported (2). Signs of clinical mastitis include swollen or painful quarters, clumps, flakes or watery appearance of milk, fever, hypothermia, inappetence, dehydration, depression, recumbency, and death (1). The lysosomal enzyme NAGase is found in milk, serum, and other body fluids. Milk NAGase increases during mastitis (4, 5, 7, 8, 9, 13, 16, 17). Most of this increase is thought to be due to leakage from damaged secretory epithelial cells (4, 5, 9, 16, 17). By the time clinical mastitis is detected, NAGase has risen to or near its peak value (3, 10). Peak activity is maintained for 24 to 36 h (3, 10). Activity of NAGase can be measured on the same day that milk is collected (15). It has been speculated that NAGase assay may be developed as a rapid test for predicting severity of clinical mastitis at the time of onset (5,7,8, 13, 14, 16, 17). The purpose of this study was to determine the effects of repeated episodes of clinical mastitis in chronically infected quarters on milk NAGase and duration of clinical signs.

Abbreviation key: CMT = California mastitis test, i.m.m. = intramammary, NAGM =

MATERIALS AND METHODS Sample Collection

Received August 9, 1990. Accepted December 6, 1990. 1991 J Dairy Sci 74:1539-1543

Milk samples were collected at clinical onset from every episode of clinical mastitis that 1539

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wasON ET AL.

occurred during a 6-mo period in a 1700-cow Holstein dairy herd in central Michigan. Mastitis control procedures included washing only the teats with planar nozzle drop hoses and drying teats with single use paper towels before milking, postmilking teat dipping using spray nozzles, prompt treatment of clinical cases, and dry cow antibiotic therapy administered to all cows at the end of lactation. Streptococcus aga/actiae had been eradicated from the herd. Prevalence of specific mastitis pathogens on this farm was unknown before the study. Somatic cell count measured at the milk plant was 230,OOO/ml at the beginning of the study and 190,OOO/ml at its conclusion. Milking personnel detected mastitis by presence of a hot, hard, or swollen quarter, abnormal flakes, and color or consistency of the milk. Clinical mastitis was further confirmed with the California mastitis test (CMT), and any CMT score ~1 was considered positive. Stripping milk was discarded from all quarters prior to sample collection. Milk samples were then collected for NAGase determination and Wisconsin mastitis test (WMT). For clinical quarters only, teat ends were swabbed using 70% isopropyl alcohol, and foremilk was aseptically collected for subsequent microbiological examination. Following collection, all samples were frozen. Samples were driven to our laboratory each week and arrived frozen. Case Definition

Some cows had two clinical episodes in the same quarter within a few days of each other. Any such episode that occurred within 5 d of recovery following the earlier episode was considered a chronic case of mastitis. Any such episode that occurred within 14 d of recovery from the earlier episode was considered chronic if the same etiologic agent was isolated from both episodes.

.1 ml from each aseptically collected milk sample and incubated at 37°C for 48 h. Colony morphology, hemolysis, and lactose fermentation were used for the first diagnosis, and confirmatory tests were used for final identification of pathogens (11). If two pathogens were isolated from the same sample, the agent was defined as mixed pathogens. Isolation of more than two agents in one sample was considered contamination. Measurement of NAGase

Activity of NAGase was determined using a commercially available test kit (12, 15). Using an eight-channel micropipette, 10 J.1l of milk per sample were placed into each test well of a 96-well plate. Substrate (50 J.1l of 4-methylumbelliferyl-N -acetyl- P- D-glucosaminide) was added to each test well. Samples were incubated for 15 min at 25°C while being agitated by a plate shaker. Then 100 J11 of stopping buffer were added to each well. The amount of NAGase in the milk sample is proportional to the amount of 4-methylumbelliferone (4-MU) released from the substrate per unit time. Because 4-MU is a fluorescent compound, the NAGase activity in each sample test well was quantified by measuring the fluorescence using a fluorometer. The fluorometer was set at excitation wavelength 355 om and emission wavelength 480 om. Calibration of the fluorometer, including correction for room temperature, was performed before every test plate was submitted. Milk NAGase was measured for each clinical quarter and for one reference quarter from the same cow. The quarter used for reference NAGase was the quarter with the lowest WMf value. Enzyme activity was reported as mean ± standard error of the mean in micromoles per minute per liter. Data Analysis

Microbiology

Milk samples were mailed frozen to a microbiology laboratory. Samples arrived at the microbiology laboratory within 24 h. One frrst episode sample leaked its contents; therefore, only 48 of the 49 first episodes were included in analysis of etiologic agent. Bovine blood agar and MacConkey agar were inoculated with Journal of Dairy Science Vol. 74, No.5, 1991

The time a cow's milk was withheld from sale was used as a measure of mastitis outcome. The variable days OUT is the number of days until return to the milking herd or until culling or drying off following each case of clinical mastitis. All cows were milked twice daily. If rectal temperature was greater than 39.7·C, 4 g of oxytetracycline were administered i.v. once

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NAGase AND CHRONIC MASTITIS

daily, and cephapirin (Cefa Lak, Bristol-Myers, Bristol, TN) was administered intramammarily (i.m.m.) twice daily. If rectal temperature was less than 39.7°C, but the quarter was swollen or milk was grossly abnormal, cephapirin only was administered Lm.m. If milk appeared normal and quarter swelling was absent, cows were returned to the milking herd. Cases that were evaluated but not treated with antibiotics were immediately returned to the lactating herd. Some treated cases returned to the lactating herd after a longer time than that required for clearance of antibiotics; these cows were judged by the herdsman not to warrant further antibiotic therapy but were in need of more recovery time before return to the lactating herd. Transformation of days OUT to the natural log (days OUT +1) was performed to achieve normal distribution. A general linear model was constructed to identify independent variables affecting variability among cases in the dependent variable days OUT. Days OUT was regressed against the effects of log [NAGase in a mastitic quarter (NAGM)], log [NAGase in a reference quarter (NAGR)], episode number, etiologic agent, WMT in mastitic quarter, and laboratory batch for NAGase determination. These independent variables were screened at P < .25 using oneway ANOVA for continuous variables or chisquare analysis for categorical values. Nonsignificant independent variables were removed in a stepwise order of increasing significance if P > .10. All variables that might be expected to influence OUT were then forced into an additional model in order to test common assumptions regarding clinical recovery from mastitis. In other models, episode number was regressed on etiologic agent, NAGase, and days OUT. A correlation matrix was created for variables NAGase and days OUT. The analyses were performed using SAS (18). RESULTS

siella spp., Enterobacter spp., and Citrobacter spp.) (22.0%), 14 other pathogens (Serratia spp., Bacillus spp., diphtheroids [Corynebacterium spp. and Actinomyces pyogenes], Pseudomonas spp., and Nocardia spp.) (11.9%), 14 mixed pathogens (2 agents isolated) (12.8%), and 8 contaminated (7.3%). Distribution of agents did not differ by episode number (P = .40, chi-square). One second episode was discovered to be more than three standard deviations from the mean in NAGM and was therefore considered an outlier and discarded. This point affected estimates of slopes and intercepts in the regression model. Relationships among variables were changed, but conclusions regarding statistical significance were not. Chronic Mastitis and NAGase

Mean NAGM for all episodes was 11.16 ± 1.09 ~/min per L. Means for NAGM were 11.09 ± 1.70 for fust episodes, 10.44 ± 1.56 for second episodes, and 14.10 ± 3.54 for third or more episodes. For all episodes, NAGR (the NAGase in reference quarter) was 1.05 ± .12 ~/min per L. There was no difference in NAGM (P = .60) or NAGR (P = .59) among episodes (one-way ANOVA) (fable 1). Clinical Recovery from Chronic Mastitis

Mean days OUT following mastitis was 5.1 ± .6 d for all episodes. Means for days OUT

TABLE 1. N-Acetyl-~D-glucosaminidase for all cases and by episode number. No. of cases

Episode l

Microbiology

As defmed herein, 49 quarters were chronically mastitic. There were 49 first episodes, 49 second episodes, and 13 episodes that were third or greater. Agents isolated were 20 Staphylococcus aureus (18.4%), 8 Staphylococcus spp. (7.3%),22 no growth (20.2%), 24 environmental pathogens (streptococci other than Strep. agalactiae, Escherichia coli, Kleb-

All cases First Second Third plus

110 49 48 13

- - (J.1M/min X SEM 11.16 1.09 11.09 1.70 10.44 1.56 14.10 3.54

per L) - X SEM 1.05 .12 1.19 .22 .96 .15 .88 .24

lChronically infected quarters, first, second, or third onset of clinical signs of mastitis. Differences in NAGase among cases were not significant. 2NAGase in the mastitic quarter.

~eference quarter NAGase. Journal of Dairy Science Vol. 74, No.5, 1991

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wn.sON ET AL.

TABLE 2. N-Acetyl-II-D-glucosaminidase and days out of herd following clinical mastitis for all cases and by episode number.

Episode 1

No. of

cases

All cases

no

First Second Third plus

49 48 13

Days OUT2

X SEM 5.1.6 4.4 1.0 5.2.8 6.7 1.6

r for log(NAGM)3 with log (days OU1)

.34··· .26 .47··· .11

lCbronically infected quarters, first, second, or, third onset of clinical signs of mastitis. Differences in NAGase among cases were not significant. 2Days until return to the lactating herd or until culled or dried off following each case of mastitis.

~AGase in the mastitic quaner. •••p < .001, simple correlation.

were 4.4 ± 1.0 d for rust episodes, 5.2 ± .8 d for second episodes, and 6.7 ± 1.6 d for third or more episodes. Values for days OUT were not different among episodes (P = .47, one-way ANOVA) (Table 2). Relationship Between NAGase and Clinical Recovery

The correlation between 10g(NAGM) and 10g(days OUT) was .34 (P < .001, simple correlation). Correlation between 10g(NAGM) and log(days OUT) was strongest (r = .47, P < .001, simple correlation) for second episodes, and the relationship was weakest for third and greater episodes (Table 2). The only variable that explained variability among log of days OUT was 10g(NAGM), (r2 = .11, P < .001, one-way ANOVA). The final general linear model to explain variability in log(days OUT), therefore, included only 10g(NAGM). The model with variables expected to influence clinical recovery forced into it shows that in addition to episode number, etiologic agent, WMI', and reference NAGase failed to predict time until clinical recovery from mastitis (Table 3). DISCUSSION

Milk NAGase and etiologic agent isolated were not significantly different among first episodes or subsequent episodes of chronic clinical Journal of Dairy Science Vol. 74, No.5, 1991

TABLE 3. Summazy of analysis of varian~ctorsof log of time until clinical recovery following mastitis. l Source of variation Log(NAGM)2 Episode2

=1s

WMTM Log(NAGR)2 Error

Independent variable Intercept Log(NAGM} Episode First Second Third Labday

FIrSt

df

Type SS

1 2 6 6

14.0 3.3 5.s 5.3

1 1

.5 .1

76 Estimate

-.2118 .3878 -.4437 -.0197

o

-.7674

Second -.6823 -.9912 Third Fourth -.5010 -1.0792 Fifth -1.3218 Sixth Seventh o Agent Staphylococcus aureus .2820 Staphylococcus spp. -.1815 No growth .2971 Contaminated -.2530 Environmental -.2146

Others Mixed pathogens WMTM Log(NAGR) JR2

-.3466 0 -.0071 .0098

m

Type m Probability

.0002 .1756 .4420 .4680 .4634 .8979

70.5 SE .7871 Jy)c)7 .3461

.3450 NA .6365 .5923 .6376 .5927 .6562 .6567 NA .3699 .5077 .3721 .4526 .3707 .4319 NA .0097 .0764

= .30, P < .05. Grand mean for time until recovery

= 5.1 d. 2NAGM = NAGase in the mastitic quarter; NAGR = NAGase in the reference quarter; Episode chronically infected quarters, first, second or third onset of clinical signs of mastitis.

=

3uworatory sample batch during which NAGase was measured. "Etiologic agent isolated.

Swisconsin mastitis test (WMT) in mastitic quarter.

mastitis. There was an apparent increase in time until recovery for chronic infections compared to new infections, but these differences were not significant. Further investigation of effect of chronicity on response to clinical mastitis may be warranted. N-Acetyl-p-D-glucosaminidase has been identified as a possible indicator of secretory cell damage and resultant severity of clinical

NAGase AND CHRONIC MASTITIS

mastitis (4, 5, 7, 8, 9, 13, 14, 16, 17). On the day of clinical onset, decisions to treat, cull, or not to treat mastitis cases based on probable economic outcome could be aided with such a test. As NAGase increased, clinical recovery from mastitis was prolonged. This relationship was true for new and chronic clinical cases. However, the association between NAGase and time until clinical recovery from mastitis was weak, which limits the value of NAGase as a prognostic test. More recently, NAGase has been found to originate also from white blood cells that increase in milk as a result of inflammation in mastitis (6). It would seem possible that chronically mastitic quarters could have elevated basal NAGase due to increased infiltration of sec into milk, even when clinical signs are absent. If so, use of milk NAGase as a prognostic test for new clinical episodes of chronic cases might be less accurate than for new infections. However, NAGase was a better prognostic test for recovery for second clinical episodes of chronic mastitis than for new infections. The correlation between NAGase and clinical recovery dropped marlt.edly only for third and subsequent clinical recurrences of chronic cases. ACKNOWLEDGMENTS

The authors wish to thank: the personnel on the farm where data were collected. Farm management has requested anonymity. REFERENCES 1 Blood, D. C., O. M. Radostits, and I. A. Henderson. 1983. Veterinary medicine. Lea and Febiger, Philadelphia, PA. 2 Erskine, R I., R. I. Eberhart, L. I. Hutchinson, S. B. Spencer, and M A. Campbell. 1988. Incidence aud types of clinical mastitis in dairy herds with high and low somatic cell counts. I. Am. Vet Med. Assoc. 192: 761. 3 Fitz-Gerald, C. M., H.-C. Deeth, aud B. I. Kitchen. 1981. The relationship between the levels of free fatty acids, lipoprotein lipase, carboxylesterase, N-acetylbeta-D-glucosaminidase, somatic cell count, and other mastitis indices in bovine milk. I. Dairy Res. 48:253. 4 Fox, L. K., and L. H. Schultz. 1985. Effect of infection

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status on quarter milk production and composition following omitted milking. 1. Dairy Sci. 68:418. 5 Fox, L. K., G. B. Shook, and L. H. Schultz. 1987. Factors related to milk loss in quarters with low s0matic cell counts. I. Dairy Sci 68:2100. 6 Kaartinen, L., P. L. Kuosa, K. Veijalainen, and M Sandholm. 1988. Compartmentalization of milk Nacetyl-~D-glucosaminidase(NAGase). Release of NAGase from the cellular compartment by storage, freezing and thawing, detergent and using cell stimulants. 1. Vet. Med. Ser. B. 35:408. 7 Kitchen, B. I. 1976. BDzymic methods for estimation of the somatic cell count in bovine milk. 1. Development of assay techniques and a study of their usefulness in evaluating the somatic cell content of milk. I. Dairy Res. 43:251. 8 Kitchen, B. I., W. S. Kwee, G. Middleton, and R I. Andrews. 1984. Relationship between the level of Nacetyl-beta-D-glucosaminidase (NAGase) in bovine milk and the presence of mastitis parhogens. I. Dairy Res. 51:11. 9 Kitchen, B. 1., G. Middleton, and M Salmon. 1978. Bovine milk N-acetyl-beta-D-glucosaminidase and its g~inthe~~~of~~oo~~

tions. I. Dairy Res. 45:15. 10 Kunkel, I. R, R B. Bushnell, I. Cnllor, and I. Aleong. 1987. Studies on induced Klebsiella mastitis with relationships among N-acetyl-~D-glucosaminidase, bacterial and somatic cell counts. Cornell Vet 77:225. 11 Natioual Mastitis Council. 1987. Laboratory and field handbook on bovine mastitis. Arlington, VA. 12Linko-Lopponen, S., and M Makinen. 1985. A microtiter plate assay for N-acetyl-beta-D-glucosaminidase using a f1uorogenic substrate. Anal. Biochem. 148:50. 13 Mattila, T., I. Syvajarvi, N. B. lensen, and M. Saudholm. 1986. N-acetyl-beta-D-glucosaminidase and antitrypsin in subclinically infected quarter-milk samples: effect of bacteria and hemolysins, lactation stage, and lactation number. Am. I. Vet. Res. 47:139. 14 Mattila, T., 1. Syvajarvi, and M. Sandholm. 1986. Milk: antitrypsin, NAGase, plasmin and bacterial replication rate in whey. Effects of lactation stage, parity and daily milk yield. Zentralbl. Veterinaenned. Reihe B 33:462. 15 Flow Laboratories Inc. 1987. Milk NAGase test instruction manual, McLean, VA. 16 Obara, Y., and M. Komatsu. 1984. Relationship be.tween N-acetyl-beta-D-glucosaminidase activity and cell count, lactose, chloride, or Iactoferrin in cow milk. 1. Dairy Sci. 67:1043. 17 Schaar, I., and H. Funke. 1986. Effect of subclinical mastitis on milk plasminogen and plasmin compared with that on sodium, antitrypsin and N-acetyl-beta-Dglucosaminidase. I. Dairy Res. 53:515. 18 Statistical Aualysis System for Personal Computers (SAS-PC). 1985. SAS Inst., Inc., Cary, NC.

Iourual of Dairy Science Vol 74, No.5, 1991

N-acetyl-beta-D-glucosaminidase, etiologic agent, and duration of clinical signs for sequential episodes of chronic clinical mastitis in dairy cows.

This study examined effects of repeated episodes of clinical mastitis in chronically infected quarters on milk N-acetyl-beta-D-glucosaminidase activit...
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