J Vet Intern Med 2001;15:394–400
Ability of Hematologic and Serum Biochemical Variables to Differentiate Gram-Negative and Gram-Positive Mastitis in Dairy Cows Geoffrey W. Smith, Peter D. Constable, and Dawn E. Morin Medical records of 142 dairy cows with clinical mastitis were examined to determine whether hematologic or serum biochemical results could be used to distinguish between mastitis episodes caused by gram-negative bacteria (n 5 78) from those caused by gram-positive bacteria (n 5 64). Signalment, historic information, hematologic and serum biochemical results, milk culture results, and outcome (discharged from hospital or died) were obtained from the medical records. Cows with gram-negative mastitis had significantly (P , .01) lower blood leukocyte, segmented neutrophil, monocyte, and lymphocyte counts and had higher blood hemoglobin concentrations and hematocrits than did cows with gram-positive mastitis. Serum urea nitrogen was the only serum biochemical result associated with pathogen type, and it was higher in cows with gram-negative mastitis than in those with grampositive mastitis. Mortality rate (25% overall) did not differ between groups. Logistic regression indicated that routine hematologic analysis (segmented neutrophil count, monocyte count, and hemoglobin concentration) was an accurate predictor of gram-negative mastitis, with a sensitivity of .93, a specificity of .89, and an overall accuracy of 91%. The values for sensitivity and specificity were higher than those previously reported for clinical tests differentiating mastitis episodes caused by gram-negative bacteria from those caused by gram-positive bacteria. Our results indicate that routine hematologic analysis is useful for predicting pathogen type in dairy cows with clinical mastitis, thereby facilitating treatment decisions. Key words: Bovine; Coliform; Neutrophil.
C
linical mastitis is a costly disease on dairy farms in the United States, with an average lactational incidence rate of 14.2% according to a retrospective analysis of 62 reports.1 In herds that have controlled the 2 major contagious mastitis pathogens (Streptococcus agalactiae and Staphylococcus aureus), mastitis primarily is caused by environmental organisms such as coliform bacteria (Escherichia coli and Klebsiella pneumoniae), Streptococcus species other than S agalactiae, and Staphylococcus species other than S aureus.2–5 Environmental bacteria are ubiquitous, and eradication of clinical mastitis is an unreasonable goal. Consequently, veterinarians and dairy producers must be able to diagnose and treat mastitis effectively. Treatment protocols for clinical mastitis must be efficacious and economical, while minimizing milk and slaughter withholding times. The ideal treatment protocol is dependent upon pathogen type and severity of clinical signs, with antibiotic selection often being different for gram-positive and gram-negative bacterial mastitis pathogens. Clinical judgement often is used to determine the treatment protocol for cows with mastitis. Bacteriologic culture of milk is not routinely performed because it is expensive, results are not available for 24–48 hours, and a pathogen is not isolated in 15–40% of cows with clinical mastitis.2–4,6–8 Various diagnostic schemes that use clinical parameters to differentiate mastitis caused by gram-negative bacteria from that caused by gram-positive bacteria have been reported to have an overall accuracy of 71–78%.9–11 However, the current use of lipopolysaccharide core antigen vaccines can From the Department of Veterinary Clinical Medicine, University of Illinois, Urbana, IL. Reprint requests: Geof Smith, DVM, MS, Department of Veterinary Clinical Medicine, University of Illinois, 1008 W. Hazelwood, Urbana, IL 61802; e-mail:
[email protected]. Submitted March 20, 2000; Revised July 19, 2000, and December 8, 2000; Accepted February 5, 2001. Copyright q 2001 by the American College of Veterinary Internal Medicine 0891-6640/01/1504-0011/$3.00/0
reduce the severity of gram-negative mastitis,12,13 potentially making it more difficult to predict the cause of mastitis based on physical examination alone. In a recent study, we confirmed that clinical observations were not sufficiently accurate to be useful for predicting gram-negative mastitis (sensitivity 5 .58; specificity 5 .80).14 The HyMast testa has been promoted as a rapid bacteriologic test system that enables the producer to differentiate gram-negative mastitis pathogens from gram-positive mastitis pathogens. This test was reported to have excellent specificity for detecting gram-negative mastitis (98%) but relatively poor sensitivity (60%).15 To achieve maximal sensitivity and specificity, the test results should not be considered accurate until after 36 hours of incubation. Decisions based on results obtained at earlier incubation times (particularly at 12 hours) have resulted in greater misclassification of cases.16 Therefore, treatment decisions are delayed until test results are available. A Limulus amoebocyte lysate cow-side test designed to detect endotoxin in milk shows promise for rapidly identifying the type of mastitis pathogen. However, this test is not widely available in the United States and had relatively low sensitivity in one report (63%).17 Both naturally occurring and experimentally induced coliform mastitis in dairy cows increase endotoxin concentrations in milk and blood.18,19 Endotoxemia has profound effects on hematologic and serum biochemical results in cattle,20,21 and we hypothesized that routine laboratory tests would be useful in predicting the gram-staining characteristics of mastitis pathogens. The purpose of the study reported here was to determine whether hematologic or serum biochemical results could accurately differentiate clinical mastitis episodes caused by gram-negative bacteria from those caused by gram-positive bacteria, thereby facilitating treatment decisions.
Materials and Methods Case Selection The medical records of all lactating dairy cows presented to the University of Illinois Large Animal Clinic between October 1989 and
Differentiation of Gram-Positive from Gram-Negative Mastitis April 1999 were examined. Medical records were used in this retrospective study if (1) clinical mastitis (defined as abnormal milk, abnormal mammary gland, or both) was present in 1 or more quarters, (2) mastitis was the major reason for hospitalization, (3) bacteria considered to be major mastitis pathogens were isolated from affected quarters, and (4) serum biochemical data or leukocyte numbers and differential leukocyte counts were available. Cows were included in the study only once.
Medical Record Analysis Demographic data, clinical history, number of days in lactation, duration of mastitis before admission, initial physical examination findings (rectal temperature, heart rate, respiratory rate), results of routine bacteriologic culture of secretions from affected quarters, clinicopathologic data (red blood cell count; hemoglobin concentration; hematocrit; total white blood cell count; numbers of segmented and band neutrophils, eosinophils, lymphocytes, and monocytes; plasma fibrinogen concentration; serum creatinine, urea nitrogen, total protein, albumin, calcium, phosphorus, sodium, potassium, magnesium, chloride, glucose, total bilirubin, and cholesterol concentrations; and creatine kinase, alkaline phosphatase, aspartate aminotransferase, g-glutamyl transferase, and sorbitol dehydrogenase activities), nature of any concurrent diseases, and outcome (discharged, euthanized, or died) were recorded.
Data Analysis Cows were divided into 2 groups based on milk culture results (those with gram-negative pathogens and those with gram-positive pathogens). Cows with a mastitis episode caused by more than 1 gramnegative or gram-positive pathogen were enrolled in the study, but cows with both gram-negative and gram-positive bacterial isolates were excluded. Data were expressed as mean 6 SD. Differences between groups initially were evaluated by analysis of variance; data that were not normally distributed were log transformed or ranked before analysis of variance was performed. A P value of ,.01 was considered significant. This conservative P value was used because of the number of potentially related comparisons performed with the same data set. Forward stepwise logistic regression was performed to identify the relative predictive value of variables, and all variables with a univariate P value of ,.25 were entered into the logistic regression procedure, with P 5 .10 and P 5 .15 used for variables to enter and exit the logistic regression procedure, respectively. Backward elimination logistic regression also was performed using P 5 .10 for leaving and reentering to examine whether the backwards procedure produced the same final model as the forward stepwise procedure. To check the assumption that the logistic function was linear over the entire data set, the range of continuous variables was divided into quartiles, and variables that did not fulfill the assumption of linearity (heart rate, days in lactation, blood fibrinogen concentration) were categorized using dummy variable coding: normal heart rate (#80 beats/min), 0; increased heart rate (.80 beats/min), 1; first 2 weeks of lactation, 0; .2 weeks of lactation, 1; normal fibrinogen concentration (#600 mg/ dL), 0; increased fibrinogen concentration (.600 mg/dL), 1. The predictive ability of each logistic model was evaluated using the HosmerLemeshow test,22 the area under the receiver operating characteristic curve, and the generalized R2 value.23 The presence of statistical outliers or influential data points was evaluated by calculating influence statistics. A software programb was used for analyses. Three logistic regression procedures were performed: (1) hematologic data, serum biochemical data, age of cow, and number of days in lactation, (2) hematologic data only, and (3) hematologic data that could be rapidly, easily, and inexpensively obtained (hematocrit and differential white blood cell percentages). After development of a logistic regression equation based on only main effects, all potential interaction terms for
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the main effects in the model were evaluated to see if they should be included in the final model. Linear regression was used to explore the relationship between segmented neutrophil count and days in lactation; data for both variables were log transformed before linear regression analysis.
Results One hundred forty-two cases of clinical mastitis were identified, including 78 cases of gram-negative mastitis and 64 cases of gram-positive mastitis. Gram-negative pathogens included E coli (56), Klebsiella spp. (13), Pseudomonas spp. (8), and Enterobacter spp. (1), and gram-positive isolates consisted of Streptococcus spp. other than S agalactiae (21), Staphylococcus aureus (19), Arcanobacterium pyogenes (13), Staphylococcus spp. other than S aureus (10), and Clostridium spp. (1).
Demographic Data and Clinical History Cows with gram-negative mastitis tended to be older than those with gram-positive mastitis (P 5 .016; Table 1) and tended (P 5 .022) to be later in lactation at the time of admission (geometric mean of 29 days in milk) than cows with gram-positive mastitis (geometric mean of 13 days in milk). The duration of clinical signs of mastitis before hospital admission was not different between groups.
Physical Examination Findings There were no differences in rectal temperature, heart rate, or respiratory rate between the 2 groups (Table 1).
Hematologic Data CBC results were available for 62 cows with gram-negative mastitis and for 46 cows with gram-positive mastitis (same distribution as for all cows in the study; x2 5 0.02, P 5 .89). Cows with gram-negative mastitis had decreased total white blood cell (Fig 1), segmented neutrophil (Fig 2), monocyte (Fig 3), and lymphocyte counts (Table 1) and increased blood hemoglobin concentration and hematocrit when compared with cows with gram-positive mastitis. No differences in blood band neutrophil counts, eosinophil counts, or plasma fibrinogen concentrations were observed between groups. Linear regression analysis indicated that in gram-positive mastitis episodes, segmented neutrophil count was not affected by stage of lactation (Fig 4). However, in gram-negative mastitis episodes, the log10 of the segmented neutrophil count was negatively correlated (R 5 2.37, P 5 .009) with the log10 of days in lactation, indicating that cows with gram-negative mastitis had a lower segmented neutrophil count in late lactation than did cows in early lactation.
Serum Biochemistry Data Serum biochemistry data were available for 56 cows with gram-negative mastitis and for 42 cows with gram-positive mastitis (same distribution as for all cows in the study, x2 5 0.18, P 5 .68). Serum urea nitrogen concentrations were higher and serum albumin (P 5 .010), creatinine (P 5 .041), and cholesterol (P 5 .013) concentrations tended to be higher in cows with gram-negative mastitis. No differ-
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Table 1. Summary of data (mean 6 SD) from cows with gram-negative mastitis and cows with gram-positive mastitis. Variable
Gram Negative
Gram Positive
P
4.3 103 102.4 91 52
6 6 6 6 6
2.3 175 1.5 23 23
.009 .35 .45 .17 .14
1.1 5.5 1.6 3,571 1,251 1,088 2,526 99 257 217
6.1 29.8 10.3 10,862 5,128 1,088 3,828 153 719 826
6 6 6 6 6 6 6 6 6 6
1.0 5.4 1.7 4,056 2,649 2,247 2,064 271 498 305
.18 ,.0001 ,.0001 ,.0001 ,.0001 .20 .0007 .77 ,.0001 .059
0.6 12 1.0 0.5 2.1 1.8 6 0.8 8 0.7 51 70 0.7 6,574 60 25 204 15
1.5 21 7.3 3.0 8.0 5.8 140 4.0 100 1.8 77 84 1.1 1,116 154 35 233 27
6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6
1.4 21 1.1 0.5 1.1 2.5 4 0.7 7 1.0 19 47 0.9 1,635 91 26 292 22
.041 .004 .095 .010 .89 .74 .82 .80 .42 .74 .26 .013 .62 .45 .39 .93 .29 .58
Historic and physical examination findings Age of cow (years) Duration of clinical signs (hours) Rectal temperature (8F) Heart rate (beats/min) Respiratory rate (breaths/min)
5.2 43 102.6 96 47
6 6 6 6 6
2.2 57 1.8 19 19
Hematologic parameters Red blood cell count (3106 cells/mL) Hematocrit (%) Hemoglobin (g/dL) White blood cell count (cells/mL) Segmented neutrophils (cells/mL) Band neutrophils (cells/mL) Lymphocytes (cells/mL) Eosinophils (cells/mL) Monocytes (cells/mL) Fibrinogen (mg/dL)
6.6 33.6 11.8 4,461 896 529 2,777 76 193 712
6 6 6 6 6 6 6 6 6 6
Serum biochemical parameters Creatininie (mg/dL) Urea nitrogen (mg/dL) Total protein (g/dL) Albumin (g/dL) Calcium (mg/dL) Phosphorus (mg/dL) Sodium (mEq/L) Potassium (mEq/L) Chloride (mEq/L) Magnesium (mEq/L) Glucose (mg/dL) Cholesterol (mg/dL) Total bilirubin (mg/dL) Creatine kinase (IU/L) Alkaline phosphatase (IU/L) Gamma-glutamyl tranferase (IU/L) Aspartate aminotransferase (IU/L) Sorbitol dehydrogenase (IU/L)
1.5 25 6.9 3.3 8.1 6.0 141 3.9 101 1.7 76 121 0.9 2,480 140 34 179 22
6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6
ences in any other serum biochemical parameters were identified between the 2 groups.
Nature of Concurrent Diseases and Outcome Twenty-one percent (30/142) of the cows in this study had concurrent problems, which included retained placenta, metritis, left-displaced abomasum, right-displaced abomasum, abomasal volvulus, uterine torsion, milk fever, traumatic reticuloperitonitis, pneumonia, and teat lacerations. No significant difference in the incidence of concurrent problems between cows with gram-negative mastitis and cows with gram-positive mastitis were identified. Similarly, mortality rate did not differ for cows with gram-negative mastitis (31%) and cows with gram-positive mastitis (22%). Overall mortality rate due to mastitis was 25% (36 of 142 cows died or were euthanized before discharge). This mortality rate represents mastitis mortality only; no cows with death (or euthanasia) attributable to concurrent diseases were included in this study.
Logistic Regression The 1st logistic regression procedure evaluated hematologic, serum biochemical, and demographic data. With this process, segmented neutrophil count was identified as an important predictor of mastitis type (Table 2). Both forward stepwise and backward elimination procedures produced the same result. The 2nd logistic regression procedure performed using hematologic data indicated that blood segmented neutrophil count, monocyte count, and blood hemoglobin concentration were of predictive value; decreased neutrophil and monocyte counts and increased hemoglobin concentration were associated with gram-negative mastitis episodes (Table 2). Both forward stepwise and backward elimination procedures produced the same result. This data set was larger than that used in the 1st logistic regression procedure because the days of lactation and serum biochemical data were not available for all cows that had white blood cell counts available. The resultant logistic regression equation produced a sensitivity of .93, a specificity of .89, and an
Differentiation of Gram-Positive from Gram-Negative Mastitis
Fig 1. Scatter plot of total white blood cell count (expressed on a logarithmic scale) for cows with mastitis. The hatched area represents normal values, and data are expressed as mean 6 SD.
397
Fig 3. Scatter plot of monocyte count (expressed on a logarithmic scale) for cows with mastitis. The hatched area represents normal values, and data are expressed as mean 6 SD.
Discussion
overall accuracy of 91%, using a probability of .5 to differentiate gram-negative from gram-positive mastitis episodes (Table 3). The 3rd logistic regression procedure was performed using hematologic data that could be rapidly, easily, and inexpensively obtained. Blood segmented neutrophil percentage and monocyte percentage were of predictive value, with a sensitivity of .87, a specificity of .71, and an overall accuracy of 79%, using a probability of .5 to differentiate gram-negative from gram-positive mastitis episodes (Table 3).
The major finding of this study was that hematologic analysis allowed accurate differentiation between gramnegative and gram-positive clinical mastitis episodes in dairy cows. Cows with gram-negative mastitis had decreased segmented neutrophil, monocyte, and lymphocyte counts, decreased segmented neutrophil percentages, and increased hematocrit and blood hemoglobin concentrations as compared with cows with gram-positive mastitis. Although not a cow-side test and therefore not ideal, a CBC is a rapid and readily available test that clinicians can use
Fig 2. Scatter plot of segmented neutrophil count (expressed on a logarithmic scale) for cows with mastitis. The hatched area represents normal values, and data are expressed as mean 6 SD.
Fig 4. Log-to-log scatter plot comparing stage of lactation to segmented neutrophil count in cows with mastitis. Regression lines (solid lines) and the 95% confidence intervals (dotted lines) are shown for gram-negative and gram-positive mastitis episodes.
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Table 2. Final logistic regression models for prediction of gram-negative versus gram-positive clinical mastitis in dairy cows. Variable Hematologic, serum biochemical, and demographic data y intercept Segmented neutrophils (cells/mL)
b value
Standard Error (b)
4.67 20.00147
1.28 0.00041
.0003
0.61 20.00116 0.0029 0.47
2.77 0.00027 0.0011 0.25
,.0001 .004 .049
4.28 20.104 20.104
0.83 0.020 0.062
,.0001 .092
P
P for Hosmer-Lemeshow goodness of fit statistic 5 .80 Area under receiver operating characteristic curve 5 20.97 Hematologic data y intercept Segmented neutrophils (cells/mL) Monocytes (cells/mL) Hemoglobin concentration (g/dL) P for Hosmer-Lemeshow goodness of fit statistic 5 .35 Area under receiver operating characteristic curve 5 0.97 Differential white blood cell (WBC) percentages and hematocrit y intercept Segmented neutrophil % of total WBC Monocyte % of total WBC P for Hosmer-Lemeshow goodness of fit statistic 5 .41 Area under receiver operating characteristic curve 5 0.91
to aid treatment decisions in clinical mastitis. In many cases, a blood smear can be made on the farm, and the segmented neutrophil percentage can be rapidly determined if a microscope is available. In clinicopathologic studies of experimentally induced and naturally occurring coliform mastitis, significant decreases in white blood cell counts have been identified.21,24–26 In a study of 44 cows with naturally occurring E. coli mastitis, affected cows frequently had leukopenia (lymphopenia and neutropenia), and a left shift.24 Experimentally, intramammary administration of endotoxin produces a consistent and profound leukopenia within 1 hour, followed by a rebound leukocytosis within 24 hours.25,27,28 Both neutropenia and lymphopenia have been observed, the neutropenia being more severe.25 Leukopenia appears to be an immediate response to endotoxin administration and has been observed as early
as 5 minutes after IV infusion.29 Endotoxin administration causes an immediate accumulation, margination, and activation of leukocytes in the microcirculation, particularly in the alveolar capillaries.30,31 Lymphopenia has been attributed to the release of endogenous corticosteroids,30 whereas neutropenia is thought to result primarily from pulmonary sequestration of neutrophils.32 Although not examined in this study, thrombocytopenia also often is observed after endotoxin administration and is thought to result from sequestration of platelets in lung, liver, and splenic capillary beds.29,33 Cows with gram-negative mastitis that were early in lactation did not have the same degree of reduction in blood neutrophil concentration as did cows in later lactation (Fig 4). This finding was consistent with reports of increased blood neutrophil concentrations around the time of partu-
Table 3. Summary of reported test sensitivity (Se), specificity (Sp), and predictive value of a positive test (PVPT), assuming a 50% prevalence of gram-negative mastitis. Values were calculated using bacteriologic culture of the milk as the gold standard. n
Se
Sp
PVPT (%)
Clinical findings Clinical prediction Numerous predictors Season, milk viscosity, rumen motility Numerous predictors
112 114 143 135
.64 .42 .58 .79
.61 .85 .80 .79
62 74 74 79
White et al11 White et al10 Morin et al14 Jones and Ward9
Clinicopathologic findings Presence of endotoxin in milk Segmented neutrophils ,35% Presence of endotoxin in milk Hematologic analysis Presence of endotoxin in milk Milk culture (HyMastt)
37 108 47 108 724 219
.64 .87 .76 .93 .72 .60
.67 .71 .90 .89 .95 .98
66 75 88 89 94 97
Mohammed et al42 This study Katholm and Andersen43 This study Waage et al17 Jansen et al15
1.00
1.00
100
Milk culture
Reference
Theoretical
Differentiation of Gram-Positive from Gram-Negative Mastitis
rition.24,34 Neutrophil CD62L and CD18 expression is important for neutrophil migration from blood into infected mammary parenchyma,35 and glucocorticoid release associated with parturition decreases neutrophil glucocorticoid receptor expression.36 Therefore, endotoxin-induced neutropenia may have occurred in early lactation because periparturient glucocorticoid release decreased the ability of neutrophils to migrate. Cows express low CD62L for several days after parturition,37 which temporally is associated with an increase in blood corticosteroid concentrations.34 Along with other alterations in neutrophil function that are known to occur during the periparturient period,38,39 the inability of neutrophils to migrate to tissues may increase susceptibility to infectious diseases during this period. Serum calcium concentrations were low (normal range, 9–11 mg/dL) regardless of pathogen type but were not different between groups. Previously, studies of experimentally induced and naturally occurring cases of coliform mastitis revealed significant decreases in serum calcium concentrations,18,27 but the decrease in calcium concentration was not correlated with the number of bacterial colonyforming units isolated from the milk.18 Although the reason for the decrease in serum calcium concentration is not fully understood, the results of the present study indicate that this decrease is not limited to cows with gram-negative mastitis. This retrospective study was performed on a population of dairy cows that were hospitalized for clinical mastitis, and the findings may be restricted to cows with moderate to severe clinical mastitis. However, the results of this study indicated that routine hematologic examination can be used to differentiate cases of gram-negative mastitis from cases of gram-positive mastitis. The test sensitivity of routine hematologic analysis is the highest reported for diagnosing gram-negative mastitis (Table 3); thus, a CBC is the best method for ruling in gram-negative bacteria as the cause of a clinical mastitis episode. Some clinicians believe antibiotics should not be administered to cows with gram-negative mastitis, despite recent evidence to the contrary,40,41 and we believe that routine hematologic analysis is useful in guiding initial treatment decisions, particularly in valuable cows or when cows are hospitalized.
Footnotes a b
HyMastt, Pharmacia & UpJohn Animal Health, Kalamazoo, MI PROC LOGISTIC, version 6.11, SAS Institute, Cary, NC
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5. Guterbock WM, Van Eenennaam AL, Anderson RJ, et al. Efficacy of intramammary antibiotic therapy for the treatment of clinical mastitis caused by environmental pathogens. J Dairy Sci 1993;76: 3437–3444. 6. Pearson JKL, Mackie DP. Factors associated with the occurrence, cause and outcome of clinical mastitis in dairy cattle. Vet Rec 1979; 105:456–463. 7. Anderson KL, Smith AR, Gustaffson BK, et al. Diagnosis and treatment of acute mastitis in a large dairy herd. J Am Vet Med Assoc 1982;181:690–693. 8. Morin DE, Constable PD. Characteristics of dairy cows during episodes of bacteriologically negative clinical mastitis or mastitis caused by Corynebacterium spp. J Am Vet Med Assoc 1998;213:855– 861. 9. Jones GF, Ward GE. Evaluation of a scheme for predicting the gram-staining reaction of organisms causing bovine mastitis. J Am Vet Med Assoc 1990;196:597–599. 10. White ME, Glickman LT, Barnes-Pallesen FD, et al. Accuracy of a discriminant analysis model for prediction of coliform mastitis in dairy cows and a comparison with clinical prediction. Cornell Vet 1986;76:342–347. 11. White ME, Glickman LT, Barnes-Pallesen FD, et al. Discriminant analysis of the clinical indicants for bovine coliform mastitis. Cornell Vet 1986;76:335–341. 12. McClure AM, Christopher EE, Wolff WA, et al. Effect of Re17 mutant Salmonella typhimurium bacterin toxoid on clinical colifom mastitis. J Dairy Sci 1994;77:2272–2280. 13. Hogan JS, Weiss WP, Smith KL, et al. Effects of an Escherichia coli J5 vaccine on mild clinical coliform mastitis. J Dairy Sci 1995; 78:285–290. 14. Morin DE, Constable PD, McCoy GC. Use of clinical parameters for differentiation of gram-positive and gram-negative mastitis in dairy cows vaccinated against lipopolysaccharide core antigens. J Am Vet Med Assoc 1998;212:1423–1431. 15. Jansen J, Leslie K, Kelton D. Utilizing and evaluating the HyMastt test on dairy farms. Regional Meeting of the National Mastitis Council, 1997. 16. Jansen JT, Kelton DF, Leslie KE, et al. Evaluation of the test characteristics of the HyMastt bacteriological test system. AABP Proc 1999;32:188–189. 17. Waage S, Jonsson P, Franklin A. Evaluation of a cow-side test for detection of gram-negative bacteria in milk from cows with mastitis. Acta Vet Scand 1994;35:207–212. 18. Katholm J, Andersen PH. Acute coliform mastitis in dairy cows: Endotoxin and biochemical changes in plasma and colony-forming units in milk. Vet Rec 1992;131:513–514. 19. Ziv G, Hartman I, Bogin E, et al. Endotoxin in blood and milk and enzymes in the milk of cows during experimental Escherichia coli endotoxin mastitis. Theriogenology 1976;6:343–352. 20. Constable PD, Schmall LM, Muir WW, et al. Respiratory, renal, hematologic, and serum biochemical effects of hypertonic saline solution on endotoxemic calves. Am J Vet Res 1991;52:990–998. 21. Saad AM, Ostensson K. Flow cytofluorometric studies on the alteration of leukocyte populations in blood and milk during endotoxin-induced mastitis in cows. Am J Vet Res 1990;51:1603–1607. 22. Lemeshow S, Hosmer DW Jr. A review of goodness of fit statistics for use in the development of logistic regression models. Am J Epidemiol 1982;115:92–106. 23. SAS Institute. SAS/STAT Software: Changes and Enhancements through Release 6.11. Cary, NC: SAS Institute; 1996:383–490. 24. Cebra CK, Garry FB, Dinsmore RP. Naturally occurring acute coliform mastitis in Holstein cows. J Vet Intern Med 1996;10:252– 257. 25. Carroll EJ, Schalm OW, Lasmanis J. Experimental coliform (Aerobacter aerogenes) mastitis: Characteristics of the endotoxin and its role in pathogenesis. Am J Vet Res 1964;25:720–726.
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26. DeGraves FJ, Anderson KL. Ibuprofen treatment of endotoxininduced mastitis in cows. Am J Vet Res 1993;54:1128–1132. 27. Griel LC, Zarkower A, Eberhart RJ. Clinical and clinicopathological effects of Escherichia coli endotoxin in mature cattle. Can J Comp Med 1975;39:1–6. 28. Pappe MJ, Schultze WD, Desjardins C, et al. Plasma corticosteroid, circulating leukocyte and milk somatic cell responses to Escherichia coli endotoxin-induced mastitis. Proc Soc Exp Biol Med 1974; 145:553–559. 29. Tennant B, Harrold D, Reina-Guerra M. Hematology of the neonatal calf. II. Responses associated with acute enteric infections, gramnegative septicemia, and experimental endotoxemia. Cornell Vet 1975; 65:457–475. 30. Deldar A, Naylor JM, Bloom JC. Effects of Escherichia coli endotoxin on leukocyte and platelet counts, fibrinogen concentrations, and blood clotting in colostrum-fed and colostrum-deficient neonatal calves. Am J Vet Res 1984;45:670–677. 31. Meyrick B, Brigham KL. Acute effects of Escherichia coli endotoxin on the pulmonary microcirculation of anesthetized sheep. Lab Invest 1983;48:458–470. 32. Warner AE, DeCamp MM, Molina RM, et al. Pulmonary removal of circulating endotoxin results in acute lung injury in sheep. Lab Invest 1988;59:219–230. 33. Emau P, Giri SN, Bruss ML. Comparative effects of smooth and rough Pasteurella hemolytica lipopolysaccharide on arachidonic acid, eiconsanoids, serotonin, and histamine in calves. Circ Shock 1986;20:239–253. 34. Guidry AJ, Paape MJ, Pearson RE. Effects of parturition and Efficacy of a therapy regimen not including antibiotics. Proceedings
lactation on blood and milk cell concentrations, corticosteroids, and neutrophil phagocytosis in the cow. Am J Vet Res 1976;37:1195–1200. 35. Burton JL, Kehrli ME Jr. Regulation of neutrophil adhesion molecules and shedding of Staphylococcus aureus in milk of cortisol and dexamethasone-treated cows. Am J Vet Res 1995;56:997–1006. 36. Preisler MT, Weber PSD, Tempelman RJ, et al. Glucocorticoid receptor down-regulation in neutrophils of periparturient cows. Am J Vet Res 2000;61:14–19. 37. Lee EK, Kehrli ME Jr. Expression of adhesion molecules on neutrophils of periparturient cows and neonatal calves. Am J Vet Res 1998 59:37–43. 38. Cai TQ, Weston PG, Lund LA, et al. Association between neutrophil functions and periparturient disorders in cows. Am J Vet Res 1994;55:934–943. 39. Kehrli ME Jr, Nonnecke BJ, Roth JA. Alterations in bovine neutrophil function during the periparturient period. Am J Vet Res 1989;50:207–214. 40. Morin DE, Shanks RD, McCoy GC. Comparison of antibiotic administration in conjunction with supportive measures versus supportive measures alone for treatment of dairy cows with clinical mastitis. J Am Vet Med Assoc 1998;213:676–684. 41. Wenz JR, Barrington GM, Dinsmore RP. Differentiation of bacteremic and non-bacteremic cows with acute coliform mastitis. J Vet Intern Med 1999;13:279. 42. Mohammed AH, McCallus DE, Norcross NL. Development and evaluation of an enzyme-linked immunosorbent assay for endotoxin in milk. Vet Microbiol 1988;18:27–39. 43. Katholm J, Andersen PH. Severe coliform mastitis in practice:
The Horse Homolog of Congenital Aniridia Conforms to Codominant Inheritance.
pedigree. Logistic regressive segregation analysis of a subset of animals (n 5 337) in which the ocular phenotypes of progeny and both parents were known indicated that the codominant inheritance model best fit the data. This model predicted cyst phenotype expression in heterozygous animals and multiple anterior segment anomalies in homozygous animals. Several cases of nonpenetrance of the cyst phenotype were detected in one lineage. The close resemblance between the inheritance and lesions observed in Small eye mice and rats, humans with congenital aniridia or anterior segment malformation, and horses with anterior segment dysgenesis syndrome supported the conclusion that anterior segment dysgenesis syndrome in the horse may be homologous to similar ophthalmic anomalies in other species.
Ewart SL, Ramsey DT, Xu J, and Meyers D. J Hered 2000;91(2):93–98 (abstract) Anterior segment dysgenesis syndrome occurs frequently in Rocky Mountain horses and has two distinct ocular phenotypes: (1) large cysts originating from the temporal ciliary body or peripheral retina and (2) multiple anterior segment anomalies including ciliary cysts, iris hypoplasia, iridocorneal adhesions and opacification, nuclear cataract, and megalocornea. To determine if anterior segment dysgenesis syndrome is heritable in horses we performed ophthalmic examinations and collected pedigree information on horses (n 5 516) in an extended Rocky Mountain horse
of the World Buiatrics Congress 1998:261–264.
