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Case-Control Study of Risk Factors for Clinical Mastitis in Postpartum Dairy Heifers
J. Dairy Sci. 84:392–399 American Dairy Science Association, 2001.
Case-Control Study of Risk Factors for Clinical Mastitis in Postpartum Dairy Heifers S. Waage,* S. A. Ødegaard,* A. Lund,† S. Brattgjerd,‡ and T. Røthe† *Department of Reproduction and Forensic Medicine, Norwegian School of Veterinary Science, P. O. Box 8146 Dep., 0033 Oslo, Norway †Veterinary Center in Sparbu, 7710 Sparbu, Norway ‡Veterinary Center in Indero/y, 7670 Sakshaug, Norway
ABSTRACT A case-control study was carried out to evaluate risk factors for clinical mastitis occurring in dairy heifers between 1 and 14 d postpartum. Case and control heifers were matched on herd; the control was the heifer that calved closest in time, before or after, the particular case. Data were analyzed by conditional logistic regression. The final multivariate model included 339 casecontrol pairs. Blood in the milk, udder edema, teat edema, and milk leakage, all recorded at the time of parturition, were significant risk factors. Purchased heifers and heifers with skin lesions between udder and thigh were not at increased risk of clinical mastitis. Separate analysis of a subgroup of case-control pairs identified teat edema, blood in the milk, and milk leakage at calving as risk factors for clinical mastitis caused by Staphylococcus aureus. (Key words: heifer, mastitis, risk factors) Abbreviation key: CI = confidence interval, OR = odds ratio. INTRODUCTION Several studies have found a high prevalence of subclinical IMI in heifers in the periparturient period (Aarestrup and Jensen, 1997; Fox et al., 1995), and episodes of clinical mastitis are not uncommon in this period (Myllys and Rautala, 1995; Nickerson et al., 1995). In a study from The Netherlands, >30% of all episodes of clinical mastitis in first lactation occurred within 14 d postpartum (Barkema et al., 1998). Risk factors for clinical mastitis in dairy cows have been identified in epidemiological studies. Some researchers have used the herd as the unit of observation and studied the association between the herd incidence rate of mastitis and various herd factors that character-
Received May 5, 2000. Accepted September 29, 2000. Corresponding author: S. Waage; e-mail: [email protected].
ized management practices, housing, feeding, and milking (Barkema et al., 1999a, 1999b; Elbers et al., 1998; O / stera˚s et al., 1995; Schukken et al., 1990). Generally, models in which the herd is the unit of observation are not particularly suitable for the study of individual animal risk factors. However, by including as explanatory variables the proportion of cows within the herd with certain characteristics, an increased incidence rate of clinical mastitis was shown to be associated with the presence in the herd of cows leaking milk (Elbers et al., 1998; Schukken et al., 1990). Some researchers have used the individual cow as the unit of observation, and one study found that cows with udder edema were at increased risk of clinical mastitis (Gro¨hn et al., 1990). Only a few studies have considered risk factors for clinical mastitis specifically in periparturient heifers (Myllys and Rautala, 1995; Waage et al., 1998). Such animals might be expected to be particularly suitable for the study of mastitis risk. In contrast to older cows, periparturient heifers have usually not experienced a previous episode of mastitis. Cows that have been treated for mastitis are at increased risk of suffering additional mastitis attacks (Bendixen et al., 1988), and confounding might be a problem in studies that include both cows that have, and cows that have not, suffered from mastitis previously. The effects of certain unfavorable udder characteristics on mastitis risk are likely to appear when machine milking begins; thus, the initial postpartum period would be the preferable time to study the relationship between udder characteristics and mastitis risk of heifers. The udder of a heifer changes during the periparturient period. To ensure uniform assessment of udder characteristics, such factors should be recorded at a specified time in relation to parturition. Although the exposure of individual animals to important risk factors concerning housing, feeding, milking procedures, and management practices may vary within herds, much greater differences are observed between herds. To some extent, such factors might be described, categorized, and included as explanatory
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variables in studies of risk factors. However, individual animal exposures related to hygiene and milking procedures, for example, are difficult to characterize and record in a precise manner and may also vary considerably over time within a herd. Consequently, substantial parts of the risk factor complex escape analysis and remain potentially confounding factors. To minimize the risk of confounding, a study design in which cases and controls are matched on herd is appropriate. This approach is efficient when the purpose is to study factors that vary between animals within herd, e.g., individual animal characteristics. A case-control study, in which case-control pairs were matched on herd, identified milk leakage as a risk factor for clinical mastitis (van de Geer et al., 1988). Another study with a similar design found periparturient udder edema to be associated with increased risk of clinical mastitis (Slettbakk et al., 1995); milk leakage was not a risk factor in this study. The purpose of the present case-control study was to identify risk factors for clinical mastitis in postpartum heifers. The main focus was on individual animal characteristics, in particular, characteristics of the udder and teats. MATERIALS AND METHODS Clinical Cases and Control Animals The study was a case-control study. The clinical cases were recruited during a 1-yr field study on clinical mastitis in heifers that was carried out in 24 veterinary districts located in different parts of Norway. In accordance with the definition of the International Dairy Federation (1987), a heifer was considered a clinical case when macroscopic changes in the secretion or palpatory abnormalities of the udder were observed. Quarter milk samples were collected and clinical data were supplied by veterinary surgeons. Microbiological examination of samples was carried out at the Mastitis Laboratory of the National Veterinary Institute in Oslo, Norway, according to recommendations of the International Dairy Federation (1981). A detailed presentation of the study and the location of the veterinary districts have been given previously (Waage et al., 1999). At the time of treatment of the heifer, the farmer received a standard form that contained questions about herd and individual animal characteristics. The farmer was asked to record information on the clinical case and also, if available, on two heifers in the herd that had not been treated for clinical mastitis within 14 d postpartum. The two heifers were to be those that calved closest in time before and after, respectively, the day of parturition of the case heifer. A letter that explained the purpose of the study and how to complete
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the questionnaire was also delivered to the farmer. The form was returned when the second control heifer had calved, or not later than 3 mo following parturition of the clinical case, even if no heifer that was eligible as control had calved by that point in time. Variables created from the questionnaire answers are shown in Table 1. The broader field study (Waage et al., 1999) involved heifers treated for clinical mastitis prepartum or within 14 d postpartum. In general, when the objective is to evaluate risk factors for disease, only those cases that occur after exposure to the risk factors should be considered. In the current study, some of the individual animal characteristics that varied over time were recorded as they appeared at the time of parturition; therefore, only those heifers that were treated between 1 and 14 d postpartum were included. Heifers (n = 717) in 553 different herds were eligible for inclusion as cases. A total of 462 forms were returned. Several forms that were distributed to herds represented by >1 case in the primary material were not returned because, according to information from the farmer, the only possible control heifer was matched to the first case. Of the forms that were returned, 63 contained data on the clinical case only, 180 supplied data on the case and one control, and 219 supplied data on the case and two controls. In some herds, the case was the first heifer that calved that season; thus, no control heifer that had calved prior to the day of parturition of the case heifer was reported. In some other herds, no heifer had calved within 3 mo of parturition of the case heifer. On some of the forms, the farmer reported that no control heifer was available, and on a few forms no reason was given for not including two controls or any control at all. When two controls were reported on the form, the one that had calved closest in time to the day of parturition of the clinical case, either before or after, was selected as the matched control. When the same heifer was reported as the only control for two or more cases in a herd, only the first form that was received was included in the study. The material available for analysis included 364 casecontrol pairs. These pairs were from 306 different herds; 258 herds were represented by one pair, 39 herds by two pairs, eight herds by three pairs, and one herd by four pairs. Two hundred ninety-three of these herds were enrolled in the National Production Recording Scheme and the Norwegian Health Card System for Cattle (Waage et al., 1998), and data on herd size, milk yield, overall incidence of clinical mastitis, and bulk milk SCC were available for those herds. Median, first, and third quartiles for these herd variables are presented in Table 2. Journal of Dairy Science Vol. 84, No. 2, 2001
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Table 1. Description of study variables, derived from producer questionnaire responses. Variable
Alternatives in questionnaire
Levels used in analyses
Origin of heifer
0 = Not purchased; 1 = purchased
Udder edema at calving
Raised in the current herd; purchased as calf; purchased when pregnant; purchased postpartum No; slight; moderate; very swollen and hard udder
Teat edema at calving Milk leakage 1 wk prepartum Milk leakage at calving Blood in the milk at calving Skin lesion at calving Insect control prepartum Teat disinfection prepartum Location during calfhood Location at breeding age Bucket milking unit
No; slight; moderate; very swollen and firm teats No; slight; moderate; pronounced No; slight; moderate; pronounced Visual presence of blood in the milk (yes or no) Lesion between udder and thigh (yes or no) Used ear tags or long-acting skin preparation (yes or no) Used teat dip or bactericide ointment (yes or no) Kept in pen together with other calves (yes or no) Kept in pen together with other heifers (yes or no) Used bucket unit in colostrum period (yes or no)
Time of treatment had been recorded and bacteriological examination of quarter milk samples had been performed for all clinical cases included in the broader field study (Waage et al., 1999). One hundred fifty-three (42%) of the cases included in the current study were treated 1 d postpartum, 73 cases (20%) were treated 2 d postpartum, 38 cases (10%) were treated 3 d postpartum, 29 cases (8%) were treated 4 d postpartum, and 71 cases (20%) were treated between d 5 and 14 postpartum. Organisms that were isolated from clinically affected quarters (n = 455) of the 364 cases were Staphylococcus aureus (46.2%), Streptococcus dysgalactiae (18.7%), a combination of Staph. aureus and Strep. dysgalactiae (0.7%), coagulase-negative staphylococci (12.5%), Escherichia coli or other coliforms (5.7%), Arcanobacterium pyogenes (2.6%), a combination of A. pyogenes and Strep. dysgalactiae (0.4%), Streptococcus uberis (3.3%), and other streptococci or enterococci (0.4%); samples from 9.5% of the quarters were either bacteriologically negative or contaminated. Of the controls, 206 (57%) calved before the matched case. Three hundred sixty-one case heifers and 356 control heifers were of Norwegian Cattle breed. The remaining heifers were
0 = No or slight edema; 1 = moderate edema or very swollen and hard udder 0 = No edema; 1 = presence of edema 0 = No leakage; 1 = leakage 0 = No leakage; 1 = leakage 0 = Not present; 1 = present 0 = Not present; 1 = present 0 = Not used; 1 = used 0 = Not used; 1 = used 0 = No; 1 = yes 0 = No; 1 = yes 0 = No; 1 = yes
cross breeds of Norwegian Cattle and other breeds (n = 3), of a local breed (n = 2), or of unknown breed (n = 6). Statistical Analysis Data were analyzed by conditional logistic regression (Hosmer and Lemeshow, 1989; SAS Institute, 1995). Separate analyses were performed for all case-control pairs and for those pairs in which at least one clinically affected quarter was infected with Staph. aureus. Explanatory variables that were graded at more than two levels in the questionnaire were dichotomized and coded as 0 or 1 (Table 1). Data on some of the explanatory variables were missing for some of the heifers. Thus, the number of case-control pairs included in the multivariate analyses varied depending on which variables that were contained in the particular model. Variables that were associated with the outcome variable at P < 0.25 in univariate analysis were included in the multivariate analysis. Variable selection methods provided by the Logistic procedure in SAS (SAS Institute, 1995) were used to aid in the process of selecting variables for the final model. Regression coefficients
Table 2. Median, first, and third quartiles for variables1 that characterized 2932 of the herds from which the 364 pairs of case and control heifers used for analysis were obtained. Variable
Median
First quartile
Third quartile
Herd size, cow-years3 Mean milk yield, kg of FPCM4/cow-year Incidence of clinical mastitis, cases/100 cow-years Geometric mean bulk milk SCC, 103 cells/ml
13.5 6485 51 122
10.4 6025 33 97
16.8 6990 76 155
1
Data recorded in 1996. Data on 13 herds were not available. 3 The number of cow-years for a herd over a 1-yr period was the sum of days each cow was present in the herd divided by 365. A cow was contributing to the sum of cow-years from the day of first parturition to the day it was culled or sold. 4 Milk corrected for fat and protein. 2
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RISK FACTORS FOR CLINICAL MASTITIS IN HEIFERS Table 3. Number of case-control pairs in which both the heifer with clinical mastitis and its matched control had recordings for the particular explanatory variable, numbers of cases and controls with level = 1 for the dichotomous variable1 used for analysis of the data, and number of case-control pairs with different variable levels for case and control.
Variable
Pairs
Cases, level = 1
Udder edema at calving Milk leakage at calving Teat edema at calving Blood in the milk at calving Milk leakage 1 wk prepartum Skin lesion between udder and thigh Origin of heifer Insect control prepartum Used bucket milking unit Location during calfhood Teat disinfection prepartum Location at breeding age
(n) 363 359 359 344 362 345 364 347 352 350 354 344
(n) 227 256 243 93 34 30 24 41 208 215 28 146
Controls, level = 1
Pairs with different levels (1, 0 or 0, 1)
(n) 158 219 187 36 33 25 29 43 205 215 30 145
(n) 155 125 108 103 53 43 25 10 9 8 6 5
1
See Table 1 for coding of variables.
were estimated by maximum likelihood. Confidence limits for the coefficients were computed by the profile likelihood method (Allison, 1999). The P values (twotailed) for the coefficients were based on Wald statistic. Odds ratio (OR) and 95% confidence limits for OR for each of the variables, adjusted for the effects of the other variables in the model, were calculated as the antilogarithme of the estimated coefficient and its confidence limits. The effects of two-factor interaction terms when added to the model were assessed using the likelihood ratio test. The significance level was set to 0.05. Goodness of fit of models was assessed by visual examination of diagnostic plots (Hosmer and Lemeshow, 1989). The fit of individual observations was studied with plots of changes in different diagnostic statistics produced by deletion of case-control pairs with the same covariate pattern, one particular pattern at a time, versus the estimated logistic probability. Diagnostic measures that were used were the deviance, the Pearson chi-square statistic, and the leverage. The predictive power of a model was measured by a rescaled R2 with the maximum value of 1 (Nagelkerke, 1991). RESULTS Table 3 shows, for each of the explanatory variables, the number of case and control heifers with variable level equal to 1 and the number of pairs of heifers with different exposure status for case and control. Results of univariate analyses are shown in Table 4. The presence at calving of udder edema, teat edema, blood in the milk, or milk leakage was associated with increased risk of postpartum clinical mastitis. For five of the variables (insect control prepartum, use of bucket
milking unit, location during calfhood, teat disinfection prepartum, and location at breeding age), ≤10 pairs had different values for case and control, and the 95% confidence interval (CI) for OR for those variables was relatively wide. Correlations between variables for which >10 pairs were discordant are shown in Table 5. Table 6 shows estimates based on the final multivariate model. Two-factor interaction terms of the explanatory variables were not significant when added to the model (0.8 < P < 1.0). The rescaled R2 of the final model was 0.24. The fit of the model was examined graphically (Figure 1). One particular covariate pattern, which included two case-control pairs, was poorly fit. Deletion of those pairs produced a large change in the Pearson chi-square statistic (12.53) and a moderate change in the deviance (5.32); however, only small changes were observed in the leverage (0.05) and in the estimated coefficients and their standard error. Similar analyses were performed for a subgroup of case-control pairs (n = 170 pairs) in which cases had mastitis caused by Staph. aureus. Significant risk factors for clinical Staph. aureus mastitis identified by the final multivariate model were teat edema (OR = 4.54; 95% CI for OR, 2.24 to 10.29; P < 0.001), blood in the milk (OR = 2.64; 95% CI for OR, 1.40 to 5.28; P = 0.004), and milk leakage (OR = 1.96; 95% CI for OR, 1.08 to 3.67; P = 0.030). Udder edema was not a significant factor when added to this model (OR = 1.43, P = 0.3). DISCUSSION The main objective of this study was to investigate whether certain individual characteristics of heifers Journal of Dairy Science Vol. 84, No. 2, 2001
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WAAGE ET AL. Table 4. Results of univariate logistic regression analyses of association between postpartum clinical mastitis in heifers and potential risk factors, showing number of pairs of matched case and control heifers for which the particular variable was recorded, and point estimates and 95% confidence intervals (CI) for odds ratios (OR). Variable1
No.
OR
95% CI for OR
P
Udder edema at calving Teat edema at calving Blood in the milk at calving Milk leakage at calving Milk leakage 1 wk prepartum Origin of heifer Skin lesion between udder and thigh Insect control prepartum Teat disinfection prepartum Location during calfhood Location at breeding age Used bucket milking unit
363 359 344 359 362 364 345 347 354 350 344 352
2.61 3.15 3.48 1.84 1.04 0.67 1.26 0.67 0.50 1.00 1.50 2.00
1.85 2.06 2.23 1.28 0.61 0.29 0.69 0.17 0.09 0.24 0.25 0.53
<0.001 <0.001 <0.001 0.001 0.89 0.32 0.45 0.53 0.42 1.00 0.66 0.42
to to to to to to to to to to to to
3.74 5.00 5.66 2.68 1.79 1.47 2.33 2.33 2.73 4.23 11.39 9.48
1
See Table 1 for description of variables.
were associated with increased risk of clinical mastitis during the first 2 wk after parturition. Also, some variables that characterized management of the heifers as calves, at breeding age, and around parturition were recorded. However, this was a case-control study in which cases and controls were matched on herd and, generally, management practices of heifers are rather constant within a herd over time. For each of the herd level management variables that were recorded in this study, ≤10 case-control pairs were discordant. Therefore, as would be expected, this study design was not very suitable for evaluating the effect of herd level management variables on mastitis risk, and these variables were included mainly for the control of potential confounding. The design of the study implied that the same farmer assessed the characteristics of a particular case and its matched control. It was therefore unlikely that re-
cordings for heifers within a pair would be influenced by variation that might arise when observations were made by different persons. Differences between pairs in choice of code level for a given manifestation of a variable might have occurred but would not be critical to the current conditional logistic regression analysis, which is equivalent to unconditional analysis of differences in variable levels between case and control within pairs (Breslow and Day, 1980). To minimize the possibility of confounding due to a seasonal effect, the study was designed so that the heifer that calved closest to the time of parturition of a particular case heifer, either before or after, was chosen as its matched control. However, when a heifer in a herd had recently been treated for clinical mastitis, those that calved later might have been subjected to special care by the farmer. Hypothetically, actions that were not among those factors that were recorded in this
Table 5. Pairwise correlation between study variables1 for which at least 25 of 364 case-control pairs had different values for the case2 and its matched control3. Variable Variable Origin of heifer Udder edema at calving Teat edema at calving Milk leakage 1 wk prepartum Milk leakage at calving Blood in the milk at calving
Udder edema at calving
Teat edema at calving
Milk leakage 1 wk prepartum
Milk leakage at calving
Blood in the milk at calving
Skin lesion between udder and thigh
(r) −0.01
(r) 0.01 0.446
(r) 0.001 0.094 0.094
(r) −0.07 0.156 0.156 0.236
(r) 0.05 0.176 0.02 −0.01 0.07
(r) 0.04 0.094 0.115 0.03 0.07 −0.01
1
Dichotomous variables (see Table 1) used in the univariate and multivariate logistic regression analyses. Heifer with postpartum clinical mastitis. 3 Heifer from the same herd as its matched case. 4 P < 0.05. 5 P < 0.01. 6 P < 0.001. 2
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RISK FACTORS FOR CLINICAL MASTITIS IN HEIFERS Table 6. Significant risk factors for postpartum clinical mastitis according to the final multivariate logistic regression model comprising 339 pairs of matched case and control heifers. The estimated coefficient (b), standard error of the coefficient (SEb), odds ratio (OR), 95% confidence interval (CI) for OR, and significance level are given for each variable. Variable1
b
SEb
OR
95% CI for OR
P
Blood in the milk Udder edema Teat edema Milk leakage at calving
1.23 0.50 0.79 0.41
0.25 0.21 0.26 0.21
3.43 1.65 2.20 1.51
2.13 1.10 1.35 1.01
<0.001 0.017 0.002 0.049
to to to to
5.74 2.49 3.68 2.30
1
See Table 1 for description of variables.
study, taken by the farmer to prevent new cases of mastitis, might, if they were effective, have introduced some bias. However, the majority of the controls (57%) calved before the corresponding case. Variables that characterized the heifers were recorded as observed at the time of parturition; additionally, the presence or not of milk leakage 1 wk prepartum was reported. Heifers that were treated for clinical mastitis prepartum or on the day of parturition were not included in this study because, obviously, the time of exposure to the potential risk factors studied here must precede the occurrence of disease. Also, the signs of clinical mastitis before calving might be confused with extreme manifestations of the physiological phenomena that were to be recorded, i.e., udder and teat edema and blood in the milk. It was assumed that by including as cases only those heifers that were treated between d 1 and 14 postpartum, it was unlikely that signs of mastitis would interfere with udder and teat characteristics at calving. Additionally, farmers were directed to record characteristics shown by nondiseased quarters and not signs in the affected quarter that could be attributed to clinical mastitis. The presence of blood in the milk at the time of parturition was a highly significant risk factor for clinical mastitis in this study. Adjusted for the effects of other variables included in the final model, heifers with blood in the milk at calving were 3.4 times more likely to experience an episode of clinical mastitis during the first 2 wk postpartum than were those without blood in the milk. The presence of blood in the milk from one or more quarters of heifers at parturition is not uncommon. In the present study, blood was observed in the milk of 36 (10%) of the 344 controls for which this variable was recorded. The reason why heifers with blood in the milk were more at risk of clinical mastitis is not clear. According to Radostits et al. (1994), presence of blood in the milk close to parturition is usually an indication of rupture of a blood vessel by direct trauma or capillary bleeding due to congestion. Lesions within the udder caused by trauma might allow tissue invasion of intramammary pathogens. Blood in the milk might stimulate growth of certain pathogens, thus in-
creasing the risk of clinical mastitis, particularly when subclinical IMI is already present. The pathogenesis of bovine mastitis varies depending on the causal organism (Bramley and Dodd, 1984). If possible, risk factors for clinical mastitis caused by different organisms should be studied separately, as was done in a previous study of cows (Barkema et al., 1999a). In almost 50% of the cases in the current study, clinical mastitis was caused by Staph. aureus. Analysis of those case-control pairs in which cases were infected with Staph. aureus revealed that blood in the milk was also a significant risk factor for clinical Staph. aureus mastitis. The estimated OR (95% CI, 1.4 to 5.3) for the effect of that factor in cases caused by Staph. aureus was not different from the corresponding OR for all cases. Udder edema was a significant risk factor for clinical mastitis in postpartum heifers in this study. Based on the final multivariate model, heifers that had moderate or severe udder edema at parturition were 1.7 times more likely to suffer an episode of clinical mastitis than were those that exhibited no signs or only a slight degree of edema. Also, previous studies in cows (Gro¨ hn et al., 1990; Slettbakk et al., 1995) found udder edema to be associated with increased risk of clinical mastitis. Udder edema may affect the teats to a varying extent. Therefore, we decided to record the degree of teat edema separately. As would be expected, udder and teat edema were positively correlated. However, in the multivariate analysis both factors were significant. The point estimate of OR for the effect of teat edema, adjusted for the effects of other significant factors (including udder edema), was 2.2. The risk of clinical mastitis caused by Staph. aureus was not associated with udder edema; however, teat edema was a significant risk factor and the OR for the effect of this factor was relatively large (4.5). Associations between increased risk of clinical mastitis and both teat edema and udder edema are biologically plausible. An edematous, rigid teat is less likely to react normally to machine milking. For example, teats in which tissue elasticity is reduced may be more vulnerable to forces generated by changes in pressure Journal of Dairy Science Vol. 84, No. 2, 2001
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Figure 1. Change in Pearson chi-square statistic due to deletion of pairs of case and control heifers sharing the same covariate pattern, one particular pattern at a time, according to the estimated logistic probability of clinical mastitis for 339 pairs of case and control heifers. Size of plotting symbol is proportional to the corresponding change in the leverage.
during pulsation; in addition, the risk of liner slip may increase. Vacuum fluctuations induced by liner slip increase the risk of IMI (Baxter et al., 1992). Severe distension of the udder caused by udder edema may change the orientation of the teats towards a lateral direction; thus, the teats will bend during machine milking, leading to a potentially harmful load on the teats. Severe edema also tends to decrease the length of the teats and, even when small liners are used, one might question whether adequate pulsation effect is achieved on short edematous teats. Additional factors that might explain the observed association between udder and teat edema and risk of clinical mastitis are impaired local blood circulation, which may decrease udder defense mechanisms and increased udder size, which makes the teats more liable to be injured. Several studies have identified milk leakage as a risk factor for clinical mastitis in heifers (Myllys and Rautala, 1995; Waage et al., 1998) or cows (Schukken et al., 1990; van de Geer et al., 1988). In the current study, a highly significant association between milk leakage and mastitis risk was found in univariate analysis. However, in the multivariate model, which included udder edema and teat edema as additional explanatory variables, the effect of milk leakage was barely significant when P ≤ 0.05 was used as significance criterion. Thus, adjusting the analysis for the effects of udder and teat edema almost eliminated the effect of milk leakage as a risk factor per se. The positive correlations between teat edema and milk leakage and between udder edema and milk leakage suggest that milk leakage might have been, to some extent, a consequence of udder and teat edema. The reduced OR for teat edema, udder edema, and milk leakage found in multivariate analysis, compared with the corresponding OR observed in Journal of Dairy Science Vol. 84, No. 2, 2001
univariate analyses, support the interpretation that these variables, to some extent, may be parts of a common pathogenetic mechanism. A study that included cows in first and second lactation (Slettbakk et al., 1995), in which milk leakage and udder edema were recorded, failed to find any effect of milk leakage when both factors were included in the multivariate analysis. The latter study did not report the univariate relationship between milk leakage and clinical mastitis. In agreement with a previous study (Waage et al., 1998), heifers purchased before first parturition were not at a greater risk of clinical mastitis than were heifers that were not purchased. Skin lesions between udder and thigh are sometimes observed in periparturient heifers. In this study, the presence of such lesions was detected in 25 (7%) of 345 control heifers and was positively correlated with the presence of udder edema. However, no association was found between the occurrence of such skin lesions and the risk of clinical mastitis. CONCLUSIONS Blood in the milk, udder edema, teat edema, and milk leakage at the time of parturition were identified as risk factors for clinical mastitis in heifers during the first 2 wk postpartum. Blood in the milk, teat edema, and milk leakage were also associated with increased risk of clinical mastitis caused by Staph. aureus. Heifers with skin lesions between udder and thigh and purchased heifers were not at increased risk of clinical mastitis. ACKNOWLEDGMENTS The authors thank the veterinary practitioners who participated in the study and the farmers that supplied information on their heifers and management practices. The study was supported by the Research Council of Norway (Oslo, Norway), grant no. 112,956. REFERENCES Aarestrup, F. M., and N. E. Jensen. 1997. Prevalence and duration of intramammary infection in Danish heifers during the peripartum period. J. Dairy Sci. 80:307–312. Allison, P. D. 1999. Page 32 in Logistic Regression Using the SAS System: Theory and Application. SAS Institute Inc., Cary, NC. Barkema, H. W., Y. H. Schukken, T.J.G.M. Lam, M. L. Beiboer, H. Wilmink, G. Benedictus, and A. Brand. 1998. Incidence of clinical mastitis in dairy herds grouped in three categories by bulk milk somatic cell counts. J. Dairy Sci. 81:411–419. Barkema, H. W., Y. H. Schukken, T.J.G.M. Lam, M. L. Beiboer, G. Benedictus, and A. Brand. 1999a. Management practices associated with the incidence rate of clinical mastitis. J. Dairy Sci. 82:1643–1654. Barkema, H. W., J. D. van der Ploeg, Y. H. Schukken, T.J.G.M. Lam, G. Benedictus, and A. Brand. 1999b. Management style and its
RISK FACTORS FOR CLINICAL MASTITIS IN HEIFERS association with bulk milk somatic cell count and incidence rate of clinical mastitis. J. Dairy Sci. 82:1655–1663. Baxter, J. D., G. W. Rogers, S. B. Spencer, and R. J. Eberhart. 1992. The effect of milking machine liner slip on new intramammary infections. J. Dairy Sci. 75:1015–1018. ˚ strand. 1988. Disease Bendixen, P. H., B. Vilson, I. Ekesbo, and D. B. A frequencies in dairy cows in Sweden. V. Mastitis. Prev. Vet. Med. 5:263–274. Bramley, A. J., and F. H. Dodd. 1984. Reviews of the progress of dairy science: mastitis control—progress and prospects. J. Dairy Res. 51:481–512. Breslow, N. E., and N. E. Day. 1980. Pages 248–279 in Statistical Methods in Cancer Research. Volume 1. The Analysis of CaseControl Studies. Int. Agency for Research on Cancer, Lyon, France. Elbers, A.R.W., J. D. Miltenburg, D. de Lange, A.P.P. Crauwels, H. W. Barkema, and Y. H. Schukken. 1998. Risk factors for clinical mastitis in a random sample of dairy herds in the southern part of The Netherlands. J. Dairy Sci. 81:420–426. Fox, L. K., S. T. Chester, J. W. Hallberg, S. C. Nickerson, J. W. Pankey, and L. D. Weaver. 1995. Survey of intramammary infections in dairy heifers at breeding age and first parturition. J. Dairy Sci. 78:1619–1628. Gro¨ hn, Y. T., H. N. Erb, C. E. McCulloch, and H. S. Saloniemi. 1990. Epidemiology of mammary gland disorders in multiparous Finnish Ayrshire cows. Prev. Vet. Med. 8:241–252. Hosmer, D. W., and S. Lemeshow. 1989. Applied Logistic Regression. John Wiley & Sons, New York, NY. International Dairy Federation. 1981. Laboratory Methods for Use in Mastitis Work. Document 132. Int. Dairy Fed., Brussels, Belgium. International Dairy Federation. 1987. Bovine Mastitis. Definition and Guidelines for Diagnosis. Bull. 211. Int. Dairy Fed., Brussels, Belgium.
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Myllys, V., and H. Rautala. 1995. Characterization of clinical mastitis in primiparous heifers. J. Dairy Sci. 78:538–545. Nagelkerke, N.J.D. 1991. A note on a general definition of the coefficient of determination. Biometrika 78:691–692. Nickerson, S. C., W. E. Owens, and R. L. Boddie. 1995. Mastitis in dairy heifers: initial studies on prevalence and control. J. Dairy Sci. 78:1607–1618. Østera˚ s, O., O. Rønningen, L. Sandvik, and S. Waage. 1995. Field studies show associations between pulsator characteristics and udder health. J. Dairy Res. 62:1–13. Radostits, O. M., D. C. Blood, and C. C. Gay. 1994. Pages 614–615 in Veterinary Medicine. 8th ed. W. B. Saunders Co., London, UK. SAS Institute. 1995. Pages 111–117 in Logistic Regression Examples Using the SAS System. Version 6. SAS Inst. Inc., Cary, NC. Schukken, Y. H., F. J. Grommers, D. van de Geer, H. N. Erb, and A. Brand. 1990. Risk factors for clinical mastitis in herds with a low bulk milk somatic cell count. 1. Data and risk factors for all cases. J. Dairy Sci. 73:3463–3471. Slettbakk, T., A. Jørstad, T. B. Farver, and J. C. Holmes. 1995. Impact of milking characteristics and morphology of udder and teats on clinical mastitis in first- and second-lactation Norwegian cattle. Prev. Vet. Med. 24:235–244. van de Geer, D., Y. H. Schukken, F. J. Grommers, and A. Brand. 1988. A matched case-control study on clinical mastitis in Holstein-Frisian dairy cows. Pages 60–64 in Proc. 6th Int. Congr. Anim. Hygiene, Skara, Sweden. Swedish Univ. of Agricultural Sci., Skara, Sweden. Waage, S., T. Mørk, A. Røros, D. Aasland, A. Hunshamar, and S. A. Ødegaard. 1999. Bacteria associated with clinical mastitis in dairy heifers. J. Dairy Sci. 82:712–719. Waage, S., S. Sviland, and S. A. Ødegaard. 1998. Identification of risk factors for clinical mastitis in dairy heifers. J. Dairy Sci. 81:1275–1284.
Journal of Dairy Science Vol. 84, No. 2, 2001
Case-Control Study of Risk Factors for Clinical Mastitis in Postpartum Dairy Heifers S. Waage,* S. A. Ødegaard,* A. Lund,† S. Brattgjerd,‡ and T. Røthe† *Department of Reproduction and Forensic Medicine, Norwegian School of Veterinary Science, P. O. Box 8146 Dep., 0033 Oslo, Norway †Veterinary Center in Sparbu, 7710 Sparbu, Norway ‡Veterinary Center in Indero/y, 7670 Sakshaug, Norway
ABSTRACT A case-control study was carried out to evaluate risk factors for clinical mastitis occurring in dairy heifers between 1 and 14 d postpartum. Case and control heifers were matched on herd; the control was the heifer that calved closest in time, before or after, the particular case. Data were analyzed by conditional logistic regression. The final multivariate model included 339 casecontrol pairs. Blood in the milk, udder edema, teat edema, and milk leakage, all recorded at the time of parturition, were significant risk factors. Purchased heifers and heifers with skin lesions between udder and thigh were not at increased risk of clinical mastitis. Separate analysis of a subgroup of case-control pairs identified teat edema, blood in the milk, and milk leakage at calving as risk factors for clinical mastitis caused by Staphylococcus aureus. (Key words: heifer, mastitis, risk factors) Abbreviation key: CI = confidence interval, OR = odds ratio. INTRODUCTION Several studies have found a high prevalence of subclinical IMI in heifers in the periparturient period (Aarestrup and Jensen, 1997; Fox et al., 1995), and episodes of clinical mastitis are not uncommon in this period (Myllys and Rautala, 1995; Nickerson et al., 1995). In a study from The Netherlands, >30% of all episodes of clinical mastitis in first lactation occurred within 14 d postpartum (Barkema et al., 1998). Risk factors for clinical mastitis in dairy cows have been identified in epidemiological studies. Some researchers have used the herd as the unit of observation and studied the association between the herd incidence rate of mastitis and various herd factors that character-
Received May 5, 2000. Accepted September 29, 2000. Corresponding author: S. Waage; e-mail: [email protected].
ized management practices, housing, feeding, and milking (Barkema et al., 1999a, 1999b; Elbers et al., 1998; O / stera˚s et al., 1995; Schukken et al., 1990). Generally, models in which the herd is the unit of observation are not particularly suitable for the study of individual animal risk factors. However, by including as explanatory variables the proportion of cows within the herd with certain characteristics, an increased incidence rate of clinical mastitis was shown to be associated with the presence in the herd of cows leaking milk (Elbers et al., 1998; Schukken et al., 1990). Some researchers have used the individual cow as the unit of observation, and one study found that cows with udder edema were at increased risk of clinical mastitis (Gro¨hn et al., 1990). Only a few studies have considered risk factors for clinical mastitis specifically in periparturient heifers (Myllys and Rautala, 1995; Waage et al., 1998). Such animals might be expected to be particularly suitable for the study of mastitis risk. In contrast to older cows, periparturient heifers have usually not experienced a previous episode of mastitis. Cows that have been treated for mastitis are at increased risk of suffering additional mastitis attacks (Bendixen et al., 1988), and confounding might be a problem in studies that include both cows that have, and cows that have not, suffered from mastitis previously. The effects of certain unfavorable udder characteristics on mastitis risk are likely to appear when machine milking begins; thus, the initial postpartum period would be the preferable time to study the relationship between udder characteristics and mastitis risk of heifers. The udder of a heifer changes during the periparturient period. To ensure uniform assessment of udder characteristics, such factors should be recorded at a specified time in relation to parturition. Although the exposure of individual animals to important risk factors concerning housing, feeding, milking procedures, and management practices may vary within herds, much greater differences are observed between herds. To some extent, such factors might be described, categorized, and included as explanatory
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variables in studies of risk factors. However, individual animal exposures related to hygiene and milking procedures, for example, are difficult to characterize and record in a precise manner and may also vary considerably over time within a herd. Consequently, substantial parts of the risk factor complex escape analysis and remain potentially confounding factors. To minimize the risk of confounding, a study design in which cases and controls are matched on herd is appropriate. This approach is efficient when the purpose is to study factors that vary between animals within herd, e.g., individual animal characteristics. A case-control study, in which case-control pairs were matched on herd, identified milk leakage as a risk factor for clinical mastitis (van de Geer et al., 1988). Another study with a similar design found periparturient udder edema to be associated with increased risk of clinical mastitis (Slettbakk et al., 1995); milk leakage was not a risk factor in this study. The purpose of the present case-control study was to identify risk factors for clinical mastitis in postpartum heifers. The main focus was on individual animal characteristics, in particular, characteristics of the udder and teats. MATERIALS AND METHODS Clinical Cases and Control Animals The study was a case-control study. The clinical cases were recruited during a 1-yr field study on clinical mastitis in heifers that was carried out in 24 veterinary districts located in different parts of Norway. In accordance with the definition of the International Dairy Federation (1987), a heifer was considered a clinical case when macroscopic changes in the secretion or palpatory abnormalities of the udder were observed. Quarter milk samples were collected and clinical data were supplied by veterinary surgeons. Microbiological examination of samples was carried out at the Mastitis Laboratory of the National Veterinary Institute in Oslo, Norway, according to recommendations of the International Dairy Federation (1981). A detailed presentation of the study and the location of the veterinary districts have been given previously (Waage et al., 1999). At the time of treatment of the heifer, the farmer received a standard form that contained questions about herd and individual animal characteristics. The farmer was asked to record information on the clinical case and also, if available, on two heifers in the herd that had not been treated for clinical mastitis within 14 d postpartum. The two heifers were to be those that calved closest in time before and after, respectively, the day of parturition of the case heifer. A letter that explained the purpose of the study and how to complete
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the questionnaire was also delivered to the farmer. The form was returned when the second control heifer had calved, or not later than 3 mo following parturition of the clinical case, even if no heifer that was eligible as control had calved by that point in time. Variables created from the questionnaire answers are shown in Table 1. The broader field study (Waage et al., 1999) involved heifers treated for clinical mastitis prepartum or within 14 d postpartum. In general, when the objective is to evaluate risk factors for disease, only those cases that occur after exposure to the risk factors should be considered. In the current study, some of the individual animal characteristics that varied over time were recorded as they appeared at the time of parturition; therefore, only those heifers that were treated between 1 and 14 d postpartum were included. Heifers (n = 717) in 553 different herds were eligible for inclusion as cases. A total of 462 forms were returned. Several forms that were distributed to herds represented by >1 case in the primary material were not returned because, according to information from the farmer, the only possible control heifer was matched to the first case. Of the forms that were returned, 63 contained data on the clinical case only, 180 supplied data on the case and one control, and 219 supplied data on the case and two controls. In some herds, the case was the first heifer that calved that season; thus, no control heifer that had calved prior to the day of parturition of the case heifer was reported. In some other herds, no heifer had calved within 3 mo of parturition of the case heifer. On some of the forms, the farmer reported that no control heifer was available, and on a few forms no reason was given for not including two controls or any control at all. When two controls were reported on the form, the one that had calved closest in time to the day of parturition of the clinical case, either before or after, was selected as the matched control. When the same heifer was reported as the only control for two or more cases in a herd, only the first form that was received was included in the study. The material available for analysis included 364 casecontrol pairs. These pairs were from 306 different herds; 258 herds were represented by one pair, 39 herds by two pairs, eight herds by three pairs, and one herd by four pairs. Two hundred ninety-three of these herds were enrolled in the National Production Recording Scheme and the Norwegian Health Card System for Cattle (Waage et al., 1998), and data on herd size, milk yield, overall incidence of clinical mastitis, and bulk milk SCC were available for those herds. Median, first, and third quartiles for these herd variables are presented in Table 2. Journal of Dairy Science Vol. 84, No. 2, 2001
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Table 1. Description of study variables, derived from producer questionnaire responses. Variable
Alternatives in questionnaire
Levels used in analyses
Origin of heifer
0 = Not purchased; 1 = purchased
Udder edema at calving
Raised in the current herd; purchased as calf; purchased when pregnant; purchased postpartum No; slight; moderate; very swollen and hard udder
Teat edema at calving Milk leakage 1 wk prepartum Milk leakage at calving Blood in the milk at calving Skin lesion at calving Insect control prepartum Teat disinfection prepartum Location during calfhood Location at breeding age Bucket milking unit
No; slight; moderate; very swollen and firm teats No; slight; moderate; pronounced No; slight; moderate; pronounced Visual presence of blood in the milk (yes or no) Lesion between udder and thigh (yes or no) Used ear tags or long-acting skin preparation (yes or no) Used teat dip or bactericide ointment (yes or no) Kept in pen together with other calves (yes or no) Kept in pen together with other heifers (yes or no) Used bucket unit in colostrum period (yes or no)
Time of treatment had been recorded and bacteriological examination of quarter milk samples had been performed for all clinical cases included in the broader field study (Waage et al., 1999). One hundred fifty-three (42%) of the cases included in the current study were treated 1 d postpartum, 73 cases (20%) were treated 2 d postpartum, 38 cases (10%) were treated 3 d postpartum, 29 cases (8%) were treated 4 d postpartum, and 71 cases (20%) were treated between d 5 and 14 postpartum. Organisms that were isolated from clinically affected quarters (n = 455) of the 364 cases were Staphylococcus aureus (46.2%), Streptococcus dysgalactiae (18.7%), a combination of Staph. aureus and Strep. dysgalactiae (0.7%), coagulase-negative staphylococci (12.5%), Escherichia coli or other coliforms (5.7%), Arcanobacterium pyogenes (2.6%), a combination of A. pyogenes and Strep. dysgalactiae (0.4%), Streptococcus uberis (3.3%), and other streptococci or enterococci (0.4%); samples from 9.5% of the quarters were either bacteriologically negative or contaminated. Of the controls, 206 (57%) calved before the matched case. Three hundred sixty-one case heifers and 356 control heifers were of Norwegian Cattle breed. The remaining heifers were
0 = No or slight edema; 1 = moderate edema or very swollen and hard udder 0 = No edema; 1 = presence of edema 0 = No leakage; 1 = leakage 0 = No leakage; 1 = leakage 0 = Not present; 1 = present 0 = Not present; 1 = present 0 = Not used; 1 = used 0 = Not used; 1 = used 0 = No; 1 = yes 0 = No; 1 = yes 0 = No; 1 = yes
cross breeds of Norwegian Cattle and other breeds (n = 3), of a local breed (n = 2), or of unknown breed (n = 6). Statistical Analysis Data were analyzed by conditional logistic regression (Hosmer and Lemeshow, 1989; SAS Institute, 1995). Separate analyses were performed for all case-control pairs and for those pairs in which at least one clinically affected quarter was infected with Staph. aureus. Explanatory variables that were graded at more than two levels in the questionnaire were dichotomized and coded as 0 or 1 (Table 1). Data on some of the explanatory variables were missing for some of the heifers. Thus, the number of case-control pairs included in the multivariate analyses varied depending on which variables that were contained in the particular model. Variables that were associated with the outcome variable at P < 0.25 in univariate analysis were included in the multivariate analysis. Variable selection methods provided by the Logistic procedure in SAS (SAS Institute, 1995) were used to aid in the process of selecting variables for the final model. Regression coefficients
Table 2. Median, first, and third quartiles for variables1 that characterized 2932 of the herds from which the 364 pairs of case and control heifers used for analysis were obtained. Variable
Median
First quartile
Third quartile
Herd size, cow-years3 Mean milk yield, kg of FPCM4/cow-year Incidence of clinical mastitis, cases/100 cow-years Geometric mean bulk milk SCC, 103 cells/ml
13.5 6485 51 122
10.4 6025 33 97
16.8 6990 76 155
1
Data recorded in 1996. Data on 13 herds were not available. 3 The number of cow-years for a herd over a 1-yr period was the sum of days each cow was present in the herd divided by 365. A cow was contributing to the sum of cow-years from the day of first parturition to the day it was culled or sold. 4 Milk corrected for fat and protein. 2
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RISK FACTORS FOR CLINICAL MASTITIS IN HEIFERS Table 3. Number of case-control pairs in which both the heifer with clinical mastitis and its matched control had recordings for the particular explanatory variable, numbers of cases and controls with level = 1 for the dichotomous variable1 used for analysis of the data, and number of case-control pairs with different variable levels for case and control.
Variable
Pairs
Cases, level = 1
Udder edema at calving Milk leakage at calving Teat edema at calving Blood in the milk at calving Milk leakage 1 wk prepartum Skin lesion between udder and thigh Origin of heifer Insect control prepartum Used bucket milking unit Location during calfhood Teat disinfection prepartum Location at breeding age
(n) 363 359 359 344 362 345 364 347 352 350 354 344
(n) 227 256 243 93 34 30 24 41 208 215 28 146
Controls, level = 1
Pairs with different levels (1, 0 or 0, 1)
(n) 158 219 187 36 33 25 29 43 205 215 30 145
(n) 155 125 108 103 53 43 25 10 9 8 6 5
1
See Table 1 for coding of variables.
were estimated by maximum likelihood. Confidence limits for the coefficients were computed by the profile likelihood method (Allison, 1999). The P values (twotailed) for the coefficients were based on Wald statistic. Odds ratio (OR) and 95% confidence limits for OR for each of the variables, adjusted for the effects of the other variables in the model, were calculated as the antilogarithme of the estimated coefficient and its confidence limits. The effects of two-factor interaction terms when added to the model were assessed using the likelihood ratio test. The significance level was set to 0.05. Goodness of fit of models was assessed by visual examination of diagnostic plots (Hosmer and Lemeshow, 1989). The fit of individual observations was studied with plots of changes in different diagnostic statistics produced by deletion of case-control pairs with the same covariate pattern, one particular pattern at a time, versus the estimated logistic probability. Diagnostic measures that were used were the deviance, the Pearson chi-square statistic, and the leverage. The predictive power of a model was measured by a rescaled R2 with the maximum value of 1 (Nagelkerke, 1991). RESULTS Table 3 shows, for each of the explanatory variables, the number of case and control heifers with variable level equal to 1 and the number of pairs of heifers with different exposure status for case and control. Results of univariate analyses are shown in Table 4. The presence at calving of udder edema, teat edema, blood in the milk, or milk leakage was associated with increased risk of postpartum clinical mastitis. For five of the variables (insect control prepartum, use of bucket
milking unit, location during calfhood, teat disinfection prepartum, and location at breeding age), ≤10 pairs had different values for case and control, and the 95% confidence interval (CI) for OR for those variables was relatively wide. Correlations between variables for which >10 pairs were discordant are shown in Table 5. Table 6 shows estimates based on the final multivariate model. Two-factor interaction terms of the explanatory variables were not significant when added to the model (0.8 < P < 1.0). The rescaled R2 of the final model was 0.24. The fit of the model was examined graphically (Figure 1). One particular covariate pattern, which included two case-control pairs, was poorly fit. Deletion of those pairs produced a large change in the Pearson chi-square statistic (12.53) and a moderate change in the deviance (5.32); however, only small changes were observed in the leverage (0.05) and in the estimated coefficients and their standard error. Similar analyses were performed for a subgroup of case-control pairs (n = 170 pairs) in which cases had mastitis caused by Staph. aureus. Significant risk factors for clinical Staph. aureus mastitis identified by the final multivariate model were teat edema (OR = 4.54; 95% CI for OR, 2.24 to 10.29; P < 0.001), blood in the milk (OR = 2.64; 95% CI for OR, 1.40 to 5.28; P = 0.004), and milk leakage (OR = 1.96; 95% CI for OR, 1.08 to 3.67; P = 0.030). Udder edema was not a significant factor when added to this model (OR = 1.43, P = 0.3). DISCUSSION The main objective of this study was to investigate whether certain individual characteristics of heifers Journal of Dairy Science Vol. 84, No. 2, 2001
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WAAGE ET AL. Table 4. Results of univariate logistic regression analyses of association between postpartum clinical mastitis in heifers and potential risk factors, showing number of pairs of matched case and control heifers for which the particular variable was recorded, and point estimates and 95% confidence intervals (CI) for odds ratios (OR). Variable1
No.
OR
95% CI for OR
P
Udder edema at calving Teat edema at calving Blood in the milk at calving Milk leakage at calving Milk leakage 1 wk prepartum Origin of heifer Skin lesion between udder and thigh Insect control prepartum Teat disinfection prepartum Location during calfhood Location at breeding age Used bucket milking unit
363 359 344 359 362 364 345 347 354 350 344 352
2.61 3.15 3.48 1.84 1.04 0.67 1.26 0.67 0.50 1.00 1.50 2.00
1.85 2.06 2.23 1.28 0.61 0.29 0.69 0.17 0.09 0.24 0.25 0.53
<0.001 <0.001 <0.001 0.001 0.89 0.32 0.45 0.53 0.42 1.00 0.66 0.42
to to to to to to to to to to to to
3.74 5.00 5.66 2.68 1.79 1.47 2.33 2.33 2.73 4.23 11.39 9.48
1
See Table 1 for description of variables.
were associated with increased risk of clinical mastitis during the first 2 wk after parturition. Also, some variables that characterized management of the heifers as calves, at breeding age, and around parturition were recorded. However, this was a case-control study in which cases and controls were matched on herd and, generally, management practices of heifers are rather constant within a herd over time. For each of the herd level management variables that were recorded in this study, ≤10 case-control pairs were discordant. Therefore, as would be expected, this study design was not very suitable for evaluating the effect of herd level management variables on mastitis risk, and these variables were included mainly for the control of potential confounding. The design of the study implied that the same farmer assessed the characteristics of a particular case and its matched control. It was therefore unlikely that re-
cordings for heifers within a pair would be influenced by variation that might arise when observations were made by different persons. Differences between pairs in choice of code level for a given manifestation of a variable might have occurred but would not be critical to the current conditional logistic regression analysis, which is equivalent to unconditional analysis of differences in variable levels between case and control within pairs (Breslow and Day, 1980). To minimize the possibility of confounding due to a seasonal effect, the study was designed so that the heifer that calved closest to the time of parturition of a particular case heifer, either before or after, was chosen as its matched control. However, when a heifer in a herd had recently been treated for clinical mastitis, those that calved later might have been subjected to special care by the farmer. Hypothetically, actions that were not among those factors that were recorded in this
Table 5. Pairwise correlation between study variables1 for which at least 25 of 364 case-control pairs had different values for the case2 and its matched control3. Variable Variable Origin of heifer Udder edema at calving Teat edema at calving Milk leakage 1 wk prepartum Milk leakage at calving Blood in the milk at calving
Udder edema at calving
Teat edema at calving
Milk leakage 1 wk prepartum
Milk leakage at calving
Blood in the milk at calving
Skin lesion between udder and thigh
(r) −0.01
(r) 0.01 0.446
(r) 0.001 0.094 0.094
(r) −0.07 0.156 0.156 0.236
(r) 0.05 0.176 0.02 −0.01 0.07
(r) 0.04 0.094 0.115 0.03 0.07 −0.01
1
Dichotomous variables (see Table 1) used in the univariate and multivariate logistic regression analyses. Heifer with postpartum clinical mastitis. 3 Heifer from the same herd as its matched case. 4 P < 0.05. 5 P < 0.01. 6 P < 0.001. 2
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RISK FACTORS FOR CLINICAL MASTITIS IN HEIFERS Table 6. Significant risk factors for postpartum clinical mastitis according to the final multivariate logistic regression model comprising 339 pairs of matched case and control heifers. The estimated coefficient (b), standard error of the coefficient (SEb), odds ratio (OR), 95% confidence interval (CI) for OR, and significance level are given for each variable. Variable1
b
SEb
OR
95% CI for OR
P
Blood in the milk Udder edema Teat edema Milk leakage at calving
1.23 0.50 0.79 0.41
0.25 0.21 0.26 0.21
3.43 1.65 2.20 1.51
2.13 1.10 1.35 1.01
<0.001 0.017 0.002 0.049
to to to to
5.74 2.49 3.68 2.30
1
See Table 1 for description of variables.
study, taken by the farmer to prevent new cases of mastitis, might, if they were effective, have introduced some bias. However, the majority of the controls (57%) calved before the corresponding case. Variables that characterized the heifers were recorded as observed at the time of parturition; additionally, the presence or not of milk leakage 1 wk prepartum was reported. Heifers that were treated for clinical mastitis prepartum or on the day of parturition were not included in this study because, obviously, the time of exposure to the potential risk factors studied here must precede the occurrence of disease. Also, the signs of clinical mastitis before calving might be confused with extreme manifestations of the physiological phenomena that were to be recorded, i.e., udder and teat edema and blood in the milk. It was assumed that by including as cases only those heifers that were treated between d 1 and 14 postpartum, it was unlikely that signs of mastitis would interfere with udder and teat characteristics at calving. Additionally, farmers were directed to record characteristics shown by nondiseased quarters and not signs in the affected quarter that could be attributed to clinical mastitis. The presence of blood in the milk at the time of parturition was a highly significant risk factor for clinical mastitis in this study. Adjusted for the effects of other variables included in the final model, heifers with blood in the milk at calving were 3.4 times more likely to experience an episode of clinical mastitis during the first 2 wk postpartum than were those without blood in the milk. The presence of blood in the milk from one or more quarters of heifers at parturition is not uncommon. In the present study, blood was observed in the milk of 36 (10%) of the 344 controls for which this variable was recorded. The reason why heifers with blood in the milk were more at risk of clinical mastitis is not clear. According to Radostits et al. (1994), presence of blood in the milk close to parturition is usually an indication of rupture of a blood vessel by direct trauma or capillary bleeding due to congestion. Lesions within the udder caused by trauma might allow tissue invasion of intramammary pathogens. Blood in the milk might stimulate growth of certain pathogens, thus in-
creasing the risk of clinical mastitis, particularly when subclinical IMI is already present. The pathogenesis of bovine mastitis varies depending on the causal organism (Bramley and Dodd, 1984). If possible, risk factors for clinical mastitis caused by different organisms should be studied separately, as was done in a previous study of cows (Barkema et al., 1999a). In almost 50% of the cases in the current study, clinical mastitis was caused by Staph. aureus. Analysis of those case-control pairs in which cases were infected with Staph. aureus revealed that blood in the milk was also a significant risk factor for clinical Staph. aureus mastitis. The estimated OR (95% CI, 1.4 to 5.3) for the effect of that factor in cases caused by Staph. aureus was not different from the corresponding OR for all cases. Udder edema was a significant risk factor for clinical mastitis in postpartum heifers in this study. Based on the final multivariate model, heifers that had moderate or severe udder edema at parturition were 1.7 times more likely to suffer an episode of clinical mastitis than were those that exhibited no signs or only a slight degree of edema. Also, previous studies in cows (Gro¨ hn et al., 1990; Slettbakk et al., 1995) found udder edema to be associated with increased risk of clinical mastitis. Udder edema may affect the teats to a varying extent. Therefore, we decided to record the degree of teat edema separately. As would be expected, udder and teat edema were positively correlated. However, in the multivariate analysis both factors were significant. The point estimate of OR for the effect of teat edema, adjusted for the effects of other significant factors (including udder edema), was 2.2. The risk of clinical mastitis caused by Staph. aureus was not associated with udder edema; however, teat edema was a significant risk factor and the OR for the effect of this factor was relatively large (4.5). Associations between increased risk of clinical mastitis and both teat edema and udder edema are biologically plausible. An edematous, rigid teat is less likely to react normally to machine milking. For example, teats in which tissue elasticity is reduced may be more vulnerable to forces generated by changes in pressure Journal of Dairy Science Vol. 84, No. 2, 2001
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Figure 1. Change in Pearson chi-square statistic due to deletion of pairs of case and control heifers sharing the same covariate pattern, one particular pattern at a time, according to the estimated logistic probability of clinical mastitis for 339 pairs of case and control heifers. Size of plotting symbol is proportional to the corresponding change in the leverage.
during pulsation; in addition, the risk of liner slip may increase. Vacuum fluctuations induced by liner slip increase the risk of IMI (Baxter et al., 1992). Severe distension of the udder caused by udder edema may change the orientation of the teats towards a lateral direction; thus, the teats will bend during machine milking, leading to a potentially harmful load on the teats. Severe edema also tends to decrease the length of the teats and, even when small liners are used, one might question whether adequate pulsation effect is achieved on short edematous teats. Additional factors that might explain the observed association between udder and teat edema and risk of clinical mastitis are impaired local blood circulation, which may decrease udder defense mechanisms and increased udder size, which makes the teats more liable to be injured. Several studies have identified milk leakage as a risk factor for clinical mastitis in heifers (Myllys and Rautala, 1995; Waage et al., 1998) or cows (Schukken et al., 1990; van de Geer et al., 1988). In the current study, a highly significant association between milk leakage and mastitis risk was found in univariate analysis. However, in the multivariate model, which included udder edema and teat edema as additional explanatory variables, the effect of milk leakage was barely significant when P ≤ 0.05 was used as significance criterion. Thus, adjusting the analysis for the effects of udder and teat edema almost eliminated the effect of milk leakage as a risk factor per se. The positive correlations between teat edema and milk leakage and between udder edema and milk leakage suggest that milk leakage might have been, to some extent, a consequence of udder and teat edema. The reduced OR for teat edema, udder edema, and milk leakage found in multivariate analysis, compared with the corresponding OR observed in Journal of Dairy Science Vol. 84, No. 2, 2001
univariate analyses, support the interpretation that these variables, to some extent, may be parts of a common pathogenetic mechanism. A study that included cows in first and second lactation (Slettbakk et al., 1995), in which milk leakage and udder edema were recorded, failed to find any effect of milk leakage when both factors were included in the multivariate analysis. The latter study did not report the univariate relationship between milk leakage and clinical mastitis. In agreement with a previous study (Waage et al., 1998), heifers purchased before first parturition were not at a greater risk of clinical mastitis than were heifers that were not purchased. Skin lesions between udder and thigh are sometimes observed in periparturient heifers. In this study, the presence of such lesions was detected in 25 (7%) of 345 control heifers and was positively correlated with the presence of udder edema. However, no association was found between the occurrence of such skin lesions and the risk of clinical mastitis. CONCLUSIONS Blood in the milk, udder edema, teat edema, and milk leakage at the time of parturition were identified as risk factors for clinical mastitis in heifers during the first 2 wk postpartum. Blood in the milk, teat edema, and milk leakage were also associated with increased risk of clinical mastitis caused by Staph. aureus. Heifers with skin lesions between udder and thigh and purchased heifers were not at increased risk of clinical mastitis. ACKNOWLEDGMENTS The authors thank the veterinary practitioners who participated in the study and the farmers that supplied information on their heifers and management practices. The study was supported by the Research Council of Norway (Oslo, Norway), grant no. 112,956. REFERENCES Aarestrup, F. M., and N. E. Jensen. 1997. Prevalence and duration of intramammary infection in Danish heifers during the peripartum period. J. Dairy Sci. 80:307–312. Allison, P. D. 1999. Page 32 in Logistic Regression Using the SAS System: Theory and Application. SAS Institute Inc., Cary, NC. Barkema, H. W., Y. H. Schukken, T.J.G.M. Lam, M. L. Beiboer, H. Wilmink, G. Benedictus, and A. Brand. 1998. Incidence of clinical mastitis in dairy herds grouped in three categories by bulk milk somatic cell counts. J. Dairy Sci. 81:411–419. Barkema, H. W., Y. H. Schukken, T.J.G.M. Lam, M. L. Beiboer, G. Benedictus, and A. Brand. 1999a. Management practices associated with the incidence rate of clinical mastitis. J. Dairy Sci. 82:1643–1654. Barkema, H. W., J. D. van der Ploeg, Y. H. Schukken, T.J.G.M. Lam, G. Benedictus, and A. Brand. 1999b. Management style and its
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