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Enterobacteriaceae bedding populations, rainfall and mastitis on a california dairy
Preventive Veterinary Medicine, 1 (1982/1983) 227--242 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands
227
ENTEROBACTERIACEAE BEDDING POPULATIONS, RAINFALL AND MASTITIS ON A C A L I F O R N I A D A I R Y
C.B. THOMAS, D.E. JASPER, M.H. ROLLINS, R.B. BUSHNELL* and E.J. CARROLL Department of Clinical Pathology, Veterinary School, University of California, Davis, CA 95616 (U.S.A.) *Cooperative Veterinary Extension, University of California, Davis, CA 95616 (U.S.A.) (Accepted 31 December 1982)
ABSTRACT Thomas, C.B., Jasper, D.E., Rollins, M.H., Bushnell, R.B. and Carroll, E.J., 1983. Enterobacteriaceae bedding populations, rainfall and mastitis on a California dairy. Prev. Vet. Med., 1: 227--242. The bedding populations of Enterobacteriaceae, rainfall and mastitis incidence due to Enterobacteriaceae were studied over 12 months on one California dairy. The bedding material used was a mixture of sawdust and wood shavings. Over the 12-month period Escherichia coli was isolated from 59 mastitic milk samples and Klebsiella pneumoniae from 44. The bedding population, rainfall and mastitis incidence data were analyzed by fitting log-linear models. The results of these analyses indicated that rainfall, bedding populations of E. coli and E. coli mastitis incidence were statistically independent, while a strong association existed between rainfall and K. pneumoniae bedding populations and separately between K. pneumoniae bedding populations and the incidence of K. pneumoniae mastitis.
INTRODUCTION Microbiological studies o f i n c i d e n t c o l i f o r m mastitis c o n d u c t e d b y M c D o n a l d et al. (1970), B r a m a n et al. ( 1 9 7 3 ) , E b e r h a r t and B u c k a l e w ( 1 9 7 7 ) and Saran ( 1 9 8 0 ) have s h o w n great s e r o t y p i c diversity a m o n g colif o r m isolates within the same dairy herd. Post-milking teat sanitization, w h i c h is an effective c o n t r o l measure for c o n t a g i o u s mastitis due to Streptococcus agalactiae a n d S t a p h y l o c o c c u s aureus, does n o t a f f e c t n e w colif o r m i n f e c t i o n rates ( C o l i f o r m s u b c o m m i t t e e , NMC, 1979). These findings s u p p o r t the h y p o t h e s i s t h a t the risk o f i n c i d e n t c o l i f o r m mastitis is o f t e n d e p e n d e n t o n e n v i r o n m e n t a l reservoirs as o p p o s e d to c o n t a g i o u s reservoirs e m a n a t i n g f r o m the i n f e c t e d u d d e r s o f herd mates, and t h a t the time o f invasion o f the u d d e r m a y be in the intermilking period. B r a m l e y ( 1 9 7 4 ) , Jasper and Dellinger ( 1 9 7 5 ) , N a t z k e and LeClair ( 1 9 7 5 ) , R e n d o s et al. ( 1 9 7 5 ) , Carroll and Jasper (1978), Carroll and Jasper (1980),
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© 1983 Elsevier Science Publishers B.V.
228 Francis et al. (1980) and Schultz and Thompson (1980) have examined these hypotheses by studying coliform bacterial populations in bedding materials and at the teat apices. Results of these studies have associated increased risk of coliform mastitis with total coliform bedding populations in excess of 1 X 106 colony forming units (CFU) per gram (wet weight) of bedding material (Bramley, 1974). An association between sawdust or wood shavings and increased incidence of mastitis due to Klebsiella pneumoniae, as a subset of the total coliform incidence has also been demonstrated by Newman and Kowalski (1973), Bramley (1974) and Rendos et al. (1975). Most of these investigations were conducted under management systems and climatic conditions quite dissimilar to those in California. The goal of this study was to examine the relationship between coliform mastitis incidence, bedding populations of mastitogenic coliform bacteria, and rainfall in a typical commercial dairy enterprise in California. An additional goal was to employ epidemiological methods of causal modelling to refine and extend hypotheses concerning the risk of coliform mastitis. MATERIALS AND METHODS The dairy herd studied was located in the northern San Joaquin valley of California and averaged 400 cows milking. The 305 day production averages were 7575 kg milk and 276 kg fat per cow. The prior mastitis history of this herd (identified as Herd C) has been reported (Jasper et al., 1975) and at the time of initiation of this study the herd was nearly free of contagious mastitis due either to S. agalactiae or S. aureus. However, sporadic coliform mastitis was occurring. Lactating cows were segregated by group into five pens. A modified production feeding plan was employed. Pen 1 contained cows with daily production levels above 30 kg. There was a mean of 84 cows in the pen over the 12-month period studied. The cows consisted predominantly of 2nd or later lactation cows in the first trimester of their current lactation. Pen 2 contained cows with milk production between 22.5 and 30 kg per day. The average size was 72 cows and contained a higher proportion of cows which had entered the 2nd trimester of lactation. Pen 3 contained cows with milk production between 18 and 22.5 kg per day. The average size of pen 3 was 72 cows and contained a high proportion of heifers. Pen 4 was the lowest production level group containing cows producing less than 18 kg milk per day. Cows in pen 4 were late in lactation and there was a rapid turnover as cows were dried off or culled. The size of pen 4 was more variable but averaged 85 cows. Pen 5 was not segregated by production but contained cows which were either slow or problem milkers. The size of pen 5 averaged 85 cows and in general contained a higher proportion of older cows than other pens. Cow groups were kept in separate pens with large centrally located shades available and concrete aprons around the feed bunkers. The bedding mate-
229 rial was sawdust and wood shavings which were d u m p e d in piles near the center of the pens or spread under the shades. The piles of bedding were scattered by the cows and were often heavily contaminated with feces and urine. Old bedding was not always removed before addition of new bedding and deep layers of bedding containing both old and fresh manure accumulated before removal at irregular intervals. Feed was not made immediately available after the morning milking and the cows often bedded down after this milking. Additional concurrent information on the bedding managem e n t and bedding populations of Enterobacteriaceae for this herd (identified as Herd C) have been reported (Carroll and Jasper, 1980).
Microbiological methods In each pen multiple samples of bedding material were collected m o n t h l y from areas recently lain upon the cows. These samples were combined and mixed thoroughly to ensure a representative sample from each pen. Using bacteriological methods recommended by the Coliform subcommittee, NMC (1975) a 10 g aliquot of each pen bedding sample was placed in a blender with 90 ml of sterile saline diluent. The contents were then blended at high speed for 1 min. Ten milliliters of the resulting slurry were taken into a 90 ml dilution blank (10 -2 dilution). A 1 ml aliquot of the 10 -2 dilution was taken into a 9 ml dilution blank and serial tenfold dilutions were carried o u t to 10 -l°. Aliquots (0.1 ml) of the 10 -2 to 10 -1° dilutions were added to tubes of MacConkey broth and incubated at 37°C overnight. Growth in MacConkey broth tubes, as indicated by acid production, was recorded and a loopfull was streaked on the surface of Tergitol-7 (T-7) agar plates (Difco) containing triphenyltetrazolium chloride. After incubation, a representative colony of each type of organism was described, picked and inoculated onto Triple Sugar Iron (TSI) agar (Difco) slants or restreaked on T-7 to assure purity before streaking on TSI. Reactions on TSI were recorded, and growth was placed in Simmon's citrate slants, MRVP broth, and Gillie's motility agar. A satisfactory identification usually could be made by the IMVIC test along with motility. All cultures were stored in the frozen state. When needed, the API-20E system (Analytab Products, Plainview, NY) was used to confirm identity of organisms n o t fitting the usual pattern. Mastitic milk samples were taken aseptically from clinically affected quarters before treatment. These samples were kept cold or frozen until streaked on blood agar plates. Coliform colonies were picked and identified as described above.
Statistical analysis As there were only coliform mastitis cases due to Escherichia coli and K. pneumoniae over the 12-month period, recording for computer analysis
230 of other coliform bacterial species from the bedding material samples was dropped. The total coliform bedding population expressed as CFU per gram wet weight, including E. coli, K. pneumoniae and other coliform species, was recorded as the absolute value of the exponent of the highest log10 dilution of MacConkey broth showing acid production or growth. Both E. coli and K. pneumoniae bedding populations were recorded similarly from the highest log10 dilution of MacConkey broth from which these species were isolated. There were 7 variables initially examined with the aid of computer programs (Dixon and Brown, 1979). They are (1) the m o n t h l y pen frequency of mastitis due to E. coli (2) the m o n t h l y pen frequency of mastitis due to K. pneumoniae (3) the average number of cows in each of the five pens for each m o n t h (4) the m o n t h l y rainfall in inches as reported by the local office of the National Weather Service (5) the total coliform population in the bedding sample (6) the population of E. coli in the bedding sample and (7) the population of K. penurnoniae in the bedding sample. With five pens and 12 m o n t h l y samples there were 60 individual data records containing these seven variables. Three additional variables were calculated on various computer runs. They were the total coliform incidence derived as the sum of the E. coli and K. pneumoniae frequency divided by the number of cow in the pen for that m o n t h , and similarly the incidence of each species separately. RESULTS Total coliform bedding populations were 1 X 106 CFU or higher in 40 of the 60 m o n t h by pen observations, 33 of 60 observations contained 1 X 106CFU or m o r e E , c o l i a n d l l of the 60 contained 1 X 1 0 6 C F U o r more K. pneumoniae. Over the 12-month period E. coli was isolated from 59 mastitic milk samples and K. pneumoniae was isolated from 44. These isolates were obtained from 103 separate quarters. Fig. 1 presents the m o n t h l y mean values over all five pens of the bedding populations of each species, the mean mastitis incidence over the five pens due to each species and the rainfall reported for each month. The total rainfall for the 12-month period was 5.67 inches (14.40 cm), which represents a very dry winter (1941--1971 30 year average annual rainfall = 16.34 inches). The m o n t h l y distribution of rainfall is typical, significant rainfall being u n c o m m o n from mid-May through September. In general the E. coli bedding populations were high with a mean equal to 1 X 10 s'~ CFU over all pens and months. The mean K. pneumoniae population was 1 X 102.2 CFU. The incidence of mastitis due to E. coli was approximately twice as frequent as that due to K. pneumoniae. The monthly average mastitis incidence due to E. coli over all pens was 1.14% while t h a t due to K. pneumoniae was 0.64%. The bedding populations of K.
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NOV DEC JAN FEBMAR APRMAY JUN JUL AUG SEPOCT
Fig. 1. The monthly mean values over all five pens of the log of the bedding populations of E. coli and K. p n e u m o n i a e in CFU/g wet weight, the monthly mean mastitis incidence over all five pens due to E. coli and K. p n e u m o n i a e and the monthly rainfall in inches.
pneumoniae were more variable than those of E. coli and tended to be higher in m o n t h s with greater rainfall. The variation by m o n t h in incidence of Klebsiella mastitis appeared to correlate more closely with the Klebsiella bedding populations, the m o n t h of June being a notable exception. E. coli mastitis incidence appeared rather more independent of E. coli bedding populations. However, it should be noted that the two highest incidence periods, November and February, also had the highest bedding populations of E. coli, perhaps reflecting a risk threshold noted by Bramley (1974). Variation in bedding populations and mastitis incidence by pen over the 12 m o n t h l y observations are presented in Table I. Differences in mean incidence and mean bedding populations between pens were tested by Kruskal-Wallis one-way analysis of variance. Only the mean incidence of E. coli mastitis was found to be significantly different (P = 0.004) between pens, although the difference in Klebsiella bedding populations attained a P = 0.072. Penwise comparisons of the means of E. coli incidence were made by the m e t h o d of Dunn (Hollander and Wolfe, 1973). The overall protection of six pen comparisons (3 vs 2, 4 vs 2, 3 vs 4, 5 vs 2 and 4 vs 1) was P = 0.3. Pen means sharing the same letter are n o t statistically significantly different.
232
TABLE I The means of the incidence and bedding populations of E. coli and K. pneumoniae by pen over the 12 monthly observations Pen
1 2 3 4 5 Grand mean
E. coli
K. pneumoniae
Incidence
Bedding
Incidence
Bedding
(%)*
Oog,0)
(%)
Oog,0)
1.73 bc 1.94 a 0.31 a 0.79 ab 0.94 abc 1.14
5.50 5.92 5.67 5.25 5.50 5.57
0.88 0.90 0.11 1.02 0.31 0.64
3.25 1.08 0.92 3.25 2.50 2.20
*Means by pen are different P = 0.004. abCMeans by pen with same superscript are not statistically different. In general, the b e d d i n g p o p u l a t i o n s o f E. coli were h o m o g e n o u s a n d little a g r e e m e n t b e t w e e n average b e d d i n g p o p u l a t i o n s a n d average incid e n c e o f E. coli mastitis was a p p a r e n t b e t w e e n t h e five pens. H o w e v e r , p e n 2, with t h e highest average b e d d i n g p o p u l a t i o n , also h a d t h e highest average incidence. With regard to t h e i n c i d e n c e o f E. coli mastitis, p e n 1 a n d 2 c o n t a i n i n g a high p r o p o r t i o n o f c o w s in early t o m i d - l a c t a t i o n h a d t h e highest incidence. H i g h e r risk o f c o l i f o r m mastitis has b e e n n o t e d b y o t h e r s at p a r t u r i t i o n a n d early in l a c t a t i o n , as r e v i e w e d b y t h e C o l i f o r m S u b c o m m i t t e e o f t h e R e s e a r c h C o m m i t t e e o f t h e N a t i o n a l Mastitis Council ( 1 9 7 5 ) . N o similar t r e n d was a p p a r e n t in t h e incidence o f Klebsiella mastitis a m o n g t h e five pens. T h e r e was m o r e v a r i a t i o n in the b e d d i n g p o p u l a t i o n s o f Klebsiella a n d also s o m e w h a t b e t t e r c o r r e l a t i o n b e t w e e n b e d d i n g p o p u l a t i o n s a n d i n c i d e n c e o f Klebsiella mastitis. Pen 3, w h i c h was c o m p r i s e d o f a high p r o p o r t i o n o f heifers, h a d t h e l o w e s t i n c i d e n c e o f mastitis d u e t o e i t h e r species. In o r d e r t o e x a m i n e the possibility o f a t h r e s h o l d e f f e c t in b e d d i n g p o p u l a t i o n s , t h e 60 m o n t h b y p e n r e c o r d s w e r e divided f o r each species i n t o t h o s e in w h i c h mastitis h a d o c c u r r e d a n d t h o s e in which it h a d n o t . T h e b e d d i n g p o p u l a t i o n f o r each species w e r e t h e n c o m p a r e d b y a MannW h i t n e y t e s t ( D i x o n a n d B r o w n , 1 9 7 9 , BMDP 3S). T a b l e II p r e s e n t s these results. T h e r e was n o d i f f e r e n c e in E. coli b e d d i n g p o p u l a t i o n s b e t w e e n t h e 34 m o n t h b y p e n r e c o r d s in w h i c h E. coli m a s t i t i s o c c u r r e d , as c o m p a r e d to t h e 26 o b s e r v a t i o n s in w h i c h mastitis h a d n o t o c c u r r e d . A v e r y highly significant d i f f e r e n c e (P ~ 0 . 0 0 0 1 ) was f o u n d b e t w e e n t h e 19 m o n t h b y p e n o b s e r v a t i o n s in w h i c h Klebsiella mastitis o c c u r r e d as c o m p a r e d t o t h e 41 o b s e r v a t i o n s in w h i c h it did n o t . S i m u l t a n e o u s e x a m i n a t i o n o f t h e rainfall, b e d d i n g p o p u l a t i o n a n d inc i d e n c e d a t a f o r t h e t o t a l c o l i f o r m a n d E. coli a n d K. p n e u m o n i a e as sub-
233 TABLE II The means of the log of the bedding population for pen by month observations with incidence of coliform mastitis by species compared to those without coliform mastitis E. coli
N Mean log Bedding
K. pneumoniae
Mastitis
N o mastitis
Mastitis
N o mastitis
34
26
19
41
5.67
5.42
4.84*
0.97
*The comparison between K. pneumoniae groups is significant (P < 0.0001). sets of the total was undertaken for the purpose of determining if any statistical interactions were present, which might represent important causal biological relationships. Evaluation of the data for appropriate statistical analysis led to an appreciation of procedural difficulties. Initially a stepwise multiple regression was applied to the data. Significant regressions were found between the incidence of mastitis due to each species, as the dependent variable and the log of the bedding population of the same species as the explanatory variable. The relationship for E. coli mastitis and E. coli bedding population was significant at the 0.05 level but n o t at the 0.025 level, while that for K. p n e u m o n i a e mastitis and K. p n e u m o n i a e bedding population was highly significant (P < 0.001). When the Klebsiella bedding population variable was moved to the dependent position, a significant regression (P < 0.001) with m o n t h l y rainfall as the explanatory variable was found. No such relationship was found between E. coli bedding population and rain or number of cows. Analysis of residuals of these regressions, as well as bivariate plots of the dependent versus independent variables, revealed problems in meeting the assumption of uniform variance and normal distribution necessary for regression analysis, in spite of efforts to transform the dependent variables. The discrete nature of the variables, such as the frequency counts of mastitis and the log values of the bedding population variables, were responsible. In order to employ fewer assumptions a categorical approach to the analysis was performed. For each species separately and for the total coliforms, a three-way contingency table was formed and analyzed by fitting log-linear models. This was partly justified by the results of the regression analysis in which there appeared to be no association between the incidence of mastitis due to one species and the bedding population of the other species. Limiting the cross-classification of the data to three variables avoided the occurrence of marginal totals with zero frequencies and simplified the subsequent selection of potential log-linear models. The m o n t h l y incidence rate was divided into two levels (negative and positive), the log of the bedding population was divided into three levels (low or negative,
234 medium, and high), and the m o n t h l y rainfall was divided into two levels (dry and wet). The cutpoints for categorizing the data were the same for all three tables with the ex cep tio n of the bedding population of K. pneumoniae. The fr e q u e n cy distribution of the total coliform bedding population and the E. coli bedding population were symmetrical, and the medium range from 1 0 4.s to 106"s CFU cont ai ned the mean, median and mode. Additionally the division between the m edi um and high bedding population levels was near the 106 CFU threshold referenced by Bramley (1974) as an elevated risk level for the incidence of coliform mastitis. The frequency distribution of the K. p n eu m oni ae bedding popul a t i on was decidedly asymmetrical with 35 o f the 60 observations being negative. The remaining 25 observations showed little central t e n d e n c y , and t he refore a linear division which yielded ap p r o x imate l y equal frequencies in the medium and high bedding p o p u l a tio n levels was applied. There were 14 observations in the m edi um range (101"s--105.s CFU) and 11 observations in the high range above 10 s's CFU. Table III presents the observed frequencies in the 2 X 3 × 2 crossclassifications for E. coli, K. pneum oni ae and total coliforms. TABLE III Cross-classified frequencies by categories for E. coli, K. pneumoniae, and total coliforms Mastitis incidence
Negative Positive
Bedding
Rainfall
population range
E. coli
Low Medium High Low Medium High
Dry 2 7 1 0 8 2
K. pneumoniae Wet 1 13 2 5 14 5
Dry 14 1 0 3 0 2
Wet
17 8 1 1 5 8
Total coliforms Dry
Wet
0 11 2 1 17 9
1 6 0 0 9 4
The basic strategy in fitting log-linear models was to identify a model of the relationship among the three categorical variables which adequately explains the observed frequencies when t hey are cross-classified. This was accomplished with the aid of a c o m p u t e r program (Dixon and Brown, 1979, BMDP 3F) which developed expect ed frequencies for successively more complex models, i.e. the inclusion in the model of terms which represented successively the addition of each categorical variable by itself, t h e n combinations of the variables and finally all combinations of twoway crossclassifications. Suitable models were identified by calculating a goodness-of-fit Chi-square statistic for each model. Acceptable models produced a Chi-square statistic with a low value relative to the degree of free-
235
dom, since the difference between the expected frequencies generated for the model and the observed frequencies should agree closely if the model fit. In general more complex models fit better b u t the preferred model was the least complex adequately fitting model. When several models fit the data, the selection of the best model was accomplished by partitioning the Chi-square statistic. To determine if a particular term was required in the model, the Chi-square was partitioned by subtracting the Chi-square value of the more complex model containing the term from the Chi-square value of the next least complex model which deleted the term. Additionally the degrees of freedom were subtracted and the value of the Chi-square, which was partitioned to the term deleted, was compared to a standard Chi-square table. If the partitioned value was significant, then the inclusion of the term in the model contributed significantly to decreasing the size of the goodness-of-fit Chi-square and it was required in the model. Table IV presents the likelihood-ratio Chi-square statistics, the degrees of freedom and the probabilities associated with all possible models of the 2 X 3 X 2 tables for E. coli and K. pneumoniae. For E. coli the first model to include all three variables, model 7, adequately fit the observed data, having a low Chi-square and the highest probability. This model represents T A B L E IV T h e l i k e l i h o o d - r a t i o goodness-of-fit Chi-square statistics, t h e degrees of f r e e d o m a n d the p r o b a b i l i t i e s associated w i t h all possible m o d e l s o f t h e 2 x 3 × 2 cross-classification of m a s t i t i s incidence, b e d d i n g p o p u l a t i o n a n d rainfall for E. coli a n d K. pneumoniae Model number
Terms included
DF
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
[R] [B] [I] [ R ] ,[B] [ R ] ,[I] [ B ] ,[I] J R ] ,[B] ,[I] [RB] [RI] [BI] [I] , [ R B ] [B] ,[RI] [R] ,[BI] [RB] ,[RI] [RB] ,[BI] [RI] ,[BI] [RB] ,[RI] ,[BI]
10 9 10 8 9 8 7 6 8 6 5 6 5 4 3 4 2
E. coli
K. pneumoniae
Chi-square
Probability
Chi-square
Probability
41.67 14.67 47.40 7.78 40.60 13.60 6.81 7.46 12.42 40.06 6.26 5.63 6.39 5.84 5.21 5.09 4.76
<0.0001 0.1004 < 0.0001 0.4458 < 0.0001 0.0928 0.4494 0.2808 0.0531 < 0.0001 0.3943 0.3440 0.2705 0.211 0.1571 0.2784 0.0928
62.30 53.06 60.83 46.26 54.04 44.80 38.00 36.01 53.41 19.71 27.75 37.37 12.91 27.12 2.66 12.28 2.02
< < < < < < < < <
R = Rainfall, B = B e d d i n g p o p u l a t i o n , I = incidence.
0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0031 < 0.0001 < 0.0001 0.0242 < 0.0001 0.4475 0.0154 0.3641
236 statistical independence among the three variables, rainfall, E. coli bedding population and E. coli incidence. For K. pneumoniae, models 15 and 17 were the only models which adequately fit the observed data. Model 15 differed from model 17, the full two-way interaction model, by n o t including an interaction term for rainfall and K. pneumoniae mastitis incidence ( [ R I ] ) . Partitioning of the Chi-square for this interaction term yielded a value of 0.64 with 1 d.f., which was not significant. Therefore the preferred model was model 15, which indicates that significant associations existed between rainfall and the bedding population of K. pneumoniae, and separately between the bedding population of K. pneumoniae and the incidence of mastitis due to K. pneumoniae. The log-linear model selected for the 2 X 3 X 2 cross-classification of rainfall, total coliform bedding population RAINFALL
BEDDING POPULATION
MASTITIS INCIDENCE
TOTAL COLIFORM
E. coli
0-
", "d5 ~'-.~6p,-, "0 " ~-'J
K. p n e u m o n i a e ~ . . .
p
Fig. 2. Path diagram showing causal interpretation of the relationship between rainfall, bedding populations and mastitis incidence for total coliforms, E. coli and K. pneumoniae. P values represent the probability of association between two variables when the third is not considered.
237 a n d t o t a l c o l i f o r m mastitis incidence was the s a m e as t h a t f i t t e d for E. coli, n a m e l y i n d e p e n d e n c e a m o n g the t h r e e variables ( d a t a n o t s h o w n ) . A p a t h d i a g r a m o f the r e l a t i o n s h i p a m o n g t h e t h r e e variables f o r t o t a l c o l i f o r m , E. coli a n d K. p n e u m o n i a e is p r e s e n t e d in Fig. 2. T h e statistical a s s o c i a t i o n s m e a s u r e d in fitting t h e log-linear m o d e l s m e r e l y assessed t h e p r o b a b i l i t y of i n t e r r e l a t e d n e s s b e t w e e n variables. A n d thus the n a t u r e of t h e r e l a t i o n s h i p b e t w e e n related variables m a y be in either d i r e c t i o n or bidirectional. N e i t h e r mastitis incidence n o r b e d d i n g p o p u l a t i o n s of bacteria c o u l d have e f f e c t e d rainfall a n d t h e r e f o r e the d i r e c t i o n of these a r r o w s b e t w e e n the variables in Fig. 2 c o u l d be set. A l t h o u g h it is possible to c o n c e i v e t h a t high mastitis p r e v a l e n c e c o u l d have seeded t h e b e d d i n g m a t e rial w i t h c o l i f o r m bacteria, the weight o f biological evidence is in the opp o s i t e d i r e c t i o n a n d thus the d i r e c t i o n o f the arrows was set f r o m b e d d i n g p o p u l a t i o n to mastitis incidence. Solid arrows indicate statistically signific a n t associations a n d d a s h e d arrows indicate i n d e p e n d e n c e . P values repr e s e n t i n g t h e p r o b a b i l i t y o f marginal association b e t w e e n the t w o variables w h e n t h e t h i r d was n o t c o n s i d e r e d are p r e s e n t e d for each arrow. T a b l e V s u m m a r i z e s t h e d i f f e r e n c e s n o t e d b e t w e e n E. coli a n d K. p n e u rnoniae in this s t u d y . TABLE V Summary of differences of epidemiological characteristics between the bedding populations and incidence of E. coli and K. pneumoniae Characteristic
E. coli
K. pneurnoniae
Grand mean of bedding populations Overall incidence rate Bedding population by time Bedding population by rainfall Mastitis incidence by bedding population Mastitis incidence by early lactation Incidence among heifers Threshold of risk for bedding populations
Higher (1 × 10 s'7) Higher ( 1.14 %/month) Constant
Lower (1 × 102':) Lower (0.64%/month) Variable
Independent
Dependent
Independent
Dependent
Trend
No Trend
Low
Low
If any above 1 × 10 6
As low as 1 × 10 4
DISCUSSION T h e results o f this s t u d y bring i n t o q u e s t i o n t h e advisability o f l u m p ing citrate-utilizing m e m b e r s o f t h e E n t e r o b a c t e r i a c e a e , a n d p a r t i c u l a r l y K. p n e u m o n i a e w i t h E. coli, u n d e r the c o m m o n d e s i g n a t i o n of c o l i f o r m ,
238 when investigating the relationship between bedding populations of these species and the incidence of mastitis associated with them. Thomas et al. (1960) indicated that the citrate-utilizing Enterobacteriaceae outlived E. coli in environments other than the intestine. Studies by Carroll and Jasper (1980) have shown the ability of K. pneumoniae to increase from undetectable levels in fresh sawdust bedding to very high levels within several days. K. pneumoniae was rarely found in bovine feces and was difficult to demonstrate in fresh sawdust in these studies, indicating that while E. coli numbers from the inoculum remained constant there was a geometric growth of K. pneumoniae. Differences in growth rates representing different facultative abilities under varying growth conditions are ignored when these organisms are classified under the same heading. Though the final c o m m o n portal of entry is the streak canal, differences in survival of various Enterobacteriaceae in bedding materials or different thresholds of risk for infectivity may be masked by such nosological malfeasance. In important epidemiological ways these results indicate that E. coli and K. pneumoniae behave very differently, as summarized in Table V. The finding of statistical independence between E. coli bedding populations and the incidence of E. coli mastitis in this study is not necessarily at odds with the findings of others. Bramley (1974) reported a direct relationship between levels of coliform contamination of bedding, contamination of the teat apex, and the coliform new infection rate. In his study Bramley determined the total coliform bedding populations by a tube dilution m e t h o d similar to that used in this study. However, he did not enumerate the subpopulations of E. coli, K. pneumoniae and other Enterobacteriaceae in the bedding samples. Though the mastitis which he observed was predominantly due to K. pneumoniae and the predominant bedding species, by frequency of isolation, was K. pneumoniae, he used the more general term, coliform, when summarizing his findings. He noted that of the seven cases of mastitis observed, six being due to K. pneumoniae, all occurred on sawdust bedding containing greater than 104 coliforms/g wet weight. Subsequent authors have widely referenced this observation as a threshold for increased risk of coliform mastitis without emphasizing the predominant role of K. pneumoniae in this study. Francis et al. (1980) studied the influence of bedding temperature in straw bedded cubicles on E. coli bedding populations and the influence of these bedding populations on udder infections. One of the conclusions of this study was that high E. coli populations in bedding were associated with contaminated teat apices and clinical E. coli mastitis. Although all of the E. coli mastitis cases in this study occurred in a group of high yielding cows, whose bedding contained higher populations of E. coli, such a conclusion may not be warranted. The high yielding group was composed of early lactation cows with nearly half of clinical mastitis occurring in the first 40 days of lactation. Thus the association of mastitis with higher E. coli bedding populations is confounded with the greater susceptibility
239 of early lactation cows. A further indictment of this conclusion was the lack of a week by week relationship between E. coli bedding populations and the frequency with which E. coli was recovered from teat apex swabs or hind quarter fore-milk samples. Similarly there was a lack of correlation between E. coli bedding populations and E. coli mastitis cases on a week by week basis, which agrees with our findings on a m o n t h by m o n t h basis as seen in Fig. 1. In a study which controlled the effect of early lactation susceptibility Natzke and LeClair (1975) were able to increase the teat end contamination by E. coli, but were unable to produce E. coli mastitis among 10 cows bedded on uncured sawdust seeded with virulent E. coli broth culture, which was maintained at approximately 106 CFU/g wet weight of E. coli over a 4-week experimental period. While we find no unexplainable discrepancies between our findings and those of others, we are aware of the hazards of bias in observational research. Certainly the difference in outcome between the 1971--1972 studies of Bramley (1974), in which no coliform mastitis was observed among sawdust bedded cows, and his studies of 1972--1973 exemplify this. The large number of cows at risk in this study gives some protection from observational bias. The risk experienced by these cows, however, represents the set of management and environmental factors found in the dairy studied. Though the authors believe this dairy was typical of well managed dairies in the San Joaquin Valley, this belief remains subjective. Certainly the rainfall during the observational period was atypically low. The relationship between bedding populations of K. pneumoniae and mastitis due to it seem well corroborated in the literature, while the lack of such a relationship for E. coli is not. Thus the scope of inference for these findings may be geographically limited or even unique to the dairy studied. With regard to the statistical methods, the authors endeavored to use methods which required the fewest assumptions. However, Chi-square analysis is n o t assumptionless. An important requirement for unbiased Chisquare analysis is independence in sampling. Sixty separate observations were made for each of the bedding population and mastitis incidence variables, while only 12 independent data were recorded for rainfall. Therefore the rainfall variable violates the requirement for independence in sampling. The effect of any bias introduced by this lack of independence was restricted to the marginal associations involving the rainfall variable (i.e. rainfall and bedding population or rainfall and mastitis incidence) but did not effect the marginal association between bedding populations and mastitis incidence for the three 2 X 3 X 2 contingency tables analyzed. With regard to the marginal associations between rainfall and bedding populations, for each pen by m o n t h data record, the E. coli and K. pneumoniae bedding population variables shared the same rainfall value, any bias due to lack of independence would effect them equally. Thus the level of significance of each marginal association may contain bias but the difference noted between them should be free of this bias.
240 In evaluating the success of the study design, the authors feel that expressing the bedding population in terms of dry weight would have been an improvement. The minor additional work of aliquoting and desiccating a second bedding sample at the time of inoculation of MacConkey broth would have yielded two benefits. The first would have been bedding population estimates free of the variability due to water content in the bedding sample. The second would have been a moisture content variable, derived by subtracting the dry weight of the sample from its wet weight, which may have been more causally relevant to the bedding populations than m o n t h l y rainfall. With the above considerations of representativeness and bias in mind the authors' interpretations of these results follow. The statistical association between K. pneumoniae bedding populations and mastitis due to K. pneumoniae represent a significant biological risk identified by Newman and Kowalski (1973). The bedding populations of K. pneumoniae necessary to constitute this risk are likely to be much lower than previously appreciated, possibly as low as 104 CFU/g wet weight. The statistical association between rainfall and the bedding populations of K. pneumoniae we believe represents a true biological phenomenon, but rainfall per se may be a surrogate measure for moisture in general. We do not conclude that the lack of a statistical association between E. coli bedding populations and E. coli mastitis represents biological independence. Though a direct relationship was not shown, interactions between E. coli bedding populations and other variables n o t measured in this study, may y e t be important in fitting a causal epidemiological model. In considering the very different behavior of these two organisms the authors have constructed a hypothesis based on our findings and those in the literature. A necessary assumption for this hypothesis is that there exists a very different ability between the two organisms to penetrate the streak canal. The highly significant relationship between K. pneumoniae bedding populations and the occurrence of K. pneumoniae mastitis indicates that very few other factors may be necessary to consider. Thus when K. pneumoniae is present it is able to penetrate the streak canal and cause mastitis at a significant rate and this may occur independently of other factors. The lack of an association between E. coli bedding populations and E. coli mastitis incidence under the same conditions suggests that though E. coli is consistently present at higher levels in the bedding, it is less able to penetrate the streak canal and that other factors axe necessary to explain the variability in occurrence of E. coli mastitis. Since post-milking teat sanitization does not affect E. coli mastitis incidence, this focuses attention on potential risk factors in the immediate pre-milking and milking periods. The lack of association between bedding populations of E. coli and E. coli mastitis coupled with the observation of increased peripartum susceptibility to E. coli suggests that the difference in the ability of E. coli to penetrate the streak canal may be a factor of resistance in the cow and
241 n o t an i n h e r e n t deficit of the organism. The lack o f an observable early l a c t a t i o n susceptibility to K. pneumoniae in this s t u d y , if c o r r e c t , suggests t h a t m e c h a n i s m s w h i c h a c c o u n t for relative late lactational resistance to E. coli mastitis m a y be d i r e c t e d at characteristics n o t shared b y the t w o organisms. If this h y p o t h e s i s is substantially c o r r e c t , f u t u r e research s h o u l d be d i r e c t e d at defining risk factors for E. coli mastitis during the i m m e d i a t e pre-milking and milking periods and q u a n t i t a t i n g their relative c o n t r i b u tions t o the variability in the o c c u r r e n c e o f E. coli mastitis. A d d i t i o n a l l y research into the n a t u r e o f late lactational resistance to E. coli mastitis m a y provide m e t h o d s t o i m p r o v e this resistance in the early lactational period. ACKNOWLEDGEMENTS The a u t h o r s wish to t h a n k Micheal Miller Ph.D. o f the Statistical Laborat o r y at t h e University o f California, Davis, f o r his c o n s u l t a t i o n . T h e a u t h o r s dedicate this w o r k to the m e m o r y o f o u r colleague E.J. Carroll in r e c o g n i t i o n o f his c o n t r i b u t i o n s to our u n d e r s t a n d i n g o f bovine mastitis. S u p p o r t e d in part b y f u n d s f r o m the California Milk A d v i s o r y Board and the U.S. D e p a r t m e n t o f Agriculture, Agricultural Research Service General Cooperative Agreement.
REFERENCES Braman, S.K., Eberhart, E.J., Asbury, M.A. and Hermann, G.J., 1973. Capsular types of Klebsiella pneumoniae associated with bovine mastitis. J. Am. Vet. Med. Assoc., 162(2): 109--111. Bramley, A.J., 1974. The aetiology and control of coliform mastitis in dairy cattle. Ph.D. Thesis, University of Reading. Bramley, A.J. and Neave, F.K., 1975. Studies on the control of coliform mastitis in dairy cows. Br. Vet. J., 131: 160--169. Carroll, E.J., 1977. Environmental factors in bovine mastitis. J. Am. Vet. Med. Assoc., 170(10): 1143--1148. Carroll, E.J. and Jasper, D.E., 1978. Distribution of Enterobacteriaceae in recycled manure bedding on California dairies. J. Dairy Sci., 61(10): 1498--1508. Carroll, E.J. and Jasper, D.E., 1980. Coliform populations in bedding materials and coliform mastitis incidence. In: Proceedings of the 19th Annual Meeting of the National Mastitis Council, Inc., 18--20 Feb. 1980, at Louisville, KY. National Mastitis Council, Inc., Arlington, VA, pp. 121--127. Coliform Subcommittee of the Research Committee of the National Mastitis Council, 1975. The problem of coliform mastitis and its implications for research. In: Proceedings of the 15th Annual Meeting of the National Mastitis Council, Inc., 16--18 Feb. 1976, at Louisville, KY. National Mastitis Council, Inc., Arlington, VA, pp. 121--180. Coliform Subcommittee of the Research Committee of the National Mastitis Council, 1979. Coliform mastitis -- a review. J. Dairy Sci., 62(1) 1--22. Dixon, W J. and Brown, M.B., 1979. Biomedical Computer Programs -- P Series. University of California Press, Berkeley, CA, 879 pp.
242 Eberhart, R.J. and Buckalew, J.M., 1977. Intramammary infections in a dairy herd with a low incidence of Streptococcus agalactiae and Staphylococcus aureus infections. J. Am. Vet. Med. Assoc., 171(7): 630--634. Francis, P.G., Sumner, J. and Joyce, D.A., 1980. The influence of the winter environment of the dairy cow on mastitis. In: Proceedings of the 11th International Congress on Diseases of Cattle, 20--23 Oct. 1980, at Tel-Aviv. Israel Association of Buiatrics, Haifa, Israel, pp. 35--41. Hollander, M. and Wolfe, D.A., 1973. Nonparametric Statistical Methods. John Wiley and Sons, New York, NY, pp. 125--126. Jasper, D.E. and Dellinger, J.D., 1975. Teat apex coliform populations and coliform mastitis -- a herd study. Cornell Vet., 65(3): 380--392. Jasper, D.E., Dellinger, J.D. and Bushnell, R.B., 1975. Herd studies on coliform mastitis. J. Am. Vet. Med. Assoc., 166(8): 778--780. McDonald, T.J., McDonald, J.S. and Rose, D.L., 1970. Aerobic gram-negative rods isolated from bovine udder infections. Am. J. Vet. Res., 31(11): 1937--1941. Natzke, R.P. and LeClair, B.J., 1975. Coliform contaminated bedding and new infections. J. Dairy Sci., 59(12): 2152--2154. Newman, L.E. and Kowalski, J.J., 1973. Fresh sawdust bedding -- a possible source of Klebsiella organisms. Am. J. Vet. Res., 34(7): 979--980. Rendos, J.J., Eberhart, R.J. and Kesler, E.M., 1975. Microbial populations of the teat ends of dairy cows, and bedding materials. J. Dairy Sci., 58(10): 1492--1500. Saran, A., 1980. Survey on E. coli strains isolated from cases of acute bovine mastitis and from the cow's environment. In: Proceedings of the 11th International Congress on Diseases of Cattle, 20--23 Oct. 1980, at Tel-Aviv. Israel Association of Buiatrics, Haifa, Israel, pp. 56--61. Schultz, W.D. and Thompson, P.D., 1980. Intramammary coliform infection after heavy external contamination of teats. Am. J. Vet. Res., 41(9): 1396--1401. Thomas, S.B., Druce, R.G. and Elson, K., 1960. An ecological study of the coli-aerogenes bacteria of surface soil. J. Appl. Bacteriol., 23(2): 169.
227
ENTEROBACTERIACEAE BEDDING POPULATIONS, RAINFALL AND MASTITIS ON A C A L I F O R N I A D A I R Y
C.B. THOMAS, D.E. JASPER, M.H. ROLLINS, R.B. BUSHNELL* and E.J. CARROLL Department of Clinical Pathology, Veterinary School, University of California, Davis, CA 95616 (U.S.A.) *Cooperative Veterinary Extension, University of California, Davis, CA 95616 (U.S.A.) (Accepted 31 December 1982)
ABSTRACT Thomas, C.B., Jasper, D.E., Rollins, M.H., Bushnell, R.B. and Carroll, E.J., 1983. Enterobacteriaceae bedding populations, rainfall and mastitis on a California dairy. Prev. Vet. Med., 1: 227--242. The bedding populations of Enterobacteriaceae, rainfall and mastitis incidence due to Enterobacteriaceae were studied over 12 months on one California dairy. The bedding material used was a mixture of sawdust and wood shavings. Over the 12-month period Escherichia coli was isolated from 59 mastitic milk samples and Klebsiella pneumoniae from 44. The bedding population, rainfall and mastitis incidence data were analyzed by fitting log-linear models. The results of these analyses indicated that rainfall, bedding populations of E. coli and E. coli mastitis incidence were statistically independent, while a strong association existed between rainfall and K. pneumoniae bedding populations and separately between K. pneumoniae bedding populations and the incidence of K. pneumoniae mastitis.
INTRODUCTION Microbiological studies o f i n c i d e n t c o l i f o r m mastitis c o n d u c t e d b y M c D o n a l d et al. (1970), B r a m a n et al. ( 1 9 7 3 ) , E b e r h a r t and B u c k a l e w ( 1 9 7 7 ) and Saran ( 1 9 8 0 ) have s h o w n great s e r o t y p i c diversity a m o n g colif o r m isolates within the same dairy herd. Post-milking teat sanitization, w h i c h is an effective c o n t r o l measure for c o n t a g i o u s mastitis due to Streptococcus agalactiae a n d S t a p h y l o c o c c u s aureus, does n o t a f f e c t n e w colif o r m i n f e c t i o n rates ( C o l i f o r m s u b c o m m i t t e e , NMC, 1979). These findings s u p p o r t the h y p o t h e s i s t h a t the risk o f i n c i d e n t c o l i f o r m mastitis is o f t e n d e p e n d e n t o n e n v i r o n m e n t a l reservoirs as o p p o s e d to c o n t a g i o u s reservoirs e m a n a t i n g f r o m the i n f e c t e d u d d e r s o f herd mates, and t h a t the time o f invasion o f the u d d e r m a y be in the intermilking period. B r a m l e y ( 1 9 7 4 ) , Jasper and Dellinger ( 1 9 7 5 ) , N a t z k e and LeClair ( 1 9 7 5 ) , R e n d o s et al. ( 1 9 7 5 ) , Carroll and Jasper (1978), Carroll and Jasper (1980),
0167-5877/83/$03.00
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228 Francis et al. (1980) and Schultz and Thompson (1980) have examined these hypotheses by studying coliform bacterial populations in bedding materials and at the teat apices. Results of these studies have associated increased risk of coliform mastitis with total coliform bedding populations in excess of 1 X 106 colony forming units (CFU) per gram (wet weight) of bedding material (Bramley, 1974). An association between sawdust or wood shavings and increased incidence of mastitis due to Klebsiella pneumoniae, as a subset of the total coliform incidence has also been demonstrated by Newman and Kowalski (1973), Bramley (1974) and Rendos et al. (1975). Most of these investigations were conducted under management systems and climatic conditions quite dissimilar to those in California. The goal of this study was to examine the relationship between coliform mastitis incidence, bedding populations of mastitogenic coliform bacteria, and rainfall in a typical commercial dairy enterprise in California. An additional goal was to employ epidemiological methods of causal modelling to refine and extend hypotheses concerning the risk of coliform mastitis. MATERIALS AND METHODS The dairy herd studied was located in the northern San Joaquin valley of California and averaged 400 cows milking. The 305 day production averages were 7575 kg milk and 276 kg fat per cow. The prior mastitis history of this herd (identified as Herd C) has been reported (Jasper et al., 1975) and at the time of initiation of this study the herd was nearly free of contagious mastitis due either to S. agalactiae or S. aureus. However, sporadic coliform mastitis was occurring. Lactating cows were segregated by group into five pens. A modified production feeding plan was employed. Pen 1 contained cows with daily production levels above 30 kg. There was a mean of 84 cows in the pen over the 12-month period studied. The cows consisted predominantly of 2nd or later lactation cows in the first trimester of their current lactation. Pen 2 contained cows with milk production between 22.5 and 30 kg per day. The average size was 72 cows and contained a higher proportion of cows which had entered the 2nd trimester of lactation. Pen 3 contained cows with milk production between 18 and 22.5 kg per day. The average size of pen 3 was 72 cows and contained a high proportion of heifers. Pen 4 was the lowest production level group containing cows producing less than 18 kg milk per day. Cows in pen 4 were late in lactation and there was a rapid turnover as cows were dried off or culled. The size of pen 4 was more variable but averaged 85 cows. Pen 5 was not segregated by production but contained cows which were either slow or problem milkers. The size of pen 5 averaged 85 cows and in general contained a higher proportion of older cows than other pens. Cow groups were kept in separate pens with large centrally located shades available and concrete aprons around the feed bunkers. The bedding mate-
229 rial was sawdust and wood shavings which were d u m p e d in piles near the center of the pens or spread under the shades. The piles of bedding were scattered by the cows and were often heavily contaminated with feces and urine. Old bedding was not always removed before addition of new bedding and deep layers of bedding containing both old and fresh manure accumulated before removal at irregular intervals. Feed was not made immediately available after the morning milking and the cows often bedded down after this milking. Additional concurrent information on the bedding managem e n t and bedding populations of Enterobacteriaceae for this herd (identified as Herd C) have been reported (Carroll and Jasper, 1980).
Microbiological methods In each pen multiple samples of bedding material were collected m o n t h l y from areas recently lain upon the cows. These samples were combined and mixed thoroughly to ensure a representative sample from each pen. Using bacteriological methods recommended by the Coliform subcommittee, NMC (1975) a 10 g aliquot of each pen bedding sample was placed in a blender with 90 ml of sterile saline diluent. The contents were then blended at high speed for 1 min. Ten milliliters of the resulting slurry were taken into a 90 ml dilution blank (10 -2 dilution). A 1 ml aliquot of the 10 -2 dilution was taken into a 9 ml dilution blank and serial tenfold dilutions were carried o u t to 10 -l°. Aliquots (0.1 ml) of the 10 -2 to 10 -1° dilutions were added to tubes of MacConkey broth and incubated at 37°C overnight. Growth in MacConkey broth tubes, as indicated by acid production, was recorded and a loopfull was streaked on the surface of Tergitol-7 (T-7) agar plates (Difco) containing triphenyltetrazolium chloride. After incubation, a representative colony of each type of organism was described, picked and inoculated onto Triple Sugar Iron (TSI) agar (Difco) slants or restreaked on T-7 to assure purity before streaking on TSI. Reactions on TSI were recorded, and growth was placed in Simmon's citrate slants, MRVP broth, and Gillie's motility agar. A satisfactory identification usually could be made by the IMVIC test along with motility. All cultures were stored in the frozen state. When needed, the API-20E system (Analytab Products, Plainview, NY) was used to confirm identity of organisms n o t fitting the usual pattern. Mastitic milk samples were taken aseptically from clinically affected quarters before treatment. These samples were kept cold or frozen until streaked on blood agar plates. Coliform colonies were picked and identified as described above.
Statistical analysis As there were only coliform mastitis cases due to Escherichia coli and K. pneumoniae over the 12-month period, recording for computer analysis
230 of other coliform bacterial species from the bedding material samples was dropped. The total coliform bedding population expressed as CFU per gram wet weight, including E. coli, K. pneumoniae and other coliform species, was recorded as the absolute value of the exponent of the highest log10 dilution of MacConkey broth showing acid production or growth. Both E. coli and K. pneumoniae bedding populations were recorded similarly from the highest log10 dilution of MacConkey broth from which these species were isolated. There were 7 variables initially examined with the aid of computer programs (Dixon and Brown, 1979). They are (1) the m o n t h l y pen frequency of mastitis due to E. coli (2) the m o n t h l y pen frequency of mastitis due to K. pneumoniae (3) the average number of cows in each of the five pens for each m o n t h (4) the m o n t h l y rainfall in inches as reported by the local office of the National Weather Service (5) the total coliform population in the bedding sample (6) the population of E. coli in the bedding sample and (7) the population of K. penurnoniae in the bedding sample. With five pens and 12 m o n t h l y samples there were 60 individual data records containing these seven variables. Three additional variables were calculated on various computer runs. They were the total coliform incidence derived as the sum of the E. coli and K. pneumoniae frequency divided by the number of cow in the pen for that m o n t h , and similarly the incidence of each species separately. RESULTS Total coliform bedding populations were 1 X 106 CFU or higher in 40 of the 60 m o n t h by pen observations, 33 of 60 observations contained 1 X 106CFU or m o r e E , c o l i a n d l l of the 60 contained 1 X 1 0 6 C F U o r more K. pneumoniae. Over the 12-month period E. coli was isolated from 59 mastitic milk samples and K. pneumoniae was isolated from 44. These isolates were obtained from 103 separate quarters. Fig. 1 presents the m o n t h l y mean values over all five pens of the bedding populations of each species, the mean mastitis incidence over the five pens due to each species and the rainfall reported for each month. The total rainfall for the 12-month period was 5.67 inches (14.40 cm), which represents a very dry winter (1941--1971 30 year average annual rainfall = 16.34 inches). The m o n t h l y distribution of rainfall is typical, significant rainfall being u n c o m m o n from mid-May through September. In general the E. coli bedding populations were high with a mean equal to 1 X 10 s'~ CFU over all pens and months. The mean K. pneumoniae population was 1 X 102.2 CFU. The incidence of mastitis due to E. coli was approximately twice as frequent as that due to K. pneumoniae. The monthly average mastitis incidence due to E. coli over all pens was 1.14% while t h a t due to K. pneumoniae was 0.64%. The bedding populations of K.
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NOV DEC JAN FEBMAR APRMAY JUN JUL AUG SEPOCT
Fig. 1. The monthly mean values over all five pens of the log of the bedding populations of E. coli and K. p n e u m o n i a e in CFU/g wet weight, the monthly mean mastitis incidence over all five pens due to E. coli and K. p n e u m o n i a e and the monthly rainfall in inches.
pneumoniae were more variable than those of E. coli and tended to be higher in m o n t h s with greater rainfall. The variation by m o n t h in incidence of Klebsiella mastitis appeared to correlate more closely with the Klebsiella bedding populations, the m o n t h of June being a notable exception. E. coli mastitis incidence appeared rather more independent of E. coli bedding populations. However, it should be noted that the two highest incidence periods, November and February, also had the highest bedding populations of E. coli, perhaps reflecting a risk threshold noted by Bramley (1974). Variation in bedding populations and mastitis incidence by pen over the 12 m o n t h l y observations are presented in Table I. Differences in mean incidence and mean bedding populations between pens were tested by Kruskal-Wallis one-way analysis of variance. Only the mean incidence of E. coli mastitis was found to be significantly different (P = 0.004) between pens, although the difference in Klebsiella bedding populations attained a P = 0.072. Penwise comparisons of the means of E. coli incidence were made by the m e t h o d of Dunn (Hollander and Wolfe, 1973). The overall protection of six pen comparisons (3 vs 2, 4 vs 2, 3 vs 4, 5 vs 2 and 4 vs 1) was P = 0.3. Pen means sharing the same letter are n o t statistically significantly different.
232
TABLE I The means of the incidence and bedding populations of E. coli and K. pneumoniae by pen over the 12 monthly observations Pen
1 2 3 4 5 Grand mean
E. coli
K. pneumoniae
Incidence
Bedding
Incidence
Bedding
(%)*
Oog,0)
(%)
Oog,0)
1.73 bc 1.94 a 0.31 a 0.79 ab 0.94 abc 1.14
5.50 5.92 5.67 5.25 5.50 5.57
0.88 0.90 0.11 1.02 0.31 0.64
3.25 1.08 0.92 3.25 2.50 2.20
*Means by pen are different P = 0.004. abCMeans by pen with same superscript are not statistically different. In general, the b e d d i n g p o p u l a t i o n s o f E. coli were h o m o g e n o u s a n d little a g r e e m e n t b e t w e e n average b e d d i n g p o p u l a t i o n s a n d average incid e n c e o f E. coli mastitis was a p p a r e n t b e t w e e n t h e five pens. H o w e v e r , p e n 2, with t h e highest average b e d d i n g p o p u l a t i o n , also h a d t h e highest average incidence. With regard to t h e i n c i d e n c e o f E. coli mastitis, p e n 1 a n d 2 c o n t a i n i n g a high p r o p o r t i o n o f c o w s in early t o m i d - l a c t a t i o n h a d t h e highest incidence. H i g h e r risk o f c o l i f o r m mastitis has b e e n n o t e d b y o t h e r s at p a r t u r i t i o n a n d early in l a c t a t i o n , as r e v i e w e d b y t h e C o l i f o r m S u b c o m m i t t e e o f t h e R e s e a r c h C o m m i t t e e o f t h e N a t i o n a l Mastitis Council ( 1 9 7 5 ) . N o similar t r e n d was a p p a r e n t in t h e incidence o f Klebsiella mastitis a m o n g t h e five pens. T h e r e was m o r e v a r i a t i o n in the b e d d i n g p o p u l a t i o n s o f Klebsiella a n d also s o m e w h a t b e t t e r c o r r e l a t i o n b e t w e e n b e d d i n g p o p u l a t i o n s a n d i n c i d e n c e o f Klebsiella mastitis. Pen 3, w h i c h was c o m p r i s e d o f a high p r o p o r t i o n o f heifers, h a d t h e l o w e s t i n c i d e n c e o f mastitis d u e t o e i t h e r species. In o r d e r t o e x a m i n e the possibility o f a t h r e s h o l d e f f e c t in b e d d i n g p o p u l a t i o n s , t h e 60 m o n t h b y p e n r e c o r d s w e r e divided f o r each species i n t o t h o s e in w h i c h mastitis h a d o c c u r r e d a n d t h o s e in which it h a d n o t . T h e b e d d i n g p o p u l a t i o n f o r each species w e r e t h e n c o m p a r e d b y a MannW h i t n e y t e s t ( D i x o n a n d B r o w n , 1 9 7 9 , BMDP 3S). T a b l e II p r e s e n t s these results. T h e r e was n o d i f f e r e n c e in E. coli b e d d i n g p o p u l a t i o n s b e t w e e n t h e 34 m o n t h b y p e n r e c o r d s in w h i c h E. coli m a s t i t i s o c c u r r e d , as c o m p a r e d to t h e 26 o b s e r v a t i o n s in w h i c h mastitis h a d n o t o c c u r r e d . A v e r y highly significant d i f f e r e n c e (P ~ 0 . 0 0 0 1 ) was f o u n d b e t w e e n t h e 19 m o n t h b y p e n o b s e r v a t i o n s in w h i c h Klebsiella mastitis o c c u r r e d as c o m p a r e d t o t h e 41 o b s e r v a t i o n s in w h i c h it did n o t . S i m u l t a n e o u s e x a m i n a t i o n o f t h e rainfall, b e d d i n g p o p u l a t i o n a n d inc i d e n c e d a t a f o r t h e t o t a l c o l i f o r m a n d E. coli a n d K. p n e u m o n i a e as sub-
233 TABLE II The means of the log of the bedding population for pen by month observations with incidence of coliform mastitis by species compared to those without coliform mastitis E. coli
N Mean log Bedding
K. pneumoniae
Mastitis
N o mastitis
Mastitis
N o mastitis
34
26
19
41
5.67
5.42
4.84*
0.97
*The comparison between K. pneumoniae groups is significant (P < 0.0001). sets of the total was undertaken for the purpose of determining if any statistical interactions were present, which might represent important causal biological relationships. Evaluation of the data for appropriate statistical analysis led to an appreciation of procedural difficulties. Initially a stepwise multiple regression was applied to the data. Significant regressions were found between the incidence of mastitis due to each species, as the dependent variable and the log of the bedding population of the same species as the explanatory variable. The relationship for E. coli mastitis and E. coli bedding population was significant at the 0.05 level but n o t at the 0.025 level, while that for K. p n e u m o n i a e mastitis and K. p n e u m o n i a e bedding population was highly significant (P < 0.001). When the Klebsiella bedding population variable was moved to the dependent position, a significant regression (P < 0.001) with m o n t h l y rainfall as the explanatory variable was found. No such relationship was found between E. coli bedding population and rain or number of cows. Analysis of residuals of these regressions, as well as bivariate plots of the dependent versus independent variables, revealed problems in meeting the assumption of uniform variance and normal distribution necessary for regression analysis, in spite of efforts to transform the dependent variables. The discrete nature of the variables, such as the frequency counts of mastitis and the log values of the bedding population variables, were responsible. In order to employ fewer assumptions a categorical approach to the analysis was performed. For each species separately and for the total coliforms, a three-way contingency table was formed and analyzed by fitting log-linear models. This was partly justified by the results of the regression analysis in which there appeared to be no association between the incidence of mastitis due to one species and the bedding population of the other species. Limiting the cross-classification of the data to three variables avoided the occurrence of marginal totals with zero frequencies and simplified the subsequent selection of potential log-linear models. The m o n t h l y incidence rate was divided into two levels (negative and positive), the log of the bedding population was divided into three levels (low or negative,
234 medium, and high), and the m o n t h l y rainfall was divided into two levels (dry and wet). The cutpoints for categorizing the data were the same for all three tables with the ex cep tio n of the bedding population of K. pneumoniae. The fr e q u e n cy distribution of the total coliform bedding population and the E. coli bedding population were symmetrical, and the medium range from 1 0 4.s to 106"s CFU cont ai ned the mean, median and mode. Additionally the division between the m edi um and high bedding population levels was near the 106 CFU threshold referenced by Bramley (1974) as an elevated risk level for the incidence of coliform mastitis. The frequency distribution of the K. p n eu m oni ae bedding popul a t i on was decidedly asymmetrical with 35 o f the 60 observations being negative. The remaining 25 observations showed little central t e n d e n c y , and t he refore a linear division which yielded ap p r o x imate l y equal frequencies in the medium and high bedding p o p u l a tio n levels was applied. There were 14 observations in the m edi um range (101"s--105.s CFU) and 11 observations in the high range above 10 s's CFU. Table III presents the observed frequencies in the 2 X 3 × 2 crossclassifications for E. coli, K. pneum oni ae and total coliforms. TABLE III Cross-classified frequencies by categories for E. coli, K. pneumoniae, and total coliforms Mastitis incidence
Negative Positive
Bedding
Rainfall
population range
E. coli
Low Medium High Low Medium High
Dry 2 7 1 0 8 2
K. pneumoniae Wet 1 13 2 5 14 5
Dry 14 1 0 3 0 2
Wet
17 8 1 1 5 8
Total coliforms Dry
Wet
0 11 2 1 17 9
1 6 0 0 9 4
The basic strategy in fitting log-linear models was to identify a model of the relationship among the three categorical variables which adequately explains the observed frequencies when t hey are cross-classified. This was accomplished with the aid of a c o m p u t e r program (Dixon and Brown, 1979, BMDP 3F) which developed expect ed frequencies for successively more complex models, i.e. the inclusion in the model of terms which represented successively the addition of each categorical variable by itself, t h e n combinations of the variables and finally all combinations of twoway crossclassifications. Suitable models were identified by calculating a goodness-of-fit Chi-square statistic for each model. Acceptable models produced a Chi-square statistic with a low value relative to the degree of free-
235
dom, since the difference between the expected frequencies generated for the model and the observed frequencies should agree closely if the model fit. In general more complex models fit better b u t the preferred model was the least complex adequately fitting model. When several models fit the data, the selection of the best model was accomplished by partitioning the Chi-square statistic. To determine if a particular term was required in the model, the Chi-square was partitioned by subtracting the Chi-square value of the more complex model containing the term from the Chi-square value of the next least complex model which deleted the term. Additionally the degrees of freedom were subtracted and the value of the Chi-square, which was partitioned to the term deleted, was compared to a standard Chi-square table. If the partitioned value was significant, then the inclusion of the term in the model contributed significantly to decreasing the size of the goodness-of-fit Chi-square and it was required in the model. Table IV presents the likelihood-ratio Chi-square statistics, the degrees of freedom and the probabilities associated with all possible models of the 2 X 3 X 2 tables for E. coli and K. pneumoniae. For E. coli the first model to include all three variables, model 7, adequately fit the observed data, having a low Chi-square and the highest probability. This model represents T A B L E IV T h e l i k e l i h o o d - r a t i o goodness-of-fit Chi-square statistics, t h e degrees of f r e e d o m a n d the p r o b a b i l i t i e s associated w i t h all possible m o d e l s o f t h e 2 x 3 × 2 cross-classification of m a s t i t i s incidence, b e d d i n g p o p u l a t i o n a n d rainfall for E. coli a n d K. pneumoniae Model number
Terms included
DF
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
[R] [B] [I] [ R ] ,[B] [ R ] ,[I] [ B ] ,[I] J R ] ,[B] ,[I] [RB] [RI] [BI] [I] , [ R B ] [B] ,[RI] [R] ,[BI] [RB] ,[RI] [RB] ,[BI] [RI] ,[BI] [RB] ,[RI] ,[BI]
10 9 10 8 9 8 7 6 8 6 5 6 5 4 3 4 2
E. coli
K. pneumoniae
Chi-square
Probability
Chi-square
Probability
41.67 14.67 47.40 7.78 40.60 13.60 6.81 7.46 12.42 40.06 6.26 5.63 6.39 5.84 5.21 5.09 4.76
<0.0001 0.1004 < 0.0001 0.4458 < 0.0001 0.0928 0.4494 0.2808 0.0531 < 0.0001 0.3943 0.3440 0.2705 0.211 0.1571 0.2784 0.0928
62.30 53.06 60.83 46.26 54.04 44.80 38.00 36.01 53.41 19.71 27.75 37.37 12.91 27.12 2.66 12.28 2.02
< < < < < < < < <
R = Rainfall, B = B e d d i n g p o p u l a t i o n , I = incidence.
0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0031 < 0.0001 < 0.0001 0.0242 < 0.0001 0.4475 0.0154 0.3641
236 statistical independence among the three variables, rainfall, E. coli bedding population and E. coli incidence. For K. pneumoniae, models 15 and 17 were the only models which adequately fit the observed data. Model 15 differed from model 17, the full two-way interaction model, by n o t including an interaction term for rainfall and K. pneumoniae mastitis incidence ( [ R I ] ) . Partitioning of the Chi-square for this interaction term yielded a value of 0.64 with 1 d.f., which was not significant. Therefore the preferred model was model 15, which indicates that significant associations existed between rainfall and the bedding population of K. pneumoniae, and separately between the bedding population of K. pneumoniae and the incidence of mastitis due to K. pneumoniae. The log-linear model selected for the 2 X 3 X 2 cross-classification of rainfall, total coliform bedding population RAINFALL
BEDDING POPULATION
MASTITIS INCIDENCE
TOTAL COLIFORM
E. coli
0-
", "d5 ~'-.~6p,-, "0 " ~-'J
K. p n e u m o n i a e ~ . . .
p
Fig. 2. Path diagram showing causal interpretation of the relationship between rainfall, bedding populations and mastitis incidence for total coliforms, E. coli and K. pneumoniae. P values represent the probability of association between two variables when the third is not considered.
237 a n d t o t a l c o l i f o r m mastitis incidence was the s a m e as t h a t f i t t e d for E. coli, n a m e l y i n d e p e n d e n c e a m o n g the t h r e e variables ( d a t a n o t s h o w n ) . A p a t h d i a g r a m o f the r e l a t i o n s h i p a m o n g t h e t h r e e variables f o r t o t a l c o l i f o r m , E. coli a n d K. p n e u m o n i a e is p r e s e n t e d in Fig. 2. T h e statistical a s s o c i a t i o n s m e a s u r e d in fitting t h e log-linear m o d e l s m e r e l y assessed t h e p r o b a b i l i t y of i n t e r r e l a t e d n e s s b e t w e e n variables. A n d thus the n a t u r e of t h e r e l a t i o n s h i p b e t w e e n related variables m a y be in either d i r e c t i o n or bidirectional. N e i t h e r mastitis incidence n o r b e d d i n g p o p u l a t i o n s of bacteria c o u l d have e f f e c t e d rainfall a n d t h e r e f o r e the d i r e c t i o n of these a r r o w s b e t w e e n the variables in Fig. 2 c o u l d be set. A l t h o u g h it is possible to c o n c e i v e t h a t high mastitis p r e v a l e n c e c o u l d have seeded t h e b e d d i n g m a t e rial w i t h c o l i f o r m bacteria, the weight o f biological evidence is in the opp o s i t e d i r e c t i o n a n d thus the d i r e c t i o n o f the arrows was set f r o m b e d d i n g p o p u l a t i o n to mastitis incidence. Solid arrows indicate statistically signific a n t associations a n d d a s h e d arrows indicate i n d e p e n d e n c e . P values repr e s e n t i n g t h e p r o b a b i l i t y o f marginal association b e t w e e n the t w o variables w h e n t h e t h i r d was n o t c o n s i d e r e d are p r e s e n t e d for each arrow. T a b l e V s u m m a r i z e s t h e d i f f e r e n c e s n o t e d b e t w e e n E. coli a n d K. p n e u rnoniae in this s t u d y . TABLE V Summary of differences of epidemiological characteristics between the bedding populations and incidence of E. coli and K. pneumoniae Characteristic
E. coli
K. pneurnoniae
Grand mean of bedding populations Overall incidence rate Bedding population by time Bedding population by rainfall Mastitis incidence by bedding population Mastitis incidence by early lactation Incidence among heifers Threshold of risk for bedding populations
Higher (1 × 10 s'7) Higher ( 1.14 %/month) Constant
Lower (1 × 102':) Lower (0.64%/month) Variable
Independent
Dependent
Independent
Dependent
Trend
No Trend
Low
Low
If any above 1 × 10 6
As low as 1 × 10 4
DISCUSSION T h e results o f this s t u d y bring i n t o q u e s t i o n t h e advisability o f l u m p ing citrate-utilizing m e m b e r s o f t h e E n t e r o b a c t e r i a c e a e , a n d p a r t i c u l a r l y K. p n e u m o n i a e w i t h E. coli, u n d e r the c o m m o n d e s i g n a t i o n of c o l i f o r m ,
238 when investigating the relationship between bedding populations of these species and the incidence of mastitis associated with them. Thomas et al. (1960) indicated that the citrate-utilizing Enterobacteriaceae outlived E. coli in environments other than the intestine. Studies by Carroll and Jasper (1980) have shown the ability of K. pneumoniae to increase from undetectable levels in fresh sawdust bedding to very high levels within several days. K. pneumoniae was rarely found in bovine feces and was difficult to demonstrate in fresh sawdust in these studies, indicating that while E. coli numbers from the inoculum remained constant there was a geometric growth of K. pneumoniae. Differences in growth rates representing different facultative abilities under varying growth conditions are ignored when these organisms are classified under the same heading. Though the final c o m m o n portal of entry is the streak canal, differences in survival of various Enterobacteriaceae in bedding materials or different thresholds of risk for infectivity may be masked by such nosological malfeasance. In important epidemiological ways these results indicate that E. coli and K. pneumoniae behave very differently, as summarized in Table V. The finding of statistical independence between E. coli bedding populations and the incidence of E. coli mastitis in this study is not necessarily at odds with the findings of others. Bramley (1974) reported a direct relationship between levels of coliform contamination of bedding, contamination of the teat apex, and the coliform new infection rate. In his study Bramley determined the total coliform bedding populations by a tube dilution m e t h o d similar to that used in this study. However, he did not enumerate the subpopulations of E. coli, K. pneumoniae and other Enterobacteriaceae in the bedding samples. Though the mastitis which he observed was predominantly due to K. pneumoniae and the predominant bedding species, by frequency of isolation, was K. pneumoniae, he used the more general term, coliform, when summarizing his findings. He noted that of the seven cases of mastitis observed, six being due to K. pneumoniae, all occurred on sawdust bedding containing greater than 104 coliforms/g wet weight. Subsequent authors have widely referenced this observation as a threshold for increased risk of coliform mastitis without emphasizing the predominant role of K. pneumoniae in this study. Francis et al. (1980) studied the influence of bedding temperature in straw bedded cubicles on E. coli bedding populations and the influence of these bedding populations on udder infections. One of the conclusions of this study was that high E. coli populations in bedding were associated with contaminated teat apices and clinical E. coli mastitis. Although all of the E. coli mastitis cases in this study occurred in a group of high yielding cows, whose bedding contained higher populations of E. coli, such a conclusion may not be warranted. The high yielding group was composed of early lactation cows with nearly half of clinical mastitis occurring in the first 40 days of lactation. Thus the association of mastitis with higher E. coli bedding populations is confounded with the greater susceptibility
239 of early lactation cows. A further indictment of this conclusion was the lack of a week by week relationship between E. coli bedding populations and the frequency with which E. coli was recovered from teat apex swabs or hind quarter fore-milk samples. Similarly there was a lack of correlation between E. coli bedding populations and E. coli mastitis cases on a week by week basis, which agrees with our findings on a m o n t h by m o n t h basis as seen in Fig. 1. In a study which controlled the effect of early lactation susceptibility Natzke and LeClair (1975) were able to increase the teat end contamination by E. coli, but were unable to produce E. coli mastitis among 10 cows bedded on uncured sawdust seeded with virulent E. coli broth culture, which was maintained at approximately 106 CFU/g wet weight of E. coli over a 4-week experimental period. While we find no unexplainable discrepancies between our findings and those of others, we are aware of the hazards of bias in observational research. Certainly the difference in outcome between the 1971--1972 studies of Bramley (1974), in which no coliform mastitis was observed among sawdust bedded cows, and his studies of 1972--1973 exemplify this. The large number of cows at risk in this study gives some protection from observational bias. The risk experienced by these cows, however, represents the set of management and environmental factors found in the dairy studied. Though the authors believe this dairy was typical of well managed dairies in the San Joaquin Valley, this belief remains subjective. Certainly the rainfall during the observational period was atypically low. The relationship between bedding populations of K. pneumoniae and mastitis due to it seem well corroborated in the literature, while the lack of such a relationship for E. coli is not. Thus the scope of inference for these findings may be geographically limited or even unique to the dairy studied. With regard to the statistical methods, the authors endeavored to use methods which required the fewest assumptions. However, Chi-square analysis is n o t assumptionless. An important requirement for unbiased Chisquare analysis is independence in sampling. Sixty separate observations were made for each of the bedding population and mastitis incidence variables, while only 12 independent data were recorded for rainfall. Therefore the rainfall variable violates the requirement for independence in sampling. The effect of any bias introduced by this lack of independence was restricted to the marginal associations involving the rainfall variable (i.e. rainfall and bedding population or rainfall and mastitis incidence) but did not effect the marginal association between bedding populations and mastitis incidence for the three 2 X 3 X 2 contingency tables analyzed. With regard to the marginal associations between rainfall and bedding populations, for each pen by m o n t h data record, the E. coli and K. pneumoniae bedding population variables shared the same rainfall value, any bias due to lack of independence would effect them equally. Thus the level of significance of each marginal association may contain bias but the difference noted between them should be free of this bias.
240 In evaluating the success of the study design, the authors feel that expressing the bedding population in terms of dry weight would have been an improvement. The minor additional work of aliquoting and desiccating a second bedding sample at the time of inoculation of MacConkey broth would have yielded two benefits. The first would have been bedding population estimates free of the variability due to water content in the bedding sample. The second would have been a moisture content variable, derived by subtracting the dry weight of the sample from its wet weight, which may have been more causally relevant to the bedding populations than m o n t h l y rainfall. With the above considerations of representativeness and bias in mind the authors' interpretations of these results follow. The statistical association between K. pneumoniae bedding populations and mastitis due to K. pneumoniae represent a significant biological risk identified by Newman and Kowalski (1973). The bedding populations of K. pneumoniae necessary to constitute this risk are likely to be much lower than previously appreciated, possibly as low as 104 CFU/g wet weight. The statistical association between rainfall and the bedding populations of K. pneumoniae we believe represents a true biological phenomenon, but rainfall per se may be a surrogate measure for moisture in general. We do not conclude that the lack of a statistical association between E. coli bedding populations and E. coli mastitis represents biological independence. Though a direct relationship was not shown, interactions between E. coli bedding populations and other variables n o t measured in this study, may y e t be important in fitting a causal epidemiological model. In considering the very different behavior of these two organisms the authors have constructed a hypothesis based on our findings and those in the literature. A necessary assumption for this hypothesis is that there exists a very different ability between the two organisms to penetrate the streak canal. The highly significant relationship between K. pneumoniae bedding populations and the occurrence of K. pneumoniae mastitis indicates that very few other factors may be necessary to consider. Thus when K. pneumoniae is present it is able to penetrate the streak canal and cause mastitis at a significant rate and this may occur independently of other factors. The lack of an association between E. coli bedding populations and E. coli mastitis incidence under the same conditions suggests that though E. coli is consistently present at higher levels in the bedding, it is less able to penetrate the streak canal and that other factors axe necessary to explain the variability in occurrence of E. coli mastitis. Since post-milking teat sanitization does not affect E. coli mastitis incidence, this focuses attention on potential risk factors in the immediate pre-milking and milking periods. The lack of association between bedding populations of E. coli and E. coli mastitis coupled with the observation of increased peripartum susceptibility to E. coli suggests that the difference in the ability of E. coli to penetrate the streak canal may be a factor of resistance in the cow and
241 n o t an i n h e r e n t deficit of the organism. The lack o f an observable early l a c t a t i o n susceptibility to K. pneumoniae in this s t u d y , if c o r r e c t , suggests t h a t m e c h a n i s m s w h i c h a c c o u n t for relative late lactational resistance to E. coli mastitis m a y be d i r e c t e d at characteristics n o t shared b y the t w o organisms. If this h y p o t h e s i s is substantially c o r r e c t , f u t u r e research s h o u l d be d i r e c t e d at defining risk factors for E. coli mastitis during the i m m e d i a t e pre-milking and milking periods and q u a n t i t a t i n g their relative c o n t r i b u tions t o the variability in the o c c u r r e n c e o f E. coli mastitis. A d d i t i o n a l l y research into the n a t u r e o f late lactational resistance to E. coli mastitis m a y provide m e t h o d s t o i m p r o v e this resistance in the early lactational period. ACKNOWLEDGEMENTS The a u t h o r s wish to t h a n k Micheal Miller Ph.D. o f the Statistical Laborat o r y at t h e University o f California, Davis, f o r his c o n s u l t a t i o n . T h e a u t h o r s dedicate this w o r k to the m e m o r y o f o u r colleague E.J. Carroll in r e c o g n i t i o n o f his c o n t r i b u t i o n s to our u n d e r s t a n d i n g o f bovine mastitis. S u p p o r t e d in part b y f u n d s f r o m the California Milk A d v i s o r y Board and the U.S. D e p a r t m e n t o f Agriculture, Agricultural Research Service General Cooperative Agreement.
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