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Bacterial Counts in Bedding Materials Used on Nine Commercial Dairies1
Bacterial Counts in Bedding Materials Used on Nine Commercial Dairies 1 J. S. HOGAN! K. l. SMITH! K. H. HOBlET.' D. A. TODHUNTER!
P. S. SCHOENBERGER! W. D. HUESTON.' D. E. PRITCHARD,2 G. l. BOWMAN. 3 L. E. HEIDER.' B. L. BROCKETT,' and H. R. CONRAD' The Ohio State University Ohio Agricultural Research and Development Center Wooster 44691
ABSTRACT
Bacterial counts were monitored for 1 yr in bedding materials used on nine commercial dairies. Organic materials used to bed lactating cows had significantly higher moisture content and gramnegative bacterial, coliform, Klebsiella species, and streptococcal cOUnts than did inorganic materials. Klebsiella species counts were higher in sawdust than in chopped straw. Streptococcal counts were higher in chopped straw than sawdust. Bacterial counts did not differ between sand and crushed limestone. Gramnegative bacterial and coliform counts were higher during summer and fall than in winter and spring months. Streptococcal counts did not differ among seasons of the year. Linear relationships were significant between total rates of clinical mastitis during lactation and both gramnegative bacterial and Klebsiella species counts in lactating cow bedding. These data indicate that bacterial populations differed between both types of bedding and among seasons of the year. Rates of clinical mastitis were related to bacterial counts in bedding. INTRODUCTION
Rates of intramammary infections (IMI) are correlated with the number of mastitis patho-
Received June 16, 1988. Accepted August 31,1988. I Salaries and research support provided by State and Federal Funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University. Manuscript Number 157.88. 'Department of Dairy Science. 3 College of Veterinary Medicine. 1989 J Dairy Sci 72:250-258
gens on the teat end (18). Therefore, management practices that reduce exposure of mastitis pathogens to the teat end should result in reduced rates of new IMI. Developing such management practices requires an understanding of the periods when teats are exposed to the pathogens and the primary sources of pathogens during these periods. During lactation, ex· posure of teat ends to coliforms and environmental streptococci occurs primarily between milkings (8, 9, 20). Bedding materials were implicated as primary sources of environmental pathogens during the intermilking periods. The number and type of bacteria in bedding are related to the microbial load on the teat end (2, 14, 17,20). However, short duration trials in experimental herds were unable to demonstrate a direct relationship between bacterial counts in bedding and rates of either coliform or environmental streptococcal IMI (l0, 17). Field surveys have shown extreme variation among rates of clinical mastitis (13) and bacterial counts in bedding (3, 6) on commercial farms. Whether these parameters are interrelated within farms has not been determined. Two survey reports implied that exposing teats to bedding with coliform populations > 10 6 cfu increases the probability of coliform IMI (4,6). However, neither this relationship nor a similar relationship between environmental streptococcal IMI and bedding counts were demonstrated. The objectives of this study were to determine 1) bacterial populations in bedding materials and 2) the associations between bacterial counts in bedding and rates of clinical mastitis cases in nine commercial herds. MATERIALS AND METHODS Cooperating Herds
Bacterial counts in bedding, rates of clinical mastitis, and IMI status of cows at calving and 250
BACTERIAL COUNTS IN BEDDING
drying off were monitored in nine commercial dairy herds for 52 consecutive wk. Herds were selected by criteria that included indications that Staphylococcus aureus and Streptococcus agalactiae mastitis had been controlled as evidenced by herd SCC. Selection criteria were 1) greater than 80% of cows within each herd had DHIA linear SCC score of < 5; 2) farm owners were willing to participate; 3) herd veterinarians were willing to cooperate; 4) herd size was between 60 and 200 lactating cows, and 5) herds were located within a 4S-km radius of the laboratory. Mean number of lactating cows per herds during the study was 132. Distribution of breeds among farms was eight Holstein herds and one J ersy herd. Mean herd average for Holstein herds was 8812 (21 SD) kg of milk and 327 (21 SD) kg of fat. Herd production for the Jersy herd was 5917 kg of milk and 283 kg of fat. Lactating cows in each herd were managed in freestall housing with limited to no access to exercise lots during the study. Two farms used inorganic material to bed lactating cows throughout the study. One herd each used sand or crushed limestone the entire year. Organic bedding materials were evenly distributed among three farms using sawdust and three farms using chopped straw. One herd bedded lactating cows with sawdust during the summer months but changed to sand for bedding during the remainder of the year. Dry cows were managed in loose housing on six farms and in freestalls on three farms. Long straw-manure pack was bedding for dry cows in loose housing. Freestalls in dry cow housing were bedded with sawdust. Dry cows had limited to unlimited access to pasture or exercise lots in each herd. Maternity areas on seven farms were either loose housing or box stalls. Bedding in all maternity areas was long straw. Bacterial counts from two herds that calved on pasture nine mo of the year were omitted from maternity bedding results. Bedding Samples
Bedding samples were collected monthly from lactating cow housing and maternity areas for determining DM content and bacteriological analyses. Samples taken from freestalls were a composite of bedding from the back one-third of 10% of the stalls. Long straw samples were
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collected from two locations in the center of the maternity area and from points approximately one-third the distance from each exterior wall to the center of the box stall or loosehousing area. Bedding samples (ca. 1 kg) were collected as described by Smith et al. (22). Straw samples were chopped to facilitate handling. Approximately 25 g of each sample were placed in a convection oven at 100°C for 24 h to determine DM content. Ten grams of sample were added to 90 ml of sterile phosphatebuffered saline (pH 7.0) in a 500-ml plastic bag and mixed in a stomacher (Tekmar Company, Cincinnati, OH) for 40 s. Contents of the bag were allowed to settle 2 to 3 min. Appropriate dilutions of the liquid phase were PBS and plated on the surface of each of the three media; MacConkey's agar (Difco Laboratories, Detroit, MI), MacConkeys - inositol-carbenicillin (MCIC) agar, and TKT/FC agar (BBL Microbiology Systems, Cockeysville, MD). Four 10J.L1 samples of each dilution were plated on onehalf plate containing each agar medium. Inositol (Sigma Chemical Company, St. Louis, MO; 10 mg/L) and carbenicillin (Pfizer, Inc., New York, NY; 75 mg/L) were added to MacConkeys agar for MCIC as described (1). Bovine plasma (50 milL) was substituted for whole sheep blood in preparing TKT/FC. Inoculated agar plates were incubated 24 h at 37°C. Bacterial counts were expressed as colony-forming units 10giO per gram dry weight of sample. Bacterial groups were identified as gram-negative bacteria (total growth on MacConkey's agar), coliforms (lactose-positive colonies on MacConkeys agar), Klebsiella species (red to pink colonies on MCIC), and streptococci (total growth on TKT/ Fe). Milk Samples
Bacteriological status of mammary quarters in each herd was determined by analyses of samples collected utilizing the following scheme. Duplicate quarter foremilk samples were collected aseptically (5) by the farmers from 1) all cows during the first 7 d of lactation; 2) all cows during the 7 d prior to drying off; and 3) all quarters of a cow with clinical mastitis prior to a course of antibiotic therapy. Samples were frozen immediately after collection and remained frozen no longer than 10 d until laboratory analyses of samples were initiated. Journal of Dairy Science Vol. 72, No. I, 1989
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HOGAN ET AL.
Primary culture of all milk samples was on trypticase blood esculin agar (.01 ml of milk) and on MacConkey agar (.1 ml of milk) to aid detection of coliform IMI (22). Bacterial isolates were identified as gram-negative bacilli, environmental streptococci, Staphylococcus species, Staphylococcus aureus, Corynebacterium bovis, and other microbes as described in work of Smith et al. (22). Gram-negative bacilli were differentiated into coliform and nonlactose fermenters by tube lactose fermentation, citrate utilization, motility, and triple-sugar iron reactions (16). Diagnosis of Intramammary Infections
An IMI was diagnosed when the same pathogen or pathogens were isolated from duplicate quarter foremilk samples. Clinical Mastitis Cases
The number of clinical mastitis cases were determined retrospectively using all reports of clinical signs and culture results from foremilk samples. To account for quarters exhibiting clinical signs multiple times over the study, a clinical case was determined to be 14-d duration based upon an average duration of environmental mastitis reported by Smith et al. (22). Guidelines for determining clinical cases were: 1) a new case of clinical mastitis was declared when a 14-d period elapsed between reports of clinical signs, regardless of the bacteriological status of the quarter; 2) a new case of clinical mastitis was declared when a different pathogen was isolated from a clinical quarter regardless of the number of days between isolation of dissimilar pathogens; 3) when two or more pairs of duplicate milk samples were cultured during a 14-d period and a pathogen was isolated from one or more pairs of samples, while the remainder of samples were bacteriologicallynegative, the pathogen isolated was determined the cause of the clinical case; and 4) a new case of clinical mastitis was not recorded if the same pathogen was isolated or if sample(s) was bacteriologically negative when less than 14 d had elapsed between reports of clinical signs. Rates of clinical mastitis were defined as number of clinical cases divided by totaled cowdays exposure (CD). Cow-days were calculated as the number of cows lactating on DHIA test day multiplied by the number of days in the Journal of Dairy Science Vol. 72, No. I, 1989
experimental period. Rates were expressed as (clinical cases/305 CD) + .001 for statistical analyses. The constant .001 was added to rates to normalize data (13). Seasons
Data were analyzed for season effects. Seasons were defined as summer = June, July, August; fall = September, October, November; winter = December, January, February, and spring = March, April, and May. Statistical Analyses
Distribution of bacterial counts was normalized by 10glO tranformation. Data expressed as percentages and analyzed by parametric test were normalized by arcsine transformation. Differences among bacterial counts in bedding were tested by analyses of variance with data blocked by herds. Independent comparisons among bedding types were 1) organic vs. inorganic, 2) sawdust vs. chopped straw, and 3) sand vs. crushed limestone. Differences in bacterial counts among seasons of the year were determined by Tukey-Kramer multiple comparison test with data blocked by herd (23). Linear relationships and correlations among bacterial counts in bedding and either rates clinical mastitis or IMI were measured by Model 2 regression (23). Linear relationships tested were among rates of clinical mastitis during lactation and bacterial counts in sawdust, chopped straw, organic, inorganic, and all beddings combined. Rates of clinical mastitis and percent quarters infected at calving were tested for linear relationships with bacterial counts in maternity area bedding. RESULTS AND DISCUSSION
Maternity Area Bedding
Mean bacterial counts in long straw used to bed maternity areas on seven farms differed among the seasons of the year (Table 1). Mean gram-negative bacterial counts were higher during summer and fall than during winter and spring (P< .05). Coliform counts were greater during summer than winter (P< .05). Neither Klebsiella species nor streptococcal counts differed among seasons. Mean (SO) OM percentage of long straw bedding were 63.7 (7.5 SO). Dry matter percentages also did not differ among season of the year (P> .05). Mean percent-
253
BACTERIAL COUNTS IN BEDDING
TABLE 1. Mean bacterial counts (cfu log,o /g dry weight) in long straw maternity stall bedding by seasons of the year. Summer (n = 7)
Bacterial count
X Gram-negative Coliform Klebsiella species Streptococcal
7.S a 6.9 a 4.6 7.6
SE
X
.1 .1 .3 .2
7.6 a 6.7 ab 4.6 7.8
Spring (n= 7)
Winter (n = 7)
Fall (n = 7)
X
SE
7.1 b 6.0 b 3.4 8.0
.1 .2 .3 .1
SE
X
SE
.1 .1 .3 .1
6.9 b 6.1 ab 3.6 7.5
.1 .3 .3 .3
a,bMeans within bacterial counts with differing superscripts differ (P<.05).
ages of quarters infected at calving are in Table 2. Intramammary infection status of quarters at calving and drying off were similar to those reported in other herds that had controlled contagious mastitis (8, 21, 22). Mean rates of clinical mastitis during the first 7 d of lactation were: total rate, .077 (.061 SD); coliform, .021 (.005 SD); and environmental streptococcal, .021 (.024 SD). Neither percentages of quarters infected at calving nor mean rates of clinical mastitis during the first 7 d of lactation were correlated with long straw bacterial counts. Bacterial counts in maternity area bedding were monitored, because earlier studies showed rates of environmental streptococcal and coliform IMI were greatest during the 14 d prior to calving compared with the remainder of the dry
period or during lactation (8, 21, 22). However, one variable that could not be accounted for in the present study was the stage of either the dry period or previous lactations from which infections present at calving originated. Coliform and environmental streptococcal IMI present at calving included those infections persisting from prior lactations, the early dry period, and those infections occurring near calving (22). Another factor that was not verified on each farm was the actual number of days prior and after calving that cows were maintained in maternity areas. Bacterial populations in long straw would have less influence on rates of IMI in herds that limited the amount of time cows were confined to maternity areas. Nevertheless, data in the present study did not
TABLE 2. Percent quarters infected at calving and drying off in seven herds using long straw bedding in maturity areas.' Intramammary infection status
Coliforms Environmental streptococci Staphylococcus species Corynebacterium bovis NLF' Staphylococcus aureus Other microbes Uninfected Contaminated samples
Drying off
Calving
X
SD
X
SD
2.7 2.4 10.1
.8 1.9
.9 3.0 12.9 9.8
.5 2.4 6.4 13.6
.3
.7
.4
.6 .5 12.1 2.0
.7
.7 .3 .1 81.3 1.1
3.4 1.0 1.1 .8 .4 5.8 1.5
.3 71.3 1.3
1 Mean number of quarters sampled among farms was 520 (308 SD)at calving, and 373 (162 SD) at drying off. Nonlactose-fermenting, gram-negative bacilli.
Journal of Dairy Science Vol. 72, No.1, 1989
HOGAN ETAL.
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imply a relationship between bacterial counts in maternity bedding and mammary health at calving.
ials. In vitro studies showed organic bedding materials contained essential nutrients capable of supporting growth of coliforms and environmental streptococci (24). Despite the inorganic characteristics of sand and crushed limestone, these bedding materials contained > 10 6 cfu/g dry weight of both gram-negative bacteria and streptococci. Organic matter such as manure and feedstuffs contaminating bedding materials may have provided an exogenous source of essential nutrients for maintenance of bacterial populations. Jasper (15) reported that manurecontaminated bedding materials increased coliform populations during in vitro trials. Therefore, frequent removal of contaminated bedding was recommended to decrease bacterial numbers in bedding (7).
Organic Versus Inorganic Bedding
Independent comparisons among bacterial counts in materials used to bed lactating cows are in Table 3. Organic bedding materials had greater (P< .05) gram-negative bacterial, coliform, Klebsiella species, and streptococcal counts than did inorganic materials. These results agree with those in earlier trials conducted on experimental farms (10, 14). Basic among the ecological requirements for colonization and multiplication of bacteria are moisture, available nutrients, and proper temperature (19). Moisture and available nutrients appeared to be the ecological factors associated with greater bacterial counts in organic than inorganic beddings. Mean and standard errors DM percentages were 70.7 ± 2.8 for organic material compared with 94.7 ± 1.8 for inorganic matrials (P< .05). Although chemical analyses were not performed, carbon and other essential nutrients were also assumed to be more available in organic bedding than in inorganic mater-
Sawdust Versus Chopped Straw
Bacterial counts also differed between organic materials used to bed freestalls (Table 3). Mean and standard error Klebsiella species count in sawdust (4.8 ± .2) was greater than that in chopped straw (3.7 ± .2). Chopped straw had a significantly higher mean streptococcal count (7.8 ± .1) than did sawdust (7.1 ± .1). Gram-
TABLE 3. Independent comparisons among mean seasonal bacterial counts (cfu log,o /g dry weight) in materials used to bed lactating cows. Independent comparisons Bacterial counts
Organic' (n = 25)
Inorganic' (n = 11)
Sawdust (n = 13)
Chopped straw (n = 12)
Sand (n = 7)
Crushed limestone (n = 4)
7.1 a 0
6.4 b .1
7.0 .1
7.1 .1
6.3 .1
6.5 .1
6.2 a .1
5.7 b .1
6.2 .2
6.3 .1
5.7 .2
5.8 .1
4.P .1
3.4 b .2
4.8 a .2
3.7 b .2
3.2 .2
3.8 .2
7.5 a .1
6.8 b .1
7.P .1
7.8 b .1
7.0 .1
6.6 .1
Gr~-negative
X SE
Coliform
X SE
Kle'!..siella species X SE
Streptococcal
X SE
a,bMeans within comparisons with differing superscripts differ (P<.05). I
Organic bedding materials were sawdust and chopped straw.
• Inorganic bedding materials were sand and crushed limestone. Journal of Dairy Science Vol. 72, No. I, 1989
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BACTERIAL COUNTS IN BEDDING TABLE 4. Mean bacterial counts (cfu log,o/g dry weight) in chopped straw bedding by seasons of the year.
Bacterial count
Gram-negative Coliform Klebsiella species Streptococcal
Summer (n = 3)
Spring (n = 3)
Winter (n = 3)
Fall (n = 3)
X
SE
X
SE
X
SE
X
SE
7.6 a 6.8 a 3.9 7.8
.0 .2
7.2 b 6.1 b 4.4 7.9
.1 .3 .2 .0
6.7 c 5.9 b 3.6 7.8
.3 .3 .3 .2
7.0 bc 6.2 b 3.2 7.8
.1 .2 .3 .2
.4
.2
a,b,cMeans within bacterial counts with differing superscripts differ (P<.05).
negative bacterial and coliform counts did not differ between organic bedding materials. Data in the present study were similar to those reported in a series of controlled experiments (20). Under controlled conditions, streptococcal counts were greater in straw than sawdust, and Klebsiella species counts were greater in fresh sawdust than in fresh chopped straw. However, Rendos et at. (20) reported Klebsiella species counts did not differ between chopped straw and sawdust after 7 d to 21 d of use in freestalls. Other researchers (6, 10, 11) have also suggested that Klebsiella species counts increased the first 7 d sawdust was used, but counts decreased after 21 d in freestalls. The number of days that bedding materials were in freestalls prior to collection for analyses was not documented in the present study. Bacterial counts in organic bedding differed among season of the year. Gram-negative bacterial (Table 4) and coliform (Table 5) counts in chopped straw were greater (P< .05) during summer than during other seasons. Although differences were not significant (P> .05), gram-
negative bacterial and coliform counts in sawdust were higher in summer and fall than in winter and spring (Table 5). Klebsiella species counts in sawdust were greater (P<.05) in summer and fall months than in winter" and spring. An interaction between seasons and bedding type on Klebsiella species counts was evidenced by greater Klebsiella species counts in sawdust were than those in chopped straw during summer and fall but not during winter and spring. Higher coliform counts in organic matter during summer and fall are probably related to higher ambient temperatures during these seasons. In vitro studies have shown coliforms in organic bedding multiplied most rapidly at temperatures between 30 and 40°C (3). Streptococcal counts did not differ among seasons of the year in either sawdust or chopped straw. Sand Versus Crushed limestone
Bacterial counts did not differ between sand and crushed limestone (Table 3). Inorganic
TABLE 5. Mean bacterial counts (cfu log,o/g dry weight) in sawdust bedding by seasons of the year.
Bacterial count
Summer (n = 3) X
Gram-negative Coliform Klebsiella species Streptococcal
7.1 6.5 5.5 a 6.9
Fall (n = 3) SE .3 .3 .2 .3
X 7.4 6.6 5.4 a 7.3
Spring (n = 3)
Winter (n = 3) SE .2 .3 .1 .1
X 6.8 5.8 4.1 b 7.4
SE .2 .1 .3 .3
X 6.6 5.7 3.9 b 6.9
SE .1 .4.4 .4
a,bMeans within bacterial counts with differing superscripts differ (P<.05). Journal of Dairy Science Vol. 72, No. 1,1989
HOGAN ETAL.
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TABLE 6. Mean bacterial counts (cfu loglo/g dry weight) in inorganic' beddings by seasons of the year. Summer (n = 2)
Bacterial count
Gram-negative Coliform Klebsiella species Streptococcal
Fall (n = 3)
Winter
Spring
(n = 3)
(n = 3)
X
SE
X
SE
X
SE
X
SE
6.9 6.3 4.0 6.9
.0 .1 1.0 .1
6.4 5.7 3.5 6.7
.1 .2 .3 .4
6.2 5.3 2.9 7.0
.1 .2 .1
6.2 5.7 3.4 6.8
.3 .2 .3 .2
.0
1 Inorganic beddings were in two herds using sand and one herd using crushed limestone during fall, winter, and spring. During summer, 1 herd used sand and 1 herd used curshed limestone.
bedding materials did not differ among seasons of the year (Table 6). An important aspect of comparisons between inorganic materials in the present study was the low number of observations. Crushed limestone data were from a single farm using limestone particles with a geometric mean diameter of 2000 Ji. Sand used on both herds was washed prior to use as bedding. Further studies are needed to confirm bacterial counts in inorganic bedding and determing if there are economic advantages between using either sand or crushed limestone to bed lactating cows. The mean of each bacterial count measured was .;;; 10 7 cfulg dry weight throughout seasons of the year. Relationships Among Rates of Clinical Mastitis and Bacterial Counts in Bedding
Mean and SD rate of total clinical maStitiS cases among herds was .399 (.174 SE)/305 CD
(f)
1.0
... 0 I
.8
'"
•
• •• ••• • • # • • • • • • .-. • • • • r=.38
on
.6
0
(")
'(f) ril
... ...., ... u
.4
(f)
U
.2
Z
::l 0 5.5
6.0
6.5
7.0
7.5
Y=.399 + 088 (x-4.06)
0 I
r=.40
8
'on"
.6
0
(")
'(f) ril
... ...., ... u
•
•••
P(05
0 U
---
.4
(f)
U
2
Z
::l U
8.0
COLONY-FORMING UNITS/GRAM DRY WEIGHT
Figure 1. Linear relationship between rates of clinical mastitis during lactation and gram-negative bacterial counts in lactating cow bedding. Journal of Dairy Science Vol. 72, No.1, 1989
,
1.0
...
>-
P(.05
0 U
U
(f)
Y=.399 + .149 (x-6.92)
>-
during lactation. Percentage distribution of clinical cases during lactation by bacteriological statuses were coliforms, 36.3%; bacteriologically negative, 26.8%; environmental streptococci, 24.0%, and other statuses, 13 .9%. Linear relationships were significant (P< .05) among total rates of clinical mastitis during lactation and counts of gram-negative bacteria and Klebsiella species in lactating cow bedding (Figures 1 and 2). These relationships support the thesis that rate of environmental mastitis increases with increasing teat end exposure to environmental pathogens in bedding. Previous studies unable to demonstrate a relationship between bacterial counts in bedding and rates of IMl were short duration trials using relatively few cows (10, 17). Rate of coliform IMI was reported to be 1 IM11770 CD when cows were bedded on organic materials containing coliform counts > 106 cfu/g (22). Therefore, total CD exposure in both previous studies (10, 17) were too few
••
0 2.5
•
3.0
t
• 35
4.0
4.5
5.0
• 5.5
60
65
COLONY-FORMING UNITS/GRAM DRY WEIGHT
Figure 2. Linear relationship between rates of clinical mastitis during lactation and Klebisella species counts in lactating cow bedding.
257
BACTERIAL COUNTS IN BEDDING
TABLE 7. Mean rates' of clinical mastitis cases during lactation' in herds using sawdust. chopped straw. and inorganic materials to bed lactating cow freestalls. Bacteriological status of clinical cases
Inorganic' (n = 3)
Sawdust (n = 3)
Chopped straw (n = 3)
.113 .016
.149 .065
.147 .010
.188 .069
.082 .035
.080 .018
.488 .148
.366 .094
.342 .055
Coliform
X SD Environmental streptococci
X SD Total
X SD 1
I (Number of clinical cases/305 cow-day) + .0011 .
• From d 8 through end of lactation. 3
Inorganic bedding materials were sand and crushed limestone.
to expect a relationship between coliform bacterial counts in bedding and rate of coliform IMI to be demonstrated. Data in the present study were obtained over 12 mo in nine wellmanaged herds with varying rates of clinical mastitis and bacterial populations in bedding. Rates of clinical mastitis were used as the dependent variable in regression equations since rates of clinical mastitis were shown to reflect more accurately the dynamics of environmental mastitis than did prevalence of IMI (22). Data in the present study did support the inferences of others (2, 10, 17) that environmental mastitis is a multifactorial disease. Coefficients of determination (r 2 ) were';; .16 for relationships between total rates of clinical mastitis and both gram-negative bacterial and Klebsiella species counts. In addition, no other relationships tested were significant among rates of clinical mastitis and bacterial counts in bedding (P> .05). Rates of clinical mastitis did not differ among herds using different bedding materials (Table 7). Two important factors must be considered when interpreting relationships among bacterial counts in bedding and mammary health. An assumption that may not be valid is that bacterial counts derived from growth on selective and differential media are reflective of the mastitis pathogen load in bedding. More detailed bacteriological analyses than were used in the present trial would be needed to validate this assumption. The second
factor to be considered is that bedding is only one potential source of mastitis pathogens to the teat end. Possible interactions among bacterial exposure in bedding, milking hygiene, nutrition, and other factors affecting rates of environmental mastitis were outlined (12). CONCLUSIONS
Bacterial counts in lactating cow bedding differed among bedding materials and seasons of the year. Organic bedding materials contained significantly higher bacterial counts than did inorganic materials. Teat end exposure to gramnegative bacteria in bedding was greatest during summer and fall, which coincided with the highest rates of clinical mastitis during the year. Therefore, these data suggest that use of inorganic bedding decreases teat end exposure to environmental mastitis pathogens. Use of inorganic bedding materials appears to be most advantageous during summer and fall when gram-negative bacterial populations were greatest in bedding for lactating cows. REFERENCES 1 Bagley, S. T., and R. J. Sheidler. 1978. Primary Klebsiella identification with MacConkey-lnositolcarbenicillin agar. App!. Environ. Microbiol. 6: 536. 2 Bishop, J. R.• J. J. Janzen, A. B. Bodine. C. A. Caldwell, and D. W. Johnson. 1981. Dairy waste solids as a possible source of bedding. J. Dairy Sci. 64:706. Journal of Dairy Science Vol. 72, No.1, 1989
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3 Bramley, A. J. 1982. Sources of Streptococcus uberis in the dairy herd. I. Isolation from bovine feces and from straw bedding of cattle. J. Dairy Res. 49:369. 4 Bramley, A. J., and F. K. Neave. 1975. Studies on the control of coliform mastitis in dairy cows. Br. Vet. J. 131 :160. 5 Brown. R. W., D. A. Barnum, D. E. Jasper, J. S. McDonald, and W. D. Schultze. 1981. Microbiology procedures for use in diagnoses of bovine mastitis. Nat!. Mastitis Counc., Arlington, VA. 6 Carroll, E. J., and D. E. Jasper. 1978. Distribution of Enterobacteriaceae in recycled manure bedding on California daries. ]. Dairy Sci. 61: 1498. 7 Dodd, F. H., T. M. Higgs, and A. J. Bramley. 1984. Cubicle management and coliform mastitis. Vet. Rec. 114:522. 8 Eberhart, R. J., and]. M. Buckalew. 1972. Evaluation of a hygiene and dry period therapy program for mastitis contro!. J. Dairy Sci. 55:1683. 9 Eberhart, R. J., P. L. LeVan, and L. C. Griel. 1983. Germicidal teat dip in a herd with low prevalence of Streptococcus agalactiae and Staphylococcus aureus mastitis. J. Dairy Sci. 66: 13 90. 10 Fairchild, T. P., B. ]. McArthur, ]. H. Moore, and W. E. Hylton. 1982. Coliform counts in various bedding materials. ]. Dairy Sci. 65:1029. 11 Grier, P. H. 1985. Dynamics of bacterial populations in bedding materials. M.S. Thesis, Ohio State Univ., Columbus. 12 Hogan, ]. S., and K. L. Smith. 1988. A practical look at environmental mastitis. Compo Continuing Educ. Pract. Vet. 9:F341. 13 Hogan, J. S., K. L. Smith, K. H. Hoblet, P. S. Schoenberger, D. A. Todhunter, W. D. Hueston, D. E. Pritchard, G. L. Bowman, L. G. Heider, B. L. Brockett, and H. R. Conrad. 1988. Bacterial and somatic cell counts in bulk tank milk from nine
Journal of Dairy Science Vol. 72, No. I, 1989
well managed herds. J. Food Protect. 51: 9 30. 14 Janzen, J. J., J. R. Bishop, A. B. Bodine, C. A. Caldwell, and D. W. Johnson. 1982. Composted dairy waste solids and crushed limestone as bedding in free stalls. J. Dairy Sci. 65:1025. 15 Jasper, D. E. 1980. The coliform mastitis enigma. Page 23 in Proc. 11th Int. Congr. Dis. Dairy Cattle, Israel Assoc. for Buiatics, Tel Aviv. 16 Koneman, E. W., S. D. Allen, V. R. Dowell, and H. M. Sommers. 1979. Color atlas and textbook of diagnostic microbiology. 2nd ed. J. B. Lippincott Co., New York, NY. 17 Natzke, R. P., and B. J. LeClair. 1976. Coliform contaminated bedding and new infections. J. Dairy Sci. 59:2152. 18 Neave, F. K., F. H. Dodd, and R. A. Kingwill. 1966. A method of controlling udder disease. Vet. Res. 78:521. 19 Pekoyar, M. J., and E.C.S. Chan. 1981. Environmental microbiology. Page 583 in Elements of microbiology. 1st ed. McGraw-Hili Book Co., New York, NY. 20 Rendos, ]. J. R. J. Eberhart, and E. M. Kesler. 1975. Microbial populations of teat ends of dairy cows, and bedding materials. J. Dairy Sci. 58:1492. 21 Smith, K. L., D. A. Todhunter, and P. S. Schoenberger. 1985. Environmental pathogens and intramammary infection during the dry period. J. Dairy Sci. 68:402. 22 Smith, K. L., D. A. Todhunter, and P. S. Schoenberger. 1985. Environmental mastitis; cause, prevalence, and prevention. J. Dairy Sci. 68: 1531. 23 Sokal, R. J., and F. J. Rohlf. 1981. Biometry. 2nd ed. W. H. Freeman and Co., San Francisco, CA. 24 Zehner, M. M., R. J. Farnsworth, R. D. Appleman, K. Larntz, and J. A. Springer. 1986. Growth of environmental mastitis pathogens in various bedding materials. J. Dairy Sci. 69: 19 32.
P. S. SCHOENBERGER! W. D. HUESTON.' D. E. PRITCHARD,2 G. l. BOWMAN. 3 L. E. HEIDER.' B. L. BROCKETT,' and H. R. CONRAD' The Ohio State University Ohio Agricultural Research and Development Center Wooster 44691
ABSTRACT
Bacterial counts were monitored for 1 yr in bedding materials used on nine commercial dairies. Organic materials used to bed lactating cows had significantly higher moisture content and gramnegative bacterial, coliform, Klebsiella species, and streptococcal cOUnts than did inorganic materials. Klebsiella species counts were higher in sawdust than in chopped straw. Streptococcal counts were higher in chopped straw than sawdust. Bacterial counts did not differ between sand and crushed limestone. Gramnegative bacterial and coliform counts were higher during summer and fall than in winter and spring months. Streptococcal counts did not differ among seasons of the year. Linear relationships were significant between total rates of clinical mastitis during lactation and both gramnegative bacterial and Klebsiella species counts in lactating cow bedding. These data indicate that bacterial populations differed between both types of bedding and among seasons of the year. Rates of clinical mastitis were related to bacterial counts in bedding. INTRODUCTION
Rates of intramammary infections (IMI) are correlated with the number of mastitis patho-
Received June 16, 1988. Accepted August 31,1988. I Salaries and research support provided by State and Federal Funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University. Manuscript Number 157.88. 'Department of Dairy Science. 3 College of Veterinary Medicine. 1989 J Dairy Sci 72:250-258
gens on the teat end (18). Therefore, management practices that reduce exposure of mastitis pathogens to the teat end should result in reduced rates of new IMI. Developing such management practices requires an understanding of the periods when teats are exposed to the pathogens and the primary sources of pathogens during these periods. During lactation, ex· posure of teat ends to coliforms and environmental streptococci occurs primarily between milkings (8, 9, 20). Bedding materials were implicated as primary sources of environmental pathogens during the intermilking periods. The number and type of bacteria in bedding are related to the microbial load on the teat end (2, 14, 17,20). However, short duration trials in experimental herds were unable to demonstrate a direct relationship between bacterial counts in bedding and rates of either coliform or environmental streptococcal IMI (l0, 17). Field surveys have shown extreme variation among rates of clinical mastitis (13) and bacterial counts in bedding (3, 6) on commercial farms. Whether these parameters are interrelated within farms has not been determined. Two survey reports implied that exposing teats to bedding with coliform populations > 10 6 cfu increases the probability of coliform IMI (4,6). However, neither this relationship nor a similar relationship between environmental streptococcal IMI and bedding counts were demonstrated. The objectives of this study were to determine 1) bacterial populations in bedding materials and 2) the associations between bacterial counts in bedding and rates of clinical mastitis cases in nine commercial herds. MATERIALS AND METHODS Cooperating Herds
Bacterial counts in bedding, rates of clinical mastitis, and IMI status of cows at calving and 250
BACTERIAL COUNTS IN BEDDING
drying off were monitored in nine commercial dairy herds for 52 consecutive wk. Herds were selected by criteria that included indications that Staphylococcus aureus and Streptococcus agalactiae mastitis had been controlled as evidenced by herd SCC. Selection criteria were 1) greater than 80% of cows within each herd had DHIA linear SCC score of < 5; 2) farm owners were willing to participate; 3) herd veterinarians were willing to cooperate; 4) herd size was between 60 and 200 lactating cows, and 5) herds were located within a 4S-km radius of the laboratory. Mean number of lactating cows per herds during the study was 132. Distribution of breeds among farms was eight Holstein herds and one J ersy herd. Mean herd average for Holstein herds was 8812 (21 SD) kg of milk and 327 (21 SD) kg of fat. Herd production for the Jersy herd was 5917 kg of milk and 283 kg of fat. Lactating cows in each herd were managed in freestall housing with limited to no access to exercise lots during the study. Two farms used inorganic material to bed lactating cows throughout the study. One herd each used sand or crushed limestone the entire year. Organic bedding materials were evenly distributed among three farms using sawdust and three farms using chopped straw. One herd bedded lactating cows with sawdust during the summer months but changed to sand for bedding during the remainder of the year. Dry cows were managed in loose housing on six farms and in freestalls on three farms. Long straw-manure pack was bedding for dry cows in loose housing. Freestalls in dry cow housing were bedded with sawdust. Dry cows had limited to unlimited access to pasture or exercise lots in each herd. Maternity areas on seven farms were either loose housing or box stalls. Bedding in all maternity areas was long straw. Bacterial counts from two herds that calved on pasture nine mo of the year were omitted from maternity bedding results. Bedding Samples
Bedding samples were collected monthly from lactating cow housing and maternity areas for determining DM content and bacteriological analyses. Samples taken from freestalls were a composite of bedding from the back one-third of 10% of the stalls. Long straw samples were
251
collected from two locations in the center of the maternity area and from points approximately one-third the distance from each exterior wall to the center of the box stall or loosehousing area. Bedding samples (ca. 1 kg) were collected as described by Smith et al. (22). Straw samples were chopped to facilitate handling. Approximately 25 g of each sample were placed in a convection oven at 100°C for 24 h to determine DM content. Ten grams of sample were added to 90 ml of sterile phosphatebuffered saline (pH 7.0) in a 500-ml plastic bag and mixed in a stomacher (Tekmar Company, Cincinnati, OH) for 40 s. Contents of the bag were allowed to settle 2 to 3 min. Appropriate dilutions of the liquid phase were PBS and plated on the surface of each of the three media; MacConkey's agar (Difco Laboratories, Detroit, MI), MacConkeys - inositol-carbenicillin (MCIC) agar, and TKT/FC agar (BBL Microbiology Systems, Cockeysville, MD). Four 10J.L1 samples of each dilution were plated on onehalf plate containing each agar medium. Inositol (Sigma Chemical Company, St. Louis, MO; 10 mg/L) and carbenicillin (Pfizer, Inc., New York, NY; 75 mg/L) were added to MacConkeys agar for MCIC as described (1). Bovine plasma (50 milL) was substituted for whole sheep blood in preparing TKT/FC. Inoculated agar plates were incubated 24 h at 37°C. Bacterial counts were expressed as colony-forming units 10giO per gram dry weight of sample. Bacterial groups were identified as gram-negative bacteria (total growth on MacConkey's agar), coliforms (lactose-positive colonies on MacConkeys agar), Klebsiella species (red to pink colonies on MCIC), and streptococci (total growth on TKT/ Fe). Milk Samples
Bacteriological status of mammary quarters in each herd was determined by analyses of samples collected utilizing the following scheme. Duplicate quarter foremilk samples were collected aseptically (5) by the farmers from 1) all cows during the first 7 d of lactation; 2) all cows during the 7 d prior to drying off; and 3) all quarters of a cow with clinical mastitis prior to a course of antibiotic therapy. Samples were frozen immediately after collection and remained frozen no longer than 10 d until laboratory analyses of samples were initiated. Journal of Dairy Science Vol. 72, No. I, 1989
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HOGAN ET AL.
Primary culture of all milk samples was on trypticase blood esculin agar (.01 ml of milk) and on MacConkey agar (.1 ml of milk) to aid detection of coliform IMI (22). Bacterial isolates were identified as gram-negative bacilli, environmental streptococci, Staphylococcus species, Staphylococcus aureus, Corynebacterium bovis, and other microbes as described in work of Smith et al. (22). Gram-negative bacilli were differentiated into coliform and nonlactose fermenters by tube lactose fermentation, citrate utilization, motility, and triple-sugar iron reactions (16). Diagnosis of Intramammary Infections
An IMI was diagnosed when the same pathogen or pathogens were isolated from duplicate quarter foremilk samples. Clinical Mastitis Cases
The number of clinical mastitis cases were determined retrospectively using all reports of clinical signs and culture results from foremilk samples. To account for quarters exhibiting clinical signs multiple times over the study, a clinical case was determined to be 14-d duration based upon an average duration of environmental mastitis reported by Smith et al. (22). Guidelines for determining clinical cases were: 1) a new case of clinical mastitis was declared when a 14-d period elapsed between reports of clinical signs, regardless of the bacteriological status of the quarter; 2) a new case of clinical mastitis was declared when a different pathogen was isolated from a clinical quarter regardless of the number of days between isolation of dissimilar pathogens; 3) when two or more pairs of duplicate milk samples were cultured during a 14-d period and a pathogen was isolated from one or more pairs of samples, while the remainder of samples were bacteriologicallynegative, the pathogen isolated was determined the cause of the clinical case; and 4) a new case of clinical mastitis was not recorded if the same pathogen was isolated or if sample(s) was bacteriologically negative when less than 14 d had elapsed between reports of clinical signs. Rates of clinical mastitis were defined as number of clinical cases divided by totaled cowdays exposure (CD). Cow-days were calculated as the number of cows lactating on DHIA test day multiplied by the number of days in the Journal of Dairy Science Vol. 72, No. I, 1989
experimental period. Rates were expressed as (clinical cases/305 CD) + .001 for statistical analyses. The constant .001 was added to rates to normalize data (13). Seasons
Data were analyzed for season effects. Seasons were defined as summer = June, July, August; fall = September, October, November; winter = December, January, February, and spring = March, April, and May. Statistical Analyses
Distribution of bacterial counts was normalized by 10glO tranformation. Data expressed as percentages and analyzed by parametric test were normalized by arcsine transformation. Differences among bacterial counts in bedding were tested by analyses of variance with data blocked by herds. Independent comparisons among bedding types were 1) organic vs. inorganic, 2) sawdust vs. chopped straw, and 3) sand vs. crushed limestone. Differences in bacterial counts among seasons of the year were determined by Tukey-Kramer multiple comparison test with data blocked by herd (23). Linear relationships and correlations among bacterial counts in bedding and either rates clinical mastitis or IMI were measured by Model 2 regression (23). Linear relationships tested were among rates of clinical mastitis during lactation and bacterial counts in sawdust, chopped straw, organic, inorganic, and all beddings combined. Rates of clinical mastitis and percent quarters infected at calving were tested for linear relationships with bacterial counts in maternity area bedding. RESULTS AND DISCUSSION
Maternity Area Bedding
Mean bacterial counts in long straw used to bed maternity areas on seven farms differed among the seasons of the year (Table 1). Mean gram-negative bacterial counts were higher during summer and fall than during winter and spring (P< .05). Coliform counts were greater during summer than winter (P< .05). Neither Klebsiella species nor streptococcal counts differed among seasons. Mean (SO) OM percentage of long straw bedding were 63.7 (7.5 SO). Dry matter percentages also did not differ among season of the year (P> .05). Mean percent-
253
BACTERIAL COUNTS IN BEDDING
TABLE 1. Mean bacterial counts (cfu log,o /g dry weight) in long straw maternity stall bedding by seasons of the year. Summer (n = 7)
Bacterial count
X Gram-negative Coliform Klebsiella species Streptococcal
7.S a 6.9 a 4.6 7.6
SE
X
.1 .1 .3 .2
7.6 a 6.7 ab 4.6 7.8
Spring (n= 7)
Winter (n = 7)
Fall (n = 7)
X
SE
7.1 b 6.0 b 3.4 8.0
.1 .2 .3 .1
SE
X
SE
.1 .1 .3 .1
6.9 b 6.1 ab 3.6 7.5
.1 .3 .3 .3
a,bMeans within bacterial counts with differing superscripts differ (P<.05).
ages of quarters infected at calving are in Table 2. Intramammary infection status of quarters at calving and drying off were similar to those reported in other herds that had controlled contagious mastitis (8, 21, 22). Mean rates of clinical mastitis during the first 7 d of lactation were: total rate, .077 (.061 SD); coliform, .021 (.005 SD); and environmental streptococcal, .021 (.024 SD). Neither percentages of quarters infected at calving nor mean rates of clinical mastitis during the first 7 d of lactation were correlated with long straw bacterial counts. Bacterial counts in maternity area bedding were monitored, because earlier studies showed rates of environmental streptococcal and coliform IMI were greatest during the 14 d prior to calving compared with the remainder of the dry
period or during lactation (8, 21, 22). However, one variable that could not be accounted for in the present study was the stage of either the dry period or previous lactations from which infections present at calving originated. Coliform and environmental streptococcal IMI present at calving included those infections persisting from prior lactations, the early dry period, and those infections occurring near calving (22). Another factor that was not verified on each farm was the actual number of days prior and after calving that cows were maintained in maternity areas. Bacterial populations in long straw would have less influence on rates of IMI in herds that limited the amount of time cows were confined to maternity areas. Nevertheless, data in the present study did not
TABLE 2. Percent quarters infected at calving and drying off in seven herds using long straw bedding in maturity areas.' Intramammary infection status
Coliforms Environmental streptococci Staphylococcus species Corynebacterium bovis NLF' Staphylococcus aureus Other microbes Uninfected Contaminated samples
Drying off
Calving
X
SD
X
SD
2.7 2.4 10.1
.8 1.9
.9 3.0 12.9 9.8
.5 2.4 6.4 13.6
.3
.7
.4
.6 .5 12.1 2.0
.7
.7 .3 .1 81.3 1.1
3.4 1.0 1.1 .8 .4 5.8 1.5
.3 71.3 1.3
1 Mean number of quarters sampled among farms was 520 (308 SD)at calving, and 373 (162 SD) at drying off. Nonlactose-fermenting, gram-negative bacilli.
Journal of Dairy Science Vol. 72, No.1, 1989
HOGAN ETAL.
254
imply a relationship between bacterial counts in maternity bedding and mammary health at calving.
ials. In vitro studies showed organic bedding materials contained essential nutrients capable of supporting growth of coliforms and environmental streptococci (24). Despite the inorganic characteristics of sand and crushed limestone, these bedding materials contained > 10 6 cfu/g dry weight of both gram-negative bacteria and streptococci. Organic matter such as manure and feedstuffs contaminating bedding materials may have provided an exogenous source of essential nutrients for maintenance of bacterial populations. Jasper (15) reported that manurecontaminated bedding materials increased coliform populations during in vitro trials. Therefore, frequent removal of contaminated bedding was recommended to decrease bacterial numbers in bedding (7).
Organic Versus Inorganic Bedding
Independent comparisons among bacterial counts in materials used to bed lactating cows are in Table 3. Organic bedding materials had greater (P< .05) gram-negative bacterial, coliform, Klebsiella species, and streptococcal counts than did inorganic materials. These results agree with those in earlier trials conducted on experimental farms (10, 14). Basic among the ecological requirements for colonization and multiplication of bacteria are moisture, available nutrients, and proper temperature (19). Moisture and available nutrients appeared to be the ecological factors associated with greater bacterial counts in organic than inorganic beddings. Mean and standard errors DM percentages were 70.7 ± 2.8 for organic material compared with 94.7 ± 1.8 for inorganic matrials (P< .05). Although chemical analyses were not performed, carbon and other essential nutrients were also assumed to be more available in organic bedding than in inorganic mater-
Sawdust Versus Chopped Straw
Bacterial counts also differed between organic materials used to bed freestalls (Table 3). Mean and standard error Klebsiella species count in sawdust (4.8 ± .2) was greater than that in chopped straw (3.7 ± .2). Chopped straw had a significantly higher mean streptococcal count (7.8 ± .1) than did sawdust (7.1 ± .1). Gram-
TABLE 3. Independent comparisons among mean seasonal bacterial counts (cfu log,o /g dry weight) in materials used to bed lactating cows. Independent comparisons Bacterial counts
Organic' (n = 25)
Inorganic' (n = 11)
Sawdust (n = 13)
Chopped straw (n = 12)
Sand (n = 7)
Crushed limestone (n = 4)
7.1 a 0
6.4 b .1
7.0 .1
7.1 .1
6.3 .1
6.5 .1
6.2 a .1
5.7 b .1
6.2 .2
6.3 .1
5.7 .2
5.8 .1
4.P .1
3.4 b .2
4.8 a .2
3.7 b .2
3.2 .2
3.8 .2
7.5 a .1
6.8 b .1
7.P .1
7.8 b .1
7.0 .1
6.6 .1
Gr~-negative
X SE
Coliform
X SE
Kle'!..siella species X SE
Streptococcal
X SE
a,bMeans within comparisons with differing superscripts differ (P<.05). I
Organic bedding materials were sawdust and chopped straw.
• Inorganic bedding materials were sand and crushed limestone. Journal of Dairy Science Vol. 72, No. I, 1989
255
BACTERIAL COUNTS IN BEDDING TABLE 4. Mean bacterial counts (cfu log,o/g dry weight) in chopped straw bedding by seasons of the year.
Bacterial count
Gram-negative Coliform Klebsiella species Streptococcal
Summer (n = 3)
Spring (n = 3)
Winter (n = 3)
Fall (n = 3)
X
SE
X
SE
X
SE
X
SE
7.6 a 6.8 a 3.9 7.8
.0 .2
7.2 b 6.1 b 4.4 7.9
.1 .3 .2 .0
6.7 c 5.9 b 3.6 7.8
.3 .3 .3 .2
7.0 bc 6.2 b 3.2 7.8
.1 .2 .3 .2
.4
.2
a,b,cMeans within bacterial counts with differing superscripts differ (P<.05).
negative bacterial and coliform counts did not differ between organic bedding materials. Data in the present study were similar to those reported in a series of controlled experiments (20). Under controlled conditions, streptococcal counts were greater in straw than sawdust, and Klebsiella species counts were greater in fresh sawdust than in fresh chopped straw. However, Rendos et at. (20) reported Klebsiella species counts did not differ between chopped straw and sawdust after 7 d to 21 d of use in freestalls. Other researchers (6, 10, 11) have also suggested that Klebsiella species counts increased the first 7 d sawdust was used, but counts decreased after 21 d in freestalls. The number of days that bedding materials were in freestalls prior to collection for analyses was not documented in the present study. Bacterial counts in organic bedding differed among season of the year. Gram-negative bacterial (Table 4) and coliform (Table 5) counts in chopped straw were greater (P< .05) during summer than during other seasons. Although differences were not significant (P> .05), gram-
negative bacterial and coliform counts in sawdust were higher in summer and fall than in winter and spring (Table 5). Klebsiella species counts in sawdust were greater (P<.05) in summer and fall months than in winter" and spring. An interaction between seasons and bedding type on Klebsiella species counts was evidenced by greater Klebsiella species counts in sawdust were than those in chopped straw during summer and fall but not during winter and spring. Higher coliform counts in organic matter during summer and fall are probably related to higher ambient temperatures during these seasons. In vitro studies have shown coliforms in organic bedding multiplied most rapidly at temperatures between 30 and 40°C (3). Streptococcal counts did not differ among seasons of the year in either sawdust or chopped straw. Sand Versus Crushed limestone
Bacterial counts did not differ between sand and crushed limestone (Table 3). Inorganic
TABLE 5. Mean bacterial counts (cfu log,o/g dry weight) in sawdust bedding by seasons of the year.
Bacterial count
Summer (n = 3) X
Gram-negative Coliform Klebsiella species Streptococcal
7.1 6.5 5.5 a 6.9
Fall (n = 3) SE .3 .3 .2 .3
X 7.4 6.6 5.4 a 7.3
Spring (n = 3)
Winter (n = 3) SE .2 .3 .1 .1
X 6.8 5.8 4.1 b 7.4
SE .2 .1 .3 .3
X 6.6 5.7 3.9 b 6.9
SE .1 .4.4 .4
a,bMeans within bacterial counts with differing superscripts differ (P<.05). Journal of Dairy Science Vol. 72, No. 1,1989
HOGAN ETAL.
256
TABLE 6. Mean bacterial counts (cfu loglo/g dry weight) in inorganic' beddings by seasons of the year. Summer (n = 2)
Bacterial count
Gram-negative Coliform Klebsiella species Streptococcal
Fall (n = 3)
Winter
Spring
(n = 3)
(n = 3)
X
SE
X
SE
X
SE
X
SE
6.9 6.3 4.0 6.9
.0 .1 1.0 .1
6.4 5.7 3.5 6.7
.1 .2 .3 .4
6.2 5.3 2.9 7.0
.1 .2 .1
6.2 5.7 3.4 6.8
.3 .2 .3 .2
.0
1 Inorganic beddings were in two herds using sand and one herd using crushed limestone during fall, winter, and spring. During summer, 1 herd used sand and 1 herd used curshed limestone.
bedding materials did not differ among seasons of the year (Table 6). An important aspect of comparisons between inorganic materials in the present study was the low number of observations. Crushed limestone data were from a single farm using limestone particles with a geometric mean diameter of 2000 Ji. Sand used on both herds was washed prior to use as bedding. Further studies are needed to confirm bacterial counts in inorganic bedding and determing if there are economic advantages between using either sand or crushed limestone to bed lactating cows. The mean of each bacterial count measured was .;;; 10 7 cfulg dry weight throughout seasons of the year. Relationships Among Rates of Clinical Mastitis and Bacterial Counts in Bedding
Mean and SD rate of total clinical maStitiS cases among herds was .399 (.174 SE)/305 CD
(f)
1.0
... 0 I
.8
'"
•
• •• ••• • • # • • • • • • .-. • • • • r=.38
on
.6
0
(")
'(f) ril
... ...., ... u
.4
(f)
U
.2
Z
::l 0 5.5
6.0
6.5
7.0
7.5
Y=.399 + 088 (x-4.06)
0 I
r=.40
8
'on"
.6
0
(")
'(f) ril
... ...., ... u
•
•••
P(05
0 U
---
.4
(f)
U
2
Z
::l U
8.0
COLONY-FORMING UNITS/GRAM DRY WEIGHT
Figure 1. Linear relationship between rates of clinical mastitis during lactation and gram-negative bacterial counts in lactating cow bedding. Journal of Dairy Science Vol. 72, No.1, 1989
,
1.0
...
>-
P(.05
0 U
U
(f)
Y=.399 + .149 (x-6.92)
>-
during lactation. Percentage distribution of clinical cases during lactation by bacteriological statuses were coliforms, 36.3%; bacteriologically negative, 26.8%; environmental streptococci, 24.0%, and other statuses, 13 .9%. Linear relationships were significant (P< .05) among total rates of clinical mastitis during lactation and counts of gram-negative bacteria and Klebsiella species in lactating cow bedding (Figures 1 and 2). These relationships support the thesis that rate of environmental mastitis increases with increasing teat end exposure to environmental pathogens in bedding. Previous studies unable to demonstrate a relationship between bacterial counts in bedding and rates of IMl were short duration trials using relatively few cows (10, 17). Rate of coliform IMI was reported to be 1 IM11770 CD when cows were bedded on organic materials containing coliform counts > 106 cfu/g (22). Therefore, total CD exposure in both previous studies (10, 17) were too few
••
0 2.5
•
3.0
t
• 35
4.0
4.5
5.0
• 5.5
60
65
COLONY-FORMING UNITS/GRAM DRY WEIGHT
Figure 2. Linear relationship between rates of clinical mastitis during lactation and Klebisella species counts in lactating cow bedding.
257
BACTERIAL COUNTS IN BEDDING
TABLE 7. Mean rates' of clinical mastitis cases during lactation' in herds using sawdust. chopped straw. and inorganic materials to bed lactating cow freestalls. Bacteriological status of clinical cases
Inorganic' (n = 3)
Sawdust (n = 3)
Chopped straw (n = 3)
.113 .016
.149 .065
.147 .010
.188 .069
.082 .035
.080 .018
.488 .148
.366 .094
.342 .055
Coliform
X SD Environmental streptococci
X SD Total
X SD 1
I (Number of clinical cases/305 cow-day) + .0011 .
• From d 8 through end of lactation. 3
Inorganic bedding materials were sand and crushed limestone.
to expect a relationship between coliform bacterial counts in bedding and rate of coliform IMI to be demonstrated. Data in the present study were obtained over 12 mo in nine wellmanaged herds with varying rates of clinical mastitis and bacterial populations in bedding. Rates of clinical mastitis were used as the dependent variable in regression equations since rates of clinical mastitis were shown to reflect more accurately the dynamics of environmental mastitis than did prevalence of IMI (22). Data in the present study did support the inferences of others (2, 10, 17) that environmental mastitis is a multifactorial disease. Coefficients of determination (r 2 ) were';; .16 for relationships between total rates of clinical mastitis and both gram-negative bacterial and Klebsiella species counts. In addition, no other relationships tested were significant among rates of clinical mastitis and bacterial counts in bedding (P> .05). Rates of clinical mastitis did not differ among herds using different bedding materials (Table 7). Two important factors must be considered when interpreting relationships among bacterial counts in bedding and mammary health. An assumption that may not be valid is that bacterial counts derived from growth on selective and differential media are reflective of the mastitis pathogen load in bedding. More detailed bacteriological analyses than were used in the present trial would be needed to validate this assumption. The second
factor to be considered is that bedding is only one potential source of mastitis pathogens to the teat end. Possible interactions among bacterial exposure in bedding, milking hygiene, nutrition, and other factors affecting rates of environmental mastitis were outlined (12). CONCLUSIONS
Bacterial counts in lactating cow bedding differed among bedding materials and seasons of the year. Organic bedding materials contained significantly higher bacterial counts than did inorganic materials. Teat end exposure to gramnegative bacteria in bedding was greatest during summer and fall, which coincided with the highest rates of clinical mastitis during the year. Therefore, these data suggest that use of inorganic bedding decreases teat end exposure to environmental mastitis pathogens. Use of inorganic bedding materials appears to be most advantageous during summer and fall when gram-negative bacterial populations were greatest in bedding for lactating cows. REFERENCES 1 Bagley, S. T., and R. J. Sheidler. 1978. Primary Klebsiella identification with MacConkey-lnositolcarbenicillin agar. App!. Environ. Microbiol. 6: 536. 2 Bishop, J. R.• J. J. Janzen, A. B. Bodine. C. A. Caldwell, and D. W. Johnson. 1981. Dairy waste solids as a possible source of bedding. J. Dairy Sci. 64:706. Journal of Dairy Science Vol. 72, No.1, 1989
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