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Microbial Populations of Teat Ends of Dairy Cows, and Bedding Materials1
Microbial Populations of Teat Ends of Dairy Cows, and Bedding Materials ~ J. J. RENDOS, R. J. E B E R H A R T , and E. M. KESLER Departments of Dairy Science and Veterinary Science The Pennsylvania State University University Park 16802
infections may be unaffected (13, 29), or in some herds apparently increased (10, 12)when infection with S. agalactiae and S. aureus is reduced. Streptococcus uberis (16) or streptococci other than agalactiae (10) may respond less favorably to these measures than do S. agalactiae and S. aureus, the common contagious mastitogens. Most organisms which establish intramammary infections are believed to enter the gland through the teat canal. There is general belief in, and some evidence of (14, 17), a relationship between numbers of organisms reaching the teat end and incidence of new intramammary infection. Bedding materials are a portion of the cow's environment in direct and prolonged contact with teat ends. Bacterial populations of beddings may, therefore, influence the type and number of bacterial infections of the udder. Sawdust bedding previously has been implicated in the epizootiology of coliform mastiffs (9) and klebsiella mastiffs (19) in particular. The object of these studies was to characterize bacterial populations of common bedding materials and to determine the relationship of these populations to numbers of bacteria recoverable from teat ends.
ABSTRACT
The relationship between total coliform, klebsiella, streptococcal, and staphylococcal populations of common bedding materials and the organisms recoverable from the teat ends was investigated. Effects of sawdust, wood shavings, and wheat straw as bedding materials were studied in a 9 wk trial of Latin square design in which each of three five-cow groups was bedded for 3 wk on each of the beddings. Cows bedded on sawdust had greatest teat end populations of total coliforms and klebsiella. Streptococci were most numerous on straw-bedded cows. Staphylococci were more numerous on straw- and sawdust-bedded cows than on those bedded on shavings. These differences appeared to be related in a general way to bacterial populations in the beddings. Estimates of bacterial populations of both fresh and used beddings revealed differences among beddings. Differences among teat-swab counts of cow groups and among experimental periods indicated the importance of individual cow factors and uncontrolled environmental factors on teat end bacteria. INTRODUCTION
M A T E R I A L S A N D METHODS
Combined programs of post-milking teat dipping and dry cow therapy now make possible control of Streptococcus agalactiae and Staphylococcus aureus on a practical basis (13, 15). However, intramammary infections due to other organisms, predominantly those which appear to be contracted from the environment, may not be as effectively controlled by these measures. Thus, the incidence of coliform
Three groups of five cows each were maintained in stalls bedded with three common bedding materials: sawdust, wood shavings, and straw. The design was a Latin square with three experimental periods, each 3 wk. Assignment of cows to the three groups was by breed, lactation number, and infection status of the udder. Each of the three groups included three Holsteins and two Guernseys. ]'here were two animals in their first lactation and three older cows in each group. All cows were lactating throughout the trial. All cows had at least one front and one rear quarter free of bacterial infection (except Corynebacterium boris infections). Infection status of each quarter was
Received January 14, 1975. 1Authorized for publication as paper 4755 in the journal series of the Pennsylvania Agricultural Experiment Station. Authorized on August 22, 1974.
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BEDDING AND TEAT END BACTERIA known from previous routine herd samplings and was confirmed by culture of quarter milk samples during weeks 1, 4, 7, and 9 of the trial. Bedd i ng
The three bedding materials were hardwood sawdust, hardwood shavings, and wheat straw. Supplies of each were obtained at the beginning of the trial, and the same batch of each was used throughout the 9 wk. The straw and shavings were dry and appeared fresh and clean. The sawdust from an outdoor storage pile was wet, more than 1 yr old, and showed decomposition. Each group of five cows was maintained in a block of five adjacent stalls in a tie-stall barn. At the end of each 3 wk, the bedding material was changed and each stall thoroughly cleaned, hosed, and allowed to dry before being bedded with new material. All stalls were equipped with rubber mats, but were bedded heavily as if there were no mats. Manure was removed from the stalls once daily, and only enough fresh bedding was added to replace that removed with the feces. Cows were kept in the stalls day and night except for two 1-h periods each day when they were taken to the milking parlor. Samples of bedding material were taken once each week during the trial. Fresh bedding material was taken from unbroken straw bales or from the bedding carts. Samples of used bedding were taken once weekly (on the 7th, 14th, and 21st day of each experimental
Bovadine. (West Chemical Co., Long Island City, NY.) 3Swubes. (Falcon, Oxnard, CA.) 4proteose-peptone No. 5. (Difco Corp., Detroit, MI.)
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period) from beneath each of five cows in a group and composited before analysis. Bedding samples were prepared for plating on selective media by dilution in sterile saline. Ten g portions of shavings and sawdust were suspended in 90 ml saline. Straw first was chopped to facilitate handling and 5 g were suspended in 95 ml saline. After thorough shaking, 1:10 serial dilutions were prepared in sterile saline. Duplicate surface platings of .05 ml (occasionally, .10 ml for straw dilutions) of dilutions most likely to give countable plates were made. Sufficient bedding material was held at 4 C to prepare new dilutions if no countable plates were obtained. If a dilution yielded a colony count of 30 to 300 on one or both plates, both plates at that dilution were used to calculate a count per gram of wet bedding material. Teat Swabs
Teat swabs were obtained weekly from one front and one rear teat of quarters free of infection, the same teats being used each week. Teats were dipped after each milking in a commercial iodophor preparation containing 10,000 ppm z iodine. Swabs were obtained on the 7th, 14th, and 21st day of each experimental period at 1600 h, approximately 10 h after morning milking and 2 h before evening milking. Teats were not washed before swabbing but were cleaned with a dry paper towel when visible amounts of bedding material adhered to the teats. Sterile swabs 3 were prepared by addition of 1.3 ml of a maintenance broth containing .85% sodium chloride and .1% proteose-peptone 4. The maintenance broth also contained .2% sodium thiosulfate as a neutralizer for iodine remaining on the teats from the
TABLE 1. Procedures for plating teat swab samples. Dilutions plated, number of plates per dilution, and arbitrary teat swab counts assigned if highest dilution had colonies too numerous to be counted.
Medium Violet Red Bile Agar Modified Edwards Agar Period 1 Periods 2, 3 Staphylococcus 110
Arbitrary teat swab counts (colony-forming units/teat swab)
Dilutions plated
No. plates per dilution
1: 5, 1: 10
1
4,000
1:10, 1:20 1:10, 1:100 1 :300
1 1 2
10,000 50,000 150,000
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post-milking dip. Excess liquid was squeezed from the swab on the side of the tube, the swab was removed from the tube, held approximately perpendicular to the teat axis, and rotated three times on the apex of the teat. Swabs were returned to the maintenance broth, and the tube was held in an ice bath until dilution and plating within 3 h. Swab samples were prepared for plating by agitation of the tube, removal of excess broth from the swab by pressure against the tube wall, and discarding of the swab. Approximately 1.0 ml of broth was retained for dilution and plating. Dilutions greater than 1:20 required sterile saline blanks. Dilutions actually plated on the various media are in Table 1. The dilutions plated on Edward's medium during the first experimental period resulted in a relatively high proportion of uncountable plates. Therefore, for the remainder of the trial, a higher dilution was used. If, at the highest dilution, colonies were too numerous to be counted, arbitrary values in Table 1 were assigned. Samples of both bedding and teat swabs were surface plated on three selective media. Violet Red Bile Agar s (VRB) was used for estimation of total coliform and presumptive klebsiella counts, Edward's medium (11) with .02% sodium azide added to inhibit cotiforms (20) for streptococcus counts, and staphylococcus 110 s agar for staphylococcus counts. VRB and 110 plates were counted after 24 h incubation at 37 C, and modified Edward's plates after 48 h. On VRB plates, all lactosefermenting (red or pink) colonies were counted as coliforms. Efforts also were made to estimate klebsiella populations because these organisms have caused frequent, sometimes severe, infections in this herd. We believed that klebsiella colonies could be distinguished presumptively from other coliforms by their morphology on VRB. Klebsiella colonies were usually larger than those of other coliforms, were raised and mucoid, and were either entirely pink or red or pink with a translucent edge. To test the reliability of this presumptive identification, approximately 25 colonies believed to be kleb-
siella were isolated each week and screened biochemically. In addition, approximately 25 coliform colonies believed not to be klebsiella were isolated and screened similarly. Organisms which fermented lactose, utilized glucose with gas formation without producing H2S on Triple Sugar Iron Agar s , utilized citrate as evidenced by growth on Simmon's citrate agar s, and gave a positive urease 6 test were probable klebsiella. However, this scheme might include strains of enterobacter species. All typical streptococcal colonies on Edward's plates were counted to give total streptococcus counts. Staphylococcus counts included all white, cream, or yellow pigmented colonies on 110 agar. Preliminary studies showed that the translucent colonies sometimes observed were catalase negative. They were not counted as staphylococci. In analysis of bacteria counts from bedding, a logarithmic transformation was employed, the values being geometric means. The model for analysis of variance of fresh bedding bacteria counts was: count = mean + bedding type + error. The model employed for the used bedding materials was: count = mean + bedding type + week within period + (bedding type x week within period) + error. We determined the appropriate transformation for teat swab data by Tukey's test of additivity (23). The transformation logarithm of (count + 1) was accepted as it improved the P value, decreased the significance associated with it, and normalized the distribution of the data. Means were geometric. Factors in the analysis of variance of teat swab counts were bedding, cow group, experimental period, week within period, and front versus rear half of the udder. Elimination of interaction terms that were confounded by experimental design left the model: count = mean + bedding + group + period + week within period + udder half + (bedding x udder half) + (bedding x week within period) + (group x udder half) + (group x week within period) + (period x udder half) + (period x week within period) + error. We tested differences between individual means from analyses with significant differences by Duncan's New Multiple Range Test (1). RESULTS
SBBL (Division of Becton, Dickson and Co., Cockeysville, MD.) 6 Key Scientific Co., Los Angeles, CA. Journal of Dairy Science Vol. S8, No. 10
Over the 9 wk of the trial, 221 colonies thought to be klebsiella from VRB plates
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TABLE 2. Geometric means a of bacterial populations in fresh and used bedding.
Material
Total coliform
Klebsiella
Streptococci
Staphylococci
(Colony-forming units/g) Fresh bedding Sawdust Shavings Straw
6.1 X 103b 1.9 × 102 b 2.0 X 106c
1.0 X 103b 2.0 c .0 c
4.7 × 10 t b 7.0 b 4.7 × 103c
8.0X 103b 3.4 × 102 c 1.7 X 10 sb
Used bedding Sawdust Shavings Straw
5.2 × 107e 6.6 × 106e 3.1 × 106e
4.4 × 106e 6.6 × l 0 se 6.6 × 104e
1.1 × 10 ~e 8.6 X 106e 5.3 × 107f
3.1 × l 0 se 4.9 X 107f 2.2 × 109g
aMeans based on 9 observations. b'CMeans within fresh bedding columns with different superscripts are different (P<.O5). e'f'gMeans within used bedding columns with different superscripts are different (P<.05).
were isolated a n d s c r e e n e d b i o c h e m i c a l l y . Of these, 148 or 67% were j u d g e d p r o b a b l e klebsiella. Of 2 2 0 isolates t h o u g h t n o t to b e klebsiella, 23 or 10% were p r o b a b l e klebsiella b y our t e c h n i q u e s . Bedd i ng
G e o m e t r i c m e a n s of b a c t e r i a c o u n t s f r o m fresh and used b e d d i n g materials are in T a b l e 2. Differences in m e a n c o u n t s for each bacterial t y p e in fresh b e d d i n g materials were significant. T o t a l coliform, s t r e p t o c o c c u s , a n d s t a p h y l o c o c cus c o u n t s were highest in straw, i n t e r m e d i a t e in sawdust, a n d low in shavings. M o d e r a t e n u m b e r s of klebsiella were in fresh s a w d u s t , f e w in w o o d shavings, and n o n e in fresh straw. C o m p a r i s o n of t h e c o u n t s f r o m fresh bedding w i t h t h o s e f r o m used b e d d i n g revealed
t h a t p o p u l a t i o n s of all bacterial types increased in each b e d d i n g m a t e r i a l after use. Differences in b a c t e r i a l p o p u l a t i o n s a m o n g used b e d d i n g s were also significant. Differences in t o t a l colif o r m and ldebsiella c o u n t s a m o n g b e d d i n g s were significant o n l y at P < . 0 1 , b u t for b o t h bacterial t y p e s s a w d u s t a p p e a r e d to s u p p o r t t h e highest c o n c e n t r a t i o n s . S t r e p t o c o c c u s c o u n t s were highest in straw, a n d s a w d u s t h a d populat i o n s higher, b u t n o t significantly so, t h a n shavings. T h e r e were significant d i f f e r e n c e s in s t a p h y l o c o c c u s n u m b e r s in each t y p e o f bedding w i t h s t r a w s u p p o r t i n g t h e largest p o p u l a tion and shavings t h e smallest. C o u n t s f r o m the various b e d d i n g s b y w e e k w i t h i n p e r i o d are in T a b l e 3. D i f f e r e n c e s were significant for s t r e p t o c o c c i a n d s t a p h y l o c o c c i b u t n o t for t o t a l c o l i f o r m s or klebsiella. T h e r e
TABLE 3. Geometric means a of bacterial populations of used bedding materials by week within period (across all three bedding materials). Week within period
Total coliforms
Klebsiella
1 2 3
2.6 X 107b 4.4 X 106b 9.3 × 106b
(Colony-forming units/g) 5.8 × 10 sb 4.4 X 107b 3.8 X 10 sb 5.1 X 1 0 6 c 8.5 X 10 sb 2.3 × 107b
Streptococci
Staphylococci
4.0 X 10sb, c 1.2 X 10 sb 7.1 X 10 sc
aMeans based on 9 observations. b'CMeans within columns with different superscripts are different (P<.05). Journal of Dairy Science Vol. 58, No. 10
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TABLE 4. Geometric means of teat swab bacterial counts by bedding.
Bedding
Total coliform
Klebsiella
Sawdust Shavings Straw
127 a 12b 8b
(Colony-forming units recovered/swab) 11 a 383 a 2b 717b 1c 2064c
Streptococci
Staphylococci
7218 a 1366 b 9064 a
a'b'CMeans within columns with different superscripts are different (P<.05).
were no significant (bedding material x w e e k within period) interaction effects. Differences are not explained, but for none of the bacterial types was there a convincing trend toward increased populations over the 3 wk. Teat Swabs
During the 9 wk of the trial, 270 teat swabs were made. F r o m these, 268 total coliform, 262 klebsiella, 268 staphylococcus, and 239 streptococcus counts were obtained. (All teat swabs streptococcus counts were lost during the 8th wk due to an error in preparing Edward's m e d i u m . ) G e o m e t r i c means of teat swab counts for each of the bedding types are in Table 4. Total coliforms were m o s t c o m m o n on sawdust-bedded cows, but the difference b e t w e e n cows on shavings and straw was not significant. Klebsiella were also most c o m m o n in cows on sawdust and m o r e c o m m o n on shavings- than on straw-bedded cows. S t r e p t o c o c c u s counts were highest on cows on straw f o l l o w e d by those on shavings and lowest on sawdust-bedded cows. S t a p h y l o c o c c u s teat swab counts were higher in cows b e d d e d on straw than on sawdust, but the difference was not significant. Shavings-bedded cows had significantly lower staphylococcus counts than cows on o t h e r
beddings. Bacteria recovered f r o m teat swabs in the three experimental groups differed significantly (Table 5). For total coliforms, klebsiella, and streptococci, the ranking of the cow groups was the same f r o m highest to lowest: 1, 3, 2. For staphylococci, the ranking was 3, 1, 2. Main effects on teat swab counts were also significant with respect to experimental period (Table 6) and udder half (Table 7). There were no significant differences b e t w e e n weeks within period for any of the bacterial types, but there were significant (period x week within period) interactions for all bacterial types. Other significant interactions were (group x w e e k within period) for streptococci and staphylococci and (bedding x week within period) for streptococci only. In Table 8, bedding materials are aligned in decreasing order of numbers of each bacterial type recovered f r o m them. Similarly, beddings are ranked by recovery of each group of organisms f r o m teats of cows b e d d e d on them. A g r e e m e n t in ranking between populations in the bedding and numbers recoverable f r o m the cows' teats supports the concept that bedding material affects the bacterial flora of the teat end.
TABLE 5. Geometric means of teat swab bacterial counts by cow group.
Group
Total coliform
Klebsiella
1 2 3
47 a 11b 24 c
(Colony4orminguni~ recovered/swab) 5a 1690 a 1b 321 b 2c 1040 c
Streptococci
a'b'CMeans within columns with different superscripts are different (P<.05). Journal of Dairy Science Vo|. 5S, No. 10
Staphylococci
4,386 a 1,576 b 12,912 c
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TABLE 6. Geometric means of teat-swab bacterial counts by experimental period.
Period
Total coliform
Klebsiella
1 2 3
41 a 20 b 15 c
(Colony-forming uni~ recovered/swab) 4a 1483 a 1b 258 b 3c 1479 a
Streptococci
Staphylococci
10,565 a 6,556 b 1,288 c
a'b'CMeans within columns with different superscripts are different (P<.05).
DISCUSSION
Our study is an early effort to evaluate the role of c o m m o n bedding materials in the mastitis complex. Results probably have little bearing on the e p i z o o t o l o g y o f S. agalactiae and S. aureus, organisms which are t h o u g h t to be contagious and spread f r o m cow to cow (18) but may be significant for other forms of mastitis. While differences in bacterial populations of c o m m o n bedding materials and in numbers of organisms recoverable f r o m teats of cows on these beddings have been demonstrated, direct extrapolation to the influence of beddings on the mastitis p r o b l e m should be avoided. The pathogenic significance of the various bacterial species, which may comprise any of the groups of bacteria considered here, is uncertain. It is accepted that e x p e r i m e n t a l l y increasing the numbers of pathogens applied to the teat skin may result in greater numbers of i n t r a m a m m a r y infections. However, the exact relationship between these variables, and the degree to which their interaction is affected by m a n a g e m e n t and individual cow factors, is u n k n o w n . N o n e t h e less, the data do justify the conclusion that type of bedding p r o f o u n d l y may affect bacterial populations on the teat skin and suggest that
further w o r k on the relationship of bedding to mastitis may be useful. At present the o n l y direct approach to the p r o b l e m appears to be through lengthy and suitably controlled studies of the incidence of new infections in cows bedded on various materials. In the herd of which the experimental cows were a part, teat dipping with an effective dip after each milking had been practiced for several years and was c o n t i n u e d during the experiment. Therefore, in view of the r e p o r t e d success of teat antisepis in reducing bacterial colonization of the teat skin and orifice (16, 21), it seems likely that m o s t of the organisms recovered f r o m teat swabs were representative of a transient rather than a resident flora. The relatively long interval of a p p r o x i m a t e l y 10 h between previous teat antisepis and teat swabbing was chosen to permit accumulation of environmental bacteria on the teat skin. Coliform counts in used bedding material and f r o m teat swabs were probably largely Escbericbia coli, a much smaller p r o p o r t i o n of klebsiella, and variable numbers of other coilforms. While some strains of E. coil, klebsiella, and o t h e r coliforms may be mastitogenic, it is possible that other strains are not. Thus, no realistic assessment of the p r o p o r t i o n of the
TABLE 7. Geometric means of teat-swab bacterial counts, front quarters versus rear quarters.
Half of udder
Total coliform
Front Rear
20 a 28a
Klebsiella
Streptococci
(Colony-forming units recovered/swab) 3a 664 a 3a 1029 b
Staphylococci
2815 a 7093b
a'bMeans within columns with different superscripts are different (P<.05). Journal of Dairy Science Vol. 58, No. 10
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TABLE 8. Ranking from highest to lowest of bacterial numbers in various used beddings and bacterial numbers recovered from teat swabs of cows bedded on these materials. Total coliforms Used bedding Teat swabs
Sawdust ~>a Shavings > Straw Sawdust >b Shavings > Straw Klebsiella
Used bedding Teat swabs
Sawdust ~>Shavings ~>Straw Sawdust > Shavings > Straw Streptococci
Used bedding Teat swabs
Straw > Sawdust > Shavings Straw > Shavings > Sawdust Staphylococci
Used bedding Teat swabs
Straw > Sawdust > Shavings Straw ~>Sawdust > Shavings
a> difference (P>~.05). b> difference (P<.05).
total coliform population which was potentially pathogenic can be made. Many of the streptococci recovered were probably "fecal streptococci," a category of uncertain significance as a cause of mastitis. However, these counts also may have included appreciable numbers of S. uberis both in bedding and from teat skin. This suggestion appears reasonable in light of the discovery of S. uberis in pasture soils (6) and on body sites in direct contact with bedding, such as teat and udder skin (22, 25) and abdomen (4). Also, S. uberis infections are more common during winter months (4, 22). The influence of the streptococci in bedding on the incidence of "nonagalactiae" streptococcus infections merits further study. Staphylococci recovered from bedding materials and teat swabs were believed to be predominantly coagulase-negative, hence, not S. aureus. This supposition is based on the rarity of colonies with yellow or gold pigmentation on 110 medium, a characteristic claimed for most strains of S. aureus (3, 27). As infected udders were major reservoirs of specific strains of S. aureus (7) and their transmission from cow to cow was controlled effectively by a system of milking hygiene (8), it is unlikely Journal of Dairy Science Vol. 58, No. 10
that the environment, the bedding in particular, is an important reservoir of S. aureus. However, little is known about reservoirs of the coagulase-negative staphylococci, which are receiving increasing attention as mastitogens (24, 26). The variety of strains of S t a p b y l o c o c c u s epidermidis recovered from udders of a newly assembled research herd (2) appears to be compatible with the thought that some of these may be of environmental origin. Sawdust tended to support the highest populations of both total coliforms and klebsiella. Cows bedded on sawdust had the highest numbers of these organisms on their teat skin. Differences between sawdust and shavings in the number of soliforms on teat skin appear to depend in part on physical differences in the material. The smaller particle size of sawdust, with its accompanying increase in surface area and its greater capacity for water retention, may favor larger bacterial populations. Differences also may be explained partially by the wet and decomposing condition of the sawdust as compared to the fresh, clean state of the wood shavings. The higher number of bacteria on teat ends of sawdust-bedded cows also may be influenced by the tendency of small sawdust particles to adhere to the teat skin, especially in the teat orifice. The large populations of streptococci and staphylococci per g of straw were unexpected and might be explained partially by the lower density of straw compared to sawdust or shavings. Yet the high numbers of these organisms recovered from teat skin of straw-bedded cows suggest a need for further studies of these cocci. Cow group was a significant factor for each of the four groups of bacteria. This occurred despite efforts to assure that the cow groups were uniform. We could conclude only that bacterial populations on the teat ends are dependent on cow factors other than those considered in assigning cows in this experiment. Significant differences in teat end bacteria between experimental periods suggest that uncontrolled environmental factors greatly influence bacterial populations of teat ends. That the mean teat swab counts for coliform and staphylococcus counts decreased with each experimental period may be related to low ambient temperatures as the experiment progressed from late summer through early fall.
BEDDING AND TEAT END BACTERIA However, klebsiella and s t r e p t o c o c c u s c o u n t s , while d i f f e r e n t in the e x p e r i m e n t a l periods, did n o t f o l l o w the same trends. T h e objective o f this s t u d y was n o t t o c o m p a r e the n u m b e r s o f organisms in t h e various bacterial groups with t h o s e in the o t h e r groups. Initially, we were r e l u c t a n t to m a k e these c o m p a r i s o n s , fearing t h a t the selective media m i g h t inhibit to an u n k n o w n degree even t h o s e organisms for w h i c h t h e y are selective. However, p r e l i m i n a r y c o m p a r i s o n s o f g r o w t h o f strains of c o l i f o r m s on V R B and s t a p h y l o c o c c i on 110 agar with their g r o w t h on bovine b l o o d agar i n d i c a t e d t h a t plate c o u n t s were n o t l o w e r on the selective m e d i a and t h a t for 10 strains o f s t r e p t o c o c c i t h e m e a n r e d u c t i o n on E d w a r d ' s m e d i u m as c o m p a r e d to b l o o d agar was less t h a n 10% (28). Thus, the teat swab d a t a m a y p e r m i t s o m e c o m m e n t on relative p o p u l a t i o n s o f various bacterial groups on teat skin. Regardless o f b e d d i n g t y p e , s t a p h y l o c o c c i w e r e m o s t n u m e r o u s and s t r e p t o c o c c i were also c o m m o n , but t h e r e were relatively few coliforrns. These findings are in general a g r e e m e n t w i t h studies of Cullen and H e b e r t (5), w h o used a m o r e detailed classification o f the organisms on t e a t skin.
ACKNOWLEDGMENT The a u t h o r s a c k n o w l e d g e the assistance o f G. L. Hargrove in analysis o f the data, the advice of P. J. Glantz on microbiological p r o c e d u r e s , and the c o n s t r u c t i v e criticism o f the m a n u s c r i p t by F. H. S. N e w b o u l d .
REFERENCES 1 Alder, H. L., and E. B. Roessler. 1972. Page 163 in Introduction to probability and statistics, 5th ed. W. H. Freeman and Co., San Francisco, CA. 2 Brown, R. W., O. Sandvik, R. L. Scherer, and D. L. Rose. 1967. Differentiation of strains of Staphylococcus epidermidis isolated from bovine udders. J. Gen. Microbiol. 47:273. 3 Chapman, G. H. 1946. A single culture medium for selective isolation of plasma-coagulating staphylococci and for improved testing of chromogenesis, plasma coagulation, mannitol fermentation and the Stone reaction. J. Bacteriol. 51:409. 4 Cullen, G. A. 1966. The ecology of Streptococcus uberis. Brit. Vet. J. 122:333. 5 Cullen, G. A., and C. N. Hebert. 1967. Some ecological observations on microorganisms inhabiting bovine skin, teat canals and milk. Brit. Vet. J. 123:14. 6 Cullen, G. A., and T. W. A. Little. 1969. Isolation of Streptococcus uberis from the rumen of cows
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and from soil. Vet. Rec. 85:115. 7 Davidson, I. 1961. Observations on the pathogenic staphylococci in a dairy herd during a period of six years. Res. Vet. Sci. 2:22. 8 Davidson, 1. 1963. Experiments on controlling staphylococcal mastiffs. Res. Vet. Sci. 4:64. 9 Dodd, F. H., F. K. Neave, R. G. Kingwill, T. K. Griffin, and D. R. Westgarth. 1970. The effect of a mastiffs control system on levels of subclinical and clinical mastitis. Page 157 in Proc. V[ Int. Conf. on Cattle Diseases. Amer. Ass. Bovine Practitioners. Heritage Press, StiUwater, OK. 10 Eberhart, R. J., and J. M. Buckalew. 1972. Evaluation of a hygiene and dry period therapy program for mastitis control. J. Dairy Sci. 55:1683. 11 Edwards, S. J. 1933. Studies on bovine mastitis. IX. A selective medium for the diagnosis of bovine mastiffs. J. Comp. Pathol. Ther. 46:211. 12 Jasper, D. E. 1973. Coliform mastitis-A cause for concern. Page 59 in Proc. Ann. Mtg. Nat. Mastitis Council. 13 Kingwill, R. G. 1973. Effect of mastiffs control system on infections by the common pathogens. Page 50 in Proc. Ann. Mtg. Nat. Mastiffs Council. 14 McDonald, J. S., and R. A. Packer. 1968. Incidence of intramammary infections during lactation in dairy cattle repeatedly exposed to Streptococcus agalactiae and Aerobacter aerogenes. Amer. J. Vet. Res. 29:1525. 15 Natzke, R. P. 1970. A practical approach to mastitis control. Page 166 in Proc. VI Int. Conf. on Cattle Diseases. Amer. Ass. Bovine Practitioners. Heritage Press, Stillwater, OK. 16 Neave, F. K. 1971. The control of mastitis by hygiene. Page 55 in Control of bovine mastiffs. British Cattle Veterinary Ass. Gresham Press, Old Working, Surrey, England. 17 Neave, F. K., and J. Oliver. 1962. Relationship between the number of mastitis pathogens placed on the teats of dry cows, their survival, and the amount of intramammary infection caused. J. Dairy Res. 29:79. 18 Newbould, F. H. S. 1965. Disinfection in the prevention of udder infections. A review. Can. Vet. J. 6:29. 19 Newman, L. E., and J. ]. Kowalski. 1973. Fresh sawdust bedding-a possible source of KlebsieUa organisms. Am. J. Vet. Res. 34:979. 20 Packer, R. A. 1943. The use of sodium azide (NaN 3) and crystal violet in a selective medium for Streptococci and Erysipelotbrix rbusiopatbiae. J. Bacteriol. 46: 343. 21 Schultze, W. D., and J. W. Smith. 1970. Effectiveness of chlorhexidine in a postmilking teat dip. J. Dairy Sci. 53:38. 22 Sharma, R. M., and R. A. Packer. 1970. Occurrence and ecological features of Streptococcus uberis in the dairy cow. Am. J. Vet. Res. 3l:1197. 23 Snedecor, G. W. 1956. Page 321 in Statistical methods, 5th ed. Iowa State College Press, Ames. 24 Stabenfeldt, G. H., and G. R. Spencer. 1966. The lesions in bovine udders shedding nonhemolytic Journal of Dairy Science Vol. 58, No. 10
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coagulase-negative staphylococci. Pathol. Vet. 3:27. 25 Sweeney, E. J. 1964. Observations on the epidemiology of mastitis due to Streptococcus uberis in a laboratory herd during a complete lactation. Res. Vet. Sci. 5:483. 26 Ward, G. E., and L. H. Schultz. 1972. Relationship of somatic cells in quarter milk to type of bacteria and production. J. Dairy Sci. 55:1428.
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28 29
Vellila, D. M., J. E. Faber, and M. J. Pelczar. 1947. A technique for the isolation of coagulaseproducing staphylococci from milk in bovine mastitis. Am. J. Vet. Res. 8:275. Zarkower, A. 1974. Manuscript in preparation. Ziv, G. 1971. Mastitis control in the moshav (family-type) dairy herd in Israel. i. Effects of milking hygiene and antibiotic t r e a t m e n t on new infections. Refuah Vet. 28 : 1.
infections may be unaffected (13, 29), or in some herds apparently increased (10, 12)when infection with S. agalactiae and S. aureus is reduced. Streptococcus uberis (16) or streptococci other than agalactiae (10) may respond less favorably to these measures than do S. agalactiae and S. aureus, the common contagious mastitogens. Most organisms which establish intramammary infections are believed to enter the gland through the teat canal. There is general belief in, and some evidence of (14, 17), a relationship between numbers of organisms reaching the teat end and incidence of new intramammary infection. Bedding materials are a portion of the cow's environment in direct and prolonged contact with teat ends. Bacterial populations of beddings may, therefore, influence the type and number of bacterial infections of the udder. Sawdust bedding previously has been implicated in the epizootiology of coliform mastiffs (9) and klebsiella mastiffs (19) in particular. The object of these studies was to characterize bacterial populations of common bedding materials and to determine the relationship of these populations to numbers of bacteria recoverable from teat ends.
ABSTRACT
The relationship between total coliform, klebsiella, streptococcal, and staphylococcal populations of common bedding materials and the organisms recoverable from the teat ends was investigated. Effects of sawdust, wood shavings, and wheat straw as bedding materials were studied in a 9 wk trial of Latin square design in which each of three five-cow groups was bedded for 3 wk on each of the beddings. Cows bedded on sawdust had greatest teat end populations of total coliforms and klebsiella. Streptococci were most numerous on straw-bedded cows. Staphylococci were more numerous on straw- and sawdust-bedded cows than on those bedded on shavings. These differences appeared to be related in a general way to bacterial populations in the beddings. Estimates of bacterial populations of both fresh and used beddings revealed differences among beddings. Differences among teat-swab counts of cow groups and among experimental periods indicated the importance of individual cow factors and uncontrolled environmental factors on teat end bacteria. INTRODUCTION
M A T E R I A L S A N D METHODS
Combined programs of post-milking teat dipping and dry cow therapy now make possible control of Streptococcus agalactiae and Staphylococcus aureus on a practical basis (13, 15). However, intramammary infections due to other organisms, predominantly those which appear to be contracted from the environment, may not be as effectively controlled by these measures. Thus, the incidence of coliform
Three groups of five cows each were maintained in stalls bedded with three common bedding materials: sawdust, wood shavings, and straw. The design was a Latin square with three experimental periods, each 3 wk. Assignment of cows to the three groups was by breed, lactation number, and infection status of the udder. Each of the three groups included three Holsteins and two Guernseys. ]'here were two animals in their first lactation and three older cows in each group. All cows were lactating throughout the trial. All cows had at least one front and one rear quarter free of bacterial infection (except Corynebacterium boris infections). Infection status of each quarter was
Received January 14, 1975. 1Authorized for publication as paper 4755 in the journal series of the Pennsylvania Agricultural Experiment Station. Authorized on August 22, 1974.
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BEDDING AND TEAT END BACTERIA known from previous routine herd samplings and was confirmed by culture of quarter milk samples during weeks 1, 4, 7, and 9 of the trial. Bedd i ng
The three bedding materials were hardwood sawdust, hardwood shavings, and wheat straw. Supplies of each were obtained at the beginning of the trial, and the same batch of each was used throughout the 9 wk. The straw and shavings were dry and appeared fresh and clean. The sawdust from an outdoor storage pile was wet, more than 1 yr old, and showed decomposition. Each group of five cows was maintained in a block of five adjacent stalls in a tie-stall barn. At the end of each 3 wk, the bedding material was changed and each stall thoroughly cleaned, hosed, and allowed to dry before being bedded with new material. All stalls were equipped with rubber mats, but were bedded heavily as if there were no mats. Manure was removed from the stalls once daily, and only enough fresh bedding was added to replace that removed with the feces. Cows were kept in the stalls day and night except for two 1-h periods each day when they were taken to the milking parlor. Samples of bedding material were taken once each week during the trial. Fresh bedding material was taken from unbroken straw bales or from the bedding carts. Samples of used bedding were taken once weekly (on the 7th, 14th, and 21st day of each experimental
Bovadine. (West Chemical Co., Long Island City, NY.) 3Swubes. (Falcon, Oxnard, CA.) 4proteose-peptone No. 5. (Difco Corp., Detroit, MI.)
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period) from beneath each of five cows in a group and composited before analysis. Bedding samples were prepared for plating on selective media by dilution in sterile saline. Ten g portions of shavings and sawdust were suspended in 90 ml saline. Straw first was chopped to facilitate handling and 5 g were suspended in 95 ml saline. After thorough shaking, 1:10 serial dilutions were prepared in sterile saline. Duplicate surface platings of .05 ml (occasionally, .10 ml for straw dilutions) of dilutions most likely to give countable plates were made. Sufficient bedding material was held at 4 C to prepare new dilutions if no countable plates were obtained. If a dilution yielded a colony count of 30 to 300 on one or both plates, both plates at that dilution were used to calculate a count per gram of wet bedding material. Teat Swabs
Teat swabs were obtained weekly from one front and one rear teat of quarters free of infection, the same teats being used each week. Teats were dipped after each milking in a commercial iodophor preparation containing 10,000 ppm z iodine. Swabs were obtained on the 7th, 14th, and 21st day of each experimental period at 1600 h, approximately 10 h after morning milking and 2 h before evening milking. Teats were not washed before swabbing but were cleaned with a dry paper towel when visible amounts of bedding material adhered to the teats. Sterile swabs 3 were prepared by addition of 1.3 ml of a maintenance broth containing .85% sodium chloride and .1% proteose-peptone 4. The maintenance broth also contained .2% sodium thiosulfate as a neutralizer for iodine remaining on the teats from the
TABLE 1. Procedures for plating teat swab samples. Dilutions plated, number of plates per dilution, and arbitrary teat swab counts assigned if highest dilution had colonies too numerous to be counted.
Medium Violet Red Bile Agar Modified Edwards Agar Period 1 Periods 2, 3 Staphylococcus 110
Arbitrary teat swab counts (colony-forming units/teat swab)
Dilutions plated
No. plates per dilution
1: 5, 1: 10
1
4,000
1:10, 1:20 1:10, 1:100 1 :300
1 1 2
10,000 50,000 150,000
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post-milking dip. Excess liquid was squeezed from the swab on the side of the tube, the swab was removed from the tube, held approximately perpendicular to the teat axis, and rotated three times on the apex of the teat. Swabs were returned to the maintenance broth, and the tube was held in an ice bath until dilution and plating within 3 h. Swab samples were prepared for plating by agitation of the tube, removal of excess broth from the swab by pressure against the tube wall, and discarding of the swab. Approximately 1.0 ml of broth was retained for dilution and plating. Dilutions greater than 1:20 required sterile saline blanks. Dilutions actually plated on the various media are in Table 1. The dilutions plated on Edward's medium during the first experimental period resulted in a relatively high proportion of uncountable plates. Therefore, for the remainder of the trial, a higher dilution was used. If, at the highest dilution, colonies were too numerous to be counted, arbitrary values in Table 1 were assigned. Samples of both bedding and teat swabs were surface plated on three selective media. Violet Red Bile Agar s (VRB) was used for estimation of total coliform and presumptive klebsiella counts, Edward's medium (11) with .02% sodium azide added to inhibit cotiforms (20) for streptococcus counts, and staphylococcus 110 s agar for staphylococcus counts. VRB and 110 plates were counted after 24 h incubation at 37 C, and modified Edward's plates after 48 h. On VRB plates, all lactosefermenting (red or pink) colonies were counted as coliforms. Efforts also were made to estimate klebsiella populations because these organisms have caused frequent, sometimes severe, infections in this herd. We believed that klebsiella colonies could be distinguished presumptively from other coliforms by their morphology on VRB. Klebsiella colonies were usually larger than those of other coliforms, were raised and mucoid, and were either entirely pink or red or pink with a translucent edge. To test the reliability of this presumptive identification, approximately 25 colonies believed to be kleb-
siella were isolated each week and screened biochemically. In addition, approximately 25 coliform colonies believed not to be klebsiella were isolated and screened similarly. Organisms which fermented lactose, utilized glucose with gas formation without producing H2S on Triple Sugar Iron Agar s , utilized citrate as evidenced by growth on Simmon's citrate agar s, and gave a positive urease 6 test were probable klebsiella. However, this scheme might include strains of enterobacter species. All typical streptococcal colonies on Edward's plates were counted to give total streptococcus counts. Staphylococcus counts included all white, cream, or yellow pigmented colonies on 110 agar. Preliminary studies showed that the translucent colonies sometimes observed were catalase negative. They were not counted as staphylococci. In analysis of bacteria counts from bedding, a logarithmic transformation was employed, the values being geometric means. The model for analysis of variance of fresh bedding bacteria counts was: count = mean + bedding type + error. The model employed for the used bedding materials was: count = mean + bedding type + week within period + (bedding type x week within period) + error. We determined the appropriate transformation for teat swab data by Tukey's test of additivity (23). The transformation logarithm of (count + 1) was accepted as it improved the P value, decreased the significance associated with it, and normalized the distribution of the data. Means were geometric. Factors in the analysis of variance of teat swab counts were bedding, cow group, experimental period, week within period, and front versus rear half of the udder. Elimination of interaction terms that were confounded by experimental design left the model: count = mean + bedding + group + period + week within period + udder half + (bedding x udder half) + (bedding x week within period) + (group x udder half) + (group x week within period) + (period x udder half) + (period x week within period) + error. We tested differences between individual means from analyses with significant differences by Duncan's New Multiple Range Test (1). RESULTS
SBBL (Division of Becton, Dickson and Co., Cockeysville, MD.) 6 Key Scientific Co., Los Angeles, CA. Journal of Dairy Science Vol. S8, No. 10
Over the 9 wk of the trial, 221 colonies thought to be klebsiella from VRB plates
BEDDING AND TEAT END BACTERIA
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TABLE 2. Geometric means a of bacterial populations in fresh and used bedding.
Material
Total coliform
Klebsiella
Streptococci
Staphylococci
(Colony-forming units/g) Fresh bedding Sawdust Shavings Straw
6.1 X 103b 1.9 × 102 b 2.0 X 106c
1.0 X 103b 2.0 c .0 c
4.7 × 10 t b 7.0 b 4.7 × 103c
8.0X 103b 3.4 × 102 c 1.7 X 10 sb
Used bedding Sawdust Shavings Straw
5.2 × 107e 6.6 × 106e 3.1 × 106e
4.4 × 106e 6.6 × l 0 se 6.6 × 104e
1.1 × 10 ~e 8.6 X 106e 5.3 × 107f
3.1 × l 0 se 4.9 X 107f 2.2 × 109g
aMeans based on 9 observations. b'CMeans within fresh bedding columns with different superscripts are different (P<.O5). e'f'gMeans within used bedding columns with different superscripts are different (P<.05).
were isolated a n d s c r e e n e d b i o c h e m i c a l l y . Of these, 148 or 67% were j u d g e d p r o b a b l e klebsiella. Of 2 2 0 isolates t h o u g h t n o t to b e klebsiella, 23 or 10% were p r o b a b l e klebsiella b y our t e c h n i q u e s . Bedd i ng
G e o m e t r i c m e a n s of b a c t e r i a c o u n t s f r o m fresh and used b e d d i n g materials are in T a b l e 2. Differences in m e a n c o u n t s for each bacterial t y p e in fresh b e d d i n g materials were significant. T o t a l coliform, s t r e p t o c o c c u s , a n d s t a p h y l o c o c cus c o u n t s were highest in straw, i n t e r m e d i a t e in sawdust, a n d low in shavings. M o d e r a t e n u m b e r s of klebsiella were in fresh s a w d u s t , f e w in w o o d shavings, and n o n e in fresh straw. C o m p a r i s o n of t h e c o u n t s f r o m fresh bedding w i t h t h o s e f r o m used b e d d i n g revealed
t h a t p o p u l a t i o n s of all bacterial types increased in each b e d d i n g m a t e r i a l after use. Differences in b a c t e r i a l p o p u l a t i o n s a m o n g used b e d d i n g s were also significant. Differences in t o t a l colif o r m and ldebsiella c o u n t s a m o n g b e d d i n g s were significant o n l y at P < . 0 1 , b u t for b o t h bacterial t y p e s s a w d u s t a p p e a r e d to s u p p o r t t h e highest c o n c e n t r a t i o n s . S t r e p t o c o c c u s c o u n t s were highest in straw, a n d s a w d u s t h a d populat i o n s higher, b u t n o t significantly so, t h a n shavings. T h e r e were significant d i f f e r e n c e s in s t a p h y l o c o c c u s n u m b e r s in each t y p e o f bedding w i t h s t r a w s u p p o r t i n g t h e largest p o p u l a tion and shavings t h e smallest. C o u n t s f r o m the various b e d d i n g s b y w e e k w i t h i n p e r i o d are in T a b l e 3. D i f f e r e n c e s were significant for s t r e p t o c o c c i a n d s t a p h y l o c o c c i b u t n o t for t o t a l c o l i f o r m s or klebsiella. T h e r e
TABLE 3. Geometric means a of bacterial populations of used bedding materials by week within period (across all three bedding materials). Week within period
Total coliforms
Klebsiella
1 2 3
2.6 X 107b 4.4 X 106b 9.3 × 106b
(Colony-forming units/g) 5.8 × 10 sb 4.4 X 107b 3.8 X 10 sb 5.1 X 1 0 6 c 8.5 X 10 sb 2.3 × 107b
Streptococci
Staphylococci
4.0 X 10sb, c 1.2 X 10 sb 7.1 X 10 sc
aMeans based on 9 observations. b'CMeans within columns with different superscripts are different (P<.05). Journal of Dairy Science Vol. 58, No. 10
RENDOS ET AL.
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TABLE 4. Geometric means of teat swab bacterial counts by bedding.
Bedding
Total coliform
Klebsiella
Sawdust Shavings Straw
127 a 12b 8b
(Colony-forming units recovered/swab) 11 a 383 a 2b 717b 1c 2064c
Streptococci
Staphylococci
7218 a 1366 b 9064 a
a'b'CMeans within columns with different superscripts are different (P<.05).
were no significant (bedding material x w e e k within period) interaction effects. Differences are not explained, but for none of the bacterial types was there a convincing trend toward increased populations over the 3 wk. Teat Swabs
During the 9 wk of the trial, 270 teat swabs were made. F r o m these, 268 total coliform, 262 klebsiella, 268 staphylococcus, and 239 streptococcus counts were obtained. (All teat swabs streptococcus counts were lost during the 8th wk due to an error in preparing Edward's m e d i u m . ) G e o m e t r i c means of teat swab counts for each of the bedding types are in Table 4. Total coliforms were m o s t c o m m o n on sawdust-bedded cows, but the difference b e t w e e n cows on shavings and straw was not significant. Klebsiella were also most c o m m o n in cows on sawdust and m o r e c o m m o n on shavings- than on straw-bedded cows. S t r e p t o c o c c u s counts were highest on cows on straw f o l l o w e d by those on shavings and lowest on sawdust-bedded cows. S t a p h y l o c o c c u s teat swab counts were higher in cows b e d d e d on straw than on sawdust, but the difference was not significant. Shavings-bedded cows had significantly lower staphylococcus counts than cows on o t h e r
beddings. Bacteria recovered f r o m teat swabs in the three experimental groups differed significantly (Table 5). For total coliforms, klebsiella, and streptococci, the ranking of the cow groups was the same f r o m highest to lowest: 1, 3, 2. For staphylococci, the ranking was 3, 1, 2. Main effects on teat swab counts were also significant with respect to experimental period (Table 6) and udder half (Table 7). There were no significant differences b e t w e e n weeks within period for any of the bacterial types, but there were significant (period x week within period) interactions for all bacterial types. Other significant interactions were (group x w e e k within period) for streptococci and staphylococci and (bedding x week within period) for streptococci only. In Table 8, bedding materials are aligned in decreasing order of numbers of each bacterial type recovered f r o m them. Similarly, beddings are ranked by recovery of each group of organisms f r o m teats of cows b e d d e d on them. A g r e e m e n t in ranking between populations in the bedding and numbers recoverable f r o m the cows' teats supports the concept that bedding material affects the bacterial flora of the teat end.
TABLE 5. Geometric means of teat swab bacterial counts by cow group.
Group
Total coliform
Klebsiella
1 2 3
47 a 11b 24 c
(Colony4orminguni~ recovered/swab) 5a 1690 a 1b 321 b 2c 1040 c
Streptococci
a'b'CMeans within columns with different superscripts are different (P<.05). Journal of Dairy Science Vo|. 5S, No. 10
Staphylococci
4,386 a 1,576 b 12,912 c
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TABLE 6. Geometric means of teat-swab bacterial counts by experimental period.
Period
Total coliform
Klebsiella
1 2 3
41 a 20 b 15 c
(Colony-forming uni~ recovered/swab) 4a 1483 a 1b 258 b 3c 1479 a
Streptococci
Staphylococci
10,565 a 6,556 b 1,288 c
a'b'CMeans within columns with different superscripts are different (P<.05).
DISCUSSION
Our study is an early effort to evaluate the role of c o m m o n bedding materials in the mastitis complex. Results probably have little bearing on the e p i z o o t o l o g y o f S. agalactiae and S. aureus, organisms which are t h o u g h t to be contagious and spread f r o m cow to cow (18) but may be significant for other forms of mastitis. While differences in bacterial populations of c o m m o n bedding materials and in numbers of organisms recoverable f r o m teats of cows on these beddings have been demonstrated, direct extrapolation to the influence of beddings on the mastitis p r o b l e m should be avoided. The pathogenic significance of the various bacterial species, which may comprise any of the groups of bacteria considered here, is uncertain. It is accepted that e x p e r i m e n t a l l y increasing the numbers of pathogens applied to the teat skin may result in greater numbers of i n t r a m a m m a r y infections. However, the exact relationship between these variables, and the degree to which their interaction is affected by m a n a g e m e n t and individual cow factors, is u n k n o w n . N o n e t h e less, the data do justify the conclusion that type of bedding p r o f o u n d l y may affect bacterial populations on the teat skin and suggest that
further w o r k on the relationship of bedding to mastitis may be useful. At present the o n l y direct approach to the p r o b l e m appears to be through lengthy and suitably controlled studies of the incidence of new infections in cows bedded on various materials. In the herd of which the experimental cows were a part, teat dipping with an effective dip after each milking had been practiced for several years and was c o n t i n u e d during the experiment. Therefore, in view of the r e p o r t e d success of teat antisepis in reducing bacterial colonization of the teat skin and orifice (16, 21), it seems likely that m o s t of the organisms recovered f r o m teat swabs were representative of a transient rather than a resident flora. The relatively long interval of a p p r o x i m a t e l y 10 h between previous teat antisepis and teat swabbing was chosen to permit accumulation of environmental bacteria on the teat skin. Coliform counts in used bedding material and f r o m teat swabs were probably largely Escbericbia coli, a much smaller p r o p o r t i o n of klebsiella, and variable numbers of other coilforms. While some strains of E. coil, klebsiella, and o t h e r coliforms may be mastitogenic, it is possible that other strains are not. Thus, no realistic assessment of the p r o p o r t i o n of the
TABLE 7. Geometric means of teat-swab bacterial counts, front quarters versus rear quarters.
Half of udder
Total coliform
Front Rear
20 a 28a
Klebsiella
Streptococci
(Colony-forming units recovered/swab) 3a 664 a 3a 1029 b
Staphylococci
2815 a 7093b
a'bMeans within columns with different superscripts are different (P<.05). Journal of Dairy Science Vol. 58, No. 10
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TABLE 8. Ranking from highest to lowest of bacterial numbers in various used beddings and bacterial numbers recovered from teat swabs of cows bedded on these materials. Total coliforms Used bedding Teat swabs
Sawdust ~>a Shavings > Straw Sawdust >b Shavings > Straw Klebsiella
Used bedding Teat swabs
Sawdust ~>Shavings ~>Straw Sawdust > Shavings > Straw Streptococci
Used bedding Teat swabs
Straw > Sawdust > Shavings Straw > Shavings > Sawdust Staphylococci
Used bedding Teat swabs
Straw > Sawdust > Shavings Straw ~>Sawdust > Shavings
a> difference (P>~.05). b> difference (P<.05).
total coliform population which was potentially pathogenic can be made. Many of the streptococci recovered were probably "fecal streptococci," a category of uncertain significance as a cause of mastitis. However, these counts also may have included appreciable numbers of S. uberis both in bedding and from teat skin. This suggestion appears reasonable in light of the discovery of S. uberis in pasture soils (6) and on body sites in direct contact with bedding, such as teat and udder skin (22, 25) and abdomen (4). Also, S. uberis infections are more common during winter months (4, 22). The influence of the streptococci in bedding on the incidence of "nonagalactiae" streptococcus infections merits further study. Staphylococci recovered from bedding materials and teat swabs were believed to be predominantly coagulase-negative, hence, not S. aureus. This supposition is based on the rarity of colonies with yellow or gold pigmentation on 110 medium, a characteristic claimed for most strains of S. aureus (3, 27). As infected udders were major reservoirs of specific strains of S. aureus (7) and their transmission from cow to cow was controlled effectively by a system of milking hygiene (8), it is unlikely Journal of Dairy Science Vol. 58, No. 10
that the environment, the bedding in particular, is an important reservoir of S. aureus. However, little is known about reservoirs of the coagulase-negative staphylococci, which are receiving increasing attention as mastitogens (24, 26). The variety of strains of S t a p b y l o c o c c u s epidermidis recovered from udders of a newly assembled research herd (2) appears to be compatible with the thought that some of these may be of environmental origin. Sawdust tended to support the highest populations of both total coliforms and klebsiella. Cows bedded on sawdust had the highest numbers of these organisms on their teat skin. Differences between sawdust and shavings in the number of soliforms on teat skin appear to depend in part on physical differences in the material. The smaller particle size of sawdust, with its accompanying increase in surface area and its greater capacity for water retention, may favor larger bacterial populations. Differences also may be explained partially by the wet and decomposing condition of the sawdust as compared to the fresh, clean state of the wood shavings. The higher number of bacteria on teat ends of sawdust-bedded cows also may be influenced by the tendency of small sawdust particles to adhere to the teat skin, especially in the teat orifice. The large populations of streptococci and staphylococci per g of straw were unexpected and might be explained partially by the lower density of straw compared to sawdust or shavings. Yet the high numbers of these organisms recovered from teat skin of straw-bedded cows suggest a need for further studies of these cocci. Cow group was a significant factor for each of the four groups of bacteria. This occurred despite efforts to assure that the cow groups were uniform. We could conclude only that bacterial populations on the teat ends are dependent on cow factors other than those considered in assigning cows in this experiment. Significant differences in teat end bacteria between experimental periods suggest that uncontrolled environmental factors greatly influence bacterial populations of teat ends. That the mean teat swab counts for coliform and staphylococcus counts decreased with each experimental period may be related to low ambient temperatures as the experiment progressed from late summer through early fall.
BEDDING AND TEAT END BACTERIA However, klebsiella and s t r e p t o c o c c u s c o u n t s , while d i f f e r e n t in the e x p e r i m e n t a l periods, did n o t f o l l o w the same trends. T h e objective o f this s t u d y was n o t t o c o m p a r e the n u m b e r s o f organisms in t h e various bacterial groups with t h o s e in the o t h e r groups. Initially, we were r e l u c t a n t to m a k e these c o m p a r i s o n s , fearing t h a t the selective media m i g h t inhibit to an u n k n o w n degree even t h o s e organisms for w h i c h t h e y are selective. However, p r e l i m i n a r y c o m p a r i s o n s o f g r o w t h o f strains of c o l i f o r m s on V R B and s t a p h y l o c o c c i on 110 agar with their g r o w t h on bovine b l o o d agar i n d i c a t e d t h a t plate c o u n t s were n o t l o w e r on the selective m e d i a and t h a t for 10 strains o f s t r e p t o c o c c i t h e m e a n r e d u c t i o n on E d w a r d ' s m e d i u m as c o m p a r e d to b l o o d agar was less t h a n 10% (28). Thus, the teat swab d a t a m a y p e r m i t s o m e c o m m e n t on relative p o p u l a t i o n s o f various bacterial groups on teat skin. Regardless o f b e d d i n g t y p e , s t a p h y l o c o c c i w e r e m o s t n u m e r o u s and s t r e p t o c o c c i were also c o m m o n , but t h e r e were relatively few coliforrns. These findings are in general a g r e e m e n t w i t h studies of Cullen and H e b e r t (5), w h o used a m o r e detailed classification o f the organisms on t e a t skin.
ACKNOWLEDGMENT The a u t h o r s a c k n o w l e d g e the assistance o f G. L. Hargrove in analysis o f the data, the advice of P. J. Glantz on microbiological p r o c e d u r e s , and the c o n s t r u c t i v e criticism o f the m a n u s c r i p t by F. H. S. N e w b o u l d .
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