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Animal Welfare Program, Faculty of Agricultural Sciences, 2357 Main Mall, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4
Corresponding author: D. M. Weary; e-mail: danweary{at}interchange.ubc.ca.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Key Words: bacterial count • sawdust • sand • teat end
Abbreviation key: MC = MacConkey, MCIC = MacConkey-inositiol-carbenicillin
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Dairy cattle can spend between 40 and 65% of their time lying down (e.g., Manninen et al., 2002; Tucker et al., 2003), and bacteria can be transferred between the lying surface and the teats (Hogan and Smith, 1997; Hogan et al., 1999). Thus, in an effort to minimize teat end exposure to pathogenic microorganisms, it is important to understand the extent to which the lying surface contributes to the proliferation of bacteria. Previous work in this area focused on the differences in bacterial counts between inorganic and organic bedding (Fairchild et al., 1982; Janzen et al., 1982). Counts of bacteria in inorganic bedding are usually lower than those in organic bedding, depending on the bacterial strain and the type of material (Fairchild et al., 1982). Availability of nutrients, amount of moisture available, pH, and stall cleanliness are the main factors thought to affect bacterial growth in bedding (National Mastitis Council, 1996). Sand bedding is becoming increasingly popular on North American dairy farms, in part, because farmers perceive an improvement in udder health and cow comfort (Bewley et al., 2001). However, there has been little work to date examining the bacterial strains prevalent in sand bedding (e.g., Fairchild et al., 1982; Hogan et al., 1989).
This experiment was designed to compare bacterial populations of mastitis-causing organisms on teats of cows housed on sand and on sawdust bedding. In addition, this experiment examined the relationship between the numbers of bacteria in bedding and on teat ends. Moreover, stall and udder cleanliness were assessed and compared for cows housed on sand and sawdust bedding. The associations between bacterial counts in bedding and the DM of the bedding and stall cleanliness were also explored.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Experimental Design
The experiment was a crossover design with 2 groups of Holstein cows. Each group rotated between 2 treatments: sand bedding and sawdust bedding. Groups were subjected to each treatment for 5 wk, but data were collected only during the last 3 wk of each period.
Animals
Sixteen lactating primaparous and multiparous Holstein cows were selected using the following criteria: 1) animals had previously been housed in stalls with sand and sawdust bedding (although the amount of previous experience with each bedding type was not measured); 2) they had no history of mastitis, and SCC were below 200,000 cells/mL on their last 6 DHI reports; and 3) were less than 60 DIM. All animals were scored prior to treatment allocation for the presence of teat damage following Britt and Farnsworth (1996). Animals were then assigned into 2 experimental groups (8 animals per group) balanced according to parity (average = 2.25), stage of lactation (average = 40 DIM), and teat end score (average = 1.3).
Udder Cleanliness
The amount of fecal matter on the rear udder of all experimental cows was recorded on d 1, 2, and 6 (at the time of teat sampling before udder cleaning in preparation for milking). A 100-cm2 metal grid, containing 16 equal-sized squares, was placed on the rear side of the udder immediately above the teats to the top of the udder, and the total number of squares containing any visible fecal matter was recorded.
Housing
Cows were housed in 2 different pens within a free-stall barn. Each pen had 2 rows of 4 deep-bedded stalls each (8 stalls per pen), facing one another (‘head-to- head’). Each stall was 240 cm long and 117.5 cm wide, and the bedding was approximately 40 cm deep. Sand bedding was stored outside and consisted of river sand purchased from Armstrong Sand and Gravel Ltd. (Rosedale, BC), which had been previously sieved over a 2-mm screen with water to wash out any silt. Kiln-dried softwood sawdust was obtained from Friesen Bros. Inc. (Burnaby, BC) and stored in a shed until needed. Visible fecal matter and urine were removed twice daily, and fresh bedding added every 7 d. Animals were fed a 50:50 forage to concentrate TMR and milked twice a day at 0600 and 1600 h.
Stall Cleanliness
The amount of fecal matter in each stall was recorded 3 d per week (at the time of bedding sampling but before daily removal of any visible fecal material) to assess stall cleanliness. A 1-m2 metal grid, containing 100 equal-sized squares, was placed along the back of the stall and centered between the stall partitions, and the total number of squares containing any visible fecal matter was recorded.
Behavioral Observations
Each pen was videotaped using a single camera (Panasonic WV-BP330) attached to a video multiplexer (Panasonic WJ-FS 216) and a time-lapse videocassette recorder (Panasonic AG-6540). Pens were recorded for 12 h before sampling on d 1, 2, and 6 of each experimental week. To enable recording during the dark period, a red light (100 W, <5 lx) was suspended over each pen. All animals were uniquely identified with a symbol marked with hair dye (black or blonde) on their backs. Videotapes were scored using scan sampling at 10-min intervals. At each scan, we recorded which cows were lying down in each stall. This sampling regime allowed us to estimate the amount of time each cow spent lying in each stall between the evening and morning milking, and thus determine the potential exposure to microorganisms found in the bedding.
Bedding Samples
Bedding samples were taken on d 0 (immediately after fresh bedding was added to stalls), 1, 2, and 6 of every week, during the morning milking. A 118-mL sterile plastic scoop (Bel-Art Products) was used to collect bedding samples from 16 randomly selected locations within the previously described metal grid in the back 1 m2 of each stall. Bedding samples were collected from the same locations within the grid at each sampling. The edge of the plastic scoop was placed below the bedding surface and pulled upward, collecting all material from 10 cm to the surface. Bedding samples collected in this manner were thought to be representative of the material dairy cattle are likely to contact while lying, as animals often reposition bedding when performing this behavior. Total amount of material (approximately 0.5 kg) collected was consistent throughout the experiment. Samples from a single stall were combined in sterile 710-mL Whirl-Pak plastic bags (Nasco, Inc.). These composite samples were immediately taken to the laboratory, where they were analyzed for bacterial counts.
Wet bedding samples were thoroughly mixed by swinging the plastic bag from side to side in 90° arc for 5 min. Sample DM was determined by placing a 25-g composite sample in a convection oven at 100°C for 24 h or until constant weight was achieved. Samples for microbiological analyses were prepared by adding 10 g of the mixed wet bedding sample plus 90 mL of sterile 0.1% peptone solution to a 118-mL Qorpak square glass bottle (All-Pak, Inc.). Bottles were then mixed thoroughly by swinging the bottles from side to side in 30° arc for 5 min. Bottle contents were allowed to settle for 2 to 3 min and appropriate dilutions (102, 103, 104, 105, 106, 107) of the liquid phase were plated for enumeration on the surface of MacConkey agar (MC) (Beckman Dickinson Microbiology Systems, Canada), MacConkey-inositiol-carbenicillin agar (MCIC) (Beckman Dickinson Microbiology Systems), and Streptosel agar (Beckman Dickinson Microbiology Systems). Prior to plating, each Petri dish was divided into 2 equal parts by drawing a line on the plastic lid of the dish using a black felt marker. This was done so that each Petri dish could be used for plating 2 dilutions. Inositol (10 mg/L BBB) and carbenicillin (BBB) were added to MC agar for MCIC as described by Bagley and Sheidler (1978). Inoculum (0.1 mL) was spread on the agar plates with a sterilized steel spreader. Inoculated agar plates were incubated for 24 h at 37°C, and bacteria were counted using standard enumeration methods (Tortora et al., 1998). Only plates containing 20 to 200 colonies were used to estimate bacterial counts, and all plates showing visible signs of cross contamination were discarded. Bacterial counts were expressed as log10 cfu per gram of sample. Bacterial groups were identified as coliforms (lactose-positive colonies on MC agar), Klebsiella spp. (pink to red colonies on MCIC), and streptococci (total growth on Streptosel agar).
Teat Samples
Teat samples were taken on d 1, 2, and 6 of each experimental week and were collected before the morning milking using a BBL Collection and Transport Culture Swab (BD Microbiology Systems, Canada). Teat swabs were collected individually from all 4 teats by rotating a swab around the exterior of the teat orifice. Teat swabs were analyzed immediately for coliforms, Klebsiella spp., and Streptococcus spp. counts. The 4 swabs from each cow were pooled by placing them in a single test tube containing 4 mL of peptone solution and shaken vigorously in a circular motion for 60 s. Rinse solution and its dilutions (102, 103, 104) were plated on the 3 types of agar media and processed as described previously for the bedding samples.
Statistical Analyses
All dependent variables were screened for normality, and the presence of outliers by visual assessment of the distributions and by calculation of kurtosis and skewness (Proc Univariate in SAS, version 8.2). Bacterial counts were normalized by logarithmic transformations.
Our experiment was not designed to allow for a statistical test of treatment differences for the bacterial counts in the stall bedding. However, differences in this measure could be compared among days within treatment. The experiment was designed to allow for treatment comparisons of cow-based measures (udder cleanliness score and bacterial counts on teat ends). These variables were averaged across the 3 wk on each treatment to calculate one value per cow, per treatment, and per experimental day. Mixed model analysis was then used to test the effect of treatment (Proc Mixed, SAS, version 8.2). In the model, day, treatment (type of bedding), and day x treatment interaction were included as fixed effects, and cow was treated as the random effect. The ARH (1) (heterogeneous first-order autoregressive) covariance structure was used to test differences for udder cleanliness between treatments. For the bacterial counts, the ANTE (1) (first-order Ante-dependence) covariance structure provided the best fit for all 3 types of bacteria. This analysis revealed significant interactions between day and treatment for all 3 bacterial types; therefore, paired t-tests were used to test the differences between treatments on each day. Pearson correlation coefficients were used to estimate the associations among bacterial counts in bedding, bacterial counts on teat ends, DM, udder, and stall cleanliness, based on the average values over all experimental weeks.
Correlations between bedding counts and teat counts were determined with the aid of behavioral observations. We calculated the amount of time each cow spent lying in each stall between the previous evening milking and morning milking (usually about 10 h) on d 1, 2, and 6. The number of hours each animal spent lying in a given stall was then multiplied by the bacterial count for that particular stall. In cases in which cows used multiple stalls, this procedure was repeated for each stall and then summed for all stalls. This summed value resulted in a ‘cow-bedding count’ for each individual cow. The ‘cow- bedding count’ was then correlated with the bacterial count found on the teat ends of each individual animal. Correlations between bedding counts and teat counts were calculated for all 6 wk of the experimental period.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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), but varied little over the rest of the week. In sawdust, coliform numbers peaked by d 2 (Figure 1B). Coliform counts in sawdust were lower on d 0 and 1 than on d 2 and 6 (P < 0.001), with 1.2 log units fewer coliforms on d 0 than on d 2. Of particular interest, the highest coliform count for sand (on d 2) was similar to the lowest count observed for sawdust (on d 0).
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). Counts were similar on d 0 and 1, and on d 2 and 6. In sawdust, Klebsiella spp. counts were lowest on d 0, and slowly increased reaching maximum levels by d 2 (P < 0.01; Figure 1D). There were 1.3 log units more Klebsiella spp. on d 2 than on d 0. The highest Klebsiella spp. counts in sand (on d 2) were numerically lower than the lowest counts in sawdust (on d 0).
Streptococci.
In sand, there were more streptococci on d 1, 2, and 6 than on d 0 (P < 0.001; Figure 1E). Counts increased by 0.6 log units from d 0 to 2 and then leveled out between d 2 and 6. In sawdust bedding, streptococcal counts increased continually from d 0 to 6 (P < 0.01; Figure 1F).
Dry Matter
The mean values for DM over the experiment were 94.7 and 79.5% for sand and sawdust bedding, respectively (Table 1). Dry matter content of sand bedding did not differ between sampling days. However, the DM in sawdust bedding decreased significantly throughout the week (P < 0.001) with the lowest DM (71.7%) occurring on d 6 (P < 0.001).
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). The mean grid count for sand and sawdust bedding was 7 and 14, respectively.
Udders of cows housed on sand had a higher grid count score than cows housed on sawdust (6 and 4, respectively; P < 0.001). However, udder cleanliness for cows housed on sand and sawdust did not change over the course of the week (Table 2).
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). In contrast, the coliform counts of cows housed on sawdust increased during the week, with the lowest count occurring on d 1 and the highest count on d 6 (P < 0.01; Figure 2A). Coliform counts on teat swabs were the same for the 2 bedding treatment groups on d 1 and 2. However, by d 6, cows bedded on sand had 1 log unit fewer bacteria than cows bedded on sawdust (P < 0.001).
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). However, Klebsiella spp. counts from teat swabs taken from cows housed on sand on d 1 were often below the sensitivity of the plating procedures (<10–2 cfu/g). Klebsiella spp. counts on teat ends of cows housed on sawdust increased steadily during the week (P < 0.001). Overall, there were 0.8 log units more Klebsiella spp. on the teat swabs collected from cows when bedded on sawdust compared with when they were housed on sand (P < 0.001). The highest count for cows bedded on sand was similar to the lowest count for cows bedded on sawdust.
Streptococci.
Streptococcal counts on teat swabs for cows bedded on sand were similar on d 1 and 2, but greater on d 6 (P < 0.01) (Figure 2 C). However, when cows were bedded on sawdust, teat swab counts increased progressively during the course of the week with highest counts occurring on d 6 (P < 0.001). Cows bedded on sand had on average 1 log unit higher streptococcal counts than those bedded on sawdust (P < 0.001). The lowest count for the cows bedded on sand was similar to the highest count for cows bedded on sawdust.
Correlations Between Bacterial Counts in Bedding and on Teat Ends
For sand bedding, cow-bedding count was correlated with bacterial counts on teat swabs for coliforms (r = 0.35; P < 0.05) and Klebsiella spp. (r = 0.40; P < 0.05), but only marginally for streptococci (r = 0.28; P = 0.06). For cows on the sawdust treatment, correlation coefficients were r = 0.47, 0.69, and 0.60, P < 0.001, for coliforms, Klebsiella spp., and streptococci, respectively.
Correlations Between Udder Cleanliness and Bacterial Counts on Teat Ends
The cleanliness of the udder and the bacterial counts on teat ends were correlated, but the strength and direction of the correlation varied with day, bacteria type, and bedding (Table 3). Overall, there was no clear relationship between these measures.
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Correlation Between Bedding Counts and Stall Cleanliness
Dirtier stalls, as measured by our stall cleanliness score, contained more of all 3 types of bacteria. Correlation coefficients between bacterial counts in sand and stall cleanliness were r = 0.46 (P < 0.001) for coliforms, r = 0.50 (P < 0.001) for Klebsiella spp., and r = 0.48 (P < 0.001) for streptococci. Correlation coefficients for bacterial counts in sawdust were r = 0.43 (P < 0.001) for coliforms, r = 0.30 (P < 0.001) for Klebsiella spp., and r = 0.35 (P < 0.001) for streptococci.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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For sand bedding, the relationship between bacterial counts in bedding and on teat ends was relatively weak, suggesting that the way teat ends become contaminated is different in this medium. The physical properties of sand (e.g., adhesion to skin and abrasiveness) may influence bacterial exposure in a more sporadic manner than is the case for sawdust. Cows in this study had dirtier rear udders when housed on sand than on sawdust, but udder cleanliness was not consistently correlated to bacteria counts on the teat end. Some measure of teat cleanliness may have been a better predictor of teat end bacterial counts. Coliforms are complex organisms found in many bedding materials and can metabolize a variety of organic compounds (Madigan et al., 2000). Counts of both coliforms and Klebsiella spp. were higher on teat ends of cows housed on sawdust compared with sand. These findings are in agreement with other studies (Bramley and Neave, 1975; Fairchild et al., 1982; Janzen et al., 1982). In the current experiment, we found between 0.8 and 1 log units more coliforms and Klebsiella spp. on the teat ends of cows when they were bedded with sawdust compared with sand. Similarly, Fairchild et al. (1982) and the National Mastitis Council (1996) found higher coliform and Klebsiella spp. counts in sawdust bedding than in sand.
Other work has found that Klebsiella outbreaks are often associated with sawdust bedding (Bramley and Neave, 1975; Fairchild et al., 1982). The degree of contamination by this pathogen can vary depending on the type of wood, the kiln-drying process and the handling and storage of sawdust on the farm (Zehner et al., 1986). In the present experiment, sawdust was stored indoors to reduce the chance of environmental contamination.
Previous work has shown that feces and urine contamination of bedding plays an important role in bacterial multiplication (Carroll and Jasper, 1978; Zehner et al., 1986; National Mastitis Council, 1996). Over the course of a week, stalls bedded with sawdust were more likely to become contaminated with manure and have lower DM levels. In contrast, the DM content of the sand bedding remained constant over the week regardless of the extent of manure contamination. Greater numbers of coliform and Klebsiella colonies associated with sawdust can be attributed to the presence of moisture and availability of nutrients. Not surprisingly, coliforms, Klebsiella spp., and streptococci counts in sawdust were positively correlated with bedding DM, and bedding counts were positively correlated with stall cleanliness.
Actual bacterial counts varied during the course of the week for both sand and sawdust bedding. Bacterial counts in sawdust increased at the beginning of the week, reaching their maximum population numbers by d 2. The initial bacterial populations may be due to the availability of nutrients in fresh sawdust. As the week progressed, the sawdust bedding became more contaminated with manure, possibly resulting in differences in nutrient availability for bacteria. However, competition between bacterial populations also likely increased over this period. Hogan and Smith (1997) and Hogan et al. (1999) reported similar time trends for bacterial growth within sawdust bedding. In the present experiment, teat ends of cows housed on sand had at least 1 log unit more streptococci compared with the teat ends of cows housed on sawdust bedding. These results are in contrast to those of Janzen et al. (1982), who reported lower bacterial counts.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Received for publication August 6, 2003. Accepted for publication September 22, 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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