The discovery of the causative agent of bovine tuberculosis (Mycobacterium bovis) in a wildlife reservoir (free-ranging white-tailed deer [Odocoileus virginianus]) in 1994, and subsequent discovery of infected cattle herds in the northeastern Lower Peninsula of Michigan, have raised questions as to how the disease is being transmitted between deer and cattle. On the basis of results of experimental infection studies,12 indirect transmission is considered to be the major route through which M bovis is transmitted between livestock and wildlife. White-tailed deer have been shown to shed viable M bovis in nasal secretions, saliva, urine, and feces that subsequently contaminate hay and pelleted feed.3 Results of risk factor analyses and statistical modeling have also implicated contaminated feedstuffs as possible vehicles for indirect transmission of M bovis.4–8
Investigations of the survival of M bovis outside the host have primarily focused on survival of the organism in feces, soil, and water.9–12 In a study13 of risk factors for bovine tuberculosis in cattle herds, investigators determined that the presence of M bovis in the environment remained a risk factor for infection over the 12-month period after infected herds were depopulated. In general, survival of M bovis in the environment is reduced by exposure to sunlight and improved by moist, cool conditions,11,14–16 although an experimental study17 revealed that the optimal temperature for recovery of M bovis bacillus Calmette-Guérin from soil samples is 37°C.
Experimental studies9,11,18–20 have also revealed that viable M bovis can exist in experimentally inoculated environmental samples for 1 to 12 months, but M bovis has not been isolated from environmental field samples from Michigan.21–23 This failure may be due to patchy distribution of the organism in the environment and difficulties arising during attempts to culture this slow-growing aerobic organism in the presence of contamination by mold and nonmycobacterial species.16,22 However, techniques are being developed to improve mycobacterial recovery from environmental samples, including immunomagnetic recovery.23
Molecular techniques have been used to successfully detect M bovis genes in environmental samples. Such techniques include rt-PCR assays to detect 16S rRNA and the genes mpb64 and mpb70,17 an rt-PCR assay and use of primers flanking region of difference 4,12,23,24 and a nested PCR assay to detect insertion sequence 6110.19 Mycobacterium bovis genes have been detected in environmental samples for at least 7 months,19 and up to 20 months,17 after experimental inoculation with the organism.
The ability of M bovis to survive in livestock feed and pasturage has been investigated, with results suggesting the organism can survive on pasture for 49 days.18 An experimental New Zealand study14 involving cotton ribbons inoculated with M bovis placed in sites frequented by brushtail possums (Trichosurus vulpecula) showed that M bovis did not survive on pastures (high UV exposure, high temperature, and low humidity) after 4 days, but the organism did survive longer in possum dens (7 days during the summer, with little UV exposure and higher humidity). Survival of M bovis in livestock feed, such as hay, carrots, and corn, has been reported,16,19,25 and evidence exists to suggest that M bovis could survive in silage given its typical pH, oxygen concentration, and optimal temperature.5 However, survival of M bovis on other substrates, such as salt and salt-mineral blocks, has not been investigated.
Salt and salt-mineral blocks are a recognized attractant for free-ranging cervids.26–28 These substances may pose a greater risk for disease transmission than forages and other concentrates provided to pastured cattle in that they are not immediately consumed at once by a single animal, but instead can be consumed over several weeks by several animals. Highly saline conditions can be harmful to mammalian and bacterial cells by removing water and causing electrolyte imbalances. Salt blocks would therefore represent a harsh environment for bacterial survival and viability. However, given the desiccation-resistant, hardy cell wall structure of Mycobacteria organisms, which contributes to survival in harsh environments,29,30 we hypothesized that M bovis may be able to survive long enough on salt and mineral blocks for the bacteria to be transferred from host to host. The objective of the study reported here was to determine the survivability of M bovis on salt and salt-mineral blocks during two 12-day periods in midsummer and midwinter, under typical weather conditions in Michigan.
Materials and Methods
Sample
Four 22.7-kg salt (NaCl) blocks (19 cm wide, 22.5 cm long, and 19 cm deep), 2 with added trace mineralsa and 2 with no additives,b were used in each of 2 portions of the study, which were conducted during the summer and winter. An isolate of the wild-type M bovis commonly recovered from Michigan deer and cattle (strain 02-5342)c was subcultured in a selective broth mediumd and incubated at 37°C for months at the biosafety level III laboratory at the Diagnostic Center for Population and Animal Health, Michigan State University.
Block inoculation
The top surface of each block was marked with a black permanent marker to provide a grid containing 45 sampling squares, each measuring approximately 2.5 × 3.8 cm (Figure 1). Squares were numbered 1 through 45 on each block.
Photograph of a salt (NaCl) block prepared for inoculation with Mycobacterium bovis, showing a numbered sampling grid.
Citation: American Journal of Veterinary Research 78, 1; 10.2460/ajvr.78.1.57
Photograph of a salt (NaCl) block prepared for inoculation with Mycobacterium bovis, showing a numbered sampling grid.
Citation: American Journal of Veterinary Research 78, 1; 10.2460/ajvr.78.1.57
Photograph of a salt (NaCl) block prepared for inoculation with Mycobacterium bovis, showing a numbered sampling grid.
Citation: American Journal of Veterinary Research 78, 1; 10.2460/ajvr.78.1.57
The M bovis culture broth was serially diluted with more of the same medium,d and loops of diluted broth were used to inoculate M bovis-selective agar platese to determine the concentration of bacteria. To determine the quantity of liquid necessary to inoculate each block, various volumes of uninoculated broth were poured over a sample block to assess rate of absorption and uniform coverage. From this process, it was determined that a minimum of 100 mL of broth/block would be needed to achieve complete coverage before the liquid was fully absorbed.
A 100-mL volume of the original, undiluted M bovis culture broth was gently poured over each block, minimizing runoff while ensuring complete coverage of all 45 squares of the sample grid. The concentration of M bovis in broth applied in the summer portion of the study was 1 × 106 CFUs/mL, and the concentration in broth applied in the winter portion was 1.8 × 105 CFUs/mL. These concentrations were consistent with those used in earlier investigations of mycobacterial survival on feed samples (1.1 × 106 CFUs/mL)25 and environmental substrates (5.0 × 104 CFUs/mL).16 At the time of this study, no reports were available of any previous studies regarding the concentration of shed M bovis in deer saliva or of the quantity of saliva produced by deer at a salt block. However, findings of 1 study1 suggested that M bovis is shed in the saliva of infected deer at a concentration of 5 × 103 CFUs/mL. This concentration is apparently sufficient to transmit infection to naïve dairy calves through contaminated feed.
After inoculation, each block was placed in a leak-proof container to catch any runoff or possible rain water. The blocks were then placed on steel tables in a special secured outdoor facility used for research on survivability of M bovis on various substrates.16 One of each block type was placed on an uncovered table to allow for direct UV sunlight exposure, and the other of each type was placed on a table under an A-framed awning covered with a plastic tarpaulin to simulate a shaded environment. The awing of the shaded environment was approximately 25 to 30 cm above the top of the table to ensure that other environmental factors such as temperature, humidity, and the elements (eg, snow, rain, or wind) were consistent between shaded and unshaded blocks.
Sample collection
The summer portion of the study began on August 20, 2012, and the winter portion began on February 4, 2013. Sample collection began within 1 hour after blocks were inoculated with M bovis culture stock inoculation. Collection was then conducted twice a day for the first 4 days (beginning on day 0) and once a day from days 7 through 11. Three sampling squares were selected from each block at each sample collection point, by means of simple random sampling without replacement from a random number table. Sterilized scouring pads were used to abrade the salt surface to collect material from 1 numbered grid square from each salt or salt-mineral block. A diagram of the sampling grid numbered in the same fashion as for the blocks was kept with each container, and the identity of each square used for sample collection from the block was marked on the printed diagram to avoid duplicate sample collection in the future. After sample collection, pads were placed in sterile centrifuge tubes containing 20 mL of sterile culture brothd for transport to the biosafety level III laboratory at the Diagnostic Center for Population and Animal Health for processing.
Sample processing
Samples were processed by use of decontamination and culture methods developed for environmental samples in Michigan.16 After surface decontamination, the centrifuge tubes containing the samples were vortexed to facilitate separation of mycobacteria from the pads. Five milliliters of the selective broth mediumd was transferred to another sterile centrifuge tube, and 5 mL of brain-heart infusion broth containing 4% phenol red as a pH indicator was added to each tube. To decontaminate the sample, 7.5 mL of 0.5 N NaOH was added to the sample. After a 10-minute decontamination period, 6 N HCl was added dropwise to stop the NaOH reaction and lower the pH to acidic, and 1 N NaOH was added to restore the pH of the sample to neutral. Samples were then centrifuged for 20 minutes at 3,000 × g to concentrate the bacteria. After centrifugation, the supernatant was removed, leaving approximately 2 mL of liquid with the concentrated pellet. The pellet was resuspended in the remaining liquid, and samples were inoculated onto selective agar slantsf by use of sterile swabs.
Samples were incubated for 8 weeks at 36°C with 5% CO2. Slants were examined weekly for the presence of microbial colonies with the morphological characteristics of M bovis. Suspected M bovis colonies were smeared on a slide for acid-fast staining and microscopic evaluation. Presence of M bovis was confirmed with a nested PCR assay used for detection of M bovis in environmental samples.19
Statistical analysis
Outcomes of interest in the study were the maximum time that M bovis could be recovered after block inoculation and recovery of viable M bovis at any point during the study period (yes or no). Significant (P ≤ 0.05) differences in survival time of M bovis on salt versus salt-mineral blocks, in summer versus winter, and in sunlight versus shade were identified by use of the nonparametric Kruskal-Wallis χ2 test.g Logistic regression modelingh was used to evaluate associations between season and light exposure with recovery of viable M bovis. Values of the Akaike information criterion and coefficient of determination were used to determine whether the fit of the multivariable models (1/block type) was superior to the fit of univariate models. Strengths of association are reported as ORs with 95% CIs.
Survival curvesi for maximum recovery times of viable M bovis were also generated for the 2 block types, 2 seasons, and element exposures (sunlight and shade). The Wilcoxon χ2 test was used to assess equality of survival curves.
Results
A total of 162 samples were collected from salt blocks, and 161 samples were collected from salt-mineral blocks. Overall, M bovis was recovered from 56 of these samples (23 [14%] from salt blocks and 33 [20%] from salt-mineral blocks). Mean environmental temperatures in the summer and winter portions of the study were 21.6°C (95% CI, 19.9°C to 23.4°C) and −3.3°C (95% CI, −5.5°C to −1.1°C), respectively, and total precipitation during the summer and winter periods were 0.46 cm and 1.65 cm, respectively.
The organism survived significantly longer during the winter versus the summer on both types of blocks (Figure 2; Table 1). Exposure to sunlight decreased the period during which viable M bovis could be recovered from both block types (Figure 3). The longest period after inoculation that viable M bovis could be recovered was 78 hours (3.25 days), and this pertained to salt-mineral blocks during the winter and in the shade.
Percentages of samples with positive results of M bovis culture over time after inoculation of salt (A) and salt-mineral (B) blocks with the organism (0 hours) and storage of the blocks outdoors during the summer (n = 78 samples for both block types; black-outlined gray bars) or winter (84 samples for salt blocks and 83 samples for salt-mineral blocks; black bars) in Michigan. For both types of blocks, M bovis survived significantly (Kruskall-Wallis χ2 test; P < 0.05) longer during the winter than during the summer.
Citation: American Journal of Veterinary Research 78, 1; 10.2460/ajvr.78.1.57
Percentages of samples with positive results of M bovis culture over time after inoculation of salt (A) and salt-mineral (B) blocks with the organism (0 hours) and storage of the blocks outdoors during the summer (n = 78 samples for both block types; black-outlined gray bars) or winter (84 samples for salt blocks and 83 samples for salt-mineral blocks; black bars) in Michigan. For both types of blocks, M bovis survived significantly (Kruskall-Wallis χ2 test; P < 0.05) longer during the winter than during the summer.
Citation: American Journal of Veterinary Research 78, 1; 10.2460/ajvr.78.1.57
Percentages of samples with positive results of M bovis culture over time after inoculation of salt (A) and salt-mineral (B) blocks with the organism (0 hours) and storage of the blocks outdoors during the summer (n = 78 samples for both block types; black-outlined gray bars) or winter (84 samples for salt blocks and 83 samples for salt-mineral blocks; black bars) in Michigan. For both types of blocks, M bovis survived significantly (Kruskall-Wallis χ2 test; P < 0.05) longer during the winter than during the summer.
Citation: American Journal of Veterinary Research 78, 1; 10.2460/ajvr.78.1.57
Percentages of samples with positive results of M bovis culture over time after inoculation of salt (A) and salt-mineral (B) blocks with the organism (0 hours) and storage of the blocks outdoors with exposure to sunlight (n = 81 samples for salt blocks and 80 samples for salt-mineral blocks; black-outlined gray bars) or shade (81 samples for both block types; black bars) in Michigan. For both types of blocks, M bovis survived significantly (Kruskall-Wallis χ2 test; P < 0.05) longer when exposed to shade versus sunlight.
Citation: American Journal of Veterinary Research 78, 1; 10.2460/ajvr.78.1.57
Percentages of samples with positive results of M bovis culture over time after inoculation of salt (A) and salt-mineral (B) blocks with the organism (0 hours) and storage of the blocks outdoors with exposure to sunlight (n = 81 samples for salt blocks and 80 samples for salt-mineral blocks; black-outlined gray bars) or shade (81 samples for both block types; black bars) in Michigan. For both types of blocks, M bovis survived significantly (Kruskall-Wallis χ2 test; P < 0.05) longer when exposed to shade versus sunlight.
Citation: American Journal of Veterinary Research 78, 1; 10.2460/ajvr.78.1.57
Percentages of samples with positive results of M bovis culture over time after inoculation of salt (A) and salt-mineral (B) blocks with the organism (0 hours) and storage of the blocks outdoors with exposure to sunlight (n = 81 samples for salt blocks and 80 samples for salt-mineral blocks; black-outlined gray bars) or shade (81 samples for both block types; black bars) in Michigan. For both types of blocks, M bovis survived significantly (Kruskall-Wallis χ2 test; P < 0.05) longer when exposed to shade versus sunlight.
Citation: American Journal of Veterinary Research 78, 1; 10.2460/ajvr.78.1.57
Survival time (h) of Mycobacterium bovis inoculated onto salt and salt-mineral blocks in various outdoor conditions in Michigan.
| Block type | Condition | No. of samples collected | No. (%) of samples with positive M bovis culture results | Mean survival time (h) | Maximum survival time (h) | P value* |
|---|---|---|---|---|---|---|
| Salt | Summer | 78 | 7 (9) | 0 | 0 | 0.01 |
| Winter | 84 | 16 (19) | 13.6 | 48 | — | |
| Salt-mineral | Summer | 78 | 7 (9) | 1.4 | 5 | 0.008 |
| Winter | 83 | 26 (31) | 25.0 | 78 | — | |
| Salt | Sunlight | 81 | 8 (10) | 0.6 | 5 | 0.02 |
| Shade | 81 | 15 (19) | 14.2 | 48 | — | |
| Salt-mineral | Sunlight | 80 | 10 (12) | 10.6 | 48 | 0.11 |
| Shade | 81 | 23 (28) | 24.0 | 78 | — |
Values represent the results of Kruskal-Wallis χ2 test. Values of P ≤ 0.05 were considered significant.
— = Not applicable.
Results of multivariable logistic regression indicated that M bovis was more likely to be recovered at any point during the study period from samples during the winter (vs summer) and from blocks exposed to shade (vs sunlight), but these associations were significant for salt-mineral blocks only (Table 2). Results of survival analysis confirmed the significant differences in survival time of M bovis by season and sunlight exposure on salt blocks (Wilcoxon χ2 test; P < 0.001) and salt-mineral blocks (Wilcoxon χ2 test; P < 0.001; Figure 4).
Product-limit survival curves for M bovis on salt (solid line) and salt-mineral (dotted line) blocks kept in the shade in the summer (A), sunlight in the summer (B), shade in the winter (C), and sunlight in the winter (D) in Michigan.
Citation: American Journal of Veterinary Research 78, 1; 10.2460/ajvr.78.1.57
Product-limit survival curves for M bovis on salt (solid line) and salt-mineral (dotted line) blocks kept in the shade in the summer (A), sunlight in the summer (B), shade in the winter (C), and sunlight in the winter (D) in Michigan.
Citation: American Journal of Veterinary Research 78, 1; 10.2460/ajvr.78.1.57
Product-limit survival curves for M bovis on salt (solid line) and salt-mineral (dotted line) blocks kept in the shade in the summer (A), sunlight in the summer (B), shade in the winter (C), and sunlight in the winter (D) in Michigan.
Citation: American Journal of Veterinary Research 78, 1; 10.2460/ajvr.78.1.57
Results of multivariable logistic regression (1 model/block type) to detect associations between various outdoor conditions in Michigan and recovery of viable M bovis from experimentally inoculated salt and salt-mineral blocks at any point during the 12-day study period.
| Block type | Condition | P value | OR | 95% CI |
|---|---|---|---|---|
| Salt-mineral | Winter (vs summer) | < 0.001 | 4.9 | 9.0–12.3 |
| Shade (vs sun) | 0.01 | 3.0 | 1.3–7.0 | |
| Salt | Winter (vs summer) | 0.07 | 2.4 | 0.9–6.3 |
| Shade (vs sun) | 0.12 | 2.1 | 0.8–5.3 |
Discussion
Results of the study reported here indicated that M bovis can survive for a few days on salt and salt-mineral blocks exposed to summer and winter environmental conditions in Michigan, with the longest survival time on shaded blocks during the winter. Previous environmental survival studies have shown that viable M bovis can survive in deer feed (apples, corn, and potatoes) for at least 7 days, and up to 112 days,25 and in environmental samples for up to 2 months.19 When molecular tools were used, M bovis DNA was detected 8 months after inoculation of soil samples, 9 months after inoculation of corn, 10 months after inoculation of hay, and 11 months after inoculation of water samples.19
Compared with the chemical environment of feedstuffs, soils, or surface water, the chemical environment on the surface of salt and salt-mineral blocks typically fed to cattle is inhospitable to bacteria, which would considerably reduce the ability of bacteria to survive. Nontuberculous mycobacteria can survive in saltwater habitats,31 and a recent analysis of M tuberculosis-complex bacteria by use of an experimental phenotype microarray revealed that M bovis was significantly more salt tolerant than most M tuberculosis isolates tested at 6% NaCl.32
The ability of mycobacteria to survive on salt and salt-mineral blocks may be related to trehalose, which is a nonreducing disaccharide that exists in bacteria, fungi, plants, and invertebrates.33 Salt stress has been associated with increasing trehalose concentrations in a variety of organisms, from Rhodobacter spp33 to rice.34 Trehalose concentrations in mycobacteria increase in conditions of abiotic stress,29,30 protect mycobacteria from desiccation,29 and may contribute to limited salt tolerance in M bovis. Given that viable M bovis could be recovered from salt-mineral blocks 3 days after inoculation in the present study, the possibility exists that these blocks could be a potential source of viable M bovis for the infection of cattle or wildlife.
The significant associations between improved survivability of M bovis on salt-mineral blocks in environments with colder temperatures and protection from sunlight were consistent with findings from other studies.11,14–16,19,25 Similar associations for blocks composed of salt alone failed to achieve significance. A study19 involving use of bacterial culture and PCR assay for detection of M bovis in environmental samples also revealed no significant differences in DNA recovery or bacterial culture results between shaded or unshaded samples, suggesting that protection from sunlight is not sufficient on its own to ensure bacterial survival but can contribute to the effects of other factors that allow bacteria to persist in the environment.
Results of the study reported here suggested that the use of salt and salt-mineral blocks should be considered as a potential pathway for spreading M bovis between cattle and white-tailed deer and increasing the risk for bovine tuberculosis in cattle herds. Salt and salt-mineral blocks are commonly used in livestock operations in Michigan, and these blocks are frequently used by wildlife.21 Although the quantities of M bovis present in the saliva of infected cattle or white-tailed deer have yet to be fully evaluated, M bovis has been detected in the saliva of experimentally infected white-tailed deer.1,3 In addition, uneaten feed from experimentally infected deer is able to transmit infection to naïve white-tailed deer2,3 and dairy calves.1
Because of salt and salt-mineral blocks being intended for frequent, sequential use by multiple animals, survival of M bovis on these blocks may pose an important vehicle for disease transmission. Given the reported associations between the presence of deer on cattle farms and bovine tuberculosis,4,6–8 livestock managers should use caution when administering salt and minerals during winter months, take steps to keep infected cattle or wildlife from salt and salt-mineral blocks intended for use by the herd, and avoid placing blocks in shaded areas, particularly during winter, which can promote the survival of M bovis.
Acknowledgments
Supported in part by the Michigan Department of Agriculture and Rural Development and the Diagnostic Center for Population and Animal Health and the Center for Comparative Epidemiology, Michigan State University.
The authors thank Marlee Richter, Sharon Steck, and Kristen Koehl for technical assistance.
ABBREVIATIONS
| CI | Confidence interval |
| rt-PCR | Reverse-transcriptase PCR |
Footnotes
Champion's Choice 50-lb trace mineral salt block, Cargill Inc, Minneapolis, Minn.
Champion's Choice white salt block, 50 lb, Cargill, Inc, Minneapolis, Minn.
Mycobacteriology Unit, Bureau of Laboratories, Michigan Department of Health and Human Services, Lansing, Mich.
Middlebrook 7H9 broth, Becton-Dickinson, Cockeysville, Md.
Selective Middlebrook 7H11 agar plate, Becton-Dickinson, Cockeysville, Md.
Middlebrook 7H11agar slant, Becton-Dickinson, Cockeysville, Md.
PROC NPAR1WAY, SAS, version 9.4, SAS Institute Inc, Cary, NC.
PROC LOGISTIC, SAS, version 9.4, SAS Institute Inc, Cary, NC.
PROC LIFETEST, SAS, version 9.4, SAS Institute Inc, Cary, NC.
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