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Short communication: Survival of Mycobacterium avium ssp. paratuberculosis in tissues of cows following low-dose exposure to electron beam irradiation
J. Dairy Sci. 99:6134–6136 http://dx.doi.org/10.3168/jds.2016-11216 © American Dairy Science Association®, 2016.
Short communication: Survival of Mycobacterium avium ssp. paratuberculosis in tissues of cows following low-dose exposure to electron beam irradiation John F. Bode and Charles O. Thoen1
Department of Veterinary Microbiology and Preventative Medicine, College of Veterinary Medicine, Iowa State University, Ames 50011
ABSTRACT
This investigation was designed to determine the effects of low-dose electron beam irradiation on the survival of Mycobacterium avium ssp. paratuberculosis in tissue samples collected at necropsy from clinically affected cows. Mycobacterium avium ssp. paratuberculosis was isolated from the ileum and ileocecal valve of one cow and from the ileum of another cow irradiated at 4.0 kGy, but was not isolated from the ileum, ileocecal valve, or mesenteric lymph node of 11 other cows irradiated at 4 kGy. Key words: Mycobacterium avium subspecies paratuberculosis, electron beam irradiation Short Communication
Mycobacterium avium ssp. paratuberculosis (Map), the causative agent of Johne’s disease, is an acid-fast bacillus with a highly lipid cellular wall. Johne’s disease is a slowly developing, insidious disease that is characterized by chronic wasting of the infected animal without loss of appetite (Thoen, 2007). Mycobacterium avium ssp. paratuberculosis inhabits the lamina propria and in severe cases the submucosa of the intestine and associated lymph nodes. Tissues from 13 cows with clinical Johne’s disease, positive on ELISA and fecal culture, were collected aseptically at necropsy. Tissues collected from each of the cows included the following: ileocecal valve, ileum, and mesenteric lymph node. The specimens were stored in individual Whirl-pak bags (Nasco, Fort Atkinson, WI) and placed on ice in transit to the laboratory. In the laboratory, each sample was aliquoted into 4 new Whirl-pak bags that were frozen at −20°C. Samples were carefully cut to ensure equal thickness and size of
Received March 24, 2016. Accepted May 2, 2016. 1 Corresponding author: [email protected]
tissue samples. Tissues specimens were sorted and labeled according to irradiation dosage groups (0.75, 2.5, and 4.0 kGy, respectively) and placed onto trays in a single layer with no sample physically touching another sample. The trays containing the specimens were then irradiated using a Thomson CIRCE III Linear Electron Accelerator (McV Industries S.A., Jouy-en-Josas, France) at appropriate irradiation levels. The actual dosages received were monitored using dosimeter alanine pellets (Bruker Analytische Messtechnik, Rheinstetten, Germany) that were placed on the top and bottom surfaces of each sample. The doses were determined by electron paramagnetic resonance on a Bruker EMS 104 EPR Analyzer. The arithmetic averages of the top and bottom surface readings were determined as average observed doses. A 10-MeV power energy level was used, but dosage levels could be altered by controlling conveyor speed of power settings. Two samples containing Map and not irradiated were included as positive controls. All samples were conducted in duplicate. Following irradiation, the specimens were processed for isolation of Map as described previously and identified using standard techniques (Thoen and Beran, 2012). The samples were ground using a mortar and pestle (5 min) and suspended in 1.5 mL of sterile PBS. The suspension was collected into 50-mL centrifugation tubes; 1.5 mL of sterile 4% sodium hydroxide was added to each tube. One drop of 0.1% sterile phenol red indicator was added, and each tube was vortexed for 5 min to mix the sample. The samples were placed at 4°C for 30 min. The pH of each sample was neutralized (pH 7.0) by adding sterile 6 N HCl until the red indicator turned to a light pink color. The samples were inoculated onto 3 slant tubes containing 7H10 medium with mycobactin, one 7H10 tube without mycobactin, 3 tubes of Herrold’s Egg Yolk Medium with mycobactin, and 1 tube of Herrold’s Egg Yolk Medium without mycobactin (Remel Labs, Atlanta, GA). The inoculated tubes were incubated at 37°C. Growth was observed at 7-d intervals for 18 wk. Identification of Map was based on colony morphology, acid-fast staining, and dependence on mycobactin.
6134
O O O O O O O O O O O O O O O X O O O O O O O O O O O O X O O O O X O O O O O 1
CT = contaminated; X = M. avium ssp. paratuberculosis positive; O = no M. avium ssp. paratuberculosis cultivated.
X X X X X X O X X X X X X O X O X X O X X X X X O X X O X O X X O O X O X O X O X X X O X O X X O CT O O X X CT X CT X X X X CT X CT CT 1 2 3 4 5 6 7 8 9 10 11 12 13
X X CT O CT X X X X X CT X CT
X X CT X X X X X X CT X X X
O X X O X X X X X O CT O X
X O O X X O X X X X O O X
Mes. Ln. ID valve IC valve IC valve Cow ID
Ileum
Mes. Ln.
Ileum
Mes. Ln.
Ileum
IC valve
Mes. Ln.
Ileum
4.0 kGy 2.5 kGy 0.75 kGy Nonirradiated
Table 1. Survival of Mycobacterium avium ssp. paratuberculosis in ileum, ileocecal (IC) valve, and mesenteric lymph node (Mes. Ln.) collected from each of 13 cows on necropsy1
SHORT COMMUNICATION: ELECTRON BEAM IRRADIATION
6135
Mycobacterium avium ssp. paratuberculosis was isolated from tissues of each of 13 nonirradiated controls, tissues irradiated at 0.75 kGy, or both (Table 1). Also, Map was isolated from the ileocecal valve, mesenteric lymph node, or ileum (or a combination of these) of each of 13 cows irradiated at 2.5 kGy. The Map was isolated from ileum and ileocecal valve of one cow and from the ileum of another cow irradiated at 4.0 kGy, but was not isolated from the ileum, ileocecal valve, or mesenteric lymph node of the other 11 cows. This study evaluated the electron beam irradiation on bovine tissues to determine if low level irradiation of meat is an efficacious method for killing Map to prevent exposure to humans in the food supply chain. The reason why Map in tissues from some cattle naturally exposed to Map survived irradiation is not clear. In a previous study, M. avium ssp. avium grown in liquid culture for 7 d and added to ground pork was killed at 4 kGy (Thoen and Beran, 2012). These organisms may have been in exponential growth phase, whereas some Map in tissues may have been in stationary phase and more resistant to killing by irradiation. A possibility that needs to be explored more closely is whether Map is located within macrophages embedded within the tissue and if this has an effect on survival following irradiation. The maximum level of irradiation allowed by the World Health Organization (10 kGy) may have killed all mycobacteria (WHO, 1994). Irradiation of food products has been shown to be a safe method of improving food safety and preventing foodborne disease in people (O’Bryan et al., 2008). Mycobacterium avium ssp. paratuberculosis, the causative agent of Johne’s disease in ruminant food animals, has been suggested to have a link with Crohn’s disease (Thoen and Kaneene, 2014). However, epidemiologic evidence fails to support Map as the cause of Crohn’s disease in humans (Qual et al., 2010). ACKNOWLEDGMENTS
The authors acknowledge the technical assistance of Gerald L. Jarnagin (Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames). Support was provided by the Iowa Livestock Health Advisory Council (College of Veterinary Medicine, Iowa State University, Ames) and the USDA, Animal and Plant Health Inspection Service, Veterinary Services (Washington, DC). REFERENCES O’Bryan, C. A., P. G. Crandall, S. C. Ricke, and D. G. Olsen. 2008. Impact of irradiation on the safety and quality of poultry and meat products: A review. Crit. Rev. Food Sci. Nutr. 48:442–457. Journal of Dairy Science Vol. 99 No. 8, 2016
6136
BODE AND THOEN
Qual, D. A., J. B. Kaneene, T. J. Varty, R. Miller, and C. O. Thoen. 2010. Lack of association between the occurrence of Crohn’s disease and occupational exposure to dairy and beef cattle herds infected with Mycobacterium avium subspecies paratuberculosis. J. Dairy Sci. 93:2371–2376. Thoen, C. O. 2007. Johne’s disease. Pages 515–516 in McGraw-Hill Encyclopedia of Science and Technology. L. Geller, ed. McGrawHill Professional, New York, NY. Thoen, C. O., and G. W. Beran. 2012. Effect of electron beam irradiation on Mycobacterium avium complex in ground pork. Int. J. Tuberc. Lung Dis. 16:1134–1135.
Journal of Dairy Science Vol. 99 No. 8, 2016
Thoen, C. O., and J. B. Kaneene. 2014. The role of mycobacteria in food safety. Pages 156–168 in Food Associated Pathogens. W. Tham and M.-L. Danielson-Tham, ed. CRC Press, Boca Raton, FL. WHO. 1994. Safety and Nutritional Adequacy of Irradiated Food. WHO Report. World Health Organization (WHO), Geneva, Switzerland.
Short communication: Survival of Mycobacterium avium ssp. paratuberculosis in tissues of cows following low-dose exposure to electron beam irradiation John F. Bode and Charles O. Thoen1
Department of Veterinary Microbiology and Preventative Medicine, College of Veterinary Medicine, Iowa State University, Ames 50011
ABSTRACT
This investigation was designed to determine the effects of low-dose electron beam irradiation on the survival of Mycobacterium avium ssp. paratuberculosis in tissue samples collected at necropsy from clinically affected cows. Mycobacterium avium ssp. paratuberculosis was isolated from the ileum and ileocecal valve of one cow and from the ileum of another cow irradiated at 4.0 kGy, but was not isolated from the ileum, ileocecal valve, or mesenteric lymph node of 11 other cows irradiated at 4 kGy. Key words: Mycobacterium avium subspecies paratuberculosis, electron beam irradiation Short Communication
Mycobacterium avium ssp. paratuberculosis (Map), the causative agent of Johne’s disease, is an acid-fast bacillus with a highly lipid cellular wall. Johne’s disease is a slowly developing, insidious disease that is characterized by chronic wasting of the infected animal without loss of appetite (Thoen, 2007). Mycobacterium avium ssp. paratuberculosis inhabits the lamina propria and in severe cases the submucosa of the intestine and associated lymph nodes. Tissues from 13 cows with clinical Johne’s disease, positive on ELISA and fecal culture, were collected aseptically at necropsy. Tissues collected from each of the cows included the following: ileocecal valve, ileum, and mesenteric lymph node. The specimens were stored in individual Whirl-pak bags (Nasco, Fort Atkinson, WI) and placed on ice in transit to the laboratory. In the laboratory, each sample was aliquoted into 4 new Whirl-pak bags that were frozen at −20°C. Samples were carefully cut to ensure equal thickness and size of
Received March 24, 2016. Accepted May 2, 2016. 1 Corresponding author: [email protected]
tissue samples. Tissues specimens were sorted and labeled according to irradiation dosage groups (0.75, 2.5, and 4.0 kGy, respectively) and placed onto trays in a single layer with no sample physically touching another sample. The trays containing the specimens were then irradiated using a Thomson CIRCE III Linear Electron Accelerator (McV Industries S.A., Jouy-en-Josas, France) at appropriate irradiation levels. The actual dosages received were monitored using dosimeter alanine pellets (Bruker Analytische Messtechnik, Rheinstetten, Germany) that were placed on the top and bottom surfaces of each sample. The doses were determined by electron paramagnetic resonance on a Bruker EMS 104 EPR Analyzer. The arithmetic averages of the top and bottom surface readings were determined as average observed doses. A 10-MeV power energy level was used, but dosage levels could be altered by controlling conveyor speed of power settings. Two samples containing Map and not irradiated were included as positive controls. All samples were conducted in duplicate. Following irradiation, the specimens were processed for isolation of Map as described previously and identified using standard techniques (Thoen and Beran, 2012). The samples were ground using a mortar and pestle (5 min) and suspended in 1.5 mL of sterile PBS. The suspension was collected into 50-mL centrifugation tubes; 1.5 mL of sterile 4% sodium hydroxide was added to each tube. One drop of 0.1% sterile phenol red indicator was added, and each tube was vortexed for 5 min to mix the sample. The samples were placed at 4°C for 30 min. The pH of each sample was neutralized (pH 7.0) by adding sterile 6 N HCl until the red indicator turned to a light pink color. The samples were inoculated onto 3 slant tubes containing 7H10 medium with mycobactin, one 7H10 tube without mycobactin, 3 tubes of Herrold’s Egg Yolk Medium with mycobactin, and 1 tube of Herrold’s Egg Yolk Medium without mycobactin (Remel Labs, Atlanta, GA). The inoculated tubes were incubated at 37°C. Growth was observed at 7-d intervals for 18 wk. Identification of Map was based on colony morphology, acid-fast staining, and dependence on mycobactin.
6134
O O O O O O O O O O O O O O O X O O O O O O O O O O O O X O O O O X O O O O O 1
CT = contaminated; X = M. avium ssp. paratuberculosis positive; O = no M. avium ssp. paratuberculosis cultivated.
X X X X X X O X X X X X X O X O X X O X X X X X O X X O X O X X O O X O X O X O X X X O X O X X O CT O O X X CT X CT X X X X CT X CT CT 1 2 3 4 5 6 7 8 9 10 11 12 13
X X CT O CT X X X X X CT X CT
X X CT X X X X X X CT X X X
O X X O X X X X X O CT O X
X O O X X O X X X X O O X
Mes. Ln. ID valve IC valve IC valve Cow ID
Ileum
Mes. Ln.
Ileum
Mes. Ln.
Ileum
IC valve
Mes. Ln.
Ileum
4.0 kGy 2.5 kGy 0.75 kGy Nonirradiated
Table 1. Survival of Mycobacterium avium ssp. paratuberculosis in ileum, ileocecal (IC) valve, and mesenteric lymph node (Mes. Ln.) collected from each of 13 cows on necropsy1
SHORT COMMUNICATION: ELECTRON BEAM IRRADIATION
6135
Mycobacterium avium ssp. paratuberculosis was isolated from tissues of each of 13 nonirradiated controls, tissues irradiated at 0.75 kGy, or both (Table 1). Also, Map was isolated from the ileocecal valve, mesenteric lymph node, or ileum (or a combination of these) of each of 13 cows irradiated at 2.5 kGy. The Map was isolated from ileum and ileocecal valve of one cow and from the ileum of another cow irradiated at 4.0 kGy, but was not isolated from the ileum, ileocecal valve, or mesenteric lymph node of the other 11 cows. This study evaluated the electron beam irradiation on bovine tissues to determine if low level irradiation of meat is an efficacious method for killing Map to prevent exposure to humans in the food supply chain. The reason why Map in tissues from some cattle naturally exposed to Map survived irradiation is not clear. In a previous study, M. avium ssp. avium grown in liquid culture for 7 d and added to ground pork was killed at 4 kGy (Thoen and Beran, 2012). These organisms may have been in exponential growth phase, whereas some Map in tissues may have been in stationary phase and more resistant to killing by irradiation. A possibility that needs to be explored more closely is whether Map is located within macrophages embedded within the tissue and if this has an effect on survival following irradiation. The maximum level of irradiation allowed by the World Health Organization (10 kGy) may have killed all mycobacteria (WHO, 1994). Irradiation of food products has been shown to be a safe method of improving food safety and preventing foodborne disease in people (O’Bryan et al., 2008). Mycobacterium avium ssp. paratuberculosis, the causative agent of Johne’s disease in ruminant food animals, has been suggested to have a link with Crohn’s disease (Thoen and Kaneene, 2014). However, epidemiologic evidence fails to support Map as the cause of Crohn’s disease in humans (Qual et al., 2010). ACKNOWLEDGMENTS
The authors acknowledge the technical assistance of Gerald L. Jarnagin (Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames). Support was provided by the Iowa Livestock Health Advisory Council (College of Veterinary Medicine, Iowa State University, Ames) and the USDA, Animal and Plant Health Inspection Service, Veterinary Services (Washington, DC). REFERENCES O’Bryan, C. A., P. G. Crandall, S. C. Ricke, and D. G. Olsen. 2008. Impact of irradiation on the safety and quality of poultry and meat products: A review. Crit. Rev. Food Sci. Nutr. 48:442–457. Journal of Dairy Science Vol. 99 No. 8, 2016
6136
BODE AND THOEN
Qual, D. A., J. B. Kaneene, T. J. Varty, R. Miller, and C. O. Thoen. 2010. Lack of association between the occurrence of Crohn’s disease and occupational exposure to dairy and beef cattle herds infected with Mycobacterium avium subspecies paratuberculosis. J. Dairy Sci. 93:2371–2376. Thoen, C. O. 2007. Johne’s disease. Pages 515–516 in McGraw-Hill Encyclopedia of Science and Technology. L. Geller, ed. McGrawHill Professional, New York, NY. Thoen, C. O., and G. W. Beran. 2012. Effect of electron beam irradiation on Mycobacterium avium complex in ground pork. Int. J. Tuberc. Lung Dis. 16:1134–1135.
Journal of Dairy Science Vol. 99 No. 8, 2016
Thoen, C. O., and J. B. Kaneene. 2014. The role of mycobacteria in food safety. Pages 156–168 in Food Associated Pathogens. W. Tham and M.-L. Danielson-Tham, ed. CRC Press, Boca Raton, FL. WHO. 1994. Safety and Nutritional Adequacy of Irradiated Food. WHO Report. World Health Organization (WHO), Geneva, Switzerland.