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6 mice inoculated orally with Mycobacterium paratuberculosis
J. Comp. Path. 1995 Vol. 1 t3, 75-80
SHORT PAPER
Histopathology of C57BL/6 Mice Inoculated Orally with Mycobaeterium paratubereulosis R. S. Veazey*, H. W. Taylor*, D. W. Horohov~, J. L. Krahenbuhl~, J. L. Oliver III* and T. G. Snider III* Departments of* Veterinary Pathology and t Veterinary Microbiology, and the ++G. W. Long, Hansens Disease Center, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA
Summary The susceptibility of C57BL/6 mice to oral inoculation with Myobacterium paratuberculosis was evaluated histopathologically. Granulomatous lesions containing acid-fast bacteria developed in the mesenteric lymph nodes in over 50% of the mice by 11 months after inoculation. The results suggest that C57BL/6 mice may be useful for studying infection, pathogenesis, and other aspects of paratuberculosis. 9 1995 Academic Press Limited
Introduction Paratuberculosis (Johne's disease) is a chronic, granulomatous, intestinal disease of ruminants caused by the acid-fast bacterium Mycobacterium paratuberculosis. Ruminants are orally infected at a young age through the ingestion of contaminated milk, but several months or years may elapse before clinical signs appear. The disease occurs throughout the world, particularly in domestic livestock (Chiodini et al., 1984). Research into the pathogenesis and immunology of paratuberculosis has been hampered by the apparent lack of a suitable laboratory animal model. Early attempts to infect laboratory animals were often unsuccessful (Lominski et al., 1956). It is now known that murine resistance to paratuberculosis and other mycobacteria is associated with the Beg gene which is expressed in two allelic forms: the dominant resistance (Bc~) allele and the recessive susceptibility (Bc~) allele (Gros et al., 1981). Mice with the B%d genotype are resistant to many mycobacterial species, whereas mycobacteria persist and replicate in Bc~ mice (Forget et al., 1981; Skamene et al., 1982; Skamene, 1989; Frelier et al., 1990; Tanaka et al., 1994). Parenterally inoculated Bc~ mice develop granulomatous lesions resembling those of ruminant paratuberculosis (Tanaka et al., 1994). However, no recent attempts to infect immunocompetent Bc~ Correspondence to: R. S. Veazey, Harvard Medical School, New England Regional Primate Research Center, Division of Comparative Pathology, One Pine Hill Drive, P.O. Box 9102, Southborough, MA 01772, USA. 0021-9975/95/050075+06 $12.00/0
9 1995 Academic Press Limited
76
R.S. Veazey e t
al.
mice orally with M. paratuberculosis have been reported. Both nude mice and mice with severe combined immunodeficiency (SCID) are susceptible to oral infection with the organism (Hamilton et al., 1989, 1991; Mutwiri et al., 1992), but their immunodeficient state may make them unsuitable for studies of the immune response to infection. Preliminary studies suggested that Bc~ mice could be infected orally with a high dose o f M . paratuberculosis. With both low (101~ and high (5 x 1011) doses of viable M. paratuberculosis organisms, small numbers of acid-fast bacteria could sometimes be detected in the intestines of C57BL mice 60 days after inoculation (Vance, 1990). These studies indicated that a large dose and an extended post-inoculation period may be required for extensive lesions to develop in susceptible mice. The objectives of the present experiment were to confirm that Bc~ mice (C57BL/6N) could be infected orally with M. paratuberculosis, and to determine whether lesions resembling those of the natural disease would develop after 7 to 11 months, gut-associated lymphoid tissues (GALT) and other tissues being examined histologically for lesions and acid-fast bacteria. Materials and Methods
Mice All mice used in the experiment were bred from a C57BL/6N breeder colony originally obtained from Harlan Sprague-Dawley Inc., Indianapolis, IN, USA. They were conventionally housed in "shoebox"-type cages with stainless-steel wire tops and heattreated, aspen-chip bedding. The animals were fed Purina Rodent Chow 5001 (Purina Mills Inc., St Louis, MO, USA) and water ad libitum, and maintained on a 12/12-h light/dark cycle. All mice were inoculated at 3 weeks of age and then weaned. Control mice were housed in separate cages but under identical conditions.
Organism and Preparation of Inoculum M. paratuberculosis ATCC strain 19698 was obtained from the USDA-ARS, National Animal Disease Center (NADC), Ames, Iowa, USA and grown in Middlebrook 7H9 broth supplemented with Middlebrook OADC Enrichment Medium (Difco, Detroit, MI, USA), glycerol (Sigma, St Louis, MO, USA) 0'2%, and Mycobactin J (Allied Laboratories, Glenwood Springs, CO, USA) 2 mg/1. Broth cultures were incubated at 37~ for 4 months and bacteria were harvested by centrifugation (10 min at 2000 g), washed three times in phosphate-buffered saline, agitated ultrasonically for 10 s at 40W (Heat Systems-Ultrasonics Inc., Plainview, NY, USA) to reduce clumping, and adjusted to 3 x 10" cells/dose as determined by a McFarland's nephelometer standard. Each dose was suspended in 0-3 ml of pasteurized cream (Borden Inc., Baton Rouge, LA, USA) and administered orally by means of a ball-tipped needle. The number of viable bacteria per inoculum was later verified by plating serial dilutions on Middlebrook 7H10 agar supplemented with OADC and MycobactinJ. Control mice (n = 6) were inoculated orally with 0"3 ml of cream alone. Groups of infected mice (n = 4 to 12) were killed by cervical dislocation 7, 8, 9 and 11 months after inoculation (Table 1). Control mice were killed and examined 11 months after inoculation.
Histology Liver, spleen, intestine and mesenteric lymph node (MLN) were fixed in neutralbuffered 10% formalin. At least one cross-section of liver (taken from the widest part
77
M. paratuberculosis i n M i c e Table 1 Histological e x a m i n a t i o n of C57BL/6 m i c e infected orally with M. paratuberculosis Number of mice with lesions and mycobacteria in Period (months) after inoculation 11 9 8 7
Number of mice examined
mesenteric lymph node
spleen
liver
intestine
6 4 12 9
4* 1I 7 6
1 1 0 1
1 l 0 1
0 0 0 0
* Only four mice examined, because of a technical mishap. ~"Only two mice examined, because of a technical mishap. Six control (uninfected) mice gave negative results.
of the left lobe) and spleen, and at least 1 cm of duodenum, jejunum, ileum, ileocaecal valve and colon were examined histologically from each mouse. MLNs from four animals were lost during processing and therefore not examined. Tissues were embedded in paraffin wax and 5-gm sections stained with haematoxylin and eosin (HE) and Ziehl-Neelsen acid-fast stain (ZN) were examined by light microscopy. Tissues containing acid-fast bacteria were additionally stained by the avidin-biotin method with rabbit anti-M, paratuberculosispolyclonal antibody (Dako Inc., Carpinteria, CA, USA). Results
No clinical signs of disease, weight loss, or gross lesions were detected in any of the infected mice. A total of three infected and four control animals were culled 30-60 days after inoculation due to an intractable idiopathic dermatitis. No histological lesions other than dermatitis were detected in these mice and they were excluded from the study. A total of 18 out of 31 (58%) orally infected mice were found to have demonstrable acid-fast bacteria, usually limited to the MLN (Table 1). O f the 27 MLNs examined, 17 (63%) were infected. Acid-fast bacteria were also found in three livers and three spleens (in five mice). Immunohistochemical staining of infected tissue sections with rabbit anti-M, paratuberculosis antibody confirmed that the organisms were mycobacteria. The severity of the MLN lesions varied, but granulomas and multinucleated giant cells were consistent findings. The most severe lesions were observed in MLNs from mice inoculated 11 months earlier (Fig. 1). In these nodes, the normal follicular architecture was effaced by pale, swollen macrophages and multinucleated giant cells. Macrophages filled with numerous acid-fast bacteria were present within granulomas (Fig. 2). Less severe lesions, containing fewer macrophages and acid-fast bacilli, were detected in MLNs from mice in the 7-month post-inoculation group (data not shown). Hepatic microgranulomas containing acid-fast bacteria were detected in three mice. Larger periportal granulomas were also sometimes present in infected mice; these consisted predominantly of lymphocytes, plasma cells and
78
R.S. Veazey
e t ai.
Fig. 1.
Mesenteric lymph node of a C57BL/6 mouse 11 months after oral infection with M. paratuberculosis. Note the extensive infiltration of macrophages and multinucleated giant cells. HE. x 400.
Fig. 2.
Same mesenteric lymph node as in Fig. 1 showing several macrophages filled with numerous acidfast bacilli. ZN. x 1000.
M. paratuberculosis in Mice
79
small numbers of macrophages, but no acid-fast bacteria. No mycobacteria or related lesions were detected in the control mice. The intestines frequently showed hyperplastic Peyer's patches and slightly to moderately increased numbers of lymphocytes and plasma cells; occasional micro-granulomas consisted of aggregates of epithelioid macrophages, but acid-fast bacteria were not detected in the intestines of any of the mice. Peyer's patches from control mice were smaller and did not contain granulomas. Discussion
This experiment showed a successful oral infection rate (over 50%) in C 5 7 B L / 6N mice with a commonly used strain of M. paratuberculosis. Almost all of the lesions and detectable mycobacteria were limited to the MLNs. Although the viability of the bacteria was not examined, the severity of the lesions was related to the duration of infection (data not shown). Furthermore, previous studies showed that bacteria were not readily detected in C57BL mice until several weeks after oral inoculation, and then only in very small numbers (Vance, 1990). This suggests that the infection was progressive. This study indicates that the C 5 7 B L / 6 mouse (a Bc~ strain) may serve as a useful model for studying infection, immunity and pathogenesis in paratuberculosis. Acknowledgments
This work was supported in part by a grant from the Louisiana State University School of Veterinary Medicine. We give special thanks to Cheryl Crowder, Mae Lopez, Del Phillips, and Cindy Berry for their excellent technical assistance and support. This manuscript fulfils, in part, the requirements for the degree of Doctor of Philosophy in the Interdepartmental Program in Veterinary Medical Sciences, Department of Veterinary Pathology, Louisiana State University. References Chiodini, g . J . , Van Kruiningen, H . J . and Merkal, R. S. (1984). Ruminant paratuberculosis (Johne's disease): the current status and future prospects. Cornell Veterinarian, 74, 218-262. Forget, A., Skamene, E., Gros, P., Miailhe, A. C. and Turcotte, R. (1981). Differences in response among inbred mouse strains to infection with small doses of Mycobacterium bovis BCG. Infection and Immunity, 32, 42-47. Frelier, P. F., Templeton, J. W., Estes, M., Whitford, H. W. and Kienle, R. D. (1990). Genetic regulation ofMycobacteriumparatuberculosisinfection in recombinant inbred mice. VeterinaryPathology, 27, 362 364. Gros, P., Skamene, E. and Forget, A. (1981). Genetic control of natural resistance to Mycobacterium bovis (BCG) in mice. Journal of Immunology, 127, 2417 2421. Hamilton, H. L., Cooley, A.J., Adams,J. L. and Czuprynski, C.J. (1991). Mycobacterium paratuberculosis-monoassociated nude mice as a paratuberculosis model. Veterinary Pathology, 28, 146-155. Hamilton, H. L., Follett, D. M., Siegfried, L. M. and Czuprynski, C.J. (1989). Intestinal multiplication of Mycobacterium paratuberculosis in athymic nude gnotobiotic mice. Infection and Immunity, 57, 225-230. Lominski, I., Cameron, J. and Roberts, G. B. S. (1956). ExperimentalJohne's disease in mice. Journal of Pathology and Bacteriology, 71, 211 222.
80
R.S. Veazey
e t al.
Mutwiri, G. K., Butler, D. G., Rosendal, S. and Yager,J. (1992). Experimental infection of severe combined immunodeficient mice with Mycobacterium paratuberculosis of bovine origin. Infection and Immunity, 60, 4074-4079. Skamene, E. (1989). Genetic control of susceptibility to mycobacterial infections. Reviews of Infectious Diseases, 11, $394-$399. Skamene, E., Philippe, G., Forget, A., Kongshavn, P. A. L., St Charles, C. and Taylor, B. A. (1982). Genetic regulation of resistance to intracellular pathogens. Nature, 297, 506-509. Tanaka, S., Sato, M., Taniguchi, T. and Yokomizo, Y. (1994). Histopathological and morphometrical comparison ofgranulomatous lesions in BALB/c and C3H/HeJ mice inoculated with Mycobacteriumparatuberculosis. Journal of Comparative Pathology, 110, 381-388. Vance, T. L. (1990). Experimental Mycobacterium paratuberculosis infection in the beige mouse. Master's Thesis, Louisiana State University, Baton Rouge, LA, USA.
Received, October25th, 1994 ] ccepted, February l Oth, 1995J
SHORT PAPER
Histopathology of C57BL/6 Mice Inoculated Orally with Mycobaeterium paratubereulosis R. S. Veazey*, H. W. Taylor*, D. W. Horohov~, J. L. Krahenbuhl~, J. L. Oliver III* and T. G. Snider III* Departments of* Veterinary Pathology and t Veterinary Microbiology, and the ++G. W. Long, Hansens Disease Center, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA
Summary The susceptibility of C57BL/6 mice to oral inoculation with Myobacterium paratuberculosis was evaluated histopathologically. Granulomatous lesions containing acid-fast bacteria developed in the mesenteric lymph nodes in over 50% of the mice by 11 months after inoculation. The results suggest that C57BL/6 mice may be useful for studying infection, pathogenesis, and other aspects of paratuberculosis. 9 1995 Academic Press Limited
Introduction Paratuberculosis (Johne's disease) is a chronic, granulomatous, intestinal disease of ruminants caused by the acid-fast bacterium Mycobacterium paratuberculosis. Ruminants are orally infected at a young age through the ingestion of contaminated milk, but several months or years may elapse before clinical signs appear. The disease occurs throughout the world, particularly in domestic livestock (Chiodini et al., 1984). Research into the pathogenesis and immunology of paratuberculosis has been hampered by the apparent lack of a suitable laboratory animal model. Early attempts to infect laboratory animals were often unsuccessful (Lominski et al., 1956). It is now known that murine resistance to paratuberculosis and other mycobacteria is associated with the Beg gene which is expressed in two allelic forms: the dominant resistance (Bc~) allele and the recessive susceptibility (Bc~) allele (Gros et al., 1981). Mice with the B%d genotype are resistant to many mycobacterial species, whereas mycobacteria persist and replicate in Bc~ mice (Forget et al., 1981; Skamene et al., 1982; Skamene, 1989; Frelier et al., 1990; Tanaka et al., 1994). Parenterally inoculated Bc~ mice develop granulomatous lesions resembling those of ruminant paratuberculosis (Tanaka et al., 1994). However, no recent attempts to infect immunocompetent Bc~ Correspondence to: R. S. Veazey, Harvard Medical School, New England Regional Primate Research Center, Division of Comparative Pathology, One Pine Hill Drive, P.O. Box 9102, Southborough, MA 01772, USA. 0021-9975/95/050075+06 $12.00/0
9 1995 Academic Press Limited
76
R.S. Veazey e t
al.
mice orally with M. paratuberculosis have been reported. Both nude mice and mice with severe combined immunodeficiency (SCID) are susceptible to oral infection with the organism (Hamilton et al., 1989, 1991; Mutwiri et al., 1992), but their immunodeficient state may make them unsuitable for studies of the immune response to infection. Preliminary studies suggested that Bc~ mice could be infected orally with a high dose o f M . paratuberculosis. With both low (101~ and high (5 x 1011) doses of viable M. paratuberculosis organisms, small numbers of acid-fast bacteria could sometimes be detected in the intestines of C57BL mice 60 days after inoculation (Vance, 1990). These studies indicated that a large dose and an extended post-inoculation period may be required for extensive lesions to develop in susceptible mice. The objectives of the present experiment were to confirm that Bc~ mice (C57BL/6N) could be infected orally with M. paratuberculosis, and to determine whether lesions resembling those of the natural disease would develop after 7 to 11 months, gut-associated lymphoid tissues (GALT) and other tissues being examined histologically for lesions and acid-fast bacteria. Materials and Methods
Mice All mice used in the experiment were bred from a C57BL/6N breeder colony originally obtained from Harlan Sprague-Dawley Inc., Indianapolis, IN, USA. They were conventionally housed in "shoebox"-type cages with stainless-steel wire tops and heattreated, aspen-chip bedding. The animals were fed Purina Rodent Chow 5001 (Purina Mills Inc., St Louis, MO, USA) and water ad libitum, and maintained on a 12/12-h light/dark cycle. All mice were inoculated at 3 weeks of age and then weaned. Control mice were housed in separate cages but under identical conditions.
Organism and Preparation of Inoculum M. paratuberculosis ATCC strain 19698 was obtained from the USDA-ARS, National Animal Disease Center (NADC), Ames, Iowa, USA and grown in Middlebrook 7H9 broth supplemented with Middlebrook OADC Enrichment Medium (Difco, Detroit, MI, USA), glycerol (Sigma, St Louis, MO, USA) 0'2%, and Mycobactin J (Allied Laboratories, Glenwood Springs, CO, USA) 2 mg/1. Broth cultures were incubated at 37~ for 4 months and bacteria were harvested by centrifugation (10 min at 2000 g), washed three times in phosphate-buffered saline, agitated ultrasonically for 10 s at 40W (Heat Systems-Ultrasonics Inc., Plainview, NY, USA) to reduce clumping, and adjusted to 3 x 10" cells/dose as determined by a McFarland's nephelometer standard. Each dose was suspended in 0-3 ml of pasteurized cream (Borden Inc., Baton Rouge, LA, USA) and administered orally by means of a ball-tipped needle. The number of viable bacteria per inoculum was later verified by plating serial dilutions on Middlebrook 7H10 agar supplemented with OADC and MycobactinJ. Control mice (n = 6) were inoculated orally with 0"3 ml of cream alone. Groups of infected mice (n = 4 to 12) were killed by cervical dislocation 7, 8, 9 and 11 months after inoculation (Table 1). Control mice were killed and examined 11 months after inoculation.
Histology Liver, spleen, intestine and mesenteric lymph node (MLN) were fixed in neutralbuffered 10% formalin. At least one cross-section of liver (taken from the widest part
77
M. paratuberculosis i n M i c e Table 1 Histological e x a m i n a t i o n of C57BL/6 m i c e infected orally with M. paratuberculosis Number of mice with lesions and mycobacteria in Period (months) after inoculation 11 9 8 7
Number of mice examined
mesenteric lymph node
spleen
liver
intestine
6 4 12 9
4* 1I 7 6
1 1 0 1
1 l 0 1
0 0 0 0
* Only four mice examined, because of a technical mishap. ~"Only two mice examined, because of a technical mishap. Six control (uninfected) mice gave negative results.
of the left lobe) and spleen, and at least 1 cm of duodenum, jejunum, ileum, ileocaecal valve and colon were examined histologically from each mouse. MLNs from four animals were lost during processing and therefore not examined. Tissues were embedded in paraffin wax and 5-gm sections stained with haematoxylin and eosin (HE) and Ziehl-Neelsen acid-fast stain (ZN) were examined by light microscopy. Tissues containing acid-fast bacteria were additionally stained by the avidin-biotin method with rabbit anti-M, paratuberculosispolyclonal antibody (Dako Inc., Carpinteria, CA, USA). Results
No clinical signs of disease, weight loss, or gross lesions were detected in any of the infected mice. A total of three infected and four control animals were culled 30-60 days after inoculation due to an intractable idiopathic dermatitis. No histological lesions other than dermatitis were detected in these mice and they were excluded from the study. A total of 18 out of 31 (58%) orally infected mice were found to have demonstrable acid-fast bacteria, usually limited to the MLN (Table 1). O f the 27 MLNs examined, 17 (63%) were infected. Acid-fast bacteria were also found in three livers and three spleens (in five mice). Immunohistochemical staining of infected tissue sections with rabbit anti-M, paratuberculosis antibody confirmed that the organisms were mycobacteria. The severity of the MLN lesions varied, but granulomas and multinucleated giant cells were consistent findings. The most severe lesions were observed in MLNs from mice inoculated 11 months earlier (Fig. 1). In these nodes, the normal follicular architecture was effaced by pale, swollen macrophages and multinucleated giant cells. Macrophages filled with numerous acid-fast bacteria were present within granulomas (Fig. 2). Less severe lesions, containing fewer macrophages and acid-fast bacilli, were detected in MLNs from mice in the 7-month post-inoculation group (data not shown). Hepatic microgranulomas containing acid-fast bacteria were detected in three mice. Larger periportal granulomas were also sometimes present in infected mice; these consisted predominantly of lymphocytes, plasma cells and
78
R.S. Veazey
e t ai.
Fig. 1.
Mesenteric lymph node of a C57BL/6 mouse 11 months after oral infection with M. paratuberculosis. Note the extensive infiltration of macrophages and multinucleated giant cells. HE. x 400.
Fig. 2.
Same mesenteric lymph node as in Fig. 1 showing several macrophages filled with numerous acidfast bacilli. ZN. x 1000.
M. paratuberculosis in Mice
79
small numbers of macrophages, but no acid-fast bacteria. No mycobacteria or related lesions were detected in the control mice. The intestines frequently showed hyperplastic Peyer's patches and slightly to moderately increased numbers of lymphocytes and plasma cells; occasional micro-granulomas consisted of aggregates of epithelioid macrophages, but acid-fast bacteria were not detected in the intestines of any of the mice. Peyer's patches from control mice were smaller and did not contain granulomas. Discussion
This experiment showed a successful oral infection rate (over 50%) in C 5 7 B L / 6N mice with a commonly used strain of M. paratuberculosis. Almost all of the lesions and detectable mycobacteria were limited to the MLNs. Although the viability of the bacteria was not examined, the severity of the lesions was related to the duration of infection (data not shown). Furthermore, previous studies showed that bacteria were not readily detected in C57BL mice until several weeks after oral inoculation, and then only in very small numbers (Vance, 1990). This suggests that the infection was progressive. This study indicates that the C 5 7 B L / 6 mouse (a Bc~ strain) may serve as a useful model for studying infection, immunity and pathogenesis in paratuberculosis. Acknowledgments
This work was supported in part by a grant from the Louisiana State University School of Veterinary Medicine. We give special thanks to Cheryl Crowder, Mae Lopez, Del Phillips, and Cindy Berry for their excellent technical assistance and support. This manuscript fulfils, in part, the requirements for the degree of Doctor of Philosophy in the Interdepartmental Program in Veterinary Medical Sciences, Department of Veterinary Pathology, Louisiana State University. References Chiodini, g . J . , Van Kruiningen, H . J . and Merkal, R. S. (1984). Ruminant paratuberculosis (Johne's disease): the current status and future prospects. Cornell Veterinarian, 74, 218-262. Forget, A., Skamene, E., Gros, P., Miailhe, A. C. and Turcotte, R. (1981). Differences in response among inbred mouse strains to infection with small doses of Mycobacterium bovis BCG. Infection and Immunity, 32, 42-47. Frelier, P. F., Templeton, J. W., Estes, M., Whitford, H. W. and Kienle, R. D. (1990). Genetic regulation ofMycobacteriumparatuberculosisinfection in recombinant inbred mice. VeterinaryPathology, 27, 362 364. Gros, P., Skamene, E. and Forget, A. (1981). Genetic control of natural resistance to Mycobacterium bovis (BCG) in mice. Journal of Immunology, 127, 2417 2421. Hamilton, H. L., Cooley, A.J., Adams,J. L. and Czuprynski, C.J. (1991). Mycobacterium paratuberculosis-monoassociated nude mice as a paratuberculosis model. Veterinary Pathology, 28, 146-155. Hamilton, H. L., Follett, D. M., Siegfried, L. M. and Czuprynski, C.J. (1989). Intestinal multiplication of Mycobacterium paratuberculosis in athymic nude gnotobiotic mice. Infection and Immunity, 57, 225-230. Lominski, I., Cameron, J. and Roberts, G. B. S. (1956). ExperimentalJohne's disease in mice. Journal of Pathology and Bacteriology, 71, 211 222.
80
R.S. Veazey
e t al.
Mutwiri, G. K., Butler, D. G., Rosendal, S. and Yager,J. (1992). Experimental infection of severe combined immunodeficient mice with Mycobacterium paratuberculosis of bovine origin. Infection and Immunity, 60, 4074-4079. Skamene, E. (1989). Genetic control of susceptibility to mycobacterial infections. Reviews of Infectious Diseases, 11, $394-$399. Skamene, E., Philippe, G., Forget, A., Kongshavn, P. A. L., St Charles, C. and Taylor, B. A. (1982). Genetic regulation of resistance to intracellular pathogens. Nature, 297, 506-509. Tanaka, S., Sato, M., Taniguchi, T. and Yokomizo, Y. (1994). Histopathological and morphometrical comparison ofgranulomatous lesions in BALB/c and C3H/HeJ mice inoculated with Mycobacteriumparatuberculosis. Journal of Comparative Pathology, 110, 381-388. Vance, T. L. (1990). Experimental Mycobacterium paratuberculosis infection in the beige mouse. Master's Thesis, Louisiana State University, Baton Rouge, LA, USA.
Received, October25th, 1994 ] ccepted, February l Oth, 1995J