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B10 mice to infection with Mycobacterium paratuberculosis
J.
Compo Path. 1993 Vol. 109,309-319
Susceptibility of Balb/c, C57/B6 and C57/BIO Mice to Infection with Mycobacterium paratuberculosis R.
J.
Chiodini and C. D. Buergelt*
Nfycobacteriology Unit, Division Qj' Gastroenterology, De/Jartment of Medicine, Rhode lfland Hospital and Division of Biological and Medical Sciences, Department if Medicine, Brown Universiry, 593 Eddy Street, Providence, R1 02903, and *Department qj' Comparative and Experimental Pathology, College of Veterinm)' Medicine, Universiry of Florida, Gainesville, FL 32610, U.S.A. Summary Balb/C, C5 7/B I 0 and C5 7/B6 mice were examined for their susceptibility to disseminated Nfycobacterium paratuberculosis inlh:tion after intraperitoneal inoculation with a suspension of organisms cOilLaining mineral oil. Animals were examined monthly by histopathology and bacterial tissue counts of liver and spleen over a 6-month period. Only Balb/e mice maintained a steady infection with an average of 4·1 x 10"±H3x]03 and 8'1 x 10"±2'6X 10'\ colony forming units (eliJ) per gram of liver and spleen, respectively, during the course of the study. In contrast, C57/BIO mice reduced the bacterial counts in the liver and spleen from 6·8 x 104 and 1·3 x 10" to 7·1 X 10 2 and 4-3 x 10\ respectively during the first 120-150 days after infection. The reduction in C!U was associated with the development of caseous necrotic lesions. C5 7/B 10 mice were of intermediate resistance, slowly reducing cfu in the liver, but not the spleen, during the 6-month period. Balblc was found to be a suitable mouse straiIl fix the study of chronic M. /Jaratuberculosis infection.
Introduction
Mycobacterium j)aratuberculosis is the aetiological agent of paratuberculosis (Johne's disease), a chronic granulomatous ileocolitis of ruminant animals (reviewed by Chiodini et al., 1984a). The disease occurs throughout the world and in all of the United States, with a prevalence ranging from 3-17 per cent in culled dairy cattle. The disease is characterized clinically by progressive weight loss associated with a diarrhoea that fails to respond to treatment. Pathologically, the affected intestinal mucosa is severely thickened and corrugated, with an accumulation of large foamy macrophages containing abundant acid-fast bacilli within the mucusa and submucosa. Diagnosis is sometimes difficult and no effective vaccine is available. Many laboratory species are said to be susceptible to infection, but the data to support such statements are often incomplete and inconclusive. In many murine studies the mouse strain was not stated (Francis, 1943; Harding, 1959) and infections were evaluated subjectively rather than quantitatively (Lominski et al., 1956; Chandler, 1961 b). Moreover, M. j)aratuberculosis can behave differently in a single murine strain depending on its source (unpublished observation). Most studies sought to establish intestinal infections 00219975/93/080309 + 11 $08,00/0
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310
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J.
Chiodini and C. D. Buergelt
similar to those of paratuberculosis in ruminants, and took advantage of the fact tha t intestinal infection resul ted from in traperi toneal or intravenous inoculation. Intestinal infections occurred in some animals after incubation periods greater than 9 months (Lominski et al., 1956; Chandler, 1961b), but generally the infections were self-limiting. The administration of suramin tended to increase intestinal infection, while the administration of cortisone diminished both intestinal and systemic infection (Chandler, 1961a). Hamilton et al. (1989) described a progressive intestinal disease in nude mice orally infected with iV1. paratuberculosis. Others have attempted to adopt the' At. avium beige mouse model' to NJ. paratuberculosis infection or have produced systemic disease in mice with severe combined immunodeficiency (Mutwiri et al., 1992; Whipple et al., 1992). We sought a less expensive model which would support the in-vivo growth of NJ. paratuberculosis and could be used in studies not requiring intestinal infection, for example, studies on chemotherapy. Materials and methods
Organisms and culture iVI. paratuberculosis strain Linda (ATCC 43015), from a patient with Crahn's disease, was originally isolated in the authors' laboratory on Herrold's egg yolk medium (HEYM) containing mycobactin J 2 j..lg per ml (Chiodini et al., 1984b). Seed lot cultures, frozen at - SO°C, were thawed and cultivated in 7H9 broth supplemented with Dubos's oleic albumin complex, Tween 80 (0.05 per cent) and mycobactinJ 2 j..lg per ml in 25 cm 3 (30ml) tissue culture flasks (Chiodini et al., 1984b). Cultures were harvested during the mid-logarithmic phase of growth by centrifugation at 4340 G and the organisms resuspended in phosphate buffered saline (PBS) containing Tween 80 (0'05 per cent) at ca 5 x 10 9 colony forming units (cfu) per ml as determined spectrophotometrically at 540 nm (Chiodini et al., 1984c). The cell suspension was then diluted I: 1 with sterile mineral oil (to prevent rapid dispersion) to give a final cell suspension of 2'5 x 1O!1 cfu per ml in a 1: I water-in-oil emulsion.
Mice A total of78 female mice (26 each ofBalb/c, C57/B6 and C57/BIO) aged 2 weeks were obtained from Jackson Laboratories (Bar Harbour, ME, USA) and inoculated intraperitoneally with 0·25 ml of the bacterial suspension (ca 6·2 x lOB cfu). Animals were caged in groups of 6-8 and housed in an Institutional Animal Care and Use Committee (IACUC) accredited animal facility at Brown University. Four animals of each strain were killed by cervical dislocation at ca 30, 60,90, 120, 150 and 180 days after inoculation. Two uninfected animals of each strain were killed at the beginning of the experimen t to establish normal liver and spleen weights.
Histological and Bacteriological Examination The liver and spleen were removed aseptically from each animal at the time of death, weighed and then divided in half One half was used for histopathology and the other for quantitative bacteriological culture. Specimens for histopathology were fixed in 10 per cent buffered formalin, sectioned at 6j..lm, and stained with haematoxylin and eosin and by the Kinyoun's method for acid-fast bacilli. The number of granulomas per field was determined under x 10 magnification. Their size was determined subjectively and graded 1+ to 4·+ in terms
M. paratuberculosis in Mice
311
of their degree of spread. Both histological and cultural examinations were performed 'blind'. Specimens for quantitative bacteriological culture were weighed and homogenized in a Ten Broeck grinder in Q. 75 per cent hexadecylpyridinium chloride (HPC). The homogenate was allowed to stand at room temperature for 18 to 24 h, mixed, and then subjected to serial lO-fold dilution in PBS. One hundred microlitres of the homogenate and of each serial IO-fold dilution were inoculated onto each of four slopes of HEYM and incubated at 36-38°C for 12 to 16 weeks. The number of cfil per gram of tissue was then calculated.
Statistical Anarysis Data were computer-analysed with the Ncwman-Keuls test, Dunnett's test and the Bonfaroni modification of the unpaired t-test.
Results Th~
changes in liver and spleen weights and the number of cfu per gram of liver and spleen for all strains during the study are presented in Figs 1-4.
Balb/c Mice There were no gross lesions in any animal and liver and spleen weights remained normal during the course of the study. Histologically, there were peritoneal surface or subcapsular granulomas composed mainly of foamy macrophages with lymphocytes or neutrophils scattered in the periphery (Fig. 5). Towards the end of the study, some of the granulomas in a few animals were lytic or coagulative, but most remained completely cellular. The number of surface granulomas remained 2-3 per field and were small to medium size throughout the study. Inflammatory foci within the liver parenchyma were composed of individual foamy macrophages with few lymphocytes and nerrotic hepatocytes (Fig. 6). These numbered from 5-10 per field and were generally graded 2 + . Acid-fast bacilli were seen within surface granulomas in most animals throughout the study. In the spleen and liver the number of cfu remained fairly constant throughout the study, averaging 5·6 x 105± 2·6 x 10 4 and 5,4 x 105± 7·9 x 103 per gram, respectively, suggesting neither bacterial proliferation nor elimination. Spleens generally harboured more organisms per gram than liver.
C57/B6 Mice At 30 days, the livers contained multiple small white focal lesions and the spleens were enlarged. At 60 days, there were only a few white focal lesions in the liver, some of which were still present at 90 days, but were completely absent by 120 days. The spleens appeared normal at 90 days. Liver weights increased sharply during the first 30 days, and spleen weights increased by ca 260 per cent by 30 days before returning to near normal at 60 days. Histologically, peritoneal and subcapsular granulomas were as described for Balb/c mice except that there was often coagulation necrosis and some
312
R.
J.
Chiodini and C. D. Buergelt
Liver weight (gms)
1.8
-0-
Balb/C
-Gr-
C57/B6
"* C57/B10 1.5
1.2
0.9
0.6 -t-----;------'f--------+----+-----+-----\--'
o
30
60
90
120
150
180
Days of Infection Fig.!'
Changes in liver weights in mice infected with M. paratuberculosis during the 6-month course of infection. C57/B6 mice had an increased liver mass during the first 30 days; in C57/BIO mice the liver mass increased during the first 90 days and then diminished. Increased mass in C57/B10 mice was associated with coagulation nodules. Each time point represents four mice.
mineralization as the study progressed. The number of granulomas per field was 2-3 and varied from small to large with some confluency. Parenchymal granulomas numbered 5-20 per field and were graded 3 +. Acid-fast bacilli were observed sporadically, particularly towards the end of the study. Bacterial counts in the liver decreased during the course of the study from 4x10 5 to 4'6xIQ3 (P<0·05). At 180 days the number of efu differed significantly from that of Balblc mice (P<0·05) but not C57/BIO mice. In contrast to the liver, bacterial counts in the spleen were not significantly
313
M. paratuberculosis in Mice
Spleen Weight (gms) -0,
0.5
•.
_.
_ •...•...
,
-,
i'
Balb/C
"* C57/86
*"" C57/B1 0 0.4
0.3
0.2
0.1
-j----.....-----,----,..------,----r----,--'
o
30
60
90
120
150
180
Days of Infection Fig. 2.
Changes in spleen mass in mice infected with M, paratuberculosis. Spleen weights increased in response to early infection in C57/BJO and C57/B6, but not in Balb/c mice,
reduced during the 6-month period. During the first 150 days, bacterial counts remained constant at 2'8 x 105±6'5 x 103 , decreasing only between days 150 and 180.
C57/BIO Mice At 30 days, the spleen and liver had multiple white focal lesions and large yellow nodules. There were multiple adhesions between the liver and peritoneum. The lesions decreased in intensity and number up to 120 days, but by
314
R. ]. Chiodini and C. D. Buergelt
CFU per gm Liver (Log 10)
6-.-----------------------,
5
3 -0-
Balb/C
-tr-
C57/B6
*C57/B10
2-+=======::;::::::====---i----.;..----i-----r----J 30
60
90
120
150
180
Days of infection Fig. 3.
Changes in bacterial counts in the liver of mice infected with M.paratuherculosis. Both C57/BIO and C57/B6 strains showed bacterial clearance while a steady-state infection was maintained in Balb/c mice. SD= <: 10 per cent of mean.
150 days both liver and spleen were macroscopically normal. However, their weights were increased during the entire course of the study. The peak liver cha.nge occurred at 30 days with a 37 per cent weight increase; a 16 per cent increase was noted after 180 days. The spleen was 250 per cent normal weight at 30 days and was still enlarged after 180 days. Histopathologically, peritoneal and subcapsular hepatic granulomas were largely coagulative during the early part of the study, changing to coagulative
315
M. paratubercltlosis in Mice
CFU per gm Spleen (Log 10)
6
5
......
4-0-
Balb/C
-t:r-
C57/B6
*" C57/B10 330
60
90
120
150
180
Days of Infection Fig. 4.
Changes in bacterial counts in the splecn of mice infectcd with M. paratuberculosis. Bactcrial clearance was observed only in C57/BlO mice, while prolileralion may have been occurring in Balb/c mice. C57/B6 mice were less capable of reducing bacterial counts in the spleen than in the liver (Fig. 3). SD= < JO per cent of mean.
and fibrous with some mineralization by 6 months. These granulomas initially numbered 4-5 per field and were large to confluent. By the end of the study, the numbers were reduced to 1-2 per Held and they were generally small in size. Parenchymal granulomas initially numbered 10-20 and were graded 34 +, being reduced to 5-10 or less per Held with a 1-2 + grading after 6 months. Acid-fast bacilli were not observed after 4- months.
316
Fig. 5.
R.
J.
Chiodini and C. D. Buergelt
Peritoneal granulomas in Balblc mice 150 days arter inocuhuion with AI. paratuberculosis. The granulomas were composed of a lytic centre that contained amorphous, fibrillar-like or dot-like material representing dumped acid-['lst bacilli. The periphery of the granulomas were composed of foam-cell maerophages, lymphocytes and individual ncutmphils. Coagulation neerosis was not a feature. HE. x 400.
Bacterial counts tended to be reduced at 30 days, as compared with those in Ba1b/e mice, and continued to decline during the first 120-150 days of the stud y. In the liver and spleen the cfu were red uced from 6·8 x 10" and 1· 3 x 105 to 7·1 X 10 2 and 4·3 x 103 , respectively, after 180 days. Although the data indicate that bacterial elimination may have ceased at around 120-150 days, the efu were then approaching the limits of detection, with increased standard deviations. Discussion Progressive infection was not observed in any of the mouse strains examined. Balb/c mice were the most susceptible to infection, but bacterial proliferation either did not occur, equalled the rate of elimination, or occurred at a rate too slow to detect during the course of this study. The general trend of cfu in the liver and spleen suggests, however, that a very slow rate of proliferation may have been occurring, particularly in the spleen. In can trast, C5 7/B 10 mice were the most resistant to infection, with gradual bacterial elimination. The greatest reduction in bacterial counts occurred within the first 120 days of infection and then appeared to level off. It is likely that the C57/BIO mice
M. paratuberculosis in Mice
Fig. G.
317
Inflammatory foci in the liver of Balblc mice 60 days after inoculation with lV[. paratuberculosis. These were mainly composed of lymphocytes plus individual macrophages and necrotic hepatocytes. The loci were "andomly distributed throughout the hcpatic lobules, but were also present in portal triads. HE. x 'fDO.
would have ultimately cleared the organism. C57/B6 mice were of intermediate resistance, showing gradual cfu reductions during the course of the study. The relation between gross and histological lesions and changes in bactcrial counts is of particular in terest. Delayed type hypersensitivity (DTH) reactions are often considered detrimental, rather than beneficial, in mycobacterial infections, since they cause most of the tissue damage. DTH is apparently not a requirement for protective immunity (Dannenberg, 1989). The rapid elimination of bacteria in the liver of C57/BIO mice during the first 120 days was associated with coagulation necrosis characteristic of a DTH reaction. In C57/B6 mice, a slower rate of bacterial clearance was associated with a later appearance of lesions and few granulomas containing coagulation necrosis. In contrast, there was bacterial persistence unaccompanied by necrosis in Balb/c mice. Thus, we observed a direct relationship between DTH (as assessed by necrosis) and bacterial clearance. Similarly to Balb/c mice, cattle with paratuberculosis do not develop necrosis and have persistent infection. The observations reported suggest that this model system may be applicable to a comparative study of bacterial clearance or persistence and DTH in Balb/c vs C5 7/B 10 mice strains. While there is no doubt that DTH reactions are often
318
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Chiodini and C. D. Buergelt
detrimental and cause extensive tissue damage, some studies have shown an inverse relation between necrosis and host survival (Flynn et al., 1992). The studies reported here support the notion that DTH may reflect the price a host must pay for rapid bacterial clearance (Yamamura et al., 1991). Of the mouse strains examined, only Balb/c was found to be susceptible to chronic M. paratuberculosis infection. This strain would provide a suitable model for the study of antimicrobial agents or other activities in vivo in which the end point was bacterial elimination or clearance. It has an advantage over other recently described models (Hamilton et al., 1989; Mutwiri et al., 1992; Whipple et al., 1992) in that Balb/c mice are not immunologically deficient and can therefore be inexpensively housed and maintained. In addition, they do not have increased susceptibility to common respiratory infections or tumours. Although intestinal infection did not occur, Balb/c mice offer a more immunologically accurate model than immunodeficient mice, since mycobacterial lesions and disease are characteristically mediated by immunological responses to mycobacterial antigens (Dannenberg, 1989). Acknowledgments
Supported in part by a grant from Adria Laboratories, Columbus, OH, U.S.A. References
Chandler, R. L. (1961a). Infection of laboratory animals with MJ1cobactcrium johnei. I. Infection in Swiss white mice and its modification by suramin and cortisone. Journal of Comparative Pathology, 71, 118-130. Chandler, R. L. (1961 b). Infection of laboratory animals with Mycobacterium johnei. Comparative susceptibility to infection of C57, C.B.A. and Swiss white mice. Journal of Comparative Pathology, 71, 233-24'2. Chiodini, R. ]., Van Kruiningen, H. ]. and Merkal, R. S. (1984a). Ruminant paratuberculosis Oohne's disease): the current status and future prospects. Cornell Vetcrinarian, 74, 218-262. Chiodini, R.]., Van Kruiningen, H.j., Merkal, R. S., Thayer, W. R. and Coutu,J. A. (1 984b). Characteristics of an unclassified Mycobacterium species isolated from patients with Crohn's disease. Journal of Clinical Microbiology) 20, 966-971. Chiodini, R.]., Van Kruiningen, H.]., Thayer, W. R., Coutu,]. A. and Merkal, R. S. (l984c). In vitro antimicrobial susceptibility of a Mycobacterium species isolated from patients with Crohn's disease. Antimicrobial Agents and Chemothcrapy, 26, 930-932. Dannenberg, A. M. (1989). Immune mechanisms in the pathogenesis of pulmonary tuberculosis. Rcviews of Infectious Diseasc, 11, S369-S378. Flynn,]. L., Goldstein, M. M., Triebold, K. j., Koller, B. and Bloom, B. R. (1992). Major histocompatibility complex class I-restricted T cells are required for resistance to Mycobacterium tubcrculosis infection. Proceedings ofthe National Academy of Sciences, 89, 12013-120 17. Francis,]. (1943). Infection oflaboratory animals with Mycobacterium johnei. Journal of Comparative Pathology, 53, 140-150. Hamilton, H. L., Follett, D. M., Siegfried, L. M. and Czuprynski, C. ]. (1989). Intestinal multiplication of Mycobacterium paratuberculosis in athymic nude gnotobiotic mice. Infection and Immunity, 57, 225-230. Harding, H. P. (1959). The histopathology of Mycobacterium johnei infection in small lab. animals. Journal of Pathology and Bacteriology, 78, 157-169.
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319
Lominski, I., Cameron,j. and Roberts, G. B. S. (1956). Experimental Johne's disease in mice. Journal of Pathology and Bacteriology, 71, 211-222. Mutwiri, G.K., Butler, D.G., Rosendal, S. and Yager, j. (1992). Experimental infection of severe combined immunodeficient beige mice with l'/I.ycobacterium paratuberculosis of bovine origin. Injection and Immunity, 60, 4074-4079. Whipple, D. 1., Klemens, S. P. and Cynamon, M. H. (1992). Development of the beige mouse as a model for M. paratuberculosis infection. In: Proceedings of the Third International Colloquium on Paratuberculosis. R. j. Chiodini and j. M. Kreeger, Eds, International Association for Paratuberculosis, Providence, RI, pp. 551-552. Yamamura, M., Uyemura, K., Deans, R.j., Weinberg, K., Rea, T. H., Bloom, B. R. and Modlin, R. 1. (1991). Defining protective responses to pathogens: cytokine profiles in leprosy lesions. Science, 245, 277-279.
Received, April 1st, 1993J [ Accepted, July 9th, 1993
Compo Path. 1993 Vol. 109,309-319
Susceptibility of Balb/c, C57/B6 and C57/BIO Mice to Infection with Mycobacterium paratuberculosis R.
J.
Chiodini and C. D. Buergelt*
Nfycobacteriology Unit, Division Qj' Gastroenterology, De/Jartment of Medicine, Rhode lfland Hospital and Division of Biological and Medical Sciences, Department if Medicine, Brown Universiry, 593 Eddy Street, Providence, R1 02903, and *Department qj' Comparative and Experimental Pathology, College of Veterinm)' Medicine, Universiry of Florida, Gainesville, FL 32610, U.S.A. Summary Balb/C, C5 7/B I 0 and C5 7/B6 mice were examined for their susceptibility to disseminated Nfycobacterium paratuberculosis inlh:tion after intraperitoneal inoculation with a suspension of organisms cOilLaining mineral oil. Animals were examined monthly by histopathology and bacterial tissue counts of liver and spleen over a 6-month period. Only Balb/e mice maintained a steady infection with an average of 4·1 x 10"±H3x]03 and 8'1 x 10"±2'6X 10'\ colony forming units (eliJ) per gram of liver and spleen, respectively, during the course of the study. In contrast, C57/BIO mice reduced the bacterial counts in the liver and spleen from 6·8 x 104 and 1·3 x 10" to 7·1 X 10 2 and 4-3 x 10\ respectively during the first 120-150 days after infection. The reduction in C!U was associated with the development of caseous necrotic lesions. C5 7/B 10 mice were of intermediate resistance, slowly reducing cfu in the liver, but not the spleen, during the 6-month period. Balblc was found to be a suitable mouse straiIl fix the study of chronic M. /Jaratuberculosis infection.
Introduction
Mycobacterium j)aratuberculosis is the aetiological agent of paratuberculosis (Johne's disease), a chronic granulomatous ileocolitis of ruminant animals (reviewed by Chiodini et al., 1984a). The disease occurs throughout the world and in all of the United States, with a prevalence ranging from 3-17 per cent in culled dairy cattle. The disease is characterized clinically by progressive weight loss associated with a diarrhoea that fails to respond to treatment. Pathologically, the affected intestinal mucosa is severely thickened and corrugated, with an accumulation of large foamy macrophages containing abundant acid-fast bacilli within the mucusa and submucosa. Diagnosis is sometimes difficult and no effective vaccine is available. Many laboratory species are said to be susceptible to infection, but the data to support such statements are often incomplete and inconclusive. In many murine studies the mouse strain was not stated (Francis, 1943; Harding, 1959) and infections were evaluated subjectively rather than quantitatively (Lominski et al., 1956; Chandler, 1961 b). Moreover, M. j)aratuberculosis can behave differently in a single murine strain depending on its source (unpublished observation). Most studies sought to establish intestinal infections 00219975/93/080309 + 11 $08,00/0
((~
1993 Academic Press Limi ted
310
R.
J.
Chiodini and C. D. Buergelt
similar to those of paratuberculosis in ruminants, and took advantage of the fact tha t intestinal infection resul ted from in traperi toneal or intravenous inoculation. Intestinal infections occurred in some animals after incubation periods greater than 9 months (Lominski et al., 1956; Chandler, 1961b), but generally the infections were self-limiting. The administration of suramin tended to increase intestinal infection, while the administration of cortisone diminished both intestinal and systemic infection (Chandler, 1961a). Hamilton et al. (1989) described a progressive intestinal disease in nude mice orally infected with iV1. paratuberculosis. Others have attempted to adopt the' At. avium beige mouse model' to NJ. paratuberculosis infection or have produced systemic disease in mice with severe combined immunodeficiency (Mutwiri et al., 1992; Whipple et al., 1992). We sought a less expensive model which would support the in-vivo growth of NJ. paratuberculosis and could be used in studies not requiring intestinal infection, for example, studies on chemotherapy. Materials and methods
Organisms and culture iVI. paratuberculosis strain Linda (ATCC 43015), from a patient with Crahn's disease, was originally isolated in the authors' laboratory on Herrold's egg yolk medium (HEYM) containing mycobactin J 2 j..lg per ml (Chiodini et al., 1984b). Seed lot cultures, frozen at - SO°C, were thawed and cultivated in 7H9 broth supplemented with Dubos's oleic albumin complex, Tween 80 (0.05 per cent) and mycobactinJ 2 j..lg per ml in 25 cm 3 (30ml) tissue culture flasks (Chiodini et al., 1984b). Cultures were harvested during the mid-logarithmic phase of growth by centrifugation at 4340 G and the organisms resuspended in phosphate buffered saline (PBS) containing Tween 80 (0'05 per cent) at ca 5 x 10 9 colony forming units (cfu) per ml as determined spectrophotometrically at 540 nm (Chiodini et al., 1984c). The cell suspension was then diluted I: 1 with sterile mineral oil (to prevent rapid dispersion) to give a final cell suspension of 2'5 x 1O!1 cfu per ml in a 1: I water-in-oil emulsion.
Mice A total of78 female mice (26 each ofBalb/c, C57/B6 and C57/BIO) aged 2 weeks were obtained from Jackson Laboratories (Bar Harbour, ME, USA) and inoculated intraperitoneally with 0·25 ml of the bacterial suspension (ca 6·2 x lOB cfu). Animals were caged in groups of 6-8 and housed in an Institutional Animal Care and Use Committee (IACUC) accredited animal facility at Brown University. Four animals of each strain were killed by cervical dislocation at ca 30, 60,90, 120, 150 and 180 days after inoculation. Two uninfected animals of each strain were killed at the beginning of the experimen t to establish normal liver and spleen weights.
Histological and Bacteriological Examination The liver and spleen were removed aseptically from each animal at the time of death, weighed and then divided in half One half was used for histopathology and the other for quantitative bacteriological culture. Specimens for histopathology were fixed in 10 per cent buffered formalin, sectioned at 6j..lm, and stained with haematoxylin and eosin and by the Kinyoun's method for acid-fast bacilli. The number of granulomas per field was determined under x 10 magnification. Their size was determined subjectively and graded 1+ to 4·+ in terms
M. paratuberculosis in Mice
311
of their degree of spread. Both histological and cultural examinations were performed 'blind'. Specimens for quantitative bacteriological culture were weighed and homogenized in a Ten Broeck grinder in Q. 75 per cent hexadecylpyridinium chloride (HPC). The homogenate was allowed to stand at room temperature for 18 to 24 h, mixed, and then subjected to serial lO-fold dilution in PBS. One hundred microlitres of the homogenate and of each serial IO-fold dilution were inoculated onto each of four slopes of HEYM and incubated at 36-38°C for 12 to 16 weeks. The number of cfil per gram of tissue was then calculated.
Statistical Anarysis Data were computer-analysed with the Ncwman-Keuls test, Dunnett's test and the Bonfaroni modification of the unpaired t-test.
Results Th~
changes in liver and spleen weights and the number of cfu per gram of liver and spleen for all strains during the study are presented in Figs 1-4.
Balb/c Mice There were no gross lesions in any animal and liver and spleen weights remained normal during the course of the study. Histologically, there were peritoneal surface or subcapsular granulomas composed mainly of foamy macrophages with lymphocytes or neutrophils scattered in the periphery (Fig. 5). Towards the end of the study, some of the granulomas in a few animals were lytic or coagulative, but most remained completely cellular. The number of surface granulomas remained 2-3 per field and were small to medium size throughout the study. Inflammatory foci within the liver parenchyma were composed of individual foamy macrophages with few lymphocytes and nerrotic hepatocytes (Fig. 6). These numbered from 5-10 per field and were generally graded 2 + . Acid-fast bacilli were seen within surface granulomas in most animals throughout the study. In the spleen and liver the number of cfu remained fairly constant throughout the study, averaging 5·6 x 105± 2·6 x 10 4 and 5,4 x 105± 7·9 x 103 per gram, respectively, suggesting neither bacterial proliferation nor elimination. Spleens generally harboured more organisms per gram than liver.
C57/B6 Mice At 30 days, the livers contained multiple small white focal lesions and the spleens were enlarged. At 60 days, there were only a few white focal lesions in the liver, some of which were still present at 90 days, but were completely absent by 120 days. The spleens appeared normal at 90 days. Liver weights increased sharply during the first 30 days, and spleen weights increased by ca 260 per cent by 30 days before returning to near normal at 60 days. Histologically, peritoneal and subcapsular granulomas were as described for Balb/c mice except that there was often coagulation necrosis and some
312
R.
J.
Chiodini and C. D. Buergelt
Liver weight (gms)
1.8
-0-
Balb/C
-Gr-
C57/B6
"* C57/B10 1.5
1.2
0.9
0.6 -t-----;------'f--------+----+-----+-----\--'
o
30
60
90
120
150
180
Days of Infection Fig.!'
Changes in liver weights in mice infected with M. paratuberculosis during the 6-month course of infection. C57/B6 mice had an increased liver mass during the first 30 days; in C57/BIO mice the liver mass increased during the first 90 days and then diminished. Increased mass in C57/B10 mice was associated with coagulation nodules. Each time point represents four mice.
mineralization as the study progressed. The number of granulomas per field was 2-3 and varied from small to large with some confluency. Parenchymal granulomas numbered 5-20 per field and were graded 3 +. Acid-fast bacilli were observed sporadically, particularly towards the end of the study. Bacterial counts in the liver decreased during the course of the study from 4x10 5 to 4'6xIQ3 (P<0·05). At 180 days the number of efu differed significantly from that of Balblc mice (P<0·05) but not C57/BIO mice. In contrast to the liver, bacterial counts in the spleen were not significantly
313
M. paratuberculosis in Mice
Spleen Weight (gms) -0,
0.5
•.
_.
_ •...•...
,
-,
i'
Balb/C
"* C57/86
*"" C57/B1 0 0.4
0.3
0.2
0.1
-j----.....-----,----,..------,----r----,--'
o
30
60
90
120
150
180
Days of Infection Fig. 2.
Changes in spleen mass in mice infected with M, paratuberculosis. Spleen weights increased in response to early infection in C57/BJO and C57/B6, but not in Balb/c mice,
reduced during the 6-month period. During the first 150 days, bacterial counts remained constant at 2'8 x 105±6'5 x 103 , decreasing only between days 150 and 180.
C57/BIO Mice At 30 days, the spleen and liver had multiple white focal lesions and large yellow nodules. There were multiple adhesions between the liver and peritoneum. The lesions decreased in intensity and number up to 120 days, but by
314
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CFU per gm Liver (Log 10)
6-.-----------------------,
5
3 -0-
Balb/C
-tr-
C57/B6
*C57/B10
2-+=======::;::::::====---i----.;..----i-----r----J 30
60
90
120
150
180
Days of infection Fig. 3.
Changes in bacterial counts in the liver of mice infected with M.paratuherculosis. Both C57/BIO and C57/B6 strains showed bacterial clearance while a steady-state infection was maintained in Balb/c mice. SD= <: 10 per cent of mean.
150 days both liver and spleen were macroscopically normal. However, their weights were increased during the entire course of the study. The peak liver cha.nge occurred at 30 days with a 37 per cent weight increase; a 16 per cent increase was noted after 180 days. The spleen was 250 per cent normal weight at 30 days and was still enlarged after 180 days. Histopathologically, peritoneal and subcapsular hepatic granulomas were largely coagulative during the early part of the study, changing to coagulative
315
M. paratubercltlosis in Mice
CFU per gm Spleen (Log 10)
6
5
......
4-0-
Balb/C
-t:r-
C57/B6
*" C57/B10 330
60
90
120
150
180
Days of Infection Fig. 4.
Changes in bacterial counts in the splecn of mice infectcd with M. paratuberculosis. Bactcrial clearance was observed only in C57/BlO mice, while prolileralion may have been occurring in Balb/c mice. C57/B6 mice were less capable of reducing bacterial counts in the spleen than in the liver (Fig. 3). SD= < JO per cent of mean.
and fibrous with some mineralization by 6 months. These granulomas initially numbered 4-5 per field and were large to confluent. By the end of the study, the numbers were reduced to 1-2 per Held and they were generally small in size. Parenchymal granulomas initially numbered 10-20 and were graded 34 +, being reduced to 5-10 or less per Held with a 1-2 + grading after 6 months. Acid-fast bacilli were not observed after 4- months.
316
Fig. 5.
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Peritoneal granulomas in Balblc mice 150 days arter inocuhuion with AI. paratuberculosis. The granulomas were composed of a lytic centre that contained amorphous, fibrillar-like or dot-like material representing dumped acid-['lst bacilli. The periphery of the granulomas were composed of foam-cell maerophages, lymphocytes and individual ncutmphils. Coagulation neerosis was not a feature. HE. x 400.
Bacterial counts tended to be reduced at 30 days, as compared with those in Ba1b/e mice, and continued to decline during the first 120-150 days of the stud y. In the liver and spleen the cfu were red uced from 6·8 x 10" and 1· 3 x 105 to 7·1 X 10 2 and 4·3 x 103 , respectively, after 180 days. Although the data indicate that bacterial elimination may have ceased at around 120-150 days, the efu were then approaching the limits of detection, with increased standard deviations. Discussion Progressive infection was not observed in any of the mouse strains examined. Balb/c mice were the most susceptible to infection, but bacterial proliferation either did not occur, equalled the rate of elimination, or occurred at a rate too slow to detect during the course of this study. The general trend of cfu in the liver and spleen suggests, however, that a very slow rate of proliferation may have been occurring, particularly in the spleen. In can trast, C5 7/B 10 mice were the most resistant to infection, with gradual bacterial elimination. The greatest reduction in bacterial counts occurred within the first 120 days of infection and then appeared to level off. It is likely that the C57/BIO mice
M. paratuberculosis in Mice
Fig. G.
317
Inflammatory foci in the liver of Balblc mice 60 days after inoculation with lV[. paratuberculosis. These were mainly composed of lymphocytes plus individual macrophages and necrotic hepatocytes. The loci were "andomly distributed throughout the hcpatic lobules, but were also present in portal triads. HE. x 'fDO.
would have ultimately cleared the organism. C57/B6 mice were of intermediate resistance, showing gradual cfu reductions during the course of the study. The relation between gross and histological lesions and changes in bactcrial counts is of particular in terest. Delayed type hypersensitivity (DTH) reactions are often considered detrimental, rather than beneficial, in mycobacterial infections, since they cause most of the tissue damage. DTH is apparently not a requirement for protective immunity (Dannenberg, 1989). The rapid elimination of bacteria in the liver of C57/BIO mice during the first 120 days was associated with coagulation necrosis characteristic of a DTH reaction. In C57/B6 mice, a slower rate of bacterial clearance was associated with a later appearance of lesions and few granulomas containing coagulation necrosis. In contrast, there was bacterial persistence unaccompanied by necrosis in Balb/c mice. Thus, we observed a direct relationship between DTH (as assessed by necrosis) and bacterial clearance. Similarly to Balb/c mice, cattle with paratuberculosis do not develop necrosis and have persistent infection. The observations reported suggest that this model system may be applicable to a comparative study of bacterial clearance or persistence and DTH in Balb/c vs C5 7/B 10 mice strains. While there is no doubt that DTH reactions are often
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detrimental and cause extensive tissue damage, some studies have shown an inverse relation between necrosis and host survival (Flynn et al., 1992). The studies reported here support the notion that DTH may reflect the price a host must pay for rapid bacterial clearance (Yamamura et al., 1991). Of the mouse strains examined, only Balb/c was found to be susceptible to chronic M. paratuberculosis infection. This strain would provide a suitable model for the study of antimicrobial agents or other activities in vivo in which the end point was bacterial elimination or clearance. It has an advantage over other recently described models (Hamilton et al., 1989; Mutwiri et al., 1992; Whipple et al., 1992) in that Balb/c mice are not immunologically deficient and can therefore be inexpensively housed and maintained. In addition, they do not have increased susceptibility to common respiratory infections or tumours. Although intestinal infection did not occur, Balb/c mice offer a more immunologically accurate model than immunodeficient mice, since mycobacterial lesions and disease are characteristically mediated by immunological responses to mycobacterial antigens (Dannenberg, 1989). Acknowledgments
Supported in part by a grant from Adria Laboratories, Columbus, OH, U.S.A. References
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Lominski, I., Cameron,j. and Roberts, G. B. S. (1956). Experimental Johne's disease in mice. Journal of Pathology and Bacteriology, 71, 211-222. Mutwiri, G.K., Butler, D.G., Rosendal, S. and Yager, j. (1992). Experimental infection of severe combined immunodeficient beige mice with l'/I.ycobacterium paratuberculosis of bovine origin. Injection and Immunity, 60, 4074-4079. Whipple, D. 1., Klemens, S. P. and Cynamon, M. H. (1992). Development of the beige mouse as a model for M. paratuberculosis infection. In: Proceedings of the Third International Colloquium on Paratuberculosis. R. j. Chiodini and j. M. Kreeger, Eds, International Association for Paratuberculosis, Providence, RI, pp. 551-552. Yamamura, M., Uyemura, K., Deans, R.j., Weinberg, K., Rea, T. H., Bloom, B. R. and Modlin, R. 1. (1991). Defining protective responses to pathogens: cytokine profiles in leprosy lesions. Science, 245, 277-279.
Received, April 1st, 1993J [ Accepted, July 9th, 1993