resistance to mycobacterial infection

veterinary microbiology Veterinary Microbiology 50 (1996) 73-79

Establishment of Beg’ congenic mice and their susceptibility/resistance to mycobacterial infection De Long Xu, Yoshitaka Goto *, Kingsley Kwaku Amoako, Toshie Nagatomo, Tomoko Fujita, Toshiharu Shinjo Department of Veterinary Microbiology, Faculty of Agriculture, Miyazaki Uniuersity, I-l, Gakuen Kibanadai Nishi, Miyazaki 889-21, Japan

Received 27 June 1995; accepted 24 October 199.5

Abstract Beg congenic mice were developed by using C57BL/6 and DBA/2 strains of mice as progenitors. They were obtained by introgressively backcrossing the Beg’ marker of DBA/2 onto C57BL/6. After twenty successive backcrossings, the heterozygous resistant mice were mated with each other to obtain homozygous mice as the Beg’ congenic mice. The results of immunogenic and genetic markers coupled with those of an mixed lymphocyte reaction, all

confirmed that the newly developed mice were highly congenic. These congenic r-nice were found to be resistant to in vivo infections by Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium

bovis BCG.

Keywords; Mycobacterial infection; Beg congenic resistant mice

1. Introduction The Beg gene of mice is known to act as the regulator of early host defense mechanisms controlling some intracellular pathogens, such as Mycobacterium bouis BCG (Denis et al., 1986; Skamene et al., 1982). Bacteria have been found to grow well in the spleen, liver and lungs of C57BL/6 and BALB/c mice which have Beg” alelle, while they grow poorly in DBA/2 and C3H/He mice. The Beg gene is therefore thought to affect the innate resistance. Both BALB/c and C57BL/6 mice have the same Beg genotype, however, after prolonged mycobacterial infection, the severity of

f Corresponding author. Tel.: 0985-5X-2811, ext. 3721, Fax: 0985-58-2884 0378-l 135/96/$15.00 SSDl 0378-

0 1996 Elsevier

1 135(95)00198-O

Science B.V.

All rights

reserved

14

D.L. Xu et al./ Veterinq

Microbiology

50 (19%) 73-74,

disease and responsiveness to various mycobacterial antigens in BALB/c mice differ from those in C57BL/6 mice (Huygen and Palfliet, 1983; Stokes et al., 1986; Huygen et al., 1992). Several mouse models have been developed to elucidate the specific role of the Beg gene in mycobacterial infections. Beg’ congenic CD2 mice have been established from backcrossing DBA/2 mice which carry the Beg’ gene onto BALB/c mice (Potter et al., 1983). When the congenic mice were challenged with M. bovis BCG (Montreal strain), the number of bacteria in the liver and spleen did not increase until six weeks after the infection, while a rapid increase was observed in BALB/c mice. These results indicate that susceptibility or resistance to infection with organisms is controlled by the Beg gene. Studies using the Fl hybrids of C57BL/6 and DBA/2 have also been previously reported (Orme and Collins, 1984). Differences in response to mycobacterial infection between the BALB/c mice and C57BL/6 have been reported (Stokes et al., 1986, Huygen et al., 1992). These differences, coupled with the wide use of the Fl hybrids as an attempt to determine the role of the Beg gene in mycobacterial infections has thus necessitated the development of C57BL/6 congenic mice so as to understand the entire M. arium-intracellulare complex infection, especially during prolonged periods of infection. In this paper, we report the development of the first C57BL/6 congenic mice model.

2. Materials and methods

2.1. Bacteria M. avium Mino strain and M. intracellulare strain K8 from human atypical mycobacteriosis and M. bovis BCG Pasteur (vaccine strain) were used in this study. These bacteria were grown on Ogawa’s egg and/or Middlebrook 7HlO media and tested by standard biochemical procedures. The identification of mycobacteria species was performed using a commercial kit (Gen-Probe Rapid Diagnostic System for Mycobacterium TB Complex and Mycobacterium avium complex; Gen-Probe, San Diego, CA, USA) (Drake et al., 1987; Saito et al., 1990). 2.2. Mice DBA/2 and C57BL/6 mice were used as progenitor strains. These were purchased from the Shizuoka Experimental Animal Station (Hamamatsu, Japan) and the Kyushu Experimental Animal Co. (Kumamoto, Japan), respectively. Beg’ congenic strains were obtained by introgressively backcrossing the Beg’ marker of DBA/2 mice onto C57BL/6 mice. After twenty successive backcrossing, the heterozygous resistant mice were mated with each other to obtain homozygous mice as the Beg’ congenic mice. In all generations, sensitivity or resistance to M. avium Mino strain in all mice were checked (Goto et al., 1989). Mice were intravenously infected with a 1 million colony forming-unit of bacteria and killed 3 weeks later. The spleens were removed and homogenized in distilled water and the number of viable bacteria was determined in

D.L. Xu et al./ Veterinary Microbiology 50 (1996) 73-79

15

homogenates by plating appropriate dilutions on Middlebrook 7HlO agar (Difco Laboratories, Detroit, MI, USA) plates and incubating at 37°C for two weeks. Immunogenic markers such as Thy-l, H-2K and H-2D were determined using their respective monoclonal antibodies (Cedar Lane Laboratories Ltd., Canada). Briefly, cell suspensions were prepared from lymphatic organ and monoclonal antibodies were added separately, mixed and incubated for 1 h at 4°C. After a brief centrifugation, the cells were resuspended in cytotoxic medium, incubated at 37°C for 1 h and the percentage cytotoxicity was determined. The detection of genetic markers was done biochemically using electrophoresis to determine the allozyme pattern of each mouse strain as cited elsewhere (Hedrich, 1981). Three mice from each strain were used for the determination of the immunogenic and genetic markers. 2.3. Mixed lymphocyte

reaction (MLR)

To determine whether major or minor histocompatibility loci of the Beg” and Beg’ congenic mice are identical or not, the MLR test was performed. Thymocytes and spleen cells were taken from each strain of mice and adjusted to the required concentration and the MLR of the cell population was estimated as described elsewhere (Kruisbeek and Shevach, 1991). Three mice from each strain were used. Briefly, responder thymocyte cells from normal mice were incubated for three days with stimulator spleen cells which were irradiated or treated with mitomycin C and the tritiated thymidine incorporation into proliferating lymphocytes was determined. 2.4. Statistical analysis Statistical significance was analysed by student’s T-test. Difference with p < 0.05 were considered statistically significant.

3. Results 3.1. Establishment

of Beg’ congenic mice and their characters

The backcross system used in this study is illustrated in Fig. 1. The pattern of bacterial growth in our backcross mice was almost the same as that in DBA/2 mice. We tested for many allelic markers located on different chromosomes that distinguish C57BL/6 from DBA/2 mice. As shown in Table 1, many genotypes were determined. This indicates that our established mice are carrying the Zty’ gene which is the same as the Beg’. In all cases the mice which we established were found to carry the C57BL/6 allele. 3.2. Growth of bacteria in Beg ’ and Beg r mice

There was no significant difference in numbers of bacteria recovered from organs of either strains on day one. The results of the growth of M. avium Mino, M. intracellu-

76

D. L. Xu et al. / Vrterimq

Microbiuiogy

SO f I Y96) 73- 79

Nl

N2

N3

(N2W

Fig. I. Production of a Beg’ congenic strain by the backcross system. The DBAl and CS7BL/6 were used as progenitors and the &,,e’ marker was introgresaively backcrossed onto the C57BL/6 after twenty successive backcrossings

In weeks

2 aiter

3

4

6

44 0

,

I

2

,

I

4

,

,

6

iJlfection

Fig. 2. Proliferatton of (A) M. wiurn Mino. (B) M. inrracellulm-e K8 and (C) M. bwi.s BCG in spleen of (0) Bq’ congenic and (0) Beg’ mice. Mice were infected with about I X IO5 viable bacteria and the growth in spleen was determined at intervals. The vertical bars represent standard errors of the mean of ten determinations.

D. L. Xu et al. / Veterinary Table 1 Stain distribution

Microbiology

50 (1996)

II

73-79

pattern markers Mouse strain

Gene symbol

Chromosome

C57/BL6

DBA/2

Beg’ a

Idh- 1

a

Beg RY Pep-3 Akp- 1 Hc Lr- 1 Car-2 Mup- 1 Gpd- 1 Pgm- I Ldr- 1 Gpl-I Hbb Es- 1 Es-2 Thy-l Mod- 1 Ttf Es-3 H-2K H-2D

S

b r

a r

b b

a a I

No. 1

9 11 17

a The newly developed

congenic

a a I r a b a a a b

0

%

a b b a a d b b b a b

S

a b b b b a b b

: d

a b a a a a S

a b b b b a b b

mice

lare and A4. bovis in the spleen and liver are shown in Fig. 2. In Beg” mice the number of M. avium strain increased progressively in both organs. However, no increase was observed in Beg’ mice even at six weeks after infection. Growth of M. intracellulare

Table 2 MLR in Beg congenic

mice

Stimulator Exp. 1

(spleen cells)

Beg’ (C57BL/61 Beg r Beg ’ Beg r

Beg’ (~57~~/6) Beg”

DBA/Z DBA/’ Exp. 2

Responder (thymocytes)

Beg

’

BALB/c BALB/c Beg r

BC$

Beg

’

BCgs

Beg

’

Beg Beg

r ’

Beg Beg

’ ’

a Not significant among unresponsive h p < 0.01 vs. unresponsive group.

groups.

cpm (S.D.) 270.2c40.6)

a

290.2C75.5) ’ 381 I-8(353.4) b 3757.2c309.4) b 3961.2(515.71 4525.4c692.1) 316.6 (51.61 353.1 (56.21 231.7 (54.11 Not done

b ’ ’ a B

KS was found to decrease progressively during the period of infection. M. hor,i.s Pasteur increased slightly in Bc,~~’ and decreased in Beg’.

The growth of

The results of the mixed lymphocyte reaction (MLR) are shown in Table 2. No proliferation occurred when Bcs’ tCS7BL/6) thymocytes were mixed with CS7BL/6 (Beg‘) or homozygous spleen cells. Significant proliferation was observed only when they were mixed with DBA/7 (parental strain) spleen cells. A similar proliferation was observed with thymocytes and spleen cells from BALB/c.

4. Discussion Bq was originally designated for the gene which controls the resistance to BCG Montreal strain (Skamene et al.. 1981). Later. this gene was found to control the resistance to various species of mycobacteria (Goto et al.. 1984: Denis et al.. 1986; Orme et al.. 1986). On the other hand, mice infected with virulent mycobacteria such as M. her is and M. tuberculosis showed no resistance even though they possessed the Beg’ allele (Huygen and Paltliet. 19X.3). The reason for this difference in sensitivity is not clear. The difference in cell surface components may influence the sensitivity. The mice we developed showed resistance to three different species of mycobacteria; M. hwis BCG, M. ctrtiuttt and M. it?trLt(.~(lI4ICtrf’. Our experiments demonstrated that the orowth of M. rtr~iwtt. M. it~tracrllulrrre and M. hol~is BCG in the mice was controlled iy the Beg gene. The Beg gene is known to control the growth of M. hol~is (BCG Montreal). but not BCG Pasteur (Orme and Collins, 1984). However, our results on the BCG Pasteur infection showed that this strain is controlled by the Bq gene and this is inconsistent with the above report. We also found that our mice expressed resistance to S. typhitmriuttz infection, but not to L. t7lorloc’~toRrnr.r infection (data not shown). This is also concordant with a previous report (Cheers and Mckenzie, 1978). A significantly low number of live M. itztrrtcdldarr bacteria was recovered from Beg ’ mice compared with susceptible mice also indicating that the growth of this bacteria is controlled by the Beg gene. However. we have not yet determined how many centimorgans of the chromosome I gene including Bq gene from DBA/? mice was crossed over with the Beg’ gene and was present in the final congenic mice. However, all the biochemical markers and other characters we examined were the same as those of C57BL/6 except the Bc,g/lty phenotype. The transfer of spleen T cells from C57BL/6 to our Beg’ mice was successful without Graft Versus Host (GVH) reaction (data not shown) and no MLR response was observed when their spleen lymphocytes were cocultured in vitro with those of C57BL/6 (Table 3). Therefore, the major histocompatibility loci. as well as the genotypes of minor histocompatibility loci in our Beg’ mice seem to be the same as those of CS7BL/6 mice. The immune responses of the present congenic mice developed may differ from those of the BALB/c congenic mice (Potter et al.. 1983) and therefore warrant investigation

79

DA. Xu ef ul. / Veferina? Microbiologv 50 119961 73-79

because several differences such as proliferation of bacteria in liver and spleen (Stokes et al., 1986) and IL-4 and IFN-y production (Huygen et al., 1992) in C57BL/6 and BALB/c have been reported. In addition. the mice model developed may be a useful experimental tool for analysing the resistance against mycobacterial infections which have been implicated not only in the medical field (Horsburgh, 1989) but also in the veterinary field (Lepper and Corner, 1983). Acknowledgements We thank Mrs. H. Kiyoyama

and Miss C. Ohta for their technical

assistance.

References Cheers, C. and Mckenzie, I.F.C., 1978. Resistance and susceptibility of mice to bacterial infection: genetics of listeriosis. Infect. Immun.. 19: 755-762. Denis, M., Forget, A.. Pelletier. M., Turcotte, R. and Skamene. E.. 1986. Control of the Beg gene of early resistance in mice to infections with BCG substrains and atypical mycobacteria. Clin. Exp. lmmunol., 63: 517-525. Drake, T.A.. Hindler, J.A.. Berlin, O.G.W. and Bruckner. D.A.. 1987. Rapid identification of Mycohcrctenctn~ at,ilrin complex in culture using DNA probes. J. Clin. Microbial., 25: 1442- 1445. Goto, Y., Buschman, E. and Skamene. E.. 1989. Regulation of host resistance to Mwobacterium intrucrllrr/are in viva and in vitro by the Beg gene. Immunogenetics, 30: 218-221. Goto. Y.. Nakamura. R.M.. Takahasi, H. and Tokunaga. T., 1984. Genetic control of resistance to Mycobacferium intracellulnre infection in mice. Infect. Immun., 46: 135-140. Hedrich, H.J., 1981. Genetic Monitoring. In: The mouse in biomedical research.. Vol. I. Academic Press, New York, pp. l59- 176. Horsburgh. C.R. 1989. The epidemiology of disseminated nontuberculous mycobacterial infection in the acquired immunodeficiency syndrome (AIDS). Am. Rev. Respir. Dis., 139: 4-7. Huygen. K., Abramowicz. D., Vandenbussche, P.. Jacobs, F.. Bruyn, J.D., Kentos. A., Drowart. A., Vooren, J.-p.V. and Goldman, M.. 1992. Spleen cell cytokine secretion in Mycobacteriurn bmix BCG-infected mice. Infect. Immun., 60: 2880-2886. Huygen, K. and Paltliet, K.. 1983. Strain variation in interferon y production of BCG-sensitized mice challenged with PPD. Cell. Immunoi.. 80: 329-334. Kruisbeek. A.M. and Shevach, E.. 1991. Proliferative assays for T cell function, In: Current protocols in immunology, Vol. 1 and 2. Wiley. New York, pp. 3.12.1-3. I?. 14. Lepper. A.W.D. and Comer, L.A., 1983. Naturally occurring mycobacterioses of animals, In: The biology of the mycobacteria, Vol. 2. Academic Press, London, pp. 418-521. Orme. I.M. and Collins, F.M., 1984. Demonstration of acquired resistance in Beg’ inbred mouse strains infected with a low dose of BCG Montreal. Clin. Exp. Immunol.. 56: 81-88. Orme, I.M., Stokes. R.W. and Collins, F.M., 1986. Genetic control of natural resistance to nontuberculous mycobacterial infections in mice. Infect. Immun., 54: 56-62. Potter, M., O’Brien, A.D.. Skamene, E.. Gros, P.. Forget, A., Kongshavn. P.A.L. and Wax, J.S.. 1983. A BALB/C congenic strain of mice that carries a genetic locus (IQ’) controlling resistance to intracellular parasites. Infect. Immun., 40: l234- 1240. Saito, H., Tomioka, H., Sato, K.. Tasaka, H. and Dawson, D.J., 1990. Identification of various strains of M.vcobacterium aaium complex by using DNA probes specific for Myobacrerium akm and My-obacferium intracellulare. J. Clin. Microbial., 28: 1694-1697. Skamene, E., Gros, P., Forget, A.. Kongshavn, P.A.L., Charles, C.S. and Taylor, B.A.. 1982. Genetic regulation of resistance to intracellular pathogens. Nature. 297: 506-509. Stokes, R.W.. Orme, I.M. and Collins, F.M., 1986. Role of mononuclear phagocytes in expression of resistance and susceptibility to Myzobacrerium a~hm infections in mice. Infect. Immun., 54: 81 l-819.