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Genetic control of resistance to trypanosomiasis
Veterinary
Veterinary immunology and immunopathoiogy
Immunology and lmmunopathology
EISEVIER
54 (1996) 239-243
Genetic control of resistance to trypanosomiasis S.J. Kemp a7*, A. Darvasi b, M. Soller ‘, A.J. Teale a aInternational b Department
Livestock Research
of Genetics, Hebrew
Institure. PO Box 30709.
Universiry ofJerusalem.
Nairobi. Kenya
IL-91904,
Jerusalem,
Israel
Abstract
To map the genetic sources of trypanotolerance in mice, a linkage analysis of survival following trypanosome challenge was performed by selective genotyping in a large F2 population produced by crossing the resistant C57BL/6 and susceptible BALB/c inbred mouse lines. We report evidence of a chromosomal region of large effect, possibly comprising more than one resistance locus, on Chromosome 17; and of further loci on Chromosomes I and 5. Together, these genes can account for all of the difference between the mean parental phenotypes. Keywds:
Genetic control; Resistance; Trypanosomiasis
Tsetse fly-transmitted trypanosomes (T~~anosoma spp. ) cause ‘sleeping sickness’ in man and have a serious impact on livestock-based agriculture in large areas of Africa (Winrock International, 1992). Resistance to the effects of trypanosomiasis (trypanotolerance) is well documented among several breeds of west African cattle, notably the N’Dama (Murray and Trail, 1984). The underlying mechanisms of trypanotolerance are poorly understood and genetic control is apparently multigenic. In order to locate the genes responsible for trypanotolerance in N’Dama cattle, a linkage analysis study is presently underway. Trypanosusceptible Boran (Bos in&us) cows have been cross-bred with trypanotolerant N’Dama (B. tuurus> bulls. F, progeny, obtained by multiple ovulation and embryo transfer, will be challenged with one of the most important parasites of livestock, T. congofense, and their response will be monitored. The bovine linkage map (Barendse et al., 1994) will then be applied and analysis will be performed to identify the quantitative trait loci (QTL) responsible. Given the long generation time of cattle this is a slow and expensive exercise. However, it has been known for some time that different inbred strains of mice vary
* Corresponding author at: Department University, Liverpool L69 3BX, UK. 01652427/96/$15.00 Copyright PII SO1 65-2427(96)05692-9
of Genetics
and Microbiology,
Donnan
0 1996 Elsevier Science B.V. All rights reserved.
Laboratories,
Liverpool
SJ. Kemp er cd. / Verrrinury Immunology und Inrmunr~~~crtl~olo~y 54 f 19961239-243
240
Survival of BALB/c x C57BL/6 mice challenged with T.congolense loo-1
L\
-I
10 r
0
L
_--1
0
~.
__i-
20
40
60
60
120
100 Days
-
F2
Bdb,C
Fig. I. Survival of C57BL/6,
posr
BALB/c
140
160
180
200
220
240
l”feCti0” ---
c57
and F? mice challenged with T. con~&n.w.
widely in their response to challenge with trypanosomes. In particular, the C57B1/6 strain is relatively resistant to T. congolense while Balb/c mice are relatively susceptible (Morrison et al., 197X; Morrison and Murray. 1979). It is possible that an understanding of the mechanisms of resistance in the mouse will have relevance to the situation in cattle and the recent development of a high-resolution murine gene map (Dietrich et al., 19941, combined with a short generation time, makes the mouse an attractive model for linkage analysis of trypanotolerance. Populations of 200 male and 200 female F2 balb/c K C57B1/6 mice were therefore generated by crossing BALB/c/Ola/Hsd and C57BL/6/01a/Hsd mice. both males and females of each strain were used. When they were lo- 12 weeks old the F, mice were challenged by intraperitoneal inoculation of 10” blood stream forms of the cloned isolate ILNat3. I of T. congolense (Nantulya et al.. 1984). At the same time, age-matched controls comprising 10 male BALB/c. 10 female BALB/c, 10 male C57BL/6, 10 female C57BL/6 were challenged. In the following 14 days all mice but one were shown to be parasitaemic by examination of tail-blood smears. The exception. a female F?. was excluded from the study. The survival profile of the F, and parental controls is shown in Fig. 1. The 50% survival time of the susceptible BALB/c parent was 68 days. while that of the C57BL/6 parent was 142 days. Thirty percent of the C57BL/6 mice survived and became aparasitaemic. F, mice showed a range of survival times. the shortest being IX days, while 10 mice (2.5%) became aparasitaemic and fully recovered.
X.J. Kemp et ul./
Veterinary Immunology and Immunopathology 54 119961239-243
241
LODscw-Trait1 (Survival)
Fig. 2. LOD score of association sequence repeats (microsatellites) 1994).
with survival of markers on mouse Chromosome 17. All markers are simple which have been ordered into a high resolution linkage map (Dietrich et al.,
Ninety informative microsatellite loci were genotyped in 20 F2 mice which had survival times of less than 44 days, and in 20 which had survived for longer than 146 days after challenge. These mice thus comprised the extreme 5% of the range of survival times. Analysis of the data suggested a number of chromosomal regions associated with survival time. Additional mice were genotyped with markers in these regions and additional markers were included, so that a total of about 120 loci were genotyped in 130 mice. These mice represented the 16% of the F2 mice with the shortest survival times and the 16% with the longest survival times, and included the 10 surviving F, mice. This selective genotyping has power equivalent to genotyping an entire population of 320 individuals (Darvasi and Soller, 1992). Analysis with MAPMAKER/QTL (Paterson et al., 1994; Lincoln et al., 1994) revealed that a region on chromosome 17 had a dramatic effect on survival time, with a peak LOD score of 11.0 (Fig. 2). This broad region of significant LOD score may represent more then one locus, a model assuming two loci on chromosome 17 is significantly more likely (with a LOD score of 15.1) than a one gene model and explains 18% of the total variance in the F2 population. Alleles in this region appear to have an additive effect. A region of Chromosome 1 also showed a highly significant association with survival time (maximum LOD score 5, variance explained lo%, Fig. 3) The third region which
243
XI. Kemp rr 01. / Vrtrrinury
Immundo~y
und Immunc~pu~holo~y 54 (1996) 239-243
Fig. 3. LOD score of associauon wtth survival ot m~crosatellite markers on mouse: Chromosome
I
showed evidence of association with ~rvivai time was on Chromosome 5 (maximum LOD score 4: variance explained 6.1%: Fig. 4) hut in this case the resistant allele was found to be fully dominant. A model assuming four loci. two on Chromosome 17 and one on Chromosomes 1 and 5, explains 32% of the total variance in the Fz mice. Assuming an additive model with a single QTL on chromosome 17, the expected difference in survival times due to the three QTL described here is approximately 90 days. The mean difference found between the two control groups of the parental lines was 82 days. Thus. the detected loci appear to account for all the genetic difference between the two parental lines. The difference between the expected 90 days and the observed 82 days may be due to the over-estimation of effects commonly obtained in marker/QTL analysis (Hoeschele and VanRaden. 1993). or by non-additive effects. Mouse Chromosome 17 carries many genes of importance to the immune response, notably those of the major histocompatibility complex (MHC). The MHC lies close to one of the two areas of highest likelihood (Fig. 2) and genes in this complex are thus candidates for involvement in this phenotype. No known disease resistance genes in mice have been mapped to within the 95% confidence limits of the putative T. congofense resistance QTL on Chromosomes 1 or 5. It is therefore likely that these represent genes not previously described or not previously shown to have an involvement in disease resistance in mice. Our results indicate that an apparently complex quantitative disease resistance trait is in large part controlled by only three or four loci on three chromosomes. Finer resolution
S.J. Kemp
et al./
Veterinary
Fig. 4. LOD score of association
Immunology
and lmmunopathology
with survival of microsatellite
54 (19961239-243
markers on mouse Chromosome
243
5.
gene mapping followed by positional cloning should now be possible. This may provide valuable insights into the molecular basis of host resistance to trypanosomes, which will have important implications for the development of strategies for control of trypanosomiasis in man and livestock. References Barendse, W., Armitage, SM., Kossarek, L.M., Shalom, A., Kirl;patricb, B.W., Ryan, A.M., Clayton, D.. Li, L., Neibergs, H.L., Zhang, N., Grosse, W.M.. Weiss, J., Creighton. P., McCarthy, F., Ron, M., Teale, A.J., Fries. R., McGraw, R.A., Moore, S.S., George% M., Soller, M., Womack, J.E. and Hetzel. D.J.S., 1994. Nat. Genet., 6: 227. Darvasi, A. and Soller, M., 1992. Theor. Appl. Genet., 85: 353. Dietrich, W.F., Miller, J.C.. Steen, R.G., Merchant, M., Damron, D., Nahf, R., Gross, A., Joyce, D.C., Wessel, M., Dredge, R.D., Marquis, A., Stein, L.D., Goodman, N., Page, D.C. and Lander, E.S., 1994. Nat. Genet., 7: 220. Hoeschele. 1. and VanRaden, P.M., 1993. Theor. Appl. Genet., 85: 953. Lincoln, S., Daly, M. and Lander, E.S., 1994. In: Whitehead Institute Technical Report. Whitehead Institute, Cambridge, MA. Morrison, W.I., Roelants, G.E.. Mayor-Withey, KS. and Murray, M., 1978. Clin. Exp. lmmunol., 32: 25. Morrison, WI. and Murray, M. 1979. Exp. Parasitol., 48: 364. Murray, M. and Trail, J.C.M., 1984. Prev. Vet. Med., 2: 541. Nanhtlya, V.M., Musoke, A.J., Rurangirwa, F.R. and Moloo, S.K., 1984. Infect. Immun., 43: 735. Paterson, E., Lander, E.S., Lincoln, S., Hewitt, J., Peterson, S. and Tanksley, S., 1994. Nature, 335: 721. Winrock International, 1992. Assessment of animal agriculture in sub-Sahara” Africa. Winrock International Institute for Agricultural Development, AK.
Veterinary immunology and immunopathoiogy
Immunology and lmmunopathology
EISEVIER
54 (1996) 239-243
Genetic control of resistance to trypanosomiasis S.J. Kemp a7*, A. Darvasi b, M. Soller ‘, A.J. Teale a aInternational b Department
Livestock Research
of Genetics, Hebrew
Institure. PO Box 30709.
Universiry ofJerusalem.
Nairobi. Kenya
IL-91904,
Jerusalem,
Israel
Abstract
To map the genetic sources of trypanotolerance in mice, a linkage analysis of survival following trypanosome challenge was performed by selective genotyping in a large F2 population produced by crossing the resistant C57BL/6 and susceptible BALB/c inbred mouse lines. We report evidence of a chromosomal region of large effect, possibly comprising more than one resistance locus, on Chromosome 17; and of further loci on Chromosomes I and 5. Together, these genes can account for all of the difference between the mean parental phenotypes. Keywds:
Genetic control; Resistance; Trypanosomiasis
Tsetse fly-transmitted trypanosomes (T~~anosoma spp. ) cause ‘sleeping sickness’ in man and have a serious impact on livestock-based agriculture in large areas of Africa (Winrock International, 1992). Resistance to the effects of trypanosomiasis (trypanotolerance) is well documented among several breeds of west African cattle, notably the N’Dama (Murray and Trail, 1984). The underlying mechanisms of trypanotolerance are poorly understood and genetic control is apparently multigenic. In order to locate the genes responsible for trypanotolerance in N’Dama cattle, a linkage analysis study is presently underway. Trypanosusceptible Boran (Bos in&us) cows have been cross-bred with trypanotolerant N’Dama (B. tuurus> bulls. F, progeny, obtained by multiple ovulation and embryo transfer, will be challenged with one of the most important parasites of livestock, T. congofense, and their response will be monitored. The bovine linkage map (Barendse et al., 1994) will then be applied and analysis will be performed to identify the quantitative trait loci (QTL) responsible. Given the long generation time of cattle this is a slow and expensive exercise. However, it has been known for some time that different inbred strains of mice vary
* Corresponding author at: Department University, Liverpool L69 3BX, UK. 01652427/96/$15.00 Copyright PII SO1 65-2427(96)05692-9
of Genetics
and Microbiology,
Donnan
0 1996 Elsevier Science B.V. All rights reserved.
Laboratories,
Liverpool
SJ. Kemp er cd. / Verrrinury Immunology und Inrmunr~~~crtl~olo~y 54 f 19961239-243
240
Survival of BALB/c x C57BL/6 mice challenged with T.congolense loo-1
L\
-I
10 r
0
L
_--1
0
~.
__i-
20
40
60
60
120
100 Days
-
F2
Bdb,C
Fig. I. Survival of C57BL/6,
posr
BALB/c
140
160
180
200
220
240
l”feCti0” ---
c57
and F? mice challenged with T. con~&n.w.
widely in their response to challenge with trypanosomes. In particular, the C57B1/6 strain is relatively resistant to T. congolense while Balb/c mice are relatively susceptible (Morrison et al., 197X; Morrison and Murray. 1979). It is possible that an understanding of the mechanisms of resistance in the mouse will have relevance to the situation in cattle and the recent development of a high-resolution murine gene map (Dietrich et al., 19941, combined with a short generation time, makes the mouse an attractive model for linkage analysis of trypanotolerance. Populations of 200 male and 200 female F2 balb/c K C57B1/6 mice were therefore generated by crossing BALB/c/Ola/Hsd and C57BL/6/01a/Hsd mice. both males and females of each strain were used. When they were lo- 12 weeks old the F, mice were challenged by intraperitoneal inoculation of 10” blood stream forms of the cloned isolate ILNat3. I of T. congolense (Nantulya et al.. 1984). At the same time, age-matched controls comprising 10 male BALB/c. 10 female BALB/c, 10 male C57BL/6, 10 female C57BL/6 were challenged. In the following 14 days all mice but one were shown to be parasitaemic by examination of tail-blood smears. The exception. a female F?. was excluded from the study. The survival profile of the F, and parental controls is shown in Fig. 1. The 50% survival time of the susceptible BALB/c parent was 68 days. while that of the C57BL/6 parent was 142 days. Thirty percent of the C57BL/6 mice survived and became aparasitaemic. F, mice showed a range of survival times. the shortest being IX days, while 10 mice (2.5%) became aparasitaemic and fully recovered.
X.J. Kemp et ul./
Veterinary Immunology and Immunopathology 54 119961239-243
241
LODscw-Trait1 (Survival)
Fig. 2. LOD score of association sequence repeats (microsatellites) 1994).
with survival of markers on mouse Chromosome 17. All markers are simple which have been ordered into a high resolution linkage map (Dietrich et al.,
Ninety informative microsatellite loci were genotyped in 20 F2 mice which had survival times of less than 44 days, and in 20 which had survived for longer than 146 days after challenge. These mice thus comprised the extreme 5% of the range of survival times. Analysis of the data suggested a number of chromosomal regions associated with survival time. Additional mice were genotyped with markers in these regions and additional markers were included, so that a total of about 120 loci were genotyped in 130 mice. These mice represented the 16% of the F2 mice with the shortest survival times and the 16% with the longest survival times, and included the 10 surviving F, mice. This selective genotyping has power equivalent to genotyping an entire population of 320 individuals (Darvasi and Soller, 1992). Analysis with MAPMAKER/QTL (Paterson et al., 1994; Lincoln et al., 1994) revealed that a region on chromosome 17 had a dramatic effect on survival time, with a peak LOD score of 11.0 (Fig. 2). This broad region of significant LOD score may represent more then one locus, a model assuming two loci on chromosome 17 is significantly more likely (with a LOD score of 15.1) than a one gene model and explains 18% of the total variance in the F2 population. Alleles in this region appear to have an additive effect. A region of Chromosome 1 also showed a highly significant association with survival time (maximum LOD score 5, variance explained lo%, Fig. 3) The third region which
243
XI. Kemp rr 01. / Vrtrrinury
Immundo~y
und Immunc~pu~holo~y 54 (1996) 239-243
Fig. 3. LOD score of associauon wtth survival ot m~crosatellite markers on mouse: Chromosome
I
showed evidence of association with ~rvivai time was on Chromosome 5 (maximum LOD score 4: variance explained 6.1%: Fig. 4) hut in this case the resistant allele was found to be fully dominant. A model assuming four loci. two on Chromosome 17 and one on Chromosomes 1 and 5, explains 32% of the total variance in the Fz mice. Assuming an additive model with a single QTL on chromosome 17, the expected difference in survival times due to the three QTL described here is approximately 90 days. The mean difference found between the two control groups of the parental lines was 82 days. Thus. the detected loci appear to account for all the genetic difference between the two parental lines. The difference between the expected 90 days and the observed 82 days may be due to the over-estimation of effects commonly obtained in marker/QTL analysis (Hoeschele and VanRaden. 1993). or by non-additive effects. Mouse Chromosome 17 carries many genes of importance to the immune response, notably those of the major histocompatibility complex (MHC). The MHC lies close to one of the two areas of highest likelihood (Fig. 2) and genes in this complex are thus candidates for involvement in this phenotype. No known disease resistance genes in mice have been mapped to within the 95% confidence limits of the putative T. congofense resistance QTL on Chromosomes 1 or 5. It is therefore likely that these represent genes not previously described or not previously shown to have an involvement in disease resistance in mice. Our results indicate that an apparently complex quantitative disease resistance trait is in large part controlled by only three or four loci on three chromosomes. Finer resolution
S.J. Kemp
et al./
Veterinary
Fig. 4. LOD score of association
Immunology
and lmmunopathology
with survival of microsatellite
54 (19961239-243
markers on mouse Chromosome
243
5.
gene mapping followed by positional cloning should now be possible. This may provide valuable insights into the molecular basis of host resistance to trypanosomes, which will have important implications for the development of strategies for control of trypanosomiasis in man and livestock. References Barendse, W., Armitage, SM., Kossarek, L.M., Shalom, A., Kirl;patricb, B.W., Ryan, A.M., Clayton, D.. Li, L., Neibergs, H.L., Zhang, N., Grosse, W.M.. Weiss, J., Creighton. P., McCarthy, F., Ron, M., Teale, A.J., Fries. R., McGraw, R.A., Moore, S.S., George% M., Soller, M., Womack, J.E. and Hetzel. D.J.S., 1994. Nat. Genet., 6: 227. Darvasi, A. and Soller, M., 1992. Theor. Appl. Genet., 85: 353. Dietrich, W.F., Miller, J.C.. Steen, R.G., Merchant, M., Damron, D., Nahf, R., Gross, A., Joyce, D.C., Wessel, M., Dredge, R.D., Marquis, A., Stein, L.D., Goodman, N., Page, D.C. and Lander, E.S., 1994. Nat. Genet., 7: 220. Hoeschele. 1. and VanRaden, P.M., 1993. Theor. Appl. Genet., 85: 953. Lincoln, S., Daly, M. and Lander, E.S., 1994. In: Whitehead Institute Technical Report. Whitehead Institute, Cambridge, MA. Morrison, W.I., Roelants, G.E.. Mayor-Withey, KS. and Murray, M., 1978. Clin. Exp. lmmunol., 32: 25. Morrison, WI. and Murray, M. 1979. Exp. Parasitol., 48: 364. Murray, M. and Trail, J.C.M., 1984. Prev. Vet. Med., 2: 541. Nanhtlya, V.M., Musoke, A.J., Rurangirwa, F.R. and Moloo, S.K., 1984. Infect. Immun., 43: 735. Paterson, E., Lander, E.S., Lincoln, S., Hewitt, J., Peterson, S. and Tanksley, S., 1994. Nature, 335: 721. Winrock International, 1992. Assessment of animal agriculture in sub-Sahara” Africa. Winrock International Institute for Agricultural Development, AK.