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Trypanosoma musculi:Compared Levels of Parasitosis in Wild and Laboratory Strains ofMus musculusMice
Experimental Parasitology 91, 196–198 (1999) Article ID expr.1998.4375, available online at http://www.idealibrary.com on
RESEARCH BRIEF Trypanosoma musculi: Compared Levels of Parasitosis in Wild and Laboratory Strains of Mus musculus Mice
Jean-Marc Derothe, Nathalie Le Brun, Claude Loubes, Marco Perriat-Sanguinet, and Catherine Moulia1 Laboratoire de Parasitologie Compare´e, UMR 5555, cc 105, Universite´ Montpellier II, Montpellier, France
Derothe, J.-M., Le Brun, N., Loubes, C., Perriat-Sanguinet, M., and Moulia, C. 1999. Trypanosoma musculi: Compared levels of parasitosis in wild and laboratory strains of Mus musculus mice. Experimental Parasitology 91, 196–198. 䉷 1999 Academic Press Index Descriptors and Abbreviations: Trypanosoma musculi; Mus musculus; parasitosis.
HEOuJIco mice infected by dividing forms (7 to 12 days postinfection). Each sample was diluted at a concentration of 105 parasites/ml in a sodium chloride solution (0.9%). A volume of 0.5 ml of the diluted blood was intraperitoneally injected to each 1-month-old tested mouse (Albright et al. 1990; Derothe et al. submitted for publication). The number of trypanosomes by milliliter of blood was determined with an hemocytometer every 3 days until the end of infection (i.e., immune clearance, 25–27 days) (Chiejina et al., 1993). The response of each mouse is then evaluated by the maximum value of parasitemia, corresponding to a plateau phase (Albright et al., 1981; Kongshawn et al., 1985), obtained during the infection course as this seems to be the most reliable parameter whatever the route of infection (Maraghi et al., 1995). Mann–Whitney U tests detected differences between males and females in 5 strains of the 17 studied (Fig. 1). In all cases, males present a higher parasitemia than females. Although some of the significant results may be due to a type 1 error because of the number of tests performed, males (m) and females (f) of the strains concerned are separated in the following analysis (Fig. 1). The Kruskal–Wallis test (1952) showed a highly significant difference among all the strains tested (Hcor, 258.04; df ⫽ 22; P ⬍ 0.0001). The Noether’s multiple comparison tests (1976) (Sherrer 1984), used as posthoc tests, reveal that the strains segregate into three groups according to their level of resistance (Fig. 1). Inbred strains were found in all the categories. This study clearly showed that the profiles of resistance to T. musculi of laboratory mice are as variable as the profiles of wild-derived mice. This is contrary to the result obtained with A. tetraptera (Derothe et al. 1997). This difference can be explained by the differential probabilities of transmission of the two parasites in the laboratory. T. musculi presents an heteroxenous life cycle. Its intermediate host, a flea (Hoare 1972; Molyneux and Ashford 1983), is systematically eliminated from
The laboratory environment can modify mouse–parasite interactions, either increasing or limiting the parasite transmission and the consequent pressure it may represent on mouse genome. In a previous work, Derothe et al. (1997) argued that breeding conditions favored the transmission of the mouse pinworm Aspiculuris tetraptera (direct cycle). This led to the selection of resistance genes in all the laboratory strains when compared to wild-derived strains. The aim of this study was to bring new data to assess the limitations of the laboratory mouse model in parasitology. We use the blood protozoan Trypanosoma musculi, another natural parasite of the mouse. Experimental infections of mice from eight inbred laboratory strains and nine wild-derived strains (see Fig. 1 for designations) were performed with a clone of the Partinico II strain (Viens et al. 1974). Wild-derived strains, recently isolated from the field (5 to 25 generations in the laboratory) and randomly bred in little colonies, represent a broad sample of Mus musculus domesticus and M. m. musculus genomes. They are used as indicators of the variability of resistance to the trypanosome in wild populations of mice (Derothe et al. submitted for publication). Standard infections are realized from a sample of blood collected from C3H/
1
To whom correspondence should be addressed. E-mail: moulia@ crit.univ-montp2.fr.
196
0014-4894/99 $30.00 Copyright 䉷 1999 by Academic Press All rights of reproduction in any form reserved.
PARASITOSIS IN WILD AND LABORATORY MICE
197
FIG. 1. Percentile distribution of parasitemia in experimental trypanosomiasis of laboratory and wild-derived strains. For each sample, bars represent the 10th, 25th, 50th (i.e., median), 75th, and 90th percentiles of each sample tested with Trypanosoma musculi. Strain designation are as follows. Laboratory strains: D2, DBA/2/Ola//Hsd; NIH, NIH/Ola//Hsd; SWR, SWR/Ola//Hsd; SJL, SJL/Ola//Hsd; CBA, CBA/J/Ola//Hsd from Harlan Olac, U. K.; B6, C57BL/6Jico; C, BALB/cByJIco from Iffa Credo, France; 129, 129/SV/Pas from Pasteur Institute of Paris, France. Wildderived strains were BZO, DMZ, DJO, DDO, MHT, MPB, MDH, MBS, and MBT from the “Wild Mouse Repository” (UPR 9060 CNRS, France). Strains showing differences in parasite loads between males and females are divided into two subsamples, adding an m or f suffix to the strain designation. Median less than 320.105 parasites/ml (p/ml) of blood, resistant strains; median more than 600.105 p/ml, susceptible strains; median from 320 to 600.105 p/ml, intermediate strains. The intermediate mice do not present any statistical difference in infection with either resistant or susceptible strains, while the two last categories are statistically different from each other.
the breeding structures. Unlike the pinworm, the mouse-specific trypanosome is supposed not to have been transmitted in laboratory mouse stocks. The consequent lack of selective pressure would have led to the random fixation of the alleles of susceptibility or resistance, explaining the wide range of responses of laboratory strains. This process was suggested for interpreting the occurrence of susceptibility to Salmonella typhymurium of some laboratory strains (Malo et al. 1994). The result of this study strongly supports the hypothesis of Derothe et al. (1997) that environmental conditions of long-term laboratory breeding have a strong impact on mouse–parasite interactions. A definite parasite could have exerted either stronger (case of A. tetraptera) or weaker (case of T. musculi) pressure on the laboratory strains when
compared to the wild populations. It is then risky to extend the interpretations of host–parasite interactions obtained from laboratory mice to natural populations without previous comparison of the natural and laboratory conditions. Since these laboratory strains do not appear to be suitable models for analyzing the host response to natural parasitoses of the mouse (i.e., present particular responses to mouse-specific parasites when compared to wild mice), the relevance of using these strains as models for nonmurine parasitosis seems to be debatable. We are especially grateful to Professor Vincendeau (University of Bordeaux, France) for providing us a sample of the Partinico II strain, Dr. Tybairenc (UMR 9926 CNRS-ORSTOM, France) for allowing us
198 to clone the parasite strain in his laboratory, and Dr. Bonhomme and A. Orth (UPR 9060 CNRS, France) for providing us wild-derived ´ mice. Thanks to Christian Barnabe for his help in cloning the parasites and to Claude Pujol for his helpful comments on the manuscript.
REFERENCES
Albright, J. W., and Albright, J. F. 1981. Differences in resistance to Trypanosoma musculi infection among strains of inbred mice. Infection and Immunity 33(2), 364–371. Albright, J. W., Holmes, K. L., and Albright, J. F. 1990. Fluctuations in subsets of splenocytes and isotypes of Ig in young adult and aged mice resulting from Trypanosoma musculi infections. Journal of Immunology 144, 3970–3979. Chiejina, S. N., Street, J., Wakelin, D., and Behnke, J. M. 1993. Response of inbred mice to infection with a new isolate of Trypanosoma musculi. Parasitology 107, 233–236.
DEROTHE ET AL.
Moulia, C. Experimental trypanosomiasis of natural hybrids between house mouse subspecies. First insight into the susceptibility of wormy mice. [submitted for publication] Hoare, C. A. 1972. “The Trypanosomes of Mammals: A Zoological Monograph.” Blackwell Sci., Edinburgh. Kongshawn, P., Vargas, F., Skamene, E., and Ghadirian, E. 1985. Genetic control of resistance to infection with Trypanosoma musculi. In “Genetic Control of Host Resistance to Infection and Malignancy” (A. R. Liss, Ed.), pp. 517–522. A. R. Liss, New York. Malo, D., Vogan, K., Vidal, S., Hu, J., Cellier, M., Schurr, E., Fucks, A., Bumstead, M., Morgan, K., and Gros, P. 1994. Haplotype mapping and sequence analysis of the mouse Nramp gene predict susceptibility to infection with intracellular parasites. Genomics 23, 51–61. Maraghi, S., Wallbanks, K. R., and Molyneux, D. H. 1995. Oral transmission of trypanosomes of the subgenus Herpetosoma from small mammals. Parasitology Research 81, 693–695. Molyneux, D. H., and Ashford, R. W. 1983. “The Biology of Trypanosoma and Leishmania, Parasites of Man and Domestic Animals.” Taylor & Francis, London. Sherrer, B. 1984. “Biostatistique.” Gaetan Morin, Paris.
Derothe, J. -M., Loubes, C., Orth, A., Renaud, F., and Moulia, C. 1997. Comparison between patterns of pinworms infection (Aspiculuris tetraptera) in wild and laboratory strains of mice, Mus musculus. International Journal for Parasitology 27, 645–651.
Viens, P., Targett, G. A. T., Leuchars, E., and Davies, A. J. S. 1974. The immunological response of CBA mice to Trypanosoma musculi. I. Initial control of the infection and the effect of T-cell deprivation. Clinical and Experimental Immunology 16, 279–294.
Derothe, J. -M., Loubes, C., Perriat-Sanguinet, M., Bonhomme, F., and
Received 9 June 1998; accepted with revision 7 October 1998
RESEARCH BRIEF Trypanosoma musculi: Compared Levels of Parasitosis in Wild and Laboratory Strains of Mus musculus Mice
Jean-Marc Derothe, Nathalie Le Brun, Claude Loubes, Marco Perriat-Sanguinet, and Catherine Moulia1 Laboratoire de Parasitologie Compare´e, UMR 5555, cc 105, Universite´ Montpellier II, Montpellier, France
Derothe, J.-M., Le Brun, N., Loubes, C., Perriat-Sanguinet, M., and Moulia, C. 1999. Trypanosoma musculi: Compared levels of parasitosis in wild and laboratory strains of Mus musculus mice. Experimental Parasitology 91, 196–198. 䉷 1999 Academic Press Index Descriptors and Abbreviations: Trypanosoma musculi; Mus musculus; parasitosis.
HEOuJIco mice infected by dividing forms (7 to 12 days postinfection). Each sample was diluted at a concentration of 105 parasites/ml in a sodium chloride solution (0.9%). A volume of 0.5 ml of the diluted blood was intraperitoneally injected to each 1-month-old tested mouse (Albright et al. 1990; Derothe et al. submitted for publication). The number of trypanosomes by milliliter of blood was determined with an hemocytometer every 3 days until the end of infection (i.e., immune clearance, 25–27 days) (Chiejina et al., 1993). The response of each mouse is then evaluated by the maximum value of parasitemia, corresponding to a plateau phase (Albright et al., 1981; Kongshawn et al., 1985), obtained during the infection course as this seems to be the most reliable parameter whatever the route of infection (Maraghi et al., 1995). Mann–Whitney U tests detected differences between males and females in 5 strains of the 17 studied (Fig. 1). In all cases, males present a higher parasitemia than females. Although some of the significant results may be due to a type 1 error because of the number of tests performed, males (m) and females (f) of the strains concerned are separated in the following analysis (Fig. 1). The Kruskal–Wallis test (1952) showed a highly significant difference among all the strains tested (Hcor, 258.04; df ⫽ 22; P ⬍ 0.0001). The Noether’s multiple comparison tests (1976) (Sherrer 1984), used as posthoc tests, reveal that the strains segregate into three groups according to their level of resistance (Fig. 1). Inbred strains were found in all the categories. This study clearly showed that the profiles of resistance to T. musculi of laboratory mice are as variable as the profiles of wild-derived mice. This is contrary to the result obtained with A. tetraptera (Derothe et al. 1997). This difference can be explained by the differential probabilities of transmission of the two parasites in the laboratory. T. musculi presents an heteroxenous life cycle. Its intermediate host, a flea (Hoare 1972; Molyneux and Ashford 1983), is systematically eliminated from
The laboratory environment can modify mouse–parasite interactions, either increasing or limiting the parasite transmission and the consequent pressure it may represent on mouse genome. In a previous work, Derothe et al. (1997) argued that breeding conditions favored the transmission of the mouse pinworm Aspiculuris tetraptera (direct cycle). This led to the selection of resistance genes in all the laboratory strains when compared to wild-derived strains. The aim of this study was to bring new data to assess the limitations of the laboratory mouse model in parasitology. We use the blood protozoan Trypanosoma musculi, another natural parasite of the mouse. Experimental infections of mice from eight inbred laboratory strains and nine wild-derived strains (see Fig. 1 for designations) were performed with a clone of the Partinico II strain (Viens et al. 1974). Wild-derived strains, recently isolated from the field (5 to 25 generations in the laboratory) and randomly bred in little colonies, represent a broad sample of Mus musculus domesticus and M. m. musculus genomes. They are used as indicators of the variability of resistance to the trypanosome in wild populations of mice (Derothe et al. submitted for publication). Standard infections are realized from a sample of blood collected from C3H/
1
To whom correspondence should be addressed. E-mail: moulia@ crit.univ-montp2.fr.
196
0014-4894/99 $30.00 Copyright 䉷 1999 by Academic Press All rights of reproduction in any form reserved.
PARASITOSIS IN WILD AND LABORATORY MICE
197
FIG. 1. Percentile distribution of parasitemia in experimental trypanosomiasis of laboratory and wild-derived strains. For each sample, bars represent the 10th, 25th, 50th (i.e., median), 75th, and 90th percentiles of each sample tested with Trypanosoma musculi. Strain designation are as follows. Laboratory strains: D2, DBA/2/Ola//Hsd; NIH, NIH/Ola//Hsd; SWR, SWR/Ola//Hsd; SJL, SJL/Ola//Hsd; CBA, CBA/J/Ola//Hsd from Harlan Olac, U. K.; B6, C57BL/6Jico; C, BALB/cByJIco from Iffa Credo, France; 129, 129/SV/Pas from Pasteur Institute of Paris, France. Wildderived strains were BZO, DMZ, DJO, DDO, MHT, MPB, MDH, MBS, and MBT from the “Wild Mouse Repository” (UPR 9060 CNRS, France). Strains showing differences in parasite loads between males and females are divided into two subsamples, adding an m or f suffix to the strain designation. Median less than 320.105 parasites/ml (p/ml) of blood, resistant strains; median more than 600.105 p/ml, susceptible strains; median from 320 to 600.105 p/ml, intermediate strains. The intermediate mice do not present any statistical difference in infection with either resistant or susceptible strains, while the two last categories are statistically different from each other.
the breeding structures. Unlike the pinworm, the mouse-specific trypanosome is supposed not to have been transmitted in laboratory mouse stocks. The consequent lack of selective pressure would have led to the random fixation of the alleles of susceptibility or resistance, explaining the wide range of responses of laboratory strains. This process was suggested for interpreting the occurrence of susceptibility to Salmonella typhymurium of some laboratory strains (Malo et al. 1994). The result of this study strongly supports the hypothesis of Derothe et al. (1997) that environmental conditions of long-term laboratory breeding have a strong impact on mouse–parasite interactions. A definite parasite could have exerted either stronger (case of A. tetraptera) or weaker (case of T. musculi) pressure on the laboratory strains when
compared to the wild populations. It is then risky to extend the interpretations of host–parasite interactions obtained from laboratory mice to natural populations without previous comparison of the natural and laboratory conditions. Since these laboratory strains do not appear to be suitable models for analyzing the host response to natural parasitoses of the mouse (i.e., present particular responses to mouse-specific parasites when compared to wild mice), the relevance of using these strains as models for nonmurine parasitosis seems to be debatable. We are especially grateful to Professor Vincendeau (University of Bordeaux, France) for providing us a sample of the Partinico II strain, Dr. Tybairenc (UMR 9926 CNRS-ORSTOM, France) for allowing us
198 to clone the parasite strain in his laboratory, and Dr. Bonhomme and A. Orth (UPR 9060 CNRS, France) for providing us wild-derived ´ mice. Thanks to Christian Barnabe for his help in cloning the parasites and to Claude Pujol for his helpful comments on the manuscript.
REFERENCES
Albright, J. W., and Albright, J. F. 1981. Differences in resistance to Trypanosoma musculi infection among strains of inbred mice. Infection and Immunity 33(2), 364–371. Albright, J. W., Holmes, K. L., and Albright, J. F. 1990. Fluctuations in subsets of splenocytes and isotypes of Ig in young adult and aged mice resulting from Trypanosoma musculi infections. Journal of Immunology 144, 3970–3979. Chiejina, S. N., Street, J., Wakelin, D., and Behnke, J. M. 1993. Response of inbred mice to infection with a new isolate of Trypanosoma musculi. Parasitology 107, 233–236.
DEROTHE ET AL.
Moulia, C. Experimental trypanosomiasis of natural hybrids between house mouse subspecies. First insight into the susceptibility of wormy mice. [submitted for publication] Hoare, C. A. 1972. “The Trypanosomes of Mammals: A Zoological Monograph.” Blackwell Sci., Edinburgh. Kongshawn, P., Vargas, F., Skamene, E., and Ghadirian, E. 1985. Genetic control of resistance to infection with Trypanosoma musculi. In “Genetic Control of Host Resistance to Infection and Malignancy” (A. R. Liss, Ed.), pp. 517–522. A. R. Liss, New York. Malo, D., Vogan, K., Vidal, S., Hu, J., Cellier, M., Schurr, E., Fucks, A., Bumstead, M., Morgan, K., and Gros, P. 1994. Haplotype mapping and sequence analysis of the mouse Nramp gene predict susceptibility to infection with intracellular parasites. Genomics 23, 51–61. Maraghi, S., Wallbanks, K. R., and Molyneux, D. H. 1995. Oral transmission of trypanosomes of the subgenus Herpetosoma from small mammals. Parasitology Research 81, 693–695. Molyneux, D. H., and Ashford, R. W. 1983. “The Biology of Trypanosoma and Leishmania, Parasites of Man and Domestic Animals.” Taylor & Francis, London. Sherrer, B. 1984. “Biostatistique.” Gaetan Morin, Paris.
Derothe, J. -M., Loubes, C., Orth, A., Renaud, F., and Moulia, C. 1997. Comparison between patterns of pinworms infection (Aspiculuris tetraptera) in wild and laboratory strains of mice, Mus musculus. International Journal for Parasitology 27, 645–651.
Viens, P., Targett, G. A. T., Leuchars, E., and Davies, A. J. S. 1974. The immunological response of CBA mice to Trypanosoma musculi. I. Initial control of the infection and the effect of T-cell deprivation. Clinical and Experimental Immunology 16, 279–294.
Derothe, J. -M., Loubes, C., Perriat-Sanguinet, M., Bonhomme, F., and
Received 9 June 1998; accepted with revision 7 October 1998