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c mice infected by Leishmania infantum
Acta Tropica 90 (2004) 123–126
Short communication
Influence of the inoculation route in BALB/c mice infected by Leishmania infantum Nuno Rolão, Cláudia Melo, Lenea Campino∗ Lenea Campino, Unidade de Leishmanioses/Centro, Malária Outras Doenças Tropicais/Instituto de Higiene e Medicina Tropical/Universidade Nova de Lisboa, Rua da Junqueira, 96, 1349-008 Lisboa, Portugal Received 31 January 2003; received in revised form 4 July 2003; accepted 1 September 2003
Abstract Mice of the BALB/c strain are frequently used, due to their high susceptibility to Leishmania infection. Most of the studies in visceral leishmaniasis use the endovenous or the intraperitoneal routes to inoculate the parasites. In this study, the development of experimental visceral leishmaniasis infection was evaluated in BALB/c mice inoculated with Leishmania infantum parasites by endovenous (EV group) or intraperitoneal (IP group) routes. The results shows that both inoculation routes were able to produce progressive infection in mice. However, a higher dispersion of the values of parasite density was detected among the animals of the EV group than the IP group. Our results indicate that the intraperitoneal inoculation results in a higher homogeneity of infections, so we suggest that this route should be preferentially used in experimental infections in the mice model, particularly when pools of samples are required for immune cellular studies. © 2003 Elsevier B.V. All rights reserved. Keywords: Leishmania; Mice; Inoculation route
Several animal models (e.g. mice, hamsters, dogs, and primates) have been used to study Leishmania infections (Hommel et al., 1995). From these, mice of the BALB/c strain are frequently adopted, due to their high susceptibility to Leishmania. Moreover, the possibility of working with “inbred” strains reduces genetic variations between individuals of the same experimental group. In addition to host immunity and strain virulence, the outcome of the experimental infection is also dependent on the number of inoculated
∗ Corresponding author. Tel.: +351-21-365-2600; fax: +351-21-363-2105. E-mail address: [email protected] (L. Campino).
parasites (Titus and Ribeiro, 1988) and the route of inoculation (Melby et al., 1998). Experimental infections try to mimic natural infections, in which the parasite is inoculated by the phlebotomine vector in the skin of the host, but it has been reported that mice inoculated with viscerotropic Leishmania species by cutaneous via do not develop the visceral form of the disease (Melby et al., 1998, 2001). Recently, Ahmed et al. (2003) showed that intradermal infection with 107 parasites appears to result in progressive visceral disease, but the evolution of the disease and subsequent immune response was slower than a high-dose endovenous (e.v.) infection, as widely used in the murine model of VL. In fact, the intradermal inoculation was consistent
0001-706X/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2003.09.010
N. Rolão et al. / Acta Tropica 90 (2004) 123–126
• EV:mice inoculated with 107 L. infantum MON-1 (MCAN/PT/94/IMT205) promastigotes by e.v. route; • IP:mice inoculated with 107 L. infantum MON-1 ((MCAN/PT/94/IMT205) promastigotes by i.p. route; • C:control healthy mice, inoculated with 0.1 ml of 0.9% saline solution, by i.p. route. Two mice of each infected group and one of the control group were sacrificed at days 7, 15, 45, and 60 after inoculation (p.i.) and three consecutive experimental infections were performed (about 1 week between each experiment). For determination of parasite density, spleens were collected and individually processed by limiting dilution assay (LDA) (Titus et al., 1985). The results were analyzed using the statistical software SPSS® (SPSS Inc.). The progression of the parasite density was identical for both infected groups (IP and EV, Figs. 1 and 2,
Group IP 1,0E+11 1,0E+09
Parasite density
with what was observed with the low-dose (104 ) e.v. infection. Most of the studies in visceral leishmaniasis (VL) use the e.v. or the intraperitoneal (i.p.) routes to inoculate the parasites (Leclercq et al., 1996; Wilson and Weinstock, 1996; Bories et al., 1998). The e.v. route usually leads to higher parasite loads in the animals. However, this inoculation is difficult to perform on small animals, such as mice, frequently resulting in loss of part of the inoculum to the adjacent tissues. On the other hand, the i.p. route causes an infection with lower parasite loads and a longer pre-patency period, but it does infer a higher accuracy of application (Ott et al., 1967). Therefore, it would be very important to standardize the administration of the inoculum in order to improve the comparison of results from different studies. In the development of VL there is an association between the number of parasites, the severity of the clinical signs and the intensity of the immune response (Campino et al., 2000). In this study, the development of experimental visceral leishmaniasis infection was evaluated in female BALB/c mice inoculated with L. infantum parasites by e.v. or i.p. routes. All the mice were 6–8 week old at the time of the parasite challenge. The follow-up was accessed by determination of parasite density in the spleen, for each of the following groups:
1,0E+07 1,0E+05 1,0E+03 1,0E+01 1,0E-01 7
15
45
60
Days p.i.
Fig. 1. IP group:( ) parasite density of individual mice, each dot representing 1–3 animals when values are identical;( ) mean values.
respectively) and no parasites were detected in control group. Non-parametric Mann-Whitney’s test was used to evaluate differences between independent samples and the values of parasite density showed no significant differences (P ≥ 0.05) in any of the experiment time points. To assess variance homogeneity, the values of parasite density of the individual mouse of each group were analyzed by the Levene’s test. The results showed a tendency to a higher dispersion of the results in the EV group (Fig. 2) than in the IP group (Fig. 1). In this work, the results shows that both inoculation routes were able to produce progressive infection in mice. Although the differences were not statistically significant, the mean values of parasite density were always higher with the e.v. inoculation, as observed by other authors (Ott et al., 1967). Group EV 1,0E+11 Parasite density
124
1,0E+09 1,0E+07 1,0E+05 1,0E+03 1,0E+01 1,0E-01 7
15
45
60
Days p.i.
Fig. 2. EV group:( ) parasite density of individual mice, each dot representing 1–3 animals when values are identical;( ) mean values.
N. Rolão et al. / Acta Tropica 90 (2004) 123–126
Inoculation by i.p. route with higher number of parasites (108 ) may result in parasite loads close to that obtained by e.v. injection with 107 parasites. However, such a great number of parasites may be difficult to obtain in experiments that use a high number of animals. Moreover, most of the VL experimental studies use 107 parasites per mouse and the application of a different inoculum will limit comparison of results. Our results showed a higher dispersion of the values of parasite density among the animals of the EV group when compared to the IP group, in each time point of the experiment. An increase of dispersion was observed at day 45 p.i. in the IP group, but still with lower values than the EV group at the same time-point. Increased variability in the number of inoculated parasites is more likely to occur with the e.v. inoculation, which is harder to perform on small animals, and part of the inoculum may be lost to the adjacent cutaneous tissues, resulting in a subcutaneous administration. Our study detected differences in parasitism among individuals of the same group, particularly between mice of the EV group, which could lead to errors in the interpretation of the results when pools of samples are used. Some methods used in studies of cellular immune responses, such as cytokine analysis, require the pooling of tissue samples from several animals of the same group in order to obtain enough lymphocytes for cell culture (Mattner et al., 1997; Melby et al., 1998). It has been shown that mice inoculated with a low number of parasites produce Th1-type cytokines, like IFN-␥ and IL-2 (Menon and Bretscher, 1996; Mattner et al., 1997), while mice inoculated with a high number of parasites produce Th2-type cytokines, like IL-4 and IL-10 (Zwingenberger et al., 1990; Menon and Bretscher, 1996). If the animals of a pool are inoculated with a variable number of parasites, they may therefore, develop different immune responses and both types of cytokines would be detected in the pool sample. This could be incorrectly interpreted as a mixed Th1/Th2 immune response. Thus, the evaluation of the results obtained by that sample might not accurately reflect the individual response of each animal. In conclusion, our results indicate that the inoculation via intraperitoneal route results in a higher homogeneity among infections, so we suggest that this route should be preferentially used in experimental in-
125
fections in the mice model, particularly when pools of samples are required. Acknowledgements This work was supported by FEDER (FCT project POCTI/CVT/35263/99). We acknowledge J. Pinto, S. Cortes, and O. Rodrigues for the English revision and advises, and L. Gonçalves for the statistical analysis. We are most grateful to the technicians of the Leishmaniasis Unit for their technical support.
References Ahmed, S., Colmenares, M., Soong, L., Goldsmith-Pestana, K., Munstermann, L., Molina, R., McMahon-Pratt, D., 2003. Intradermal infection model for pathogenesis and vaccine studies of murine visceral leishmaniasis. Infect. Immun. 71, 401–410. Bories, C., Coffin, C., Mathieu, D., Bories, P.N., Scherman, E., Rivollet, D., Deniau, M., 1998. Lack of a nitric-oxide response during the course of Leishmania infantum infection in the golden hamster (Mesocricetus auratus), with or without treatment with liposomal amphotericin. B. Ann. Trop. Med. Parasitol. 92, 685–692. Campino, L., Santos-Gomes, G., Riça-Capela, M.J., Cortes, S., Abranches, P., 2000. Infectivity of promastigotes and amastigotes of Leishmania infantum in a canine model for leishmaniosis. Vet. Parasitol. 92, 269–275. Hommel, M., Jaffe, C.L., Travi, B., Milon, G., 1995. Experimental models for leishmaniasis and for testing anti-leishmanial vaccines. Ann. Trop. Med. Parasitol. 89, 55–73. Leclercq, V., Lebastard, M., Belkaid, Y., Louis, J., Milon, G., 1996. The outcome of the parasitic process initiated by Leishmania infantum in laboratory mice: a tissue-dependent pattern controlled by the Lsh and MHC loci. J. Immunol. 157, 4537–4545. Mattner, F., Di Padova, K., Alber, G., 1997. Interleukin-12 is indispensable for protective immunity against Leishmania major. Infect. Immun. 65, 4378–4383. Melby, P.C., Yang, Y.Z., Cheng, J., Zhao, W., 1998. Regional differences in the cellular immune response to experimental cutaneous or visceral infection with Leishmania donovani. Infect. Immun. 66, 18–27. Melby, P.C., Tabares, A., Restrepo, B.I., Cardona, A.E., McGuff, H.S., Teale, J.M., 2001. Leishmania donovani: evolution and architecture of the splenic cellular immune response related to control of infection. Exp. Parasitol. 99, 17–25. Menon, J.N., Bretscher, P.A., 1996. Characterization of the immunological memory state generated in mice susceptible to Leishmania major following exposure to low doses of L. major and resulting in resistance to a normally pathogenic challenge. Eur. J. Immunol. 26, 243–249.
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N. Rolão et al. / Acta Tropica 90 (2004) 123–126
Ott, K.J., Hanson, W.L., Stauber, L.A., 1967. Course of infection of Leishmania donovani in hamsters inoculated by the intraperitoneal route. J. Parasitol. 53, 641–645. Titus, R.G., Marchand, M., Boon, T., Louis, J., 1985. A limiting dilution assay for quantifying Leishmania major in tissues of infected mice. Parasite Immunol. 7, 545–555. Titus, R.G., Ribeiro, J.M., 1988. Salivary gland lysates from the sand fly Lutzomyia longipalpis enhance Leishmania infectivity. Science 239, 1306–1308.
Wilson, M.E., Weinstock, J.V., 1996. Hepatic granulomas in murine visceral leishmaniasis caused by Leishmania chagasi. Methods 9, 248–254. Zwingenberger, K., Harms, G., Pedrosa, C., Sandkamp, B., Neifer, S., 1990. Determinants of the immune response in visceral leishmaniasis: evidence of predominance of endogenous interleukin-4 over IFN-␥ production. Clin. Immunol. Immunopathol. 57, 242–249.
Short communication
Influence of the inoculation route in BALB/c mice infected by Leishmania infantum Nuno Rolão, Cláudia Melo, Lenea Campino∗ Lenea Campino, Unidade de Leishmanioses/Centro, Malária Outras Doenças Tropicais/Instituto de Higiene e Medicina Tropical/Universidade Nova de Lisboa, Rua da Junqueira, 96, 1349-008 Lisboa, Portugal Received 31 January 2003; received in revised form 4 July 2003; accepted 1 September 2003
Abstract Mice of the BALB/c strain are frequently used, due to their high susceptibility to Leishmania infection. Most of the studies in visceral leishmaniasis use the endovenous or the intraperitoneal routes to inoculate the parasites. In this study, the development of experimental visceral leishmaniasis infection was evaluated in BALB/c mice inoculated with Leishmania infantum parasites by endovenous (EV group) or intraperitoneal (IP group) routes. The results shows that both inoculation routes were able to produce progressive infection in mice. However, a higher dispersion of the values of parasite density was detected among the animals of the EV group than the IP group. Our results indicate that the intraperitoneal inoculation results in a higher homogeneity of infections, so we suggest that this route should be preferentially used in experimental infections in the mice model, particularly when pools of samples are required for immune cellular studies. © 2003 Elsevier B.V. All rights reserved. Keywords: Leishmania; Mice; Inoculation route
Several animal models (e.g. mice, hamsters, dogs, and primates) have been used to study Leishmania infections (Hommel et al., 1995). From these, mice of the BALB/c strain are frequently adopted, due to their high susceptibility to Leishmania. Moreover, the possibility of working with “inbred” strains reduces genetic variations between individuals of the same experimental group. In addition to host immunity and strain virulence, the outcome of the experimental infection is also dependent on the number of inoculated
∗ Corresponding author. Tel.: +351-21-365-2600; fax: +351-21-363-2105. E-mail address: [email protected] (L. Campino).
parasites (Titus and Ribeiro, 1988) and the route of inoculation (Melby et al., 1998). Experimental infections try to mimic natural infections, in which the parasite is inoculated by the phlebotomine vector in the skin of the host, but it has been reported that mice inoculated with viscerotropic Leishmania species by cutaneous via do not develop the visceral form of the disease (Melby et al., 1998, 2001). Recently, Ahmed et al. (2003) showed that intradermal infection with 107 parasites appears to result in progressive visceral disease, but the evolution of the disease and subsequent immune response was slower than a high-dose endovenous (e.v.) infection, as widely used in the murine model of VL. In fact, the intradermal inoculation was consistent
0001-706X/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2003.09.010
N. Rolão et al. / Acta Tropica 90 (2004) 123–126
• EV:mice inoculated with 107 L. infantum MON-1 (MCAN/PT/94/IMT205) promastigotes by e.v. route; • IP:mice inoculated with 107 L. infantum MON-1 ((MCAN/PT/94/IMT205) promastigotes by i.p. route; • C:control healthy mice, inoculated with 0.1 ml of 0.9% saline solution, by i.p. route. Two mice of each infected group and one of the control group were sacrificed at days 7, 15, 45, and 60 after inoculation (p.i.) and three consecutive experimental infections were performed (about 1 week between each experiment). For determination of parasite density, spleens were collected and individually processed by limiting dilution assay (LDA) (Titus et al., 1985). The results were analyzed using the statistical software SPSS® (SPSS Inc.). The progression of the parasite density was identical for both infected groups (IP and EV, Figs. 1 and 2,
Group IP 1,0E+11 1,0E+09
Parasite density
with what was observed with the low-dose (104 ) e.v. infection. Most of the studies in visceral leishmaniasis (VL) use the e.v. or the intraperitoneal (i.p.) routes to inoculate the parasites (Leclercq et al., 1996; Wilson and Weinstock, 1996; Bories et al., 1998). The e.v. route usually leads to higher parasite loads in the animals. However, this inoculation is difficult to perform on small animals, such as mice, frequently resulting in loss of part of the inoculum to the adjacent tissues. On the other hand, the i.p. route causes an infection with lower parasite loads and a longer pre-patency period, but it does infer a higher accuracy of application (Ott et al., 1967). Therefore, it would be very important to standardize the administration of the inoculum in order to improve the comparison of results from different studies. In the development of VL there is an association between the number of parasites, the severity of the clinical signs and the intensity of the immune response (Campino et al., 2000). In this study, the development of experimental visceral leishmaniasis infection was evaluated in female BALB/c mice inoculated with L. infantum parasites by e.v. or i.p. routes. All the mice were 6–8 week old at the time of the parasite challenge. The follow-up was accessed by determination of parasite density in the spleen, for each of the following groups:
1,0E+07 1,0E+05 1,0E+03 1,0E+01 1,0E-01 7
15
45
60
Days p.i.
Fig. 1. IP group:( ) parasite density of individual mice, each dot representing 1–3 animals when values are identical;( ) mean values.
respectively) and no parasites were detected in control group. Non-parametric Mann-Whitney’s test was used to evaluate differences between independent samples and the values of parasite density showed no significant differences (P ≥ 0.05) in any of the experiment time points. To assess variance homogeneity, the values of parasite density of the individual mouse of each group were analyzed by the Levene’s test. The results showed a tendency to a higher dispersion of the results in the EV group (Fig. 2) than in the IP group (Fig. 1). In this work, the results shows that both inoculation routes were able to produce progressive infection in mice. Although the differences were not statistically significant, the mean values of parasite density were always higher with the e.v. inoculation, as observed by other authors (Ott et al., 1967). Group EV 1,0E+11 Parasite density
124
1,0E+09 1,0E+07 1,0E+05 1,0E+03 1,0E+01 1,0E-01 7
15
45
60
Days p.i.
Fig. 2. EV group:( ) parasite density of individual mice, each dot representing 1–3 animals when values are identical;( ) mean values.
N. Rolão et al. / Acta Tropica 90 (2004) 123–126
Inoculation by i.p. route with higher number of parasites (108 ) may result in parasite loads close to that obtained by e.v. injection with 107 parasites. However, such a great number of parasites may be difficult to obtain in experiments that use a high number of animals. Moreover, most of the VL experimental studies use 107 parasites per mouse and the application of a different inoculum will limit comparison of results. Our results showed a higher dispersion of the values of parasite density among the animals of the EV group when compared to the IP group, in each time point of the experiment. An increase of dispersion was observed at day 45 p.i. in the IP group, but still with lower values than the EV group at the same time-point. Increased variability in the number of inoculated parasites is more likely to occur with the e.v. inoculation, which is harder to perform on small animals, and part of the inoculum may be lost to the adjacent cutaneous tissues, resulting in a subcutaneous administration. Our study detected differences in parasitism among individuals of the same group, particularly between mice of the EV group, which could lead to errors in the interpretation of the results when pools of samples are used. Some methods used in studies of cellular immune responses, such as cytokine analysis, require the pooling of tissue samples from several animals of the same group in order to obtain enough lymphocytes for cell culture (Mattner et al., 1997; Melby et al., 1998). It has been shown that mice inoculated with a low number of parasites produce Th1-type cytokines, like IFN-␥ and IL-2 (Menon and Bretscher, 1996; Mattner et al., 1997), while mice inoculated with a high number of parasites produce Th2-type cytokines, like IL-4 and IL-10 (Zwingenberger et al., 1990; Menon and Bretscher, 1996). If the animals of a pool are inoculated with a variable number of parasites, they may therefore, develop different immune responses and both types of cytokines would be detected in the pool sample. This could be incorrectly interpreted as a mixed Th1/Th2 immune response. Thus, the evaluation of the results obtained by that sample might not accurately reflect the individual response of each animal. In conclusion, our results indicate that the inoculation via intraperitoneal route results in a higher homogeneity among infections, so we suggest that this route should be preferentially used in experimental in-
125
fections in the mice model, particularly when pools of samples are required. Acknowledgements This work was supported by FEDER (FCT project POCTI/CVT/35263/99). We acknowledge J. Pinto, S. Cortes, and O. Rodrigues for the English revision and advises, and L. Gonçalves for the statistical analysis. We are most grateful to the technicians of the Leishmaniasis Unit for their technical support.
References Ahmed, S., Colmenares, M., Soong, L., Goldsmith-Pestana, K., Munstermann, L., Molina, R., McMahon-Pratt, D., 2003. Intradermal infection model for pathogenesis and vaccine studies of murine visceral leishmaniasis. Infect. Immun. 71, 401–410. Bories, C., Coffin, C., Mathieu, D., Bories, P.N., Scherman, E., Rivollet, D., Deniau, M., 1998. Lack of a nitric-oxide response during the course of Leishmania infantum infection in the golden hamster (Mesocricetus auratus), with or without treatment with liposomal amphotericin. B. Ann. Trop. Med. Parasitol. 92, 685–692. Campino, L., Santos-Gomes, G., Riça-Capela, M.J., Cortes, S., Abranches, P., 2000. Infectivity of promastigotes and amastigotes of Leishmania infantum in a canine model for leishmaniosis. Vet. Parasitol. 92, 269–275. Hommel, M., Jaffe, C.L., Travi, B., Milon, G., 1995. Experimental models for leishmaniasis and for testing anti-leishmanial vaccines. Ann. Trop. Med. Parasitol. 89, 55–73. Leclercq, V., Lebastard, M., Belkaid, Y., Louis, J., Milon, G., 1996. The outcome of the parasitic process initiated by Leishmania infantum in laboratory mice: a tissue-dependent pattern controlled by the Lsh and MHC loci. J. Immunol. 157, 4537–4545. Mattner, F., Di Padova, K., Alber, G., 1997. Interleukin-12 is indispensable for protective immunity against Leishmania major. Infect. Immun. 65, 4378–4383. Melby, P.C., Yang, Y.Z., Cheng, J., Zhao, W., 1998. Regional differences in the cellular immune response to experimental cutaneous or visceral infection with Leishmania donovani. Infect. Immun. 66, 18–27. Melby, P.C., Tabares, A., Restrepo, B.I., Cardona, A.E., McGuff, H.S., Teale, J.M., 2001. Leishmania donovani: evolution and architecture of the splenic cellular immune response related to control of infection. Exp. Parasitol. 99, 17–25. Menon, J.N., Bretscher, P.A., 1996. Characterization of the immunological memory state generated in mice susceptible to Leishmania major following exposure to low doses of L. major and resulting in resistance to a normally pathogenic challenge. Eur. J. Immunol. 26, 243–249.
126
N. Rolão et al. / Acta Tropica 90 (2004) 123–126
Ott, K.J., Hanson, W.L., Stauber, L.A., 1967. Course of infection of Leishmania donovani in hamsters inoculated by the intraperitoneal route. J. Parasitol. 53, 641–645. Titus, R.G., Marchand, M., Boon, T., Louis, J., 1985. A limiting dilution assay for quantifying Leishmania major in tissues of infected mice. Parasite Immunol. 7, 545–555. Titus, R.G., Ribeiro, J.M., 1988. Salivary gland lysates from the sand fly Lutzomyia longipalpis enhance Leishmania infectivity. Science 239, 1306–1308.
Wilson, M.E., Weinstock, J.V., 1996. Hepatic granulomas in murine visceral leishmaniasis caused by Leishmania chagasi. Methods 9, 248–254. Zwingenberger, K., Harms, G., Pedrosa, C., Sandkamp, B., Neifer, S., 1990. Determinants of the immune response in visceral leishmaniasis: evidence of predominance of endogenous interleukin-4 over IFN-␥ production. Clin. Immunol. Immunopathol. 57, 242–249.