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Increased parasitaemia and delayed parasite clearance in Schistosoma mansoni and Plasmodium berghei co-infected mice
Acta Tropica 91 (2004) 161–166
Increased parasitaemia and delayed parasite clearance in Schistosoma mansoni and Plasmodium berghei co-infected mice Mengistu Legesse∗ , Berhanu Erko, Fekede Balcha Institute of Pathobiology, Addis Ababa University, P.O. Box 1176, Addis Ababa, Ethiopia Received 1 September 2003; received in revised form 16 March 2004; accepted 6 April 2004 Available online 10 June 2004
Abstract Identifying factors that contribute to malaria susceptibility, severity and treatment failure remains one of the major research areas in malaria control strategies. In the present study, we superinfected Schistosoma mansoni infected mice with a lethal strain Plasmodium berghei ANKA to assess whether or not infection with S. mansoni affects parasite development, parasitaemia and parasite reduction or clearance following antimalarial treatment. Mice infected with P. berghei alone were used as control. The mice were followed for parasite development and parasitaemia between days 4 and 9 post-infection. On day 9, after taking blood samples, the mice were orally treated with 100 mg/kg of chloroquine and then with 10 mg/kg for three consecutive days. Parasite reduction/clearance and mortality were followed between days 10 and 13 post-treatment. The results showed, that superinfection with S. mansoni enhanced P. berghei parasite development, increased parasitaemia and mortality, and delayed reduction/clearance in parasitaemia. Hence, the results postulate that co-infections with schistosome and malaria parasites would aggravate malarial severity and prolong parasite reduction or clearance after chemotherapy in humans. This would necessitate the need for considering schistosome infection in clinical as well as therapeutic management of malaria patients in areas where the two diseases are co-endemic. © 2004 Published by Elsevier B.V. Keywords: Schistosoma mansoni; Plasmodium berghei; Concurrent infections; Parasitaemia; Treatment; Parasite clearance; Mortality
1. Introduction Malaria is one of the leading causes of morbidity and mortality in people living in sub-Saharan Africa. However, the severity of the disease varies from individual to individual. Population living in the same epidemiological context may show variation in susceptibility to Plasmodium infection and development of severe malaria. Age, pregnancy, host genetic fac∗
Corresponding author. Fax: +251 1 755296/550655. E-mail address: [email protected] (M. Legesse).
0001-706X/$ – see front matter © 2004 Published by Elsevier B.V. doi:10.1016/j.actatropica.2004.04.002
tors, immunity, inoculation dose and virulence of the parasite strains are factors that have been considered to influence the clinical outcome of the disease (Hill et al., 1992; Gupta et al., 1994; Timms et al., 2001). Furthermore, chronic intestinal helminthic infections have also become the subject of speculation and investigation in malarial severity. On one hand, it is claimed that intestinal helminthic infections are associated with protection from cerebral malaria, malaria-related acute renal failure and jaundice (Nacher et al., 2000, 2001a). On the other hand, it has been proposed that intestinal helminthic infections are
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associated with increased malarial severity (Nacher et al., 2001b). In malaria patients, the effectiveness of antimalarial drugs is also highly variable as the clinical outcome. This problem has mainly been attributable to the emergence of drug-resistant Plasmodium strains. Nevertheless, evidences have indicated that the effectiveness of antimalarial drugs can be affected by the immune status of the host. In other words, impaired immune responses contribute to rapid evolving of drug-resistant parasite strains (Lwin et al., 1987; Wernsdorfer, 1991; Targett, 1992). Intestinal helminthic infections are one of the factors that are known to impair host immune responses to other infectious agents (Pearlman et al., 1993; Bentwich et al., 1995; Diniz et al., 2001; Elias et al., 2001). Intestinal schistosomiasis is a helminthic infection that ranks second to malaria in terms of socio-economic and public health importance in tropical and subtropical areas of the world. Despite the wide distribution of the two diseases in the tropics, little effort has been made to assess possible effects of co-infection with the two parasites on malarial severity or vice versa (Lewinsohn, 1975; Lwin et al., 1982; Helmby et al., 1998; Yoshida et al., 2000). Moreover, most of the findings from the previous works are controversial. Therefore, this study was aimed to assess the effect of co-infection with Schistosoma mansoni on Plasmodium berghei development, parasitaemia, and parasite reduction or clearance following chloroquine treatment in mice. 2. Material and methods 2.1. Infection of mice with Schistosoma mansoni A total of 50 laboratory-bred male Swiss albino mice (8–10 weeks old) were selected and randomly grouped into two. The first group consisting of 30 mice was exposed to 50 cercariae of S. mansoni by the tail immersion method. Faecal samples from the exposed mice were microscopically examined to confirm establishment of infection six weeks post-exposure.
105 P. berghei ANKA parasitized erythrocytes. To inoculate approximately the same amount of parasitized erythrocytes into each mouse, blood sample was collected from previously infected mouse by cardiac puncture and the required dose was calculated as previously described (Timms et al., 2001). Fifteen free mice were also infected interaperitoneally with 5×105 P. berghei ANKA parasitized erythrocytes and used as control.
2.3. Follow-up for parasite development and parasitaemia Thick and thin blood films were prepared daily from each mouse starting from day 4 post-infection up to day 9 by taking blood from the tip of the tail. The films were stained with Giemsa and examined microscopically for the appearance of the parasite in the peripheral blood of the infected mice. The number of infected and non-infected erythrocytes was counted per 10 thin microscopic fields and parasitaemia was computed out of 1000 non-infected erythrocytes for each mouse.
2.4. Treatment and follow-up for parasite reduction or clearance After collecting blood films on day 9, all P. berghei infected mice were treated orally with 100 mg/kg of chloroquine dissolved in tap water, and then with 10 mg/kg daily for three consecutive days. Parasite reduction or clearance was checked on days 10–13 post-treatment by making thin and thick blood films daily. As in the case of the first follow-up, the number of infected and non-infected erythrocytes was counted in 10 thin microscopic fields between days 10–12 post-treatment and parasitaemia was computed out of 1000 non-infected erythrocytes for each mouse. During the follow-up, mice mortality was recorded.
2.5. Data analysis
2.2. Infection of mice with Plasmodium berghei Seven weeks after infection with S. mansoni, 15 mice were superinfected interaperitoneally with 5 ×
Average parasitaemia was compared using Student’s t-test assuming equal variances. Difference was considered significant when P-value was less than 0.05.
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The study received ethical approval from the Ethical Clearance Committee of the Institute of Pathobiology, Addis Ababa University, before its implementation. The mice were maintained in the animal house and supplied with food and water ad libitum.
fected with P. berghei alone (13.2 ± 2.5% versus 6.5 ± 0.8%, P = 0.023). Similarly, mean parasitaemia measured on day 10 (11.5 ± 2.4% versus 4.4 ± 0.7%, P = 0.007) and day 11 (4.6 ± 1.1% versus 1.2 ± 0.3%, P = 0.005) post-treatment was higher in S. mansoni and P. berghei co-infected mice than in mice infected with P. berghei alone (Fig. 2).
3. Results
3.2. Parasite reduction/clearance during the course of treatment
2.6. Ethical consideration
3.1. Parasite appearance and parasitaemia Blood-stage of P. berghei parasite was not observed in the thick and thin blood films collected either from mice co-infected with S. mansoni and P. berghei or P. berghei alone on days 4 and 5 post-infection. The parasite was observed in the blood films of 10 double-infected and five single-infected mice on day 6 post-infection. Most of the mice became positive on day 7 post-infection (Fig. 1). In mice co-infected with S. mansoni and P. berghei, rise in P. berghei parasitaemia was observed on day 7 and peaked on day 9 (Fig. 2). The mean parasitaemia values recorded for days 6, 7 and 8 were not significantly different between the two groups (P = 0 .144, 0.109 and 0.153, respectively). However, the mean parasitaemia determined on day 9 was higher in S. mansoni and P. berghei co-infected mice than mice in-
Reduction in parasitaemia was faster in mice infected with P. berghei alone than those in co-infected with the two parasites. In mice infected with P. berghei alone, reduction in parasitaemia was 39.7 and 82.1% two days (on days 10 and 11) post-treatment, respectively while it was 10.6 and 65.0% in S. mansoni and P. berghei co-infected mice on the same days post-treatment, respectively. Most of the co-infected mice (66.7%) died during the course of treatment while only three mice (20%) died from mice infected with P. berghei alone. No mice died from those infected with S. mansoni alone during follow-up. Fig. 3 shows percentage survival rate of co-infected as well as single infected mice either with P. berghei or S. mansoni alone during the course of treatment. Parasite clearance was also faster in mice infected with P. berghei alone than those co-infected with P. berghei and S. mansoni. Among the remaining 12 mice infected with P. berghei alone, nine (75%) mice were negative on day 3 post-treatment and only one mouse was positive on day 4 post-treatment. On the contrary, all the remaining 10 (100%) mice were positive on day 3 post-treatment from those co-infected with the two parasites.
4. Discussion
Fig. 1. Percentage of mice positivity between days 4 and 9 post-infection with Plasmodium berghei. Pb: Plasmodium berghei, Sm: Schistosoma mansoni.
Results of the present study showed that co-infection with P. berghei and S. mansoni in mice appeared to favor rapid P. berghei development and increase in parasitaemia, and delayed parasite clearance or reduction following chloroquine treatment. A number of previous studies have also demonstrated higher parasitaemia and severe conditions in mice co-infected with various species/strains of rodent malaria and
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Fig. 2. Percentage of malaria parasitaemia in the peripheral blood of mice co-infected with Schistosoma mansoni and Plasmodium berghei (䊏) or P. berghei alone (䉱). Results are shown as mean ± standard error of the mean (S.E.M.).
other parasites than mice infected with plasmodium alone. Strickland et al. (1972) observed higher mortality and persistent parasitaemia in T. gondii and P. berghei yoelii co-infected mice than in mice infected with P. berghei yoelii alone. In S. mansoni and Plas-
Fig. 3. Percentage of mice survival rate during the course of treatment. (䊏) Mice co-infected with Schistosoma mansoni and Plasmodium berghei, (䉬) Mice infected with P. berghei alone and (䉱) Mice infected with S. mansoni alone.
modium chabaudi co-infected mice, Helmby et al. (1998) observed remarkably higher parasitaemia than mice infected with P. chabaudi alone. Similarly, recent study by Yoshida et al. (2000) has demonstrated higher susceptibility to P. chabaudi, increased mortality and elevated P. chabaudi parasitaemia in S. mansoni and P. chabaudi co-infected resistant strain mice than mice infected with P. chabaudi alone. Contrary to our findings and also observations by others, Lewinsohn (1975) and Lwin et al. (1982) did not find increased parasitaemia in mice co-infected with S. mansoni and less virulent strains of P. berghei. Lwin et al. (1982) also claimed to have observed suppressed parasitaemia in mice co-infected with S. mansoni and P. chabaudi. Such disparity in observations might have probably been arisen from differences in plasmodium species/strains or differences in number of S. mansoni cercariae used in the experiment. For instance, study by Lwin et al. (1982) has shown enhanced and prolonged malaria in mice co-infected with virulent strain of P. yoelii and S. mansoni. In our case, we also used a virulent strain P. berghei ANKA that usually kills mice between nine and fourteen4 days after infection. In addition, differences in time course of the
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co-infection with S. mansoni and plasmodium should not also be undermined (Lewinsohn, 1975). Actor et al. (1993) have reported delayed Vaccinia virus clearance in S. mansoni and Vaccinia virus co-infected mice. Similarly, our results showed delayed P. berghei clearance in S. mansoni and P. berghei co-infected mice as compared to the mice infected with P. berghei alone following treatment with chloroquine. Mice infected with P. berghei alone showed a 4-fold parasite reduction on day 1 (i.e. on day 10) post-treatment than the co-infected mice. Moreover, after receiving the first three doses of treatment, 75% of P. berghei alone infected mice cleared parasitaemia while none of the co-infected mice cleared parasitaemia on the same day post-treatment. This may cause a very serious concern because treatment failure might contribute to the emergence of drug-resistant malaria parasites. In this study, about 66.7% of the co-infected mice and 20% of mice infected with P. berghei alone died when the parasitaemia peaked on day 9 post-infection and shortly thereafter during the course of treatment. Although the observed high mortality rate in mice with the concomitant infections could be due to P. berghei persistent parasitaemia, other possible factors such as severe anemia and S. mansoni infection induced/suppressed immune responses (Actor et al., 1993; Helmby et al., 1998; Yoshida et al., 2000) might contribute to the increased mortality rate. Mice mortality at a low parasitaemia might be also due to the pathogenesis caused by a lethal strain of P. berghei ANKA that is known to kill susceptible mice within 10 days post-infection when parasitaemia was between 15–20% (Hanum et al., 2003). Hsu and Geiman (1952) found a synergistic effect of Haemobartonella muris and P. berghei in which P. berghei infection activated latent infection of H. muris and in turn, H. muris increased P. berghei parasitaemia in white rats co-infected with the two parasites. In the present study, the high mortality of the mice co-infected with P. berghei and S. mansoni might also possibly be attributed to increased severity of schistosomiasis, which could be exacerbated by the superinfection with P. berghei. Nevertheless, further investigations are needed to determine the interplay between the two infections as well as the mechanism by which S. mansoni and P. berghei concurrent infec-
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tions affects the therapeutic efficacy of antimalarial drugs and mice mortality In conclusion, the present study shows that co-infection with P. berghei and S. mansoni in mice favored rapid P. berghei development and high parasitaemia, and delayed parasite clearance or reduction following chloroquine treatment. These observations have important public health implication since people are at risk for mixed parasitic infections in many parts of Africa. Hence, it is desirable to consider intestinal helminthic infections in clinical and therapeutic management of malaria patients in areas where the two diseases are co-endemic.
Acknowledgements This work was financial supported by Professor Aklilu Lemma Fund for Scholarships and Research Grants. The technical assistance provided by Ms Kokobe Gebere-Michae in blood film preparation and microscope examination is duly acknowledged.
References Actor, J.K., Shirai, M., Kullberg, M.C., Buller, R.M.L., Sher, A., Berzofsky, J.A., 1993. Helminth infection results in decreased virus specific CD8+ cytotoxic T-cell and TH1 cytokine responses as well as delayed virus clearance. Proc. Nation. Acad. Sci. USA 90, 948–952. Bentwich, Z., Kalinkovich, A., Weisman, Z., 1995. Immune activation is a dominant factor in pathogenesis of African AIDS. Immunol. Today 16, 187–191. Diniz, L.M., Zandonade, E., Dietze, R., Pereira, F.E.L., Rodrigues, R.R., 2001. Short report: Do intestinal nematodes increase the risk for multibacillary leprosy? Am. J. Trop. Med. Hyg. 65, 852–854. Elias, D., Wolday, D., Akuffo, H., Petros, B., Bronner, U., Britton, S., 2001. Effect of deworming on human T cell responses to mycobacterial antigens in helminth exposed individuals before and after bacille calmette guerin (BCG) vaccination. Clin. Exp. Immunol. 123, 219–225. Gupta, S., Hill, A.V., Kwiatkowski, D., Greenwood, A.M., Greenwood, B.M., Day, K.P., 1994. Parasite virulence and disease patterns in Plasmodium falciparum malaria. Proc. Natl. Acad. Sci. USA 91, 3715–3719. Hanum, P.S., Hayano, M., Kojima, S., 2003. Cytokine and chemokine responses in a cerebral malaria-susceptible or–resistant strain of mice to Plasmodium berghei ANKA infection: early chemokine expression in the brain. Int. Immunol. 15, 633–640.
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Helmby, H., Kullberg, M., Troye-Blomberg, M., 1998. Altered immune responses in mice with concomitant Schistosma mansoni and Plasmodium chabaudi infections. Infect. Immun. 66, 5167–5174. Hill, A.V., Willis, A.C., Aidoo, M., Allsopp, C.E., Gotch, F.M., Gao, X.M., Takiguchi, M., Greenwood, B.M., Townsend, A.R., McMichael, A.J., Whittle, H.C., 1992. Molecular analysis of the association of HLA-B53 and resistance to severe malaria. Nature 360, 434–439. Hsu, D.Y.M., Geiman, Q.M., 1952. Synergistic effect of Haemobartonella muris onP. berghei in white rats. Am. J. Trop. Med. Hyg. 1, 747–760. Lewinsohn, R., 1975. Anaemia in mice with concomitant S. mansoni and Plasmodium berghei yoelii infection. Trans. R. Soc. Trop. Med. Hyg. 69, 51–56. Lwin, M., Targett, G.A., Doefnhoff, M.J., 1987. Reduced efficacy of chemotherapy of Plasmodium Chabaudi in Tcell-deprived mice. Trans. R. Soc. Trop. Med. Hyg. 81, 899–902. Lwin, M., Last, C., Targett, G.A.T., Doenhoff, M.J., 1982. Infection of mice concurrently with Schistosoma mansoni and rodent malarias: contrasting effects of patent S. mansoni infections on Plasmodium chabaudi, P. yoelii and P. berghei. Ann. Trop. Med. Parasitol. 76, 265–273. Nacher, M., Gay, F., Singhasivanov, P., Krudsood, S., Treeprasertsuk, S., Mazier, D., Vouldoukis, I., Looareesuwan, S., 2000. Ascaris lumbricoides infection is associated with protection from cerebral malaria. Parasite Immunol. 22, 107–114. Nacher, M., Singhasivanov, P., Silachamroon, U., Vannaphan, S., Traore, B., Gay, F., Looareesuwan, S., 2001a. Helminth
infections are associated with protection from malaria-related acute renal failure and Jaundice in Thailand. Am. J. Trop. Med. Hyg. 65, 834–836. Nacher, M., Singhasivanov, P., Gay, F., Phumratanaprapin, W., Silachamroon, U., Looareesuwan, S., 2001b. Association of helminth infection with decreased reticulocyte counts and hemoglobin concentration in Thai falciparum malaria. Am. J. Trop. Med. Hyg. 65, 335–337. Pearlman, E., Kazura, J.W., Hazlett, F.E., Boom, W.H., 1993. Modulation of murine cytokine responses to mycobacterial antigens by helminth-induced T helper two cells responses. J. Immunol. 151, 4857–4864. Strickland, G.T., Voller, A., Pettit, L.E., Fleck, D.G., 1972. Immunodepression associated with concomitant toxoplasma and malarial infections in mice. J. Infect. Dis. 126, 54– 60. Targett, G.A.T., 1992. Malaria: drug use and the immune response. Parasitolology 105 (Suppl.), S61–S70. Timms, R., Colegrave, N., Chan, B.H.K., Read, A.F., 2001. The effect of parasite dose on disease severity in the rodent malaria Plasmodium chabaudi. Parasitology 123, 1–11. Wernsdorfer, W.H., 1991. The development and spread of drug-resistant malaria. Parasitol. Today 7, 297–303. Yoshida, A., Maruyama, H., Kumagai, T., Amano, T., Kobayashi, F., Zhang, M., Himeno, K., Ohta, N., 2000. S. mansoni infection cancels the susceptibility to Plasmodium chabaudi through induction of type 1 immune responses in A/J mice. Int. Immunol. 12, 1117–1125.
Increased parasitaemia and delayed parasite clearance in Schistosoma mansoni and Plasmodium berghei co-infected mice Mengistu Legesse∗ , Berhanu Erko, Fekede Balcha Institute of Pathobiology, Addis Ababa University, P.O. Box 1176, Addis Ababa, Ethiopia Received 1 September 2003; received in revised form 16 March 2004; accepted 6 April 2004 Available online 10 June 2004
Abstract Identifying factors that contribute to malaria susceptibility, severity and treatment failure remains one of the major research areas in malaria control strategies. In the present study, we superinfected Schistosoma mansoni infected mice with a lethal strain Plasmodium berghei ANKA to assess whether or not infection with S. mansoni affects parasite development, parasitaemia and parasite reduction or clearance following antimalarial treatment. Mice infected with P. berghei alone were used as control. The mice were followed for parasite development and parasitaemia between days 4 and 9 post-infection. On day 9, after taking blood samples, the mice were orally treated with 100 mg/kg of chloroquine and then with 10 mg/kg for three consecutive days. Parasite reduction/clearance and mortality were followed between days 10 and 13 post-treatment. The results showed, that superinfection with S. mansoni enhanced P. berghei parasite development, increased parasitaemia and mortality, and delayed reduction/clearance in parasitaemia. Hence, the results postulate that co-infections with schistosome and malaria parasites would aggravate malarial severity and prolong parasite reduction or clearance after chemotherapy in humans. This would necessitate the need for considering schistosome infection in clinical as well as therapeutic management of malaria patients in areas where the two diseases are co-endemic. © 2004 Published by Elsevier B.V. Keywords: Schistosoma mansoni; Plasmodium berghei; Concurrent infections; Parasitaemia; Treatment; Parasite clearance; Mortality
1. Introduction Malaria is one of the leading causes of morbidity and mortality in people living in sub-Saharan Africa. However, the severity of the disease varies from individual to individual. Population living in the same epidemiological context may show variation in susceptibility to Plasmodium infection and development of severe malaria. Age, pregnancy, host genetic fac∗
Corresponding author. Fax: +251 1 755296/550655. E-mail address: [email protected] (M. Legesse).
0001-706X/$ – see front matter © 2004 Published by Elsevier B.V. doi:10.1016/j.actatropica.2004.04.002
tors, immunity, inoculation dose and virulence of the parasite strains are factors that have been considered to influence the clinical outcome of the disease (Hill et al., 1992; Gupta et al., 1994; Timms et al., 2001). Furthermore, chronic intestinal helminthic infections have also become the subject of speculation and investigation in malarial severity. On one hand, it is claimed that intestinal helminthic infections are associated with protection from cerebral malaria, malaria-related acute renal failure and jaundice (Nacher et al., 2000, 2001a). On the other hand, it has been proposed that intestinal helminthic infections are
162
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associated with increased malarial severity (Nacher et al., 2001b). In malaria patients, the effectiveness of antimalarial drugs is also highly variable as the clinical outcome. This problem has mainly been attributable to the emergence of drug-resistant Plasmodium strains. Nevertheless, evidences have indicated that the effectiveness of antimalarial drugs can be affected by the immune status of the host. In other words, impaired immune responses contribute to rapid evolving of drug-resistant parasite strains (Lwin et al., 1987; Wernsdorfer, 1991; Targett, 1992). Intestinal helminthic infections are one of the factors that are known to impair host immune responses to other infectious agents (Pearlman et al., 1993; Bentwich et al., 1995; Diniz et al., 2001; Elias et al., 2001). Intestinal schistosomiasis is a helminthic infection that ranks second to malaria in terms of socio-economic and public health importance in tropical and subtropical areas of the world. Despite the wide distribution of the two diseases in the tropics, little effort has been made to assess possible effects of co-infection with the two parasites on malarial severity or vice versa (Lewinsohn, 1975; Lwin et al., 1982; Helmby et al., 1998; Yoshida et al., 2000). Moreover, most of the findings from the previous works are controversial. Therefore, this study was aimed to assess the effect of co-infection with Schistosoma mansoni on Plasmodium berghei development, parasitaemia, and parasite reduction or clearance following chloroquine treatment in mice. 2. Material and methods 2.1. Infection of mice with Schistosoma mansoni A total of 50 laboratory-bred male Swiss albino mice (8–10 weeks old) were selected and randomly grouped into two. The first group consisting of 30 mice was exposed to 50 cercariae of S. mansoni by the tail immersion method. Faecal samples from the exposed mice were microscopically examined to confirm establishment of infection six weeks post-exposure.
105 P. berghei ANKA parasitized erythrocytes. To inoculate approximately the same amount of parasitized erythrocytes into each mouse, blood sample was collected from previously infected mouse by cardiac puncture and the required dose was calculated as previously described (Timms et al., 2001). Fifteen free mice were also infected interaperitoneally with 5×105 P. berghei ANKA parasitized erythrocytes and used as control.
2.3. Follow-up for parasite development and parasitaemia Thick and thin blood films were prepared daily from each mouse starting from day 4 post-infection up to day 9 by taking blood from the tip of the tail. The films were stained with Giemsa and examined microscopically for the appearance of the parasite in the peripheral blood of the infected mice. The number of infected and non-infected erythrocytes was counted per 10 thin microscopic fields and parasitaemia was computed out of 1000 non-infected erythrocytes for each mouse.
2.4. Treatment and follow-up for parasite reduction or clearance After collecting blood films on day 9, all P. berghei infected mice were treated orally with 100 mg/kg of chloroquine dissolved in tap water, and then with 10 mg/kg daily for three consecutive days. Parasite reduction or clearance was checked on days 10–13 post-treatment by making thin and thick blood films daily. As in the case of the first follow-up, the number of infected and non-infected erythrocytes was counted in 10 thin microscopic fields between days 10–12 post-treatment and parasitaemia was computed out of 1000 non-infected erythrocytes for each mouse. During the follow-up, mice mortality was recorded.
2.5. Data analysis
2.2. Infection of mice with Plasmodium berghei Seven weeks after infection with S. mansoni, 15 mice were superinfected interaperitoneally with 5 ×
Average parasitaemia was compared using Student’s t-test assuming equal variances. Difference was considered significant when P-value was less than 0.05.
M. Legesse et al. / Acta Tropica 91 (2004) 161–166
163
The study received ethical approval from the Ethical Clearance Committee of the Institute of Pathobiology, Addis Ababa University, before its implementation. The mice were maintained in the animal house and supplied with food and water ad libitum.
fected with P. berghei alone (13.2 ± 2.5% versus 6.5 ± 0.8%, P = 0.023). Similarly, mean parasitaemia measured on day 10 (11.5 ± 2.4% versus 4.4 ± 0.7%, P = 0.007) and day 11 (4.6 ± 1.1% versus 1.2 ± 0.3%, P = 0.005) post-treatment was higher in S. mansoni and P. berghei co-infected mice than in mice infected with P. berghei alone (Fig. 2).
3. Results
3.2. Parasite reduction/clearance during the course of treatment
2.6. Ethical consideration
3.1. Parasite appearance and parasitaemia Blood-stage of P. berghei parasite was not observed in the thick and thin blood films collected either from mice co-infected with S. mansoni and P. berghei or P. berghei alone on days 4 and 5 post-infection. The parasite was observed in the blood films of 10 double-infected and five single-infected mice on day 6 post-infection. Most of the mice became positive on day 7 post-infection (Fig. 1). In mice co-infected with S. mansoni and P. berghei, rise in P. berghei parasitaemia was observed on day 7 and peaked on day 9 (Fig. 2). The mean parasitaemia values recorded for days 6, 7 and 8 were not significantly different between the two groups (P = 0 .144, 0.109 and 0.153, respectively). However, the mean parasitaemia determined on day 9 was higher in S. mansoni and P. berghei co-infected mice than mice in-
Reduction in parasitaemia was faster in mice infected with P. berghei alone than those in co-infected with the two parasites. In mice infected with P. berghei alone, reduction in parasitaemia was 39.7 and 82.1% two days (on days 10 and 11) post-treatment, respectively while it was 10.6 and 65.0% in S. mansoni and P. berghei co-infected mice on the same days post-treatment, respectively. Most of the co-infected mice (66.7%) died during the course of treatment while only three mice (20%) died from mice infected with P. berghei alone. No mice died from those infected with S. mansoni alone during follow-up. Fig. 3 shows percentage survival rate of co-infected as well as single infected mice either with P. berghei or S. mansoni alone during the course of treatment. Parasite clearance was also faster in mice infected with P. berghei alone than those co-infected with P. berghei and S. mansoni. Among the remaining 12 mice infected with P. berghei alone, nine (75%) mice were negative on day 3 post-treatment and only one mouse was positive on day 4 post-treatment. On the contrary, all the remaining 10 (100%) mice were positive on day 3 post-treatment from those co-infected with the two parasites.
4. Discussion
Fig. 1. Percentage of mice positivity between days 4 and 9 post-infection with Plasmodium berghei. Pb: Plasmodium berghei, Sm: Schistosoma mansoni.
Results of the present study showed that co-infection with P. berghei and S. mansoni in mice appeared to favor rapid P. berghei development and increase in parasitaemia, and delayed parasite clearance or reduction following chloroquine treatment. A number of previous studies have also demonstrated higher parasitaemia and severe conditions in mice co-infected with various species/strains of rodent malaria and
164
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Fig. 2. Percentage of malaria parasitaemia in the peripheral blood of mice co-infected with Schistosoma mansoni and Plasmodium berghei (䊏) or P. berghei alone (䉱). Results are shown as mean ± standard error of the mean (S.E.M.).
other parasites than mice infected with plasmodium alone. Strickland et al. (1972) observed higher mortality and persistent parasitaemia in T. gondii and P. berghei yoelii co-infected mice than in mice infected with P. berghei yoelii alone. In S. mansoni and Plas-
Fig. 3. Percentage of mice survival rate during the course of treatment. (䊏) Mice co-infected with Schistosoma mansoni and Plasmodium berghei, (䉬) Mice infected with P. berghei alone and (䉱) Mice infected with S. mansoni alone.
modium chabaudi co-infected mice, Helmby et al. (1998) observed remarkably higher parasitaemia than mice infected with P. chabaudi alone. Similarly, recent study by Yoshida et al. (2000) has demonstrated higher susceptibility to P. chabaudi, increased mortality and elevated P. chabaudi parasitaemia in S. mansoni and P. chabaudi co-infected resistant strain mice than mice infected with P. chabaudi alone. Contrary to our findings and also observations by others, Lewinsohn (1975) and Lwin et al. (1982) did not find increased parasitaemia in mice co-infected with S. mansoni and less virulent strains of P. berghei. Lwin et al. (1982) also claimed to have observed suppressed parasitaemia in mice co-infected with S. mansoni and P. chabaudi. Such disparity in observations might have probably been arisen from differences in plasmodium species/strains or differences in number of S. mansoni cercariae used in the experiment. For instance, study by Lwin et al. (1982) has shown enhanced and prolonged malaria in mice co-infected with virulent strain of P. yoelii and S. mansoni. In our case, we also used a virulent strain P. berghei ANKA that usually kills mice between nine and fourteen4 days after infection. In addition, differences in time course of the
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co-infection with S. mansoni and plasmodium should not also be undermined (Lewinsohn, 1975). Actor et al. (1993) have reported delayed Vaccinia virus clearance in S. mansoni and Vaccinia virus co-infected mice. Similarly, our results showed delayed P. berghei clearance in S. mansoni and P. berghei co-infected mice as compared to the mice infected with P. berghei alone following treatment with chloroquine. Mice infected with P. berghei alone showed a 4-fold parasite reduction on day 1 (i.e. on day 10) post-treatment than the co-infected mice. Moreover, after receiving the first three doses of treatment, 75% of P. berghei alone infected mice cleared parasitaemia while none of the co-infected mice cleared parasitaemia on the same day post-treatment. This may cause a very serious concern because treatment failure might contribute to the emergence of drug-resistant malaria parasites. In this study, about 66.7% of the co-infected mice and 20% of mice infected with P. berghei alone died when the parasitaemia peaked on day 9 post-infection and shortly thereafter during the course of treatment. Although the observed high mortality rate in mice with the concomitant infections could be due to P. berghei persistent parasitaemia, other possible factors such as severe anemia and S. mansoni infection induced/suppressed immune responses (Actor et al., 1993; Helmby et al., 1998; Yoshida et al., 2000) might contribute to the increased mortality rate. Mice mortality at a low parasitaemia might be also due to the pathogenesis caused by a lethal strain of P. berghei ANKA that is known to kill susceptible mice within 10 days post-infection when parasitaemia was between 15–20% (Hanum et al., 2003). Hsu and Geiman (1952) found a synergistic effect of Haemobartonella muris and P. berghei in which P. berghei infection activated latent infection of H. muris and in turn, H. muris increased P. berghei parasitaemia in white rats co-infected with the two parasites. In the present study, the high mortality of the mice co-infected with P. berghei and S. mansoni might also possibly be attributed to increased severity of schistosomiasis, which could be exacerbated by the superinfection with P. berghei. Nevertheless, further investigations are needed to determine the interplay between the two infections as well as the mechanism by which S. mansoni and P. berghei concurrent infec-
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tions affects the therapeutic efficacy of antimalarial drugs and mice mortality In conclusion, the present study shows that co-infection with P. berghei and S. mansoni in mice favored rapid P. berghei development and high parasitaemia, and delayed parasite clearance or reduction following chloroquine treatment. These observations have important public health implication since people are at risk for mixed parasitic infections in many parts of Africa. Hence, it is desirable to consider intestinal helminthic infections in clinical and therapeutic management of malaria patients in areas where the two diseases are co-endemic.
Acknowledgements This work was financial supported by Professor Aklilu Lemma Fund for Scholarships and Research Grants. The technical assistance provided by Ms Kokobe Gebere-Michae in blood film preparation and microscope examination is duly acknowledged.
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