Haemobartonellosis in Squirrel Monkeys (Saimiri sciureus): Antagonism betweenHaemobartonellasp. and ExperimentalPlasmodium falciparumMalaria

Experimental Parasitology 91, 297–305 (1999) Article ID expr.1998.4337, available online at http://www.idealibrary.com on

Haemobartonellosis in Squirrel Monkeys (Saimiri sciureus): Antagonism between Haemobartonella sp. and Experimental Plasmodium falciparum Malaria

Hugues Contamin and Jean-Claude Michel Unite´ de Primatologie et Laboratoire d’ Immunologie Parasitaire, Institut Pasteur de la Guyane Franc¸aise, 97306 Cayenne Cedex, France

Contamin, H., and Michel, J.-C. 1999. Haemobartonellosis in squirrel monkeys (Saimiri sciureus): Antagonism between Haemobartonella sp. and experimental Plasmodium falciparum malaria. Experimental Parasitology 91, 297–305. A hemotropic parasite of the genus Haemobartonella (rickettsial parasite of the Family Anaplasmataceae) is responsible for latent asymptomatic infection in colony-born Saimiri monkeys. Indeed, many of these animals develop a patent Haemobartonella infection following splenectomy. Such patent parasitism is characterized by an intense Haemobartonella parasitemia which peaks between days 12 and 14 after removal of the spleen and then decreases to become undetectable between days 25 and 30. During the resolving phase of parasitemia, a moderate anemia associated with monocytosis and erythrophagocytosis is observed. In certain Saimiri monkeys, Haemobartonella parasitemia remains latent following removal of the spleen. This indicates that the spleen plays a role but is not necessary to maintain latent Haemobartonella parasitism. It also suggests the existence of heterogeneity in the host immune reactivity to the parasite. Latent or patent haemobartonellosis might raise a problem when Saimiri monkeys are used as experimental hosts of Plasmodium falciparum asexual blood stages, as already noticed with “rodent malaria.” Thus we investigated the relationship between Haemobartonella and P. falciparum in splenectomized monkeys. When animals harboring latent Haemobartonella sp. were infected with P. falciparum, the former remained latent and exerted no influence on the course of the P. falciparum parasitemia. In constrast, when P. falciparum was initiated in animals which were in the process of developing patent haemobartonellosis, the course of the former was protracted and either the animal resisted longer, or it self-cleared the P. falciparum infection. Conversely, patent haemobartonellosis was delayed when splenectomy was performed at different times after initiation of P. falciparum infection in intact monkeys. Our results do not allow us to draw conclusions as to the mechanism(s) of the antagonism between the two parasites, but they emphasize the need to monitor the presence of Haemobartonella when splenectomized Saimiri monkeys are used as experimentals hosts for P. falciparum parasitism. q 1999 Academic Press

0014-4894/99 $30.00 Copyright q 1999 by Academic Press All rights of reproduction in any form reserved.

Index Descriptors and Abbreviations: Squirrel monkey; Saimiri sciureus; breeding colony; splenectomy; Haemobartonella sp.; anemia; monocytosis; latent haemobartonellosis; patent haemobartollnenosis; Plasmodium falciparum.

INTRODUCTION

A wide range of animals is susceptible to infection by Haemobartonellae. Hemotropic prokaryotes, small obligate parasitic bacteria lacking cell wall, Haemobartonellae are currently classified in the family Anaplasmataceae and order Rickettsiales (Kreier et al. 1992). They initiate latent asymptomatic processes that can evolve toward pathogenicity as soon as the hosts are coinfected by other microorganisms or are manipulated, splenectomy being the most frequent intervention. Clinical disease is often detected as anemia which varies from mild to severe. Many species have been described in small animals such as Haemobartonella muris in rats, mice, and hamsters; H. felis in cats and H. canis in dogs. In nonhuman primates Haemobartonellae parasitism has been reported in South American monkeys Cebus ˆ (5Pseudocebus) appella (Pessoa and Prado 1927) and Saimiri sciureus (Aikawa and Nussensweig 1972; Adams et al. 1984) but also in one Asian monkey Macaca mulatta (Peters et al. 1974). Another South American monkey Aotus trivirgatus was found parasitized with a closely related organism,

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298 Eperythrozoon (Peters et al. 1974). In humans, there are rare reports on Haemobartonella-like parasitism (Kallick et al. 1972; Archer et al. 1979; Duarte et al. 1992). The squirrel monkey S. sciureus is an experimental host for a wide range of human pathogens and especially for Plasmodium spp. (WHO 1988; Gysin 1991; Garraud 1992). Indeed, this animal sustains the development of asexual blood stages of Plasmodium falciparum and Plasmodium vivax parasites once the “strains” under study have been “adapted” to this experimental host (Young and Rossan 1969; Rossan et al. 1972; Gysin et al. 1980). Splenectomized animals are prone to develop high and reproducible P. falciparum parasitemia, in contrast to intact animals in which parasitemia is much more irregular, with reduced peaks. Therefore, splenectomized monkeys have been extensively used as a model for vaccination experiments. A breeding colony of squirrel monkeys was established in 1980 at the Pasteur Institute in Cayenne (French Guiana) from wild-born animals originating from the Guianese Plateau. After splenectomy, animals often develop a mild anemia associated with the presence of gram-negative microorganisms on red blood cells, identified as Haemobartonella in accordance with a previous report (Aikawa and Nussenweig 1972; Michel 1980, personal communication; Adams et al. 1984). Inasmuch as the presence of Haemobartonella and other closely related microorganisms are known to interfere with Plasmodium spp. asexual blood stage infection in rodents (Hsu and Geiman 1952; Peters 1965; Ott and Stauber 1967), splenectomized Saimiri monkeys are submitted to oxytetracycline therapy before challenge with Plasmodium spp.-parasitized red blood cells in order to prevent a possible, spontaneous Haemobartonella infection (Roussilhon et al. 1988). However, we have observed that oxytetracycline treatment sometimes fail to prevent the appearance of Haemobartonella upon splenectomy. In the present study, the course of patent Haemobartonella infection in splenectomized animals is described. Experiments were undertaken to evaluate possible intereference between Haemobartonella and P. falciparum infections.

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Plasmodium falciparum parasites. The Uganda Palo-Alto (FUP-1 alias FUP/SP) strain of P. falciparum adapted to splenectomized Saimiri monkeys was used to initiate parasitism (Gysin et al. 1980). This strain has been maintained in our laboratory by serial blood passages; it induces reproducible parasitemia in splenectomized monkeys and is lethal if these animals do not receive proper chemotherapy. Clone FUP-1/89-F5, recently adapted to intact Saimiri monkey by serial blood transfer, was derived from the FUP-1 strain of P. falciparum. The 28th blood transfer of the adapted clone was used to initiate parasitism in intact monkeys. The course of parasitemia was much more irregular with a parasitemia peak always lower than in splenectomized animals, leading to self-cure. Fresh asynchronous viable PRBC were obtained by venipuncture from infected donor monkeys (intact or splenectomized) and injected intravenously into the recipient monkeys. An inoculum of 2 3 105 PRBC was used in splenectomized monkeys, and 106 were injected in intact monkeys. The evolution of P. falciparum parasitemia was monitored daily by microscopic examination of Giemsa-stained thin blood smears. Animals with a parasitemia above 15 to 20% were treated by 20 mg/kg Mefloquine (Lariam, Roche, France) per os for 5 consecutive days. Measurement of Haemobartonella parasitemia. Haemobartonella parasitemia was followed daily on Giemsa-stained thin blood smears under light microscopic examination. Haemobartonella appears as small coccoid organisms stained reddish on the erythrocyte. At the acute phase of parasitemia, large numbers of organisms are present on erythrocytes rendering their assessement difficult. For this reason, parasitemia was estimated as the percentage of Haemobartonellabearing red blood cells (HbRBCs) on Giemsa-stained blood smears and scored as follows: 0, undetectable; 1, 1 to 20% HbRBCs; 2, 20 to 40% HbRBC; 3, 40 to 60% HbRBCs; and 4, 60 to 100% HbRBCs. For detection of latent parasitemia, a blood sample (25 ml) taken on EDTA was incubated 15 min at room temperature in 25 ml of RPMI containing 20% fetal calf serum and ethidium bromide (5 mg/ml final concentration) before being examined under ultraviolet illumination on a microscope slide under a coverslip (Leitz Laborlux). Measurement of hematological parameters. Before and during the course of parasite infection, blood samples (0.5 ml) were collected on EDTA (Capiject, Terumo) by femoral venipuncture and used to establish individual values for standard hematological parameters. An apparatus ABX Cobas Minos STE (Roche) was used for major hematologic determinations such as leukocyte count, erythrocyte count, and hematocrit determination. The leukocyte pattern was established on Giemsastained thin blood smears. The Brilliant cresyl blue dye method was used for determination of peripheral blood reticulocyte numbers.

RESULTS MATERIALS AND METHODS Animals. Three-year-old squirrel monkeys (S. sciureus, of karyotype14-7 and of Guyanese phenotype) of both sexes, from our breeding colony, were used in these studies. Conditions of maintenance of the animals as well as the parasite challenge with P. falciparum-parasitized Saimiri red blood cell (PRBC) were previously described (7). Splenectomy was performed after lateral laparotomy under anesthesia with ketamine (20 mg/kg).

Pattern of patent Haemobartonella parasitemia following splenectomy. Eight Saimiri monkeys (four males and four females) were selected, at random, from the animal facilities of our breeding colony. At day 0, once splenectomy was performed, microscopic examination of Giemsa-stained blood smears from individual animals did not allow us to detect the presence of Haemobartonella on erythrocytes.

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Two to four days later, three animals in each group developed patent Haemobartonella parasitemia with a peak 12 to 14 days later (Fig. 1). Thereafter, parasitemia decreased to become undetectable 25 to 29 days after splenectomy. Parasite clearance was characterized both by a decrease of the parasitized erythrocyte rate and of parasite density on erythrocytes. It is noteworthy that parasitemia remained latent in the case of two animals (monkeys 93042 and 93037). Thereafter, these animals developed patent Haemobartonella parasitemia following P. falciparum challenge (data not shown).

FIG. 1. Course of Haemobartonella sp. parasitemia in two groups of 3-year-old Saimiri monkeys (number keyed to symbol) splenectomized at day 0. Parasitemia was followed daily on Giemsa-stained blood smears and scored as the percentage of Haemobartonella sp.parasitized red blood cells (HbRBCs): 0, undetectable; 1, 1 to 20% HbRBCs; 2, 20 to 40% HbRBCs; 3, 40 to 60% HbRBCs; 4, 60 to 100% HbRBSs.

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The same pattern of Haemobartonella parasitemia was subsequently observed following the splenectomy of 20 other Saimiri monkeys: 15 developed patent transient Haemobartonella parasitemia while infection remained latent in the 5 others. Despite high levels of parasitemia, clinical signs of illness were not observed in the six animals who developed patent infection. In these animals a mild anemia and an increase of the monocyte percentage were observed. Neither of these phenomena occurred in the two animals with latent parasitemia (Fig. 2). Erythrocyte drop became obvious at the resolving phase of parasitemia from day 16 to day 28. Monocyte percentage increased progressively from 4 6 0.5% at day 0 to reach 27 6 10% at day 20 and then declined to normal values by day 30. Atypical large monocytes with frequent pictures of erythrophagocytosis were seen at the resolving phase of parasitemia. These results demonstrate that the majority if not all Saimiri monkeys from our breeding colony are potential carriers of Haemobartonella, since in most cases they develop patent parasitemia following splenectomy. Effect of patent Haemobartonella parasitemia on the course of P. falciparum parasitemia. In order to test an eventual concurrent effect of patent Haemobartonella parasitemia on the course of P. falciparum infection, four Saimiri monkeys (93069, 93077, 93124, and 93126) were splenectomized at day 0 and inoculated at day 13 with 2 3 105 PRBC from a splenectomized donor infected with the FUP-1 strain of P. falciparum. Monkeys 93077 and 93126 developed typical Haemobartonella parasitemia which reached a maximum level at day 14 and day 15, respectively, while it remained latent in monkeys 93069 and 93124 (Fig. 3). As a control, we infected with the same P. falciparum inoculum three Saimiri monkeys (92081, 93009, and 93016) which had been splenectomized 2 months earlier. Haemobartonella infection remained latent in these control animals, although some of them had indeed developed patent Haemobartonella parasitemia upon removal of the spleen. As shown in Fig. 3, the course of P. falciparum parasitemia in monkeys 93069 and 93124 did not differ significantly from that observed in monkeys used as controls (monkeys 92081, 93016, and 93009), i.e., a prepatent period from 6 to 7 days followed by a patent period leading to the need for treatment between the 10th and 12th day postinoculation (Fig. 5). Among the two monkeys that presented patent Haemobartonella infection at the time of P. falciparum inoculation, the course of P. falciparum parasitemia was hampered. In the first monkey (93126), a slight increase of the prepatent period (9 days) was observed, leading to a patent period followed by treatment on day 15 after inoculation.

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FIG. 2. Monocyte and erythrocyte counts as a function of time in Saimiri monkeys splenectomized at day 0. The data in this figure are derived from the monkeys described in the legend to Fig. 1. Haemobartonella sp. parasitemia was the mean values from the six monkeys which had developed patent Haemobartonella infection. Percentage of monocytes was respectively the mean values from the six monkeys which had developed patent Haemobartonella infection (H sp. 1) and the two monkeys where Haemobartonella sp. infection remained latent (H sp. 2). Percentage of erythrocyte drop was the mean values from the six monkeys which had developed patent Haemobartonella infection (H sp. 1) and the two monkeys where Haemobartonella infection remained latent (H sp. 2). Percentage of erythrocytes drop was calculated as follows: 100 2 (red blood cells counts at T1 x / red blood cells counts at T 0). x 5 time in days.

In the second monkey (93077) the course of P. falciparum was not different from that of controls until day 10. Then, after a slight decrease at day 12, parasitemia rose again to reach a maximum level of 8.6% on day 19, followed by a decrease to subpatent level by the 26th day postinoculation. Morphologically altered pyknotic intraerythrocytic parasites, i.e., crisis forms (Taliaferro and Taliaferro 1944; Ockenhouse et al. 1984), were observed from day 14 to day 26; they represented about 35% of parasites at day 15, 53% at day 21, and 70% at day 23 (Fig. 5). Finally, a second wave of low-level parasitemia (maximum rate 0.65%) occured from the 50th day to the 59th day postinoculation. It is interesting to note that PRBC from the late wave of parasitemia did not present the same surface phenotype as those that were injected (data not shown). The concurrent effect of Haemobartonella burden on the outcome of P. falciparum infection might indicate either a competition between the two organisms for substrate or the

effect of toxic products potentially produced by the Haemobartonella. Under the condition of our experiment, patent Haemobartonella parasitemia exerted a slight plasmodistatic effect in one monkey (93126) and no apparent effect in the second (93077). The inhibition of the increase of parasitemia (delayed prepatent period) noted in monkey 93126 does not seem to be related to a direct effect of Haemobartonella but to an indirect effect associated with the rise of monocytes which occurs at the resolving phase of Haemobartonella parasitemia (Fig. 4). In monkey 93077, there was no direct effect of the Haemobartonella, since increase of malaria parasites until day 9 postinoculation was not different from that of monkey 93124 whose Haemobartonella carriage remained latent. The slow growth of P. falciparum noticed in monkey 93077 occurred after clearance of Haemobartonella and correlated with a monocytosis associated with the appearence of degenerated intraerythrocytic parasites, i.e., crisis forms (Fig. 4). An other effect of Haemobartonella

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FIG. 3. Course of Haemobartonella sp. and P. falciparum parasitemia in four Saimiri monkeys splenectomized at day 0 and then infected intravenously with 2 3 105 P. falciparum-parasitized red blood cells at day 13. Identification number of each animal is indicated. T, drug treatment; P.f, P. falciparum.

burden in this animal was the occurrence of a moderate anemia (hemoglobin 14.0 g/dl the day of splenectomy vs 10.7 g/dl at time of PRBC inoculation), followed by a compensatory erythropoiesis assessed by the presence of reticulocytes early in the development of P. falciparum parasitemia. These features were not observed in the two other P. falciparum-infected monkeys (93059, 93124) who did not develop patent Haemobartonella parasitemia (Fig. 4) and in monkeys (92081, 93009, 93016) who were infected with P. falciparum after their recovery from a Haemobartonella parasitemia (Fig. 5). Effect of P. falciparum infection on the pattern of patent Haemobartonella infection. In order to observe whether P. falciparum infection would exert an action on the course of patent Haemobartonella infection, 6 intact monkeys were injected simultaneously at day 0 with 1 3 106 PRBC from an intact donor infected with the adapted FUP-1/89F5 clone of P. falciparum. These animals were thereafter splenectomized at various time intervals. At day 9 for monkeys 93010 and 93014; at day 14 for monkeys 93025 and 93038; at day 19 for monkey 93041; and at day 30 for monkey 93067. The pattern of Haemobartonella parasitemia from 15 splenectomized monkeys was used as reference. It is noteworthy

that we never observed the burden of Haemobartonella parasitemia in intact monkeys parasitized with the adapted FUP1/89F5 clone of P. falciparum. The expected pattern of Haemobartonella parasitemia was modified when splenectomy was performed in P. falciparuminfected monkeys (Fig. 6). The prepatent period was delayed (i.e., the time elapsed between splenectomy and the appearance of Haemobartonella parasitemia) particularly when the spleen was removed during or early after resolution of P. falciparum infection (monkeys 93010, 93014, 93025, and 93028). Thereafter, the prepatent period returned to expected values depending on the time elapsed between P. falciparum infection and splenectomy (monkeys 93041 and 90367). For all monkeys, splenectomy affects neither the level of the peak nor the duration of the Haemobartonella parasitemia.

DISCUSSION

Saimiri monkeys can be natural hosts for blood parasites of the genus Haemobartonella (Aikawa and Nussensweig

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FIG. 4. Evolution of monocyte and reticulocyte counts in four Saimiri monkeys splenectomized at day 0 and then infected intravenously with 2 3 105 P. falciparum-parasitized red blood cells at day 13. The data in this figure are derived from the monkeys described in the legend to Fig. 3. Identification number of each monkey is indicated. T, drug treatment; P.f, P. falciparum.

1972; Adams et al. 1984). The data presented here demonstrate that Saimiri monkeys born in captivity in our breeding colony are asymptomatic carriers of Haemobartonella sp. since, in many cases, they develop patent parasitemia following splenectomy. Such observations do lead to questions concerning the transmission and the maintainance of this microorganism in our breeding colony between 1979 and

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today. Direct and vectorial-born transmission has been described for many parasites of the family Anaplasmataceae. In addition transplacental transmission may occur in Saimiri monkeys as we were able to detect Haemobartonella on fetal erythrocytes. Moreover the regular detection of Haemobartonella on erythrocytes (under ultraviolet examination following ethidium bromide label) in blood samples recovered from young or aged monkeys indicates the existence of long-term and sustained parasitism in animals of the breeding colony. Taken together these observations could explain the high prevalence of haemobartonellosis which still actually occurs in Saimiri monkeys born in captivity. From our data it appears that splenectomy is not a sufficient prerequisite for Haemobartonella patency, since infection can remain latent following removal of the spleen. This indicates that the spleen contributes, but is not necessary, to the maintenance of latent Haemobartonella parasitism. This also suggests the existence of heterogeneity in the host’s immune reactivity to the parasite. Anemia is the most remarkable pathological effect of the processes driven by Haemobartonella and Eperythrozoa in differents animals species (cattle, sheep, pig, cat, dog, rodents). In Saimiri monkeys which develop patent Haemobartonella parasitemia following splenectomy, the course of parasitemia is remarkuably reproducible and despite high peaks, the related pathological effects remain mild. They are characterized by a moderate drop of erythrocyte number associated with erythrophagocytosis at the resolving phase of parasitemia. Clearance of parasites includes a drop both in parasitized erythrocytes and of the parasite load per erythrocyte. This suggests the occurrence of antiparasite immune mechanisms, such as erythrophagocytosis of parasitized erythrocytes by an increased number of monocytes, or the inhibition of growth and/or decrease of adhesion of the parasite on the erythrocyte membrane through still undefined mechanisms. The mechanisms involved in clearance of haemobartonellae or eperythrozoa in animals are poorly understood. An inverse relationship between the disappearance of E. coccoides and the rise in antibody titers detected by the indirect fluorescent antibody technique has been described in mice (Hyde et al. 1972). However, passive transfer of serum from carrier animals had no effect on the parasitemia in nonimmune animals (Thurston 1955). An increase of carbon particle uptake in mice parasitized with E. coccoides has been reported (Gledhill et al. 1965), suggesting that phagocytic cells may contribute to parasite clearance. Thus, the control of haemobartonellae or eperythrozoa infections could depend more on cellular mechanisms, e.g., phagocytosis and/ ¨ or secretory products from lymphomyeloıd cells, than on

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FIG. 5. Course of P. falciparum parasitemia and evolution of monocyte and reticulocyte counts in splenectomized Saimiri monkeys following intravenous inoculation of 2 3 105 P. falciparum-parasitized red blood cells. Splenectomy was performed 2 months before P. falciparum infection. Identification number of each monkey is indicated. T, drug treatment.

humoral antibodies per se. The occurrence of natural Haemobartonella infection in Saimiri monkeys might offer a useful system to investigate specific and nonspecific immune mechanisms involved in control of this model rickettsial agent in a nonhuman primate. It has been observed that haemobartonellae or eperythrozoa affect the ability of the host to resist to other pathogenic organisms. Specifically, Hsu and Geiman (1952) showed that rats harboring patent infection with H. muris developed a more severe parasitemia with Plasmodium berghei. The synergistic effect of H. muris on P. berghei infection was attributed to the fact that H. muris infection leads to a severe anemia that is followed by a compensatory erythropoiesis with a consequent rapid rise of polychromatophilic erythrocytes: indeed P. berghei preferentially invades immature red blood cells and the polychromatophilia induced by H. muris results in higher rate of parasitemia with P. berghei. Other results indicate that mice coinfected with E. coccoides and P. berghei develop a transient patent E. coccoides parasitemia

which is not followed by significant anemia, but which triggers plasmodistatic activities that lead to a prolonged survival of the coinfected mice compared to the mice infected with only P. berghei. It is suggested that there is competition between the two parasite species for an unknown substrate in the host (Peters 1965). Similarly, concurrent E. coccoides infections suppress the development of Babesia rodhaini in mice (Peters 1965). Finally, E. coccoides reduce pathogenicity of P. chabaudi in mice. P. chabaudi does not preferentially invade reticulocytes and the effect of the eperythrozoa in this case seems related both to a direct plasmodistatic effect and to an indirect effect associated with early anemia and subsequent reticulocytosis. (Ott and Stauber 1967). In Saimiri monkeys there are no data available on the relationship between haemobartonellae and malaria, apart from the previous report of Aikawa and Nussenweig (1972). These authors described Haemobartonella sp. during an experiment on one splenectomized squirrel monkey infected

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FIG. 6. Course of Haemobartonella sp. parasitemia in Saimiri monkeys (number keyed to symbol) splenectomized at various time intervals following intravenous inoculation of 13106 P. falciparum-parasitized red blood cells. The expected pattern of Haemobartonella parasitemia (mean value) used as control was established from 15 splenectomized Saimiri monkeys. T, drug treatment; P.f, P. falciparum.

with Plasmodium brasilianum. They noted that the presence of the parasite did not have any effect on the course of the concomitant P. brasilianum infection. Under our experimental conditions, when splenectomized asymptomatic Saimiri monkeys carrying Haemobartonella are infected with P. falciparum, the former remains latent and exerts no influence on the course of the parasitemia which requires drug treatment to prevent death of the animals. In contrast when P. falciparum infection was initiated in splenectomized Saimiri monkeys developing a patent Haemobartonella infection, the course of the former was protracted and the animals survived longer or self-cleared their parasite load. On the other hand, when splenectomy was performed in Saimiri monkeys developing P. falciparum infection, the Haemobartonella parasitemia burden was delayed. P. falciparum infection in intact Saimiri monkey does seem to temporarily activate some mechanisms involved in

the partial clearance and/or growth inhibition of Haemobartonella, as the removal of the spleen from these animals delays the Haemobartonella burden and not the haemobartonella parasitemia peak. Thus, it appears that antagonism between Haemobartonella and P. falciparum in Saimiri monkey depends on parasite burden. The competitive relationship between the two parasites in the host might be related, first, to a direct effect, such as competition for substrate in the host or even to the production by the haemobartonella of products active against the protozoa and vice versa and, second, to the existence of cross-reactivities at the level of specific and nonspecific mechanisms of the immune response of the host against the two parasites. We are now investigating these hypotheses. In view of the frequency of latent haemobartonella infection in squirrel monkey, our results emphasize the need to sustain the monitoring of Haemobartonella sp. carriage in splenectomized Saimiri

ANTAGONISM BETWEEN Haemobartonella sp. AND P. falciparum IN Saimiri

monkeys used as experimental hosts of P. falciparum malaria, notably in vaccination experiments.

ACKNOWLEDGMENTS

We express our gratitude to B. Bonnemains, R. Planel, and B. de Thoisy for providing veterinary assistance. We are particulary indebted to C. Behr and G. Milon for helpful comments on the manuscript and to P. H. David for the English version. This work was supported in ` part by a grant from the Ministere de la Recherche et de l’Enseigne´ ment Superieur.

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