Chemotherapy of haemobartonellosis in squirrel monkeys (Saimiri sciureus)

Splenectomised Saimiri sciureus squirrel monkeys are being used increasingly as an experimental host for human malarial studies, notably for the assessment of candidate vaccines against Plasmodium falciparum blood-stage infection. Recently, we have reported that colony-reared S. sciureus monkeys are asymptomatic carriers of Haemobartonella sp. and that patent Haemobartonella infection, activated following splenectomy, may interfere with the course of P. falciparum parasitaemia in these animals. For several years, splenectomised S. sciureus monkeys were routinely submitted to oxytetracycline therapy before their use in malarial studies in order to prevent a possible spontaneous Heamobartonella infection. However, we report here that such antibiotic therapy is often ineffective and that neoarsphenamine chemotherapy may be considered as an alternative to cure both latent and patent haemobartonellosis in S. sciureus monkeys.     

The putative haemobartonella that influences Plasmodium falciparum parasitaemia in squirrel monkeys is a haemotrophic mycoplasma

Splenectomised squirrel monkeys (Saimiri sciureus) are increasingly being used as an experimental host for human malaria studies, notably for the assessment of candidate vaccines against Plasmodium falciparum blood-stage infection. Recently, S. sciureus monkeys in our primate-breeding colony were reported to be asymptomatic carriers of a putative Haemobartonella species. Patent haemobartonella infection is frequently activated following splenectomy, and may interfere with studies on the course of P. falciparum parasitaemia in these animals. Here, we show by 16S rRNA gene sequence analysis that this wall-less bacterium is not a rickettsia but, instead, is a haemotrophic mycoplasma. Haemotrophic mycoplasmas are a newly identified group of mycoplasmas that parasitise the surfaces of erythrocytes of a wide variety of vertebrate hosts.     

Haemobartonella canis infection in research dogs.

During 1970-1972 haemobartonellosis occurred in research canines at 2 widely separated institutions. Clinical anemia occurred in a splenectomized dog at a Maryland facility, and subsequent screening disclosed an infection rate of 65% in a group of 20 splenectomized subjects. Treatment was successful, and the animals were used in research. A research institution in Texas encountered a number of dogs with fever (to 106 degrees F) and eosinophilia (to 42%) following minor surgery. Blood from affected animals was injected iv into splenectomized dogs, and 3 of 6 recipients developed haemobartonellosis. Further study was conducted, with some success, to establish a relationship between fever and eosinophilia and Haemobartonella canis infection in nonsplenectomized subjects. Our experiences suggest that haemobartonellosis is a widespread, latent disease of dogs and that significant potential exists for the infection to adversely affect research results.     

Susceptibility of Macaca fascicularis monkeys from Mauritius to different species of Plasmodium.

Macaca fascicularis monkeys from Mauritius were shown to be susceptible via sporozoite inoculation to 7 species of Plasmodium (P. fragile, P. coatneyi, P. gonderi, P. inui, P. cynomolgi, P. knowlesi, and P. fieldi), indigenous to macaques in southeastern Asia. Four monkeys were sequentially infected with different species of Plasmodium to determine maximum and course of parasitemia. In 2 nonsplenectomized monkeys, P. fragile developed maximum parasite counts of only 134 and 155/microliters. For Plasmodium knowlesi, a parasite that is life-threatening to rhesus monkeys, maximum parasite counts were 4,278 and 7,440/microliters. Plasmodium coatneyi developed to what must be considered as moderate levels. After animals underwent splenectomy, parasite counts of P. coatneyi were 58,280, 89,094, 4,464, and 43,524/microliters. The maximum parasite counts for P. gonderi (13,508 and 21,576/microliters) and P. fieldi (1,767 and 17,836/microliters) were lower than would be expected in M. mulatta. In 2 monkeys that developed patent parasitemia with P. inui, the maximum parasite counts (95,046 and 728,748/microliters) indicated that this parasite may be the best adapted species for development in these animals once infection is established. Finally, the reinfection of 2 monkeys with P. cynomolgi suggested that some animals may be basically more resistant than others, whether splenectomized or not, to the production of high-density parasitemia.     

Severe anemia affects both splenectomized and non-splenectomized Plasmodium falciparum-infected Aotus infulatus monkeys

Severe anemia is the earliest and a frequently fatal complication of Plasmodium falciparum infection. Here we describe Aotus infulatus as a primate model suitable to study this malaria complication. Both non-splenectomized and splenectomized monkeys receiving different inocula of P. falciparum FVO strain presented large (> 50%) decreases in hematocrit values during infection. Non-splenectomized animals were able to control parasite growth (parasitemia did not exceed 4%), but they had to be treated because of severe anemia. Three of 4 splenectomized monkeys did not control parasitemia and were treated, but developed severe anemia after treatment when presenting a negative blood film. Destruction of parasitized red blood cells alone cannot account for the degree of anemia. Non-splenectomized monkeys repeatedly infected with homologous parasites became rapidly and progressively resistant to reinfection and to the development of severe anemia. The data presented here point to A. infulatus as a suitable model for studying the pathogenesis of severe malarial infection.     

Experimental Plasmodium falciparum cerebral malaria in the squirrel monkey Saimiri sciureus.

Infection of the squirrel monkey, Saimiri sciureus, with several strains of Plasmodium falciparum leads in a proportion of animals to neurological symptoms with a fatal outcome. This first simian model for human cerebral malaria was studied with three strains of parasites, the uncloned Palo Alto(FUP-1) strain, the Palo AltoPLF3 clone MHB11, and the recently monkey-adapted P. falciparum strain IPC/RAY. Cerebral malaria could develop during primo infection of monkeys, whether the animals had been splenectomized or not. It did not occur in all animals and the appearance of neurological symptoms could not be predicted, as it was not related to the degree of parasitemia or duration of parasite infections.     

The Uganda I/CDC strain of Plasmodium malariae in Saimiri sciureus boliviensis

The Uganda I/CDC strain of Plasmodium malariae, initially adapted to monkeys of the genus Aotus, was studied in splenectomized Saimiri sciureus boliviensis. Mean maximum parasitemia ranged from 248 to 22,134/mm3. Only 1 mosquito was infected of 2,238 examined. After the parasite was adapted to this host, infections were characterized by periods of detectable parasitemia extending up to 269 days and by sustained periods when parasite counts were greater than 1,000/mm3. After 4 linear passages, the developmental time required before the primary peak parasite count was approximately 2 mo.     

Reproducible infection of intact Aotus lemurinus griseimembra monkeys by Plasmodium falciparum sporozoite inoculation

Aotus lemurinus griseimembra is considered one of the best nonhuman primate species for malarial studies because of its susceptibility to infection by Plasmodium falciparum asexual blood stages. However, reproducible transmission of infective P. falciparum sporozoites by mosquito inoculation has been difficult to achieve even in splenectomized monkeys. Characterization of an Aotus-P. falciparum cyclical transmission model has become a top priority as a result of the significant progress toward the development of preerythrocytic malaria vaccines. Herein, we describe a reproducible model developed using intact A. lemurinus griseimembra monkeys intravenously inoculated with sporozoites from a monkey-adapted P. falciparum (Santa Lucia) strain and a wild Falciparum-Cali-Colombia-4 (FCC-4) strain. Sporozoites were obtained by salivary gland dissection of laboratory-reared Anopheles albimanus mosquitoes. Parasitemia was monitored by thick-smear microscopy, parasite lactate dehydrogenase (pLDH) determination, and mosquito xenodiagnosis. The last method proved to be the most sensitive method for monitoring parasitemias. Infection with the Santa Lucia strain showed a mean prepatent period of 16 days (range 6-21 days), whereas infection with the wild FCC-4 strain resulted in a 24-day prepatent period. Mean peak parasite density was approximately 900 parasites/microliter for both parasite strains. The prepatent period, the peak of parasitemia, and the duration of patency were independent of the size of the sporozoite inoculum and the presence of spleen in the host. This model is being successfully used to test the protective efficacy of P. falciparum preerythrocytic vaccine candidates.     

Comparative infectivity of knobless and knobby clones of Plasmodium falciparum in splenectomized and intact Aotus trivirgatus monkeys

In two experiments, two knobless (K-) and two knob-producing (K+) clone-cultures of Plasmodium falciparum, FCR-3/Gambia strain, were injected into four Aotus trivirgatus monkeys. The parasitemia in the K(-)-infected splenectomized (S-) monkey rose to a peak of 2.1% on the 16th day, while it reached only 0.7% at the same time in the K+ infected S- animal. Passage from these animals (karyotype VI) into two intact (S+), naive monkeys of karyotype III resulted in very light infections somewhat higher with K+ than with K-. This experiment was repeated with two different clones in two other S- monkeys of karyotype III. Again, the parasitemia of the K+ infected monkey was appreciably below that of the K- monkey. Transfer of parasites into S+ animals of karyotype II resulted in very light infection and, as before, the K+ did somewhat better. About 2 months after its initial infection, the K(+)-infected S- animal from the second experiment came down with a recurrent malaria infection. Electron-microscopic observations on blood from this monkey revealed that the previously K+ parasites had become knobless (K-). Transfer of this material into an S+, naive monkey, again, gave a barely detectable infection. After splenectomy a recrudescence occurred. The results strongly indicate that K- clones of P. falciparum are more infectious to S- Aotus monkeys than K+ clones, whereas in S+ monkeys the situation is reversed.     

Platelet kinetics and other hematological profiles in experimental Plasmodium falciparum infection: a comparative study between Saimiri and Aotus monkeys.

Levels of platelets and other hematological values were monitored in 21 Saimiri and 12 Aotus monkeys over a period of three weeks post-infection with monkey-adapted Indochina CDC-1 strain of Plasmodium falciparum. In both Saimiri sciureus boliviensis and Aotus nancymai karyotype-1 monkeys the severest thrombocytopenia was observed at 14 days post-infection coinciding with peak parasitemia, neutropenia, lymphocytosis, and anemia associated with severe hemoglobinemia and elevated fibrinogen degeneration products(FDP's). MCH and MCV profiles in Aotus monkeys decreased with ascending parasitemia. In contrast, these parameters in Saimiri were characterized by a significant compensatory increase correlating with parasitemia. In general, thrombocytopenia was one of the earliest clinical manifestations of the infection with the platelets returning to normal levels shortly after peak parasitemia at 14 days. Platelet kinetics had a strong correlation with hematologic and parasitologic values in the Aotus model. No consistent associations were observed between platelet kinetics and other parameters in the Saimiri model. These data indicate that the Aotus model for malaria is more predictable than the Saimiri. Further, platelet turnover rates and recovery provide a useful prognostic parameter during malaria infection. The results are discussed in relation to the value of the two species of monkeys as models for the pathogenesis of human malaria.     

Observations on two strains of plasmodium falciparum from Haiti in Aotus monkeys

Two strains of Plasmodium falciparum originating in Haiti were studied in the Aotus monkey. The Haitian I/CDC strain was first adapted to in vitro cultivation and subsequently inoculated into monkeys. The Haitian III/CDC strain was inoculated directly from a human patient into the Aotus monkey. The strains varied in their levels of pathogenicity to the animals. The Haitian I/CDC strain was highly virulent in six splenectomized animals; in one intact animal, the infection could be controlled but not eliminated with periodic doses of quinine and chloroquine. After subsequent splenectomy, the animal developed high parasitemias and died. No gametocytes developed in any of the Haitian I infections. The Haitian III strain was lethal to five of the 14 splenectomized monkeys inoculated, but some were able to control their infections without drug intervention. Gametocytes developed in all infections that persisted for an adequate length of time, and infections of mosquitoes were obtained both during the primary attack and the first recrudescence of the parasitemia. Of the mosquitoes tested, Anopheles freeborni was most susceptible to infection, followed by An. culicifacies, An. dirus, An. maculatus, and An. albimanus. The Haitian III strain was successfully transmitted to four other splenectomized Aotus monkeys via sporozoite inoculation using An. freeborni.     

Studies on the Cambodian I strain of Plasmodium falciparum in Aotus monkeys

The Cambodian I strain of Plasmodium falciparum, originally from Kampuchea was adapted for development in three different types of Aotus monkeys. High-level parasitemias were readily produced in splenectomized Colombian A. trivirgatus griseimembra monkeys. Initially, only minimal parasitemias developed in A. t. trivirgatus monkeys from Colombia. However, in one animal, adaptation occurred and high-level parasitemias were obtained during the second recrudescence of the infection. Passage to other A. t. trivirgatus monkeys indicated that the parasite was well adapted for development in splenectomized animals; low to moderate parasitemias were still produced in intact animals. This line of the parasite produced high level parasitemias when inoculated into splenectomized Aotus monkeys from Peru. Infections in Anopheles freeborni mosquitoes were obtained as late as the 7th passage in A. t. griseimembra monkeys and as late as the 7th recrudescence of the infection in an individual monkey (348 days after inoculation). The sporogonic cycle was completed in An. freeborni mosquitoes, and one transmission to an A. t. griseimembra monkey via the bites of infected mosquitoes was obtained.     

Sporozoite-induced infections of the Salvador I strain of Plasmodium vivax in Saimiri sciureus boliviensis monkeys

Twenty Saimiri sciureus boliviensis monkeys from Bolivia were inoculated intravenously with sporozoites of the Salvador I strain of Plasmodium vivax. All animals were splenectomized 7 days after inoculation. Inoculation of 100,000 sporozoites resulted in prepatent periods averaging 16.6 days; all monkeys developed high-level parasitemias with an average maximum of 103,000 per mm3. Inoculation of 10,000 sporozoites resulted in average prepatent periods of 19.4 days; one of the resulting infections produced a transient low-level parasitemia. Of 5 monkeys inoculated with 1,000 sporozoites, 4 had prepatent periods of from 24 to 35 days and 1 failed to demonstrate any parasitemia; 1 monkey supported a low-level transient parasitemia, whereas the other 3 monkeys had high-level parasitemias. It is proposed that by using a minimum inoculum of 10,000 sporozoites, the model system may be useful in the testing of anti-sporozoite vaccines directed against P. vivax.     

Rosetting is associated with increased Plasmodium falciparum in vivo multiplication rate in the Saimiri sciureus monkey

Severe Plasmodium falciparum malaria in African children is associated with high peripheral parasite densities and high rate of rosette-forming parasites. To explore the relationship between rosette formation and parasite density in vivo, we compared the multiplication rate of a rosette-forming variant (varO) of the Palo Alto line with a sibling non-rosetting variant (varR) in splenectomized Saimiri monkeys. The multiplication rate of varO parasites was 1.5-fold higher than that of the varR variant. This indicates that rosetting is indeed associated with high parasite multiplication efficiency in vivo and, as such, may contribute to the high parasite densities observed in severe malaria.     

Studies on sporozoite-induced and chronic infections with Plasmodium fragile in Macaca mulatta and New World monkeys

Plasmodium fragile continues to be investigated because of its biologic similarities to the human malaria parasite, Plasmodium falciparum. Two strains of P. fragile are available for study; one strain is able to infect mosquitoes, whereas the other strain is transmissible only by blood inoculation. The Sri Lanka strain of P. fragile was transmitted to Macaca mulatta, Macaca fascicularis, Aotus lemurinus griseimembra, Aotus nancymaae, Aotus vociferans, and Saimiri boliviensis monkeys via sporozoites that developed to maturity only in Anopheles dirus mosquitoes. The prepatent periods ranged from 12 to 35 days for macaques and from 15 to 30 days for New World monkeys after intravenous injection of sporozoites. Eight rhesus monkeys were infected with the Nilgiri strain and followed for 482 days. Parasitemia in 6 animals persisted at relatively high density through the period of observation. Erythrocyte, hematocrit, and hemoglobin values reached their lowest levels 3 wk after infection and slowly recovered; however, the values did not approach preinfection levels as long as parasitemia persisted in the monkeys. The mean corpuscular volume and corpuscular hemoglobin concentration reached their peak and lowest values, respectively, at day 38 and then returned to the preinfection level. The mean corpuscular hemoglobin value decreased to its lowest level at day 87 and then returned to preinfection level.     

Observations on the Vietnam Palo Alto strain of Plasmodium vivax in two species of Aotus monkeys

Thirty-three splenectomized Aotus lemurinus griseimembra monkeys with no previous experience with malaria were infected with the Vietnam Palo Alto strain of Plasmodium vivax. The median maximum parasite count was 280,000/microl. Nine splenectomized monkeys with previous infection with Plasmodium falciparum had median maximum parasite counts of 120,000/microl. Splenectomized Aotus nancymai monkeys supported infections at a lower level. Transmission via the bites of Anopheles dirus mosquitoes was obtained in a splenectomized A. lemurinus griseimembra, with a prepatent period of 31 days. It is estimated that between 1.5 x 10(8) and 1.6 x 10(9) parasites can be removed from an infected animal for molecular or diagnostic antigenic studies.     

Aotus infulatus monkey is susceptible to Plasmodium falciparum infection and may constitute an alternative experimental model for malaria

Aotus is one of the WHO-recommended primate models for studies in malaria, and several species can be infected with Plasmodium falciparum or P. vivax. Here we describe the successful infection of the species A. infulatus from eastern Amazon with blood stages of P. falciparum. Both intact and splenectomized animals were susceptible to infection; the intact ones were able to keep parasitemias at lower levels for several days, but developed complications such as severe anemia; splenectomized monkeys developed higher parasitemias but no major complications. We conclude that A. infulatus is susceptible to P. falciparum infection and may represent an alternative model for studies in malaria.     

Hemobartonellosis in squirrel monkeys (Saimiri sciureus) in a domestic breeding colony: case report and preliminary study

Infection with Hemobartonella sp was diagnosed in a colony-born squirrel monkey with normocytic, normochromic anemia and pronounced punctate erythrocytic basophilic stippling on Wright's-Giemsa stained blood films. The diagnosis was confirmed using transmission electron microscopy. Two randomly selected colony-born squirrel monkeys were splenectomized in an effort to activate and detect possible latent hemobartonellosis . One monkey became parasitemic 12 days following splenectomy. The second monkey was inoculated on day 14 with 1 ml of whole blood from an infected, but nonparasitemic monkey and developed overt parasitemia 3 days later (day 17 following splenectomy). Infections in the latter two monkeys were confirmed using scanning electron microscopy.     

Infection and transmission studies with Plasmodium simiovale in the Macaca mulatta monkey.

Six different anophelines--Anopheles freeborni, An. b. balabacensis, An. maculatus, An. stephensi, An. atroparvus, and An. quadrimaculatus--were shown to be susceptible to infection with Plasmodium simiovale when fed upon splenectomized Macaca mulatta monkeys between the 1st and 26th days of patent parasitemia. Transmission was obtained via the bites of An. b. balabacensis and An. maculatus mosquitoes. Prepatent periods in M. mulatta monkeys following sporozoite inoculation ranged from 11 to 20 days with a median of 14 days.     

Chondroitin-4-sulfate impairs in vitro and in vivo cytoadherence of Plasmodium falciparum infected erythrocytes.

BACKGROUND: Chondroitin-4-sulfate (CSA) was recently described as a Plasmodium falciparum cytoadherence receptor present on Saimiri brain microvascular and human lung endothelial cells. MATERIALS AND METHODS: To specifically study chondroitin-4-sulfate-mediated cytoadherence, a parasite population was selected through panning of the Palo-Alto (FUP) 1 P. falciparum isolate on monolayers of Saimiri brain microvascular endothelial cells (SBEC). Immunofluorescence showed this SBEC cell line to be unique for its expression of CSA-proteoglycans, namely CD44 and thrombomodulin, in the absence of CD36 and ICAM-1. RESULTS: The selected parasite population was used to monitor cytoadherence inhibition/dissociating activities in Saimiri sera collected at different times after intramuscular injection of 50 mg CSA/kg of body weight. Serum inhibitory activity was detectable 30 min after injection and persisted for 8 hr. Furthermore, when chondroitin-4-sulfate was injected into monkeys infected with Palo-Alto (FUP) 1 P. falciparum, erythrocytes containing P. falciparum mature forms were released into the circulation. The cytoadherence phenotype of circulating infected red blood cells (IRBC) was determined before and 8 hr after inoculation of CSA. Before inoculation, in vitro cytoadherence of IRBCs was not inhibited by CSA. In contrast, in vitro cytoadherence of circulating infected erythrocytes obtained 8 hr after CSA inoculation was inhibited by more than 90% by CSA. CONCLUSIONS: In the squirrel monkey model for infection with P. falciparum, chondroitin-4-sulfate impairs in vitro and in vivo cytoadherence of parasitized erythrocytes.