Aminoquinolines that circumvent resistance in Plasmodium falciparum in vitro.

Aminoquinoline (AQ) resistance is one of the most important factors in the worldwide resurgence of malaria due to Plasmodium falciparum. We synthesized a series of AQs to define the structure-activity relationships responsible for AQ action against chloroquine-susceptible and -resistant P. falciparum. The AQs with ethyl, propyl, isopropyl, butyl, pentyl, isopentyl (chloroquine), hexyl, octyl, decyl, or dodecyl side chains were equally active against chloroquine-susceptible P. falciparum (50% inhibitory concentrations [IC50s] = 5-15 nM). The AQs with ethyl, propyl, isopropyl, decyl, or dodecyl side chains were also active against chloroquine-, mefloquine- and multiply-resistant P. falciparum (IC50s = 5-20 nM). Verapamil, which enhances the activity of chloroquine against chloroquine-resistant parasites, had no effect on the activity of AQs that were active against resistant parasites. These results indicate that AQs with 2-12 carbon side chains are as active as chloroquine against chloroquine-susceptible P. falciparum, and that AQs with side chains shorter or longer than chloroquine are often active against chloroquine-, mefloquine-, and multiply-resistant P. falciparum.

Hypnozoites of Plasmodium simiovale.

Hypnozoites of Plasmodium simiovale were detected in liver biopsies from a rhesus monkey (Macaca mulatta) inoculated eight days previously with sporozoites from heavily infected anopheline mosquitoes. The tissue forms, 6 mu in diameter, were found within the cytoplasm of hepatic parenchymal cells by immunofluorescence and restained with Giemsa. This is the first report of latent, pre-erythrocytic stages from an ovale-type relapsing malaria.

Enzootic schistosomiasis in a Louisiana armadillo.

Trematode eggs compatible in characteristics with those of Heterobilharzia americana, a probable cause of human schistosomal dermatitis in the Gulf States of the U.S., were found in the cirrhotic liver and other viscera of a wild-caught armadillo in Louisiana. Although adult worms were not found, the current observation suggests a need to extend the known host range of this common schistosome parasite of carnivores and other wild animals in the South, and may have additional implications in the context of the public health importance of the armadillo.

Identification of hypnozoites and tissue schizonts of Plasmodium vivax and P. cynomolgi by the immunoperoxidase method.

An immunoperoxidase technique was used to detect hypnozoites and liver schizonts of the primate malaria species Plasmodium vivax and P. cynomolgi bastianellii in Carnoy's-fixed sections. Anti-P. cynomolgi serum and a peroxidase-conjugated anti-monkey IgG serum rendered 7-day pre-erythrocytic forms clearly visible. The technique retains the specificity of the immunofluorescence method while having the advantage of a permanent preparation. Detection of hyponozoites by this alternative method provides further evidence for their plasmodial nature.

Placental changes associated with fetal outcome in the Plasmodium coatneyi/rhesus monkey model of malaria in pregnancy.

Term placentas collected surgically from seven Plasmodium coatneyi-infected rhesus monkeys, one abortion, and five controls were evaluated histopathologically. The placentas from Plasmodium-infected dams had more significant pathologic changes than those from controls for six parameters (P < 0.05) and higher numbers of activated (LN5 + Zymed) macrophages in the intervillous space (IVS) (P = 0.0173). Total parasite load (TPL) was defined as the sum of all weekly peripheral infected red blood cell counts for each trimester and for the entire pregnancy. High first trimester PLs were more likely to result in fetal demise (P = 0.0476) or increased placental damage in surviving infants. As trimester 2-3 TPL increased, so did the number of activated macrophages (P < 0.05) and the total malaria pigment scores (P < 0.05). Low birth weight (LBW) and intrauterine growth retardation (IUGR) were associated with high pigment scores and high numbers of activated macrophages in the IVS. High placental damage scores were not associated with IUGR, LBW, or early infant mortality.

Plasmodium coatneyi in the rhesus monkey (Macaca mulatta) as a model of malaria in pregnancy.

Pregnant women with Plasmodium falciparum infection are at increased risk for complications such as anemia and cerebral malaria. In addition, the infants of these women suffer intrauterine growth retardation (IUGR), low birth weight (LBW), congenital infection, and high infant mortality. Although much has been learned from studies of malaria during human pregnancy, progress has been limited by the lack of a suitable animal model. Nonhuman primates are of particular interest because, other than the armadillo, they are the only animals with a discoidal, villous, hemochorial placenta like that of humans. We have established a model of malaria during human pregnancy by inoculating pregnant rhesus monkeys (Macaca mulatta) with Plasmodium coatneyi (a sequestering parasite) during the first trimester. In our initial experiment, four monkeys were inoculated with a fresh inoculum containing 10(8) viable parasites from an infected donor monkey. All four monkeys became parasitemic seven days postinoculation (PI) and three monkeys aborted 7-10 days PI coincident with high peak parasitemias (41,088-374,325 parasites/mm3). Although abortion is one of the outcomes observed in Plasmodium-infected women, the intent of this study was to examine the effects of Plasmodium infection throughout gestation. Since the rapid onset of high parasitemia may have been responsible for the abortions, a decision was made to reduce the size of the effective inoculum. Six additional pregnant monkeys were inoculated with a frozen isolate taken from the same donor containing 10(6) parasites. These six animals became parasitemic by 14 days PI and, along with monkey E412, carried their infants to term. These seven infants weighed significantly less at term than the infants of uninfected mothers (P = 0.0355). Symmetrical IUGR was detected by ultrasound in one fetus with an LBW of 334 g. Another LBW infant (300 g) had asymmetrical growth retardation, which has been associated with uteroplacental insufficiency and was consistent with the lower placental weights found in infected dams compared with controls (P = 0.0455). The infant with symmetric IUGR died at five days of age, while the other is alive but congenitally infected. The IUGR, LBW, congenital infection, postnatal infant mortality, and early abortions observed in these animals suggest that P. coatneyi in pregnant rhesus monkeys is a valid model of malaria in human pregnancy. This model should provide the opportunity to study questions about malaria in pregnancy that have been difficult to study in humans.

Observations on early and late post-sporozoite tissue stages in primate malaria. IV. Pre-erythrocytic schizonts and/or hypnozoites of Chesson and North Korean strains of Plasmodium vivax in the chimpanzee.

In a continuing reexamination of plasmodial tissue stages within the context of the hypnozoite theory of malarial relapse, 2 strains of Plasmodium vivax with distinct and disparate relapse characteristics in humans were studied in chimpanzees. Following intravenous inoculation of massive numbers of salivary gland sporozoites, both the frequently relapsing Chesson strain and a North Korean strain characterized by predominantly delayed relapses exhibited relapse patterns and antimalarial sensitivity in the splenectomized chimpanzee essentially indistinguishable from those seen in humans. Examination of hepatic biopsies obtained at 7 and 10 days after infection revealed both pre-erythrocytic (pre-e) schizonts and hypnozoites in tissue obtained from the animal infected with the Chesson strain, but only rare hypnozoites (no pre-e schizonts) at 7 days in the animal infected with the North Korean strain. These findings, combined with the comparability of relapse behavior--which indicates the suitability of the chimpanzee as a model for the natural (human) host-parasite relationship--are essentially as predicted by the hypnozoite theory, despite the small numbers of tissue forms seen. Pre-erythrocytic schizogony of the Chesson strain in the liver was essentially indistinguishable from that of other strains studied, also underlining the suitability of this model system for tissue stage studies of P. vivax.

Demonstration of hypnozoites in sporozoite-transmitted Plasmodium vivax infection.

Hypnozoites of two strains of the human relapsing malaria parasite, Plasmodium vivax, have been detected among maturing 7- and 10-day pre-erythrocytic schizonts in liver biopsies of chimpanzees infected by intravenous inoculation of sporozoites obtained from dissected salivary glands of heavily infected anopheline mosquitoes. As in the simian relapsing species, P. cynomolgi, the hypnozoites of P. vivax at 7 and 10 days are uninucleate forms of approximately 4-5 micrometers diameter, lying within the cytoplasm of individual hepatocytes. Their presence in this relapsing human species is added support for the hypnozoite theory of malarial relapse.

Observations on early and late post-sporozoite tissue stages in primate malaria. II. The hypnozoite of Plasmodium cynomolgi bastianellii from 3 to 105 days after infection, and detection of 36- to 40-hour pre-erythrocytic forms.

Confirmation of the existence of a persistent, uninucleate, dormant pre-erythrocytic stage, the hypnozoite, of the relapsing simian malaria parasite, Plasmodium cynomolgi bastianellii, has been obtained by means of experiments involving the intravenous injection into susceptible monkeys of 48 to 85 x 10(6) sporozoites derived from mosquitoes of a different species and source than employed previously. The development of these hypnozoites was traced from 3 days until 105 days after sporozoite inoculation, employing a sensitive immunofluorescence technique followed by restaining with Giemsa. From an average mean diameter of 4 micrometers at 3 and 5 days, uninucleate hypnozoites grow to 5 micrometers at 7 days, then persist with little change until at least 105 days after infection. Strong evidence for the viability of these persistent forms was obtained by treatment of a host monkey with primaquine, which eliminated all trace of hypnozoites present 2 weeks before. Examination of hepatic tissue from a monkey injected with sporozoites 36 and 40 hours earlier revealed rare uninucleate pre-erythrocytic forms of 2.5-micrometers diameter. These early forms were present in hepatocytes in a density only approximately 1/30th of that expected on the basis of numbers of pre-erythrocytic stages found in the same animal's liver 7 days after infection. Nevertheless, subinoculation experiments appeared to rule out the circulation as a vehicle for dissemination of any putative early intermediate hepatotropic forms from another site.

Observations on early and late post-sporozoite tissue stages in primate malaria. III. Further attempts to find early forms and to correlate hypnozoites with growing exo-erythrocytic schizonts and parasitaemic relapses in Plasmodium cynomolgi bastianellii infections.

Rhesus monkeys were heavily infected with sporozoites of Plasmodium cynomolgi bastianellii in an attempt to demonstrate the site of invasion of sporozoites into tissue cells and their growth there. Further attempts were made to correlate the appearance and loss of hypnozoites with parasitaemic relapses. Hypnozoites were demonstrated and once again shown to decrease in numbers over 229 days during which time the infection showed parasitaemic relapses. Liver biopsies taken at two-day intervals for 12 days showed that hypnozoites decreased in numbers over-all and growing schizonts were demonstrated in the liver. At this time a parasite the size of a hypnozoite was seen with two nuclei and another was seen with an elongate, possibly dividing nucleus in one monkey. an attempt to find the location of the early intracellular exoerythrocytic forms in the liver at various times less than 40 hours after infection using smears and immunological staining with newly prepared anti-sera failed. Large numbers of sporozoites of P. knowlesi were also injected into a rhesus monkey the liver of which on the fifth day after infection showed no hypnozoites among 157 sections of growing schizonts and no parasites at all on the 42nd day after infection. In P. cynomolgi bastianellii infections parasites, mostly hypnozoites, were found in the liver up to 229 days after infection.

Studies on the North Korean strain of Plasmodium vivax in Aotus monkeys and different anophelines.

Twenty-two Aotus monkeys of different karyotypes were infected with the North Korean strain of Plasmodium vivax. Aotus lemurinus griseimembra animals from Colombia produced higher maximum parasitemias and more readily infected mosquitoes than did Aotus monkeys from Bolivia (K-VI) or Peru (K-V and K-X). Comparative feedings indicated that the most susceptible mosquito species was Anopheles stephensi, followed by An. gambiae, An. dirus, An. freeborni, An. quadrimaculatus, An. culicifacies, and An. maculatus.

The hypnozoite and relapse in primate malaria.

Although the phenomenon of malarial relapse was known to the ancients, the mechanism has only recently been explained satisfactorily. The long-held hypothesis of a tissue "cycle" in primate malaria as a cause of relapse did not fit clinical and experimental observations. A latent stage for Plasmodium spp. in the liver, for which there is now extensive morphological and experimental confirmation, best explains both the relapse phenomenon and the long prepatent periods seen with some strains of Plasmodium vivax. These latent stages (hypnozoites) have been detected in three relapsing malarias and have been found to persist in the liver as uninucleate parasites for up to 229 days after sporozoite inoculation. They have been found in in vitro cultures of two species of Plasmodium, and their ultrastructure has been partially described.

Host DNA can interfere with detection of Borrelia burgdorferi in skin biopsy specimens by PCR.

It is demonstrated that a diagnostic PCR for Borrelia burgdorferi can be inhibited in the presence of more than 500 ng of host (monkey skin) DNA. The inhibitor is the host DNA itself. An acceptable value for analytical sensitivity can be obtained by diluting the skin-B. burgdorferi proteinase K lysate to a level below the inhibitory concentration of the host DNA. Dilution of the lysate may obviate the need for further DNA purification.

Chronic lyme disease in the rhesus monkey.

BACKGROUND: We have previously reported the clinical, pathologic, and immunologic features of "early" Borrelia burgdorferi infection in rhesus monkeys (3). We have now evaluated these features during the chronic phase of Lyme disease in this animal model. EXPERIMENTAL DESIGN: Clinical signs, and pathologic changes at the gross and microscopic levels, were investigated 6 months post-infection in several organ systems of five rhesus macaques (Macaca mulatta), which were infected with Borrelia burgdorferi by allowing infected Ixodes scapularis nymphal ticks to feed on them. A sixth animal was used as an uninfected control. Borrelia antigens recognized by serum antibody were identified longitudinally by Western blot analysis, and C1q-binding immune complexes were quantified. Localization of the spirochete in the tissues was achieved by immunohistochemistry and in vitro culture. The species of spirocheta cultured was confirmed by the polymerase chain reaction. RESULTS: Chronic arthritis was observed in five out of five animals. The knee and elbow joints were the most consistently affected. Articular cartilage necrosis and/or degenerative arthropathy were the most severe joint structural changes. Synovial cell hyperplasia and a mononuclear/lymphocyte infiltrate were commonly seen. Nerve lesions were also observed, including nerve sheath fibrosis and focal demyelinization of the spinal cord. Peripheral neuropathy was observed in five out of five animals and could be correlated in the most severely affected monkey with the presence of higher levels of circulating immune complexes. Differences in disease severity did not correlate with differences in the antigens recognized on Western blot analysis. CONCLUSIONS: B. burgdorferi infection in rhesus macaques mirrors several aspects of both the early and chronic phases of the disease in humans. This animal model will facilitate the study of the pathogenesis of Lyme arthritis and neuroborreliosis.

Immunogenicity of the recombinant serine rich Entamoeba histolytica protein (SREHP) amebiasis vaccine in the African green monkey.

We report the first study in non-human primates of the safety and immunogenicity of a recombinant vaccine designed to prevent amebic liver abscess. In a pilot study, a recombinant vaccine containing the serine rich Entamoeba histolytica protein (SREHP) attached to a maltose binding protein (SREHP/MBP), which has been shown to be effective in preventing amebic liver abscess in rodent models of infection, was used to immunize two African Green Monkeys. Vaccination with SREHP/MBP resulted in no systemic side-effects. The monkeys receiving the SREHP/MBP protein developed antibodies that recognized the recombinant SREHP/MBP molecule, the native SREHP protein, and the surface of amebic trophozoites. Antiserum from SREHP/MBP-vaccinated monkeys could block the adhesion of E. histolytica trophozoites to mammalian cells, a feature that may correlate with vaccine efficacy. Attempts to produce amebic liver abscess in naive African Green Monkeys by direct hepatic inoculation with virulent E. histolytica trophozoites was not successful, suggesting this species is probably not suitable for vaccine efficacy studies.

Early and early disseminated phases of Lyme disease in the rhesus monkey: a model for infection in humans.

We demonstrate that Borrelia burgdorferi infection in the rhesus monkey mimics the early and early disseminated phases of human Lyme disease. Clinical, bacteriological, immunological, and pathological signs of infection were investigated during 13 weeks after inoculation of the spirochete. Three animals were given B. burgdorferi (strain JD1) by needle inoculations, six animals were exposed to the bite of B. burgdorferi-infected Ixodes dammini ticks, and three animals were uninfected controls. B. burgdorferi could be recovered from all animals that were given the spirochete. Bacteria were detectable until week 6 postinoculation (p.i.) in blood, until week 8 p.i. in skin biopsies, and at 10 weeks p.i. in the conjunctiva of one of two animals which developed conjunctivitis. Erythema migrans (EM) appeared in one of the three animals infected by needle inoculation and in five of the six animals infected by ticks. Deep dermal perivascular lymphocytic infiltrations (characteristic of human EM) were observed in all animals showing EM clinically. Both EM and conjunctivitis were documented concomitantly with the presence of the spirochete. Lethargy, splenomegaly, and cerebrospinal fluid pleocytosis were also noted in some animals, but the direct connection of these signs with the infection was not shown. The appearance rate of immunoglobulin M and immunoglobulin G antibodies to B. burgdorferi, as well as the antigen spectra recognized, were remarkably similar to those seen in humans. Serum antibodies from infected animals were able to kill B. burgdorferi in vitro in the presence of rhesus complement. The rhesus monkey model appears to be useful for the investigation of the immunology and pathogenesis of Lyme disease and for the development of immunoprophylactic, diagnostic, and chemotherapeutic protocols.