A novel haemoplasma species identified in archived primate blood smears.

In order to confirm a microscopic diagnosis of 'eperythrozoonosis' made over 40 years ago in a captive owl monkey (Aotus trivirgatus), DNA was extracted from archived fixed and stained blood smears and subjected to generic haemotropic mycoplasma (haemoplasma) quantitative real-time PCR (qPCR) and a human glyceraldehyde-3-phosphate dehydrogenase qPCR as an amplification control. The qPCRs confirmed the extraction of host DNA from the samples and the presence of a haemoplasma species. Partial 16S rRNA and ribonuclease P ribosomal gene fragments were amplified by PCR, cloned and sequenced. Sequence data and phylogeny showed the owl monkey haemoplasma to lie in the haemominutum clade of haemoplasmas, most closely related to 'Candidatus Mycoplasma kahaneii'. This study confirms the use of generic haemoplasma qPCRs to successfully amplify haemoplasma DNA from fixed, stained and archived blood smears from the early 1970s and provides molecular confirmation of the existence of a novel haemoplasma species in an owl monkey, for which the name 'Candidatus Mycoplasma aoti' sp. nov. is proposed.

Serum and urine biochemical diversity among adult wild-caught Aotus nancymae and Saimiri peruviensis.

Serum and urine analytes were compared between adult wild-caught owl monkeys (Aotus nancymae) and adult wild-caught squirrel monkeys (Saimiri peruviensis) to determine if normative clinical pathology data were similar. An objective of the study was to confirm that species of neotropical primates are distinct with regard to physiologic parameters, and should not be considered interchangeable in biomedical research. Significant differences (P < 0.05) were noted in many serum and urine analytes between the two groups. The results suggest that reference data for wild-caught owl monkeys are not applicable to squirrel monkeys, and the differences are sufficiently large to be of clinical significance. These findings illuminate the diversity among species of neotropical primates.

In vivo-in vitro model to assess chloroquine activity in monkeys.

The bioassay technique and high-performance liquid chromatography (HPLC) method were used to establish chloroquine (CQ) concentration equivalents in serum samples collected from Aotus and Saimiri monkeys after administration of CQ. The results indicate some differences in CQ metabolism between the two simian species. They also indicate a strong positive relationship (rs = 0.96) between data obtained by the bioassay and HPLC technique. The findings suggest that the use of the bioassay in these small primates, using only a fraction of the serum required for HPLC analysis, should provide a useful mean for obtaining preliminary information about the degree and duration of serum antimalarial activity of promising experimental drugs. Apart from reducing the number of monkeys required for drug evaluation, this in vivo-in vitro model should also decrease the overall cost and duration of developing new antimalarial agents.

Immunophenotypic characterization of owl monkey peripheral blood mononuclear cells.

A two-phase study was initiated to delineate the peripheral blood lymphocyte populations present in owl monkeys and to correlate those populations with immune response and parasitism during malaria infection. The goal of phase I of the study was to elucidate a monoclonal antibody panel that could be used to characterize peripheral blood mononuclear cell (PBMC) populations with flow cytometric techniques. Forty-two monoclonal antibodies (reported to be reactive with human and macaque lymphocyte antigens) were screened for activity to owl monkey PBMC. Eleven monoclonals were found to react: anti-H42A (MHC Class II DP-like); anti-TH14B (MHC Class II DR-like); and anti-TH81A5 (MHC Class II DQ-like); anti-H58A (MHC Class I); anti-DH59B (granulocyte and monocyte); anti-B1 (B cell); anti-T4 (CD4); anti-Leu3a (CD4); anti-Leu11a (CD16); anti-60.3 (CD18); and anti-OKM1 (NK and monocyte). In a preliminary retrospective study correlating antibody titers, parasitemia values, and MHC Class I and Class II marker profiles on PBMC to test antigens used in malaria vaccine trials, a significant negative association was observed between cells bearing MHC Class II molecules and the other elements of the comparison. In summary, an appropriate panel of monoclonal antibodies has been identified for characterizing PBMC in owl monkeys, and preliminary studies indicate a possible association between clinical outcome and expressed phenotypic PBMC markers.

South American monkeys in the development and testing of malarial vaccines--a review.

South American Aotus and Saimiri monkeys, which are susceptible to infection with human malarias, have been used to develop models for the testing of human malaria vaccines. Studies indicate that blood-stage and sporozoite vaccines can be tested in these monkeys using appropriate strains of parasites.

Plasmodium vivax interaction with the human Duffy blood group glycoprotein: identification of a parasite receptor-like protein.

The interaction between merozoites of the human pathogen, Plasmodium vivax, and the Duffy blood group glycoprotein on the surface of human erythrocytes is essential for the invasion of erythrocytes and the survival of the parasite. We have identified a P. vivax protein of 135 to 140 kDa which binds with receptor-like specificity to the human Duffy blood group glycoprotein. This interaction can be specifically inhibited by purified Duffy glycoprotein and by pretreating erythrocytes with a monoclonal antibody directed against a novel Duffy determinant. A protein with similar specificity for the Duffy glycoprotein from the phylogenetically related simian malaria, P. knowlesi, is shown to be immunologically related by the generation of cross-reactive antibodies. Despite their shared properties, these two Duffy associating proteins from P. vivax and P. knowlesi differ in some aspects of their interaction with the Duffy glycoprotein. The identification of these proteins will help elucidate the molecular mechanisms of erythrocyte invasion by Plasmodium.

Two approximately 300 kilodalton Plasmodium falciparum proteins at the surface membrane of infected erythrocytes.

Two very large Plasmodium falciparum proteins are identified as constituents of the infected erythrocyte membrane. Sera were obtained from Aotus monkeys that had been repeatedly infected with asexual P. falciparum from one of four strains. The capacity of these sera to block in vitro cytoadherence of infected erythrocytes and agglutinate intact infected cells was determined. The sera were also used to immunoprecipitate protein antigens from detergent extracts of 125I-surface labeled or biosynthetically radiolabeled infected erythrocytes. For each serum/antigen combination, precipitation of only one protein correlated with the ability of the serum to interfere with cytoadherence and agglutinate infected cells. This malarial protein, denoted Pf EMP 1 (P. falciparum-erythrocyte-membrane-protein 1) bore strain-specific epitope(s) on the cell surface and displayed size heterogeneity (Mr approximately 220,000-350,000). Pf EMP 1 was strongly labeled by cell-surface radioiodination but was a quantitatively very minor malarial protein. Pf EMP 1 was distinguished by its size, surface accessibility and antigenic properties from a more predominant malarial protein in the same size range (Pf EMP 2) that is under the infected erythrocyte membrane at knobs. Monoclonal antibodies and rabbit antisera raised against Pf EMP 2 were used to show that this size heterogeneous antigen was indistinguishable from the previously described MESA (mature parasite infected erythrocyte surface antigen), identified by precipitation with rabbit antisera raised against the MESA hexapeptide repeats. Antibodies raised against Pf EMP 2/MESA did not precipitate Pf EMP 1. We conclude that Pf EMP 1 is either directly responsible for the cytoadherence phenomenon, or is very closely associated with another as yet unidentified functional molecule. Pf EMP 2/MESA must have a structural property/function that is important under the host cell membrane.

Erythrocytes and erythrocyte morphologies of healthy and colony-born owl monkeys (Aotus lemurinus griseimembra).

Inadequate availability of hematological reference data seriously restricts optimal utilization of the owl monkey (Aotus lemurinus griseimembra) as an experimental model. The current study investigated erythrocytic morphology in peripheral blood of healthy, colony-born owl monkeys. The blood of the subjects contained discoid erythrocytes, poikilocytes, and showed considerable anisocytosis. Also observed were nucleated erythrocytes, erythrocytes with Howell-Jolly bodies, and reticulocyte types I, II, and III. Heinz bodies were not detected.

Reference values for peripheral blood lymphocytes from Aotus lemurinus ssp. griseimembra (owl monkey).

The reactivities of several monoclonal antibodies that define human lymphocyte cell-surface antigens have been tested with peripheral blood lymphocytes of Aotus lemurinus ssp. griseimembra. Based on reactivity patterns in humans, reactive MoAb were identified that mark pan-T, helper/inducer, suppressor/cytotoxic, pan-B, and natural killer cells. Reference values of these subsets in Aotus are presented. These MoAb should provide a useful tool for further phenotypic and functional dissection of the immune system in this simian model of human disease.

Plasmodium brasilianum in the common marmoset Callithrix jacchus.

Chronic quartan malarial infection has been established in the common marmoset (Callithrix jacchus). Plasmodium brasilianum from a douroucouli monkey (Aotus trivirgatus) was used to infect splenectomized twin animals, passed to an intact animal, and then to 4 other intact adults, 2 pairs of twins. In 2 of the 4 latter animals there was continuing patency with parasitaemias of less than or equal to 0.5% parasitized erythrocytes for 30 weeks. The other 2 had lower initial levels of parasitaemia; in 1 of these parasitaemias remained low or subpatent. All marmosets developed lymphocytosis. One animal became ill 30 weeks after infection with anaemia, weight loss and mild proteinurea, the other 3 remained well. Histological examination showed minor changes in the kidneys; spleens of infected animals showed marked follicular hyperplasia and phagocytosis of pigment. The livers showed sinusoidal hypercellularity and pigment deposition and in splenectomized animals, a marked lymphoid follicular hyperplasia in the portal tracts.

Diurnal variation of several blood parameters in the owl monkey, Aotus trivirgatus griseimembra.

Diurnal variations in normal hemogram, total serum protein and serum iron are documented in 6 adult owl monkeys, Aotus trivirgatus griseimembra, maintained in an artificial LD 12:12 (35:0.08 lx) with light phase from 03.00 to 15.00 h Central European time. Statistically significant high amplitude rhythms occurred in total leukocyte and in eosinophil numbers with acrophases at 06.35 and 09.53 h, respectively. Erythrocyte numbers and hemoglobin concentration showed statistically significant low amplitude rhythms with almost identical acrophases. Total serum protein exhibited a 10% daily fluctuation. Serum iron concentration showed high amplitude daily variations with a 60% mean range of oscillation.

Structural alteration of the membrane of erythrocytes infected with Babesia bovis.

Babesia bovis, causative agent of cattle babesiosis, induces characteristic alterations on the membrane of infected erythrocytes. Elliptical protrusions measuring about 320 nm in long axis and about 160 nm in short axis appear on the membrane of infected erythrocytes, both in vitro and in vivo. Freeze-fracture demonstrated alignment of intramembrane particles (IMP) along the long axis of both the P and E faces of the protrusions. The number of IMP on the endoplasmic face increases, but the number of IMP on the protoplasmic face of the protrusions is not statistically altered from that of uninfected erythrocytes. In vitro, there are more protrusions per erythrocyte infected with the virulent form (low passage form) of B. bovis than with the avirulent form (high passage form). This suggests that the number of protrusions which appear on the membrane of infected erythrocytes may have a direct relationship to the virulence of the parasites. These protrusions may be attached to the capillary endothelial cells, which causes fatal cerebral babesiosis.

Activated partial thromboplastin time of owl monkey (Aotus trivirgatus) plasma.

Owl monkey plasma samples produced short, reproducible activated partial thromboplastin times, similar to those obtained with samples from many other mammalian species. This was an apparent contradiction to an earlier report of long irreproducible activated partial thromboplastin times from owl monkey samples. The discrepant data could not be explained by differences in anticoagulants (citrate or oxalate), assay reagents (partial thromboplastin with either diatomaceous earth or ellagic acid), or activation incubation times (2, 5, or 10 minutes); nor could they be explained by differences in the monkeys' sex, age or previous experimental exposure to Plasmodium falciparum malaria.

Long term cultivation of Plasmodium falciparum in Aotus trivirgatus erythrocytes.

The erythrocytes of the Colombian owl monkey Aotus trivirgatus griseimembra can be used for the long-term in vitro cultivation of Plasmodium falciparum employing a modified Trager -Jensen method. Cultures are grown in HEPES-buffered RPMI-1640 using a 4% suspension of monkey erythrocytes and 10% pooled heat-inactivated human AB serum, with initial parasitemias in a range between 0.2 and 0.5%. Adaptation of new strains from human erythrocytes cultures can be performed by simply subculturing from human to owl monkey erythrocytes in a stepwise manner. When 5% human AB serum is included in cultures to support growth, as much as 5% monkey serum can be added in order to investigate serum effects, such as antibody activity against P. falciparum. The Aotus trivirgatus continuous culture system has provided a stable, consistent source of infected erythrocytes for in vitro experimentation, and the techniques developed have been used to further refine and support the animal experiments in progress.

Erythrocyte characteristics in vitamin E-responsive anemia of the owl monkey (Aotus trivirgatus).

To characterize the vitamin E-responsive anemia occurring in owl monkeys (Aotus trivirgatus), osmotic fragility, and H2O2-induced and time-dependent hemolysis, as well as RBC lipid peroxidation, were compared in anemic and nonanemic owl monkeys. Whereas vitamin E serves as a lipid-soluble antioxidant, the glutathione peroxidase system functions in the water-soluble phase of the cell. Thus, activity of glutathione peroxidase, glutathione reductase, and glucose-6-phosphate dehydrogenase, as well as reduced glutathione concentrations in owl monkeys' RBC, were compared with those of rhesus macaques and cebus and squirrel monkeys fed the same diet and maintained under the same management scheme. Osmotic fragility did not differ between anemic and nonanemic owl monkeys. The H2O2-induced and time-dependent hemolysis was approximately 10-fold greater among anemia owl monkeys than among their nonanemic counterparts, and lipid peroxidation values tended to be higher in the anemic monkeys. Owl monkeys, as a species and independent of anemia, exhibited higher RBC peroxidation than did 2 other New World species, cebus and squirrel monkeys. The glutathione peroxidase system was not depressed in owl monkey RBC. The only observed difference in this system was in the glucose-6-phosphate dehydrogenase activity, which was 3- to 6-fold higher in the owl monkey than in the other species, indicating an increased activity of the peroxidase system. Thus, a defect in the glutathione peroxidase system could not be identified.

Inhibition of in vitro growth of Plasmodium falciparum by immune serum from monkeys.

Three owl monkeys that had been immunized against the Camp strain of Plasmodium falciparum by infection were treated with chloroquine and rechallenged with parasites. Immune serum caused a dose-dependent, time-dependent inhibition of in vitro parasite growth. Heat-inactivation eliminated nonspecific inhibition by normal monkey serum without diminishing immune inhibition. Purified IgG from immune serum inhibited parasite growth. Serum taken immediately before the second challenge did not inhibit growth in vitro at a 1:10 dilution, although the monkeys successfully resisted the in vivo challenge. However, immune sera from all three monkeys taken two to four weeks after in vivo challenges were inhibitory, but sometimes detection required 20% serum. Growth inhibition in vitro by 10% serum was a poor predictor of in vivo protective immunity. Undiluted blood containing higher antibody levels (which are boosted by challenge), combined with additional immune mechanisms, may explain the protection observed in vivo.

Diurnal variations in plasma testosterone in a male nocturnal primate, the owl monkey (Aotus trivirgatus).

Six adult male owl monkeys were kept in conditions of controlled lighting (12 h white light alternating with 12 h of very dim reg light) and blood samples were taken at different times of day, once a week, for 10 weeks. Plasma testosterone levels correlated with phases of the lighting cycle: highest levels (mean +/- s.e.m.) occurred in the light (24.8 +/- 5.3 ng/ml) and lowest levels during periods of darkness (4.7 +/- 1.2 ng/ml). The owl monkey is nocturnal, and these daily changes in circulating testosterone are the reverse of those reported for some diurnal primates, although the time of the high and low levels in relation to activity patterns is similar.