DNA sequencing of 13 cytokine gene fragments of Aotus infulatus and Saimiri sciureus,two non-human primate models for malaria

Aotus and Saimiri are non-human primate models recommended by the World Health Organization for experimental studies in malaria, especially for vaccine pre-clinical trials. However, research using these primates is hindered by the lack of specific reagents to evaluate immune responses to infection or vaccination. As a step toward developing molecular tools for cytokine expression studies in these species, primer pairs for 18 cytokine gene fragments were designed based on human DNA sequences and used to amplify the corresponding genes in Aotus infulatus and Saimiri sciureus genomic DNA samples. IFNγ, TNFα, LTA, IL2, IL3, IL4, IL5, IL6, IL10, IL12, IL13, CSF2 and TGFβ2 gene fragments were amplified and sequenced. Primer pairs for IL8, IL17, IL18, IL27 and MIF failed to generate amplification products. When compared to the available corresponding human and non-human primate sequences, most – except IL3 and IL4 – showed identity degrees above 90%. Small variations in sequence can help to explain the failure to amplify certain genes or the amplification only at lower annealing temperatures as compared to human DNA samples for several primer pairs. The sequences made available provide the basis for designing molecular tools such as primers for real time PCR specific for A. infulatus and/or S. sciureus.The nucleotide sequences reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned accession numbers DQ985386 to DQ985389, DQ989356 to DQ989369, FJ89020 to FJ89024 and FJ89029.     

ABO blood groups in the primate species of Cebidae from the Amazon region

The ABO blood groups were determined in blood and saliva collected from 40 Aotus infulatus, 74 Saimiri sciureus, and 96 Cebus apella from the Amazonian region along the Tocantins river. Saliva samples were tested for human ABH antigens by a standard hemagglutination inhibition test. Aotus infulatus showed monomorphism, exhibiting only the B blood group. Saimiri sciureus exhibited the A (67) and AB (7) phenotypes. All four phenotypes have been found in C. apella: O (8), A (52), B (19) and AB (17). The observed distribution was as expected assuming Hardy-Weinberg equilibrium. The titers of ABH substances varied among the species and phenotypes. The B-like agglutinogen, common to all New World monkey species tested, was detected in the red blood cells. Sera were used to detect naturally occurring antibodies and the results showed discrepancies between serum and saliva phenotypes in all species studied.     

A survey for helminth parasites in feral New World non-human primate populations and its comparison with parasitological data from man in the region

A survey for helminth parasites in feral New World non-human primates was conducted and compared with parasitological data from man in the region. A total of 835 fecal samples were collected from feral Aotus nancymae, A. vociferans, Saguinus labiatus, S. mystax, Saimiri boliviensis peruviensis, S. sciureus macrodon, Lagothrix lagotricha and Cacajao calvus rubicundus. In addition, adult parasites were collected from necropsies performed on monkeys that died during quarantine and in captivity. Helminth parasites shared by non-human primates and man in Peru were Ancylostoma braziliensis and Ascaris lumbricoides, found in a captive L. lagotricha; Necator americanus, found in a captive C. calvus rubicundus; Hymenolepis diminuta, found in feral Aotus spp. and S. mystax; and a single Trichuris spp. specimen found in a feral S. sciureus macrodon.     

Development of a polymorphic strain of Plasmodium vivax in monkeys.

A strain of Plasmodium vivax from Thailand with a polymorphic repeat unit of the circumsporozoite protein was established in Saimiri sciureus boliviensis and 3 species of Aotus monkeys. All 11 attempts to transmit infection via sporozoite inoculation, 4 times to splenectomized S. sciureus boliviensis, 2 times to splenectomized Aotus nancymai, and 5 times to intact Saimiri monkeys, were successful. Anopheles freeborni, Anopheles stephensi, Anopheles dirus, and Anopheles gambiae mosquitoes were infected by feeding on parasitemic blood from a chimpanzee and an Aotus azarae boliviensis monkey. Our results indicate that this strain may be useful in antisporozoite vaccine trials.     

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.     

Sequence length polymorphisms within primate amelogenin and amelogenin-like genes: usefulness in sex determination

Sequence length polymorphisms between the amelogenin (AMELX) and the amelogenin-like (AMELY) genes both within and between several mammalian species have been identified and utilized for sex determination, species identification, and to elucidate evolutionary relationships. Sex determination via polymerase chain reaction (PCR) assays of the AMELX and AMELY genes has been successful in greater apes, prosimians, and two species of old world monkeys. To date, no sex determination PCR assay using AMELX and AMELY has been developed for new world monkeys. In this study, we present partial AMELX and AMELY sequences for five old world monkey species (Mandrillus sphinx, Macaca nemestrina, Macaca fuscata, Macaca mulatta, and Macaca fascicularis) along with primer sets that can be used for sex determination of these five species. In addition, we compare the sequences we generated with other primate AMELX and AMELY sequences available on GenBank and discuss sequence length polymorphisms and their usefulness in sex determination within primates. The mandrill and four species of macaque all share two similar deletion regions with each other, the human, and the chimpanzee in the region sequenced. These two deletion regions are 176-181 and 8 nucleotides in length. In analyzing existing primate sequences on GenBank, we also discovered that a separate six-nucleotide polymorphism located approximately 300 nucleotides upstream of the 177 nucleotide polymorphism in sequences of humans and chimps was also present in two species of new world monkeys (Saimiri boliviensis and Saimiri sciureus). We designed primers that incorporate this polymorphism, creating the first AMELX and AMELY PCR primer set that has been used successfully to generate two bands in a new world monkey species.     

DNA fingerprinting in three species of neotropical primates

DNA fingerprinting allows the simultaneous detection of a large number of hypervariable loci consisting of highly polymorphic tandem repeat units that are extensively dispersed in the genome. With the 33.6 human minisatellite probe, hypervariable fragments were detected, for the first time, in the genome of three different species of wild-caught neotropical primates: Aotus infulatus, Aotus azarae, and Cebus apella. As in the human, these species were highly polymorphic, showing distinctive, individual-specific patterns. Estimates of relatedness within each group were calculated from interspecific comparisons based on the number of shared fragments between individuals. This work shows that the 33.6 human minisatellite probe can be very useful for increasing our understanding of population dynamics and behavior of these species in their natural habitat. © 1996 Wiley-Liss, Inc.     

Studies on a newly isolated strain of Plasmodium brasilianum in Aotus and Saimiri monkeys and different anophelines

A strain of Plasmodium brasilianum was isolated from an Aotus vociferans monkey from Peru. The parasite readily infected Aotus monkeys from Bolivia and Columbia and Saimiri sciureus monkeys from Peru and Bolivia. Highest level mosquito infections were obtained by feeding on the Saimiri monkeys. The most susceptible mosquito was Anopheles freeborni, followed by Anopheles dirus, Anopheles stephensi, Anopheles gambiae, Anopheles culicifacies, Anopheles maculatus and Anopheles albimanus. Anopheles quadrimaculatus were also susceptible to infection. Degenerating oocysts were observed in An. dirus mosquitoes infected with this parasite. Transmission via the bites of infected An. maculatus mosquitoes was obtained to 3 Bolivian Saimiri monkeys; prepatent periods were 27, 27, and 29 days.     

Hypometabolism in Aotus trivirgatus (Primates, Plathyrhini, Cebidae)]

The standard metabolism of Aotus trivirgatus (Night monkey, Owl monkey) is 22.5 to 46.2 per cent below Kleiber's prevision curve for mammals, which applies to other cebid monkeys like Saimiri sciureus and Alouatta. However the metabolic rate of Aotus is not reduced to the extent found in two hypometabolic prosimians Perodicticus potto and Nycticebus coucang. The low metabolism in Aotus is associated with a normal body temperature and a thick fur of high insulating power. These results are discussed.     

Chromosomal distribution of interstitial telomeric sequences in nine neotropical primates (Platyrrhini): possible implications in evolution and phylogeny

To localize interstitial telomeric sequences (ITSs) and to test whether their pattern of distribution could be linked to chromosomal evolution, we hybridized telomeric sequence probes (peptide nucleic acid, PNA) on metaphases of New World monkeys: Callithrix argentata, Callithrix jacchus, Cebuella pygmaea, Saguinus oedipus, Saimiri sciureus, Aotus lemurinus griseimembra, Aotus nancymaae (Cebidae), Lagothrix lagotricha (Atelidae) and Callicebus moloch (Pithecidae), characterized by a rapid radiation and a high rate of chromosomal rearrangements. Our analysis of the probe signal localization allowed us to show in all the species analysed, as normally, the telomeric location at the terminal ends of chromosomes and unexpected signal distributions in some species. Indeed, in three species among the nine studied, Aotus lemurinus griseimembra, Aotus nancymaae (Cebidae) and Lagothrix lagotricha (Atelidae), we showed a high variability in terms of localization and degree of amplification of interstitial telomeric sequences, especially for the ones found at centromeric or pericentromeric positions (het‐ITS). A comparative analysis, between species, of homologous chromosomes to human syntenies, on which we have found positive interspersed PNA signals, allowed us to explain the observed pattern of ITS distribution as results of chromosomal rearrangements in the neotropical primates analysed. This evidence permitted us to discuss the possible implication of ITSs as phylogenetic markers for closely related species. Moreover, reviewing previous literature data of ITSs distribution in Primates and in the light of our results, we suggest an underestimation of ITSs and highlight the importance of the molecular cytogenetics approach in characterizing ITSs, which role is still not clarified.     

Conversion of AFLP markers to sequence-specific markers for closely related lines in rice by use of the rice genome sequence

DNA polymorphism between two major japonica rice cultivars, Nipponbare and Koshihikari, was identified by AFLP. Eighty-four polymorphic AFLP markers were obtained by analysis with 360 combinations of primer pairs. Nucleotide sequences of 73 markers, 29 from Nipponbare and 44 from Koshihikari, were determined, and 46 AFLP markers could be assigned to rice chromosomes based on sequence homology to the rice genome sequence. Specific primers were designed for amplification of the regions covering the AFLP markers and the flanking sequences. Out of the 46 primer pairs, 44 amplified single DNA fragments, six of which showed different sizes between Nipponbare and Koshihikari, yielding codominant SCAR markers. Eight primer pairs amplified only Nipponbare sequences, providing dominant SCAR markers. DNA fragments amplified by 13 primer pairs showed polymorphism by CAPS, and polymorphism of those amplified by 13 other primer pairs were detected by PCR-RF-SSCP (PRS). Nucleotide sequences of the other four DNA fragments were determined in Koshihikari, but no difference was found between Koshihikari and Nipponbare. In total, 40 sequence-specific markers for the combination of Nipponbare and Koshihikari were produced. All the SNPs identified by AFLP were detectable by CAPS and PRS. The same method was applicable to a combination of Kokoromachi and Tohoku 168, and 23 polymorphic markers were identified using these two rice cultivars. The procedure of conversion of AFLP-markers to the sequence-specific markers used in this study enables efficient sequence-specific marker production for closely related cultivars.     

Anatomical distribution of developing trophozoites and schizonts of Plasmodium vivax in Aotus lemurinus lemurinus and Saimiri sciureus

Organ distribution of developing trophozoite- and schizont-infected erythrocytes of Plasmodium vivax and night monkeys, Aotus lemurinus lemurinus, and squirrel monkeys, Saimiri sciureus, was determined. The primary site for the infection in both species was the splenic vasculature. Secondary organ involvement differed between hosts although some overlapping did occur.     

Multiple methods for the identification of polymorphic simple sequence repeats (SSRs) in sorghum [Sorghum bicolor (L.) Moench]

Simple sequence repeats (SSRs), also known as microsatellites, are highly variable DNA sequences that can be used as markers for the genetic analysis of plants. Three approaches were followed for the development of PCR primers for the amplification of DNA fragments containing SSRs from sorghum [Sorghum bicolor (L.) Moench]: a search for sorghum SSRs in public DNA databases; the use of SSR-specific primers developed in the Poaceae species maize (Zea mays L.) and seashore paspalum grass (Paspalum vaginatum Swartz); and the screening of sorghum genomic libraries by hybridization with SSR oligonucleotides. A total of 49 sorghum SSR-specific PCR primer pairs (two designed from GenBank SSR-containing sequences and 47 from the sequences of genomic clones) were screened on a panel of 17 sorghum and one maize accession. Ten primer pairs from paspalum and 90 from maize were also screened for polymorphism in sorghum. Length polymorphisms among amplification products were detected with 15 of these primer pairs, yielding diversity values ranging from 0.2 to 0.8 with an average diversity of 0.56. These primer pairs are now available for use as markers in crop improvement and conservation efforts.     

Can molecular data place each neotropical monkey in its own branch?

Four different DNA datasets, representative of all extant neotropical primate genera, were tandemly aligned, comprising some 6,763 base pairs (bp) with 2,086 variable characters and 674 informative sites. Maximum Parsimony, Maximum Likelihood and Neighbor-Joining analyses suggested three monophyletic families (Atelidae, Pitheciidae and Cebidae) that emerged almost at the same time during primate radiation. Combined molecular data showed congruent branching inside the atelid clade, placing Alouatta as the most basal lineage followed by Ateles and a more derived branch including Brachyteles and Lagothrix as sister groups. In the Pitheciidae, Callicebus was the most basal lineage with respect to Pithecia and to the more derived sister groups (Cacajao and Chiropotes). Conjoint analysis strongly supported the monophyly of the Cebidae, grouping Aotus, Cebus and Saimiri with the small callitrichines. Within callitrichines, Cebuella merged with Callithrix, Callimico appeared as a sister group of Callithrix/Cebuella, Leontopitecus as a sister group of the previous clade, and Saguinus was the earliest callitrichine offshoot. Two major points remained to be clarified in platyrrhine phylogeny: (i) the exact branching pattern of Aotus, Cebus, Saimiri and the callitrichines, and (ii), which two of these three families (Atelidae, Pitheciidae and Cebidae) are more closely related to one another.     

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.     

Environmental whole-genome amplification to access microbial populations in contaminated sediments

Low-biomass samples from nitrate and heavy metal contaminated soils yield DNA amounts that have limited use for direct, native analysis and screening. Multiple displacement amplification (MDA) using phi29 DNA polymerase was used to amplify whole genomes from environmental, contaminated, subsurface sediments. By first amplifying the genomic DNA (gDNA), biodiversity analysis and gDNA library construction of microbes found in contaminated soils were made possible. The MDA method was validated by analyzing amplified genome coverage from approximately five Escherichia coli cells, resulting in 99.2% genome coverage. The method was further validated by confirming overall representative species coverage and also an amplification bias when amplifying from a mix of eight known bacterial strains. We extracted DNA from samples with extremely low cell densities from a U.S. Department of Energy contaminated site. After amplification, small-subunit rRNA analysis revealed relatively even distribution of species across several major phyla. Clone libraries were constructed from the amplified gDNA, and a small subset of clones was used for shotgun sequencing. BLAST analysis of the library clone sequences showed that 64.9% of the sequences had significant similarities to known proteins, and "clusters of orthologous groups" (COG) analysis revealed that more than half of the sequences from each library contained sequence similarity to known proteins. The libraries can be readily screened for native genes or any target of interest. Whole-genome amplification of metagenomic DNA from very minute microbial sources, while introducing an amplification bias, will allow access to genomic information that was not previously accessible. The reported SSU rRNA sequences and library clone end sequences are listed with their respective GenBank accession numbers, DQ 404590 to DQ 404652, DQ 404654 to DQ 404938, and DX 385314 to DX 389173.     

Polyspecific associations between squirrel monkeys (Saimiri sciureus) and other primates in eastern Amazonia

One of the most common types of polyspecific association observed in Neotropical primate communities is that between squirrel monkeys (Saimiri) and capuchins (Cebus). The present study focused on association patterns in two Saimiri sciureus groups in eastern Brazilian Amazonia, between March and October, 2009. The associations were analyzed in terms of the species involved, the degree of association, and niche breadth and overlap. The study involved two S. sciureus groups (B4 and GI) on the right and left bank of the Tocantins River, respectively, within the area of the Tucuruí reservoir in southeastern Pará. Relations between species were classified as associations (individuals within 50 m and moving in the same direction), and encounters (individuals within 50 m and no coordinated movement). Group B4 was in association with Cebus apella during 100% of monitoring, and with Chiropotes satanas in 20.2%. By contrast, Group GI associated with Cebus 54.8% of the time, and with Chiropotes utahickae 2.5%. Encounters with Alouatta belzebul and Saguinus niger were recorded at both sites, with Aotus azarae and Dasyprocta prymnolopha at B4, and with Callicebus moloch, Dasyproct aleporina, Mazama gouazoubira, and Nasua nasua at GI. Overall, Saimiri had a broader niche than Cebus in terms of vertical spacing and diet, but not for substrate use. This pattern did not appear to be affected by association. While group GI spent significantly (P < 0.05) more time in association with Cebus during the wet season, group B4 associated with Chiropotes more during the dry season. Despite the higher association rates, niche overlap was greater for all variables at B4. This may reflect differences in the ranging and foraging patterns at the two sites, and the varying potential benefits of association for Saimiri.     

Brightness preference in nocturnal and diurnal South American monkeys.

In a test situation where choice was not tied to reinforcement, both squirrel monkeys (Saimiri sciureus) and owl monkeys (Aotus trivirgatus) consistently chose to view the more luminant of a pair of lights. Over a two log unit range of luminance, the degree of preference in both species for the brighter of two lights was found to depend on their difference in luminance and not on their actual luminances.     

Experimental infections of simians with human malaria: attempts to infect Callithrix penicillata with Plasmodium falciparum

After reviewing the use of non-human primates of the Old and New Worlds for human malaria research, we concluded that another experimental animal which is easily available to use and possible to rear indoors is needed. Thus, we studied the susceptibility of the marmoset Callithrix penicillata to Plasmodium falciparum erythrocytic infections. The marmosets received various P. falciparum human isolates, directly from a patient and from continuous cultures. The Palo Alto strain, which has been adapted to the night monkey Aotus trivirgatus and further maintained in the squirrel monkey Saimiri sciureus was also used. In a total of 20 marmosets we performed 31 inoculations, with 10(5) to 10(9) parasites, intraperitoneally, intracardiacly or intravenously. Blood samples from each animal were examined daily up to day 90 post-inoculation. None of the intact marmosets developed patent infections. Four out of 19 C. penicillata, previously splenectomized, showed circulating parasites for up to five days after intravenous inoculation with the Palo Alto strain, becoming negative thereafter. Neither the addition to the simian diet of p-aminobenzoic acid, essential for the parasite metabolism, nor drug-immunosuppression, improved the marmoset susceptibility to P. falciparum.