Anopheles hackeri, a mosquito commonly found breeding in nipa palm leaf bases along the Malayan coast, was demonstrated to be infected with Plasmodium knowlesi by the inoculation of sporozoites into an uninfected rhesus monkey. This was the first demonstration of a natural vector of any monkey malaria.
Purified schizonts (6--10 nuclei) and membranes of schizont-infected erythrocytes from the Malaysian and Philippine strain of Plasmodium knowlesi are analyzed immunochemically using immunoglobulin of rhesus monkey hyperimmune sera against schizonts and of sera from naturally immune monkeys. The anti-schizont Ig identifies less than 20 immune components in Triton X-100-solubilized schizonts and membranes of infected cells. Of these antigens, 9 (component 1, 3, 4, 5, 6, 10, 11, 18, and 20) are common to parasites and membranes of infected erythrocytes, and 12 (2A,B, 6, 8, 9, 12, 13p, 14, 16A,B, 19 A,Bp, 21, 22p, and 23) are predominantly found in the parasite; 4 components (13i, 19A,Bi, 22A, B, and 24) are unique to the membrane of infected erythrocytes. Only three parasite-specific components (1, 13, and 19) are exposed on the surface of parasitized erythrocytes as revealed by both lactoperoxidase-catalyzed radioiodination and extensive absorption of anti-schizont Ig using intact infected erythrocytes. Two plasmodium-specific antigens (1 and 13) on the surface of infected erythrocytes are recognized by sera of rhesus monkeys rendered naturally immune against P. knowlesi infections and, therefore, represent antigens in vivo. Analyses of schizonts and membranes of parasitized erythrocytes of the two different strains of P. knowlesi yields only some minor quantitative, but no qualitative differences when analyzed with both types of antisera. Importantly, components 1 and 13 appear identical in both strains.
In the past decade, many researchers have published papers about hybridization between long-tailed and rhesus macaques. These previous works have proposed unidirectional gene flow with the Isthmus of Kra as the zoogeographical barrier of hybridization. However, these reports analyzed specimens of unknown origin and/or did not include specimens from Thailand, the center of the proposed area of hybridization. Collected specimens of long-tailed and rhesus macaques representing all suspected hybridization areas were examined. Blood samples from four populations each of long-tailed and rhesus macaques inhabiting Thailand, Myanmar, and Laos were collected and analyzed with conspecific references from China (for rhesus macaques) and multiple countries from Sundaic regions (for long-tailed macaques). Ninety-six single nucleotide polymorphism (SNP) markers specifically designed to interrogate admixture and ancestry were used in genotyping. We found genetic admixture maximized at the hybrid zone (15-20°N), as well as admixture signals of varying strength in both directions outside of the hybrid zone. These findings show that the Isthmus of Kra is not a barrier to gene flow from rhesus to long-tailed populations. However, to precisely identify a southernmost barrier, if in fact a boundary rather than simple isolation by distance exists, the samples from peninsular Malaysia must be included in the analysis. Additionally, a long-tailed to rhesus gene flow boundary was found between northern Thailand and Myanmar. Our results suggest that selection of long-tailed and rhesus macaques, the two most commonly used non-human primates for biomedical research, should take into account not only the species identification but also the origin of and genetic admixture within and between the species.
Finite element analysis (FEA) is a commonly used tool in musculoskeletal biomechanics and vertebrate paleontology. The accuracy and precision of finite element models (FEMs) are reliant on accurate data on bone geometry, muscle forces, boundary conditions and tissue material properties. Simplified modeling assumptions, due to lack of in vivo experimental data on material properties and muscle activation patterns, may introduce analytical errors in analyses where quantitative accuracy is critical for obtaining rigorous results. A subject-specific FEM of a rhesus macaque mandible was constructed, loaded and validated using in vivo data from the same animal. In developing the model, we assessed the impact on model behavior of variation in (i) material properties of the mandibular trabecular bone tissue and teeth; (ii) constraints at the temporomandibular joint and bite point; and (iii) the timing of the muscle activity used to estimate the external forces acting on the model. The best match between the FEA simulation and the in vivo experimental data resulted from modeling the trabecular tissue with an isotropic and homogeneous Young's modulus and Poisson's value of 10GPa and 0.3, respectively; constraining translations along X,Y, Z axes in the chewing (left) side temporomandibular joint, the premolars and the m1; constraining the balancing (right) side temporomandibular joint in the anterior-posterior and superior-inferior axes, and using the muscle force estimated at time of maximum strain magnitude in the lower lateral gauge. The relative strain magnitudes in this model were similar to those recorded in vivo for all strain locations. More detailed analyses of mandibular strain patterns during the power stroke at different times in the chewing cycle are needed.
The effect of oxytocin on testicular function was examined in the adult male long-tailed macaques (Macaca fascicularis). The monkeys were either infused with increasing concentrations of synthetic oxytocin (16-128 m.i.u./min for 3 h) or injected daily for a week with the same hormone (20 i.u., i.v.) and the plasma testosterone levels measured. The results of the present study show that acute infusion or chronic injection of oxytocin does not significantly affect the plasma testosterone levels, suggesting that systemic control of testicular endocrine function by oxytocin may be unimportant.
Plasmodium knowlesi poses a health threat throughout Southeast Asian communities and currently causes most cases of malaria in Malaysia. This zoonotic parasite species has been studied in Macaca mulatta (rhesus monkeys) as a model for severe malarial infections, chronicity, and antigenic variation. The phenomenon of Plasmodium antigenic variation was first recognized during rhesus monkey infections. Plasmodium-encoded variant proteins were first discovered in this species and found to be expressed at the surface of infected erythrocytes, and then named the Schizont-Infected Cell Agglutination (SICA) antigens. SICA expression was shown to be spleen dependent, as SICA expression is lost after P. knowlesi is passaged in splenectomized rhesus. Here we present data from longitudinal P. knowlesi infections in rhesus with the most comprehensive analysis to date of clinical parameters and infected red blood cell sequestration in the vasculature of tissues from 22 organs. Based on the histopathological analysis of 22 tissue types from 11 rhesus monkeys, we show a comparative distribution of parasitized erythrocytes and the degree of margination of the infected erythrocytes with the endothelium. Interestingly, there was a significantly higher burden of parasites in the gastrointestinal tissues, and extensive margination of the parasites along the endothelium, which may help explain gastrointestinal symptoms frequently reported by patients with P. knowlesi malarial infections. Moreover, this margination was not observed in splenectomized rhesus that were infected with parasites not expressing the SICA proteins. This work provides data that directly supports the view that a subpopulation of P. knowlesi parasites cytoadheres and sequesters, likely via SICA variant antigens acting as ligands. This process is akin to the cytoadhesive function of the related variant antigen proteins, namely Erythrocyte Membrane Protein-1, expressed by Plasmodium falciparum.
This report summarizes the results of a comparative study of the virulence of the "S-M," H, and C strains of P. knowlesi for Indian rhesus monkeys (Macaca mulatta) and cynomolgus monkeys [M. irus (fascicularis)] of Malayan (West Malaysia) and Philippine origins. Each of the above strains produced fulminating, uniformly fatal infections in the rhesus monkey and mild, chronic infections, characterized by relatively low level parasitemias in cynomolgus monkeys of Philippine origin. In striking contrast, the H and C strains produced infections in cynomolgus monkeys of Malayan origin which were indistinguishable in severity from infections produced in M. mulatta. The circumstances of the study precluded evaluation of the virulence of the "S-M" strain for M. irus of Malayan origin. Even so, the available data make it necessary to qualify the long-held belief that infections with P. knowlesi in M. irus invariably follow a benign course.
BACKGROUND: Most cynomolgus macaques (Macaca fascicularis) used in the United States as animal models are imported from Chinese breeding farms without documented ancestry. Cynomolgus macaques with varying rhesus macaque ancestry proportions may exhibit differences, such as susceptibility to malaria, that affect their suitability as a research model.
METHODS: DNA of 400 cynomolgus macaques from 10 Chinese breeding farms was genotyped to characterize their regional origin and rhesus ancestry proportion. A nested PCR assay was used to detect Plasmodium cynomolgi infection in sampled individuals.
RESULTS: All populations exhibited high levels of genetic heterogeneity and low levels of inbreeding and genetic subdivision. Almost all individuals exhibited an Indochinese origin and a rhesus ancestry proportion of 5%-48%. The incidence of P. cynomolgi infection in cynomolgus macaques is strongly associated with proportion of rhesus ancestry.
CONCLUSIONS: The varying amount of rhesus ancestry in cynomolgus macaques underscores the importance of monitoring their genetic similarity in malaria research.
Mirror neurons are visuo-motor neurons found in primates and thought to be significant for imitation learning. The proposition that mirror neurons result from associative learning while the neonate observes his own actions has received noteworthy empirical support. Self-exploration is regarded as a procedure by which infants become perceptually observant to their own body and engage in a perceptual communication with themselves. We assume that crude sense of self is the prerequisite for social interaction. However, the contribution of mirror neurons in encoding the perspective from which the motor acts of others are seen have not been addressed in relation to humanoid robots. In this paper we present a computational model for development of mirror neuron system for humanoid based on the hypothesis that infants acquire MNS by sensorimotor associative learning through self-exploration capable of sustaining early imitation skills. The purpose of our proposed model is to take into account the view-dependency of neurons as a probable outcome of the associative connectivity between motor and visual information. In our experiment, a humanoid robot stands in front of a mirror (represented through self-image using camera) in order to obtain the associative relationship between his own motor generated actions and his own visual body-image. In the learning process the network first forms mapping from each motor representation onto visual representation from the self-exploratory perspective. Afterwards, the representation of the motor commands is learned to be associated with all possible visual perspectives. The complete architecture was evaluated by simulation experiments performed on DARwIn-OP humanoid robot.
Despite increasing conflict at human-wildlife interfaces, there exists little research on how the attributes and behavior of individual wild animals may influence human-wildlife interactions. Adopting a comparative approach, we examined the impact of animals' life-history and social attributes on interactions between humans and (peri)urban macaques in Asia. For 10 groups of rhesus, long-tailed, and bonnet macaques, we collected social behavior, spatial data, and human-interaction data for 11-20 months on pre-identified individuals. Mixed-model analysis revealed that, across all species, males and spatially peripheral individuals interacted with humans the most, and that high-ranking individuals initiated more interactions with humans than low-rankers. Among bonnet macaques, but not rhesus or long-tailed macaques, individuals who were more well-connected in their grooming network interacted more frequently with humans than less well-connected individuals. From an evolutionary perspective, our results suggest that individuals incurring lower costs related to their life-history (males) and resource-access (high rank; strong social connections within a socially tolerant macaque species), but also higher costs on account of compromising the advantages of being in the core of their group (spatial periphery), are the most likely to take risks by interacting with humans in anthropogenic environments. From a conservation perspective, evaluating individual behavior will better inform efforts to minimize conflict-related costs and zoonotic-risk.
In 1960, American parasitologist Don Eyles was unexpectedly infected with a malariaparasite isolated from a macaque. He and his supervisor, G. Robert Coatney of the National Institutes of Health, had started this series of experiments with the assumption that humans were not susceptible to "monkey malaria." The revelation that a mosquito carrying a macaque parasite could infect a human raised a whole range of public health and biological questions. This paper follows Coatney's team of parasitologists and their subjects: from the human to the nonhuman; from the American laboratory to the forests of Malaysia; and between the domains of medical research and natural history. In the course of this research, Coatney and his colleagues inverted Koch's postulate, by which animal subjects are used to identify and understand human parasites. In contrast, Coatney's experimental protocol used human subjects to identify and understand monkey parasites. In so doing, the team repeatedly followed malaria parasites across the purported boundary separating monkeys and humans, a practical experience that created a sense of biological symmetry between these separate species. Ultimately, this led Coatney and his colleagues make evolutionary inferences, concluding "that monkeys and man are more closely related than some of us wish to admit." In following monkeys, men, and malaria across biological, geographical, and disciplinary boundaries, this paper offers a new historical narrative, demonstrating that the pursuit of public health agendas can fuel the expansion of evolutionary knowledge.
A vaccine against human enterovirus 71 (EV-A71) is urgently needed to combat outbreaks of EV-A71 and in particular, the serious neurological complications that manifest during these outbreaks. In this study, an EV-A71 virus-like-particle (VLP) based on a B5 subgenogroup (EV-A71-B5 VLP) was generated using an insect cell/baculovirus platform. Biochemical analysis demonstrated that the purified VLP had a highly native procapsid structure and initial studies in vivo demonstrated that the VLPs were immunogenic in mice. The impact of VLP immunization on infection was examined in non-human primates using a VLP prime-boost strategy prior to EV-A71 challenge. Rhesus macaques were immunized on day 0 and day 21 with VLPs (100 μg/dose) containing adjuvant or with adjuvant alone (controls), and were challenged with EV-A71 on day 42. Complete blood counts, serum chemistry, magnetic resonance imaging (MRI) scans, and histopathology results were mostly normal in vaccinated and control animals after virus challenge demonstrating that the fatal EV-A71-B3 clinical isolate used in this study was not highly virulent in rhesus macaques. Viral genome and/or infectious virus were detected in blood, spleen or brain of two of three control animals, but not in any specimens from the vaccinated animals, indicating that VLP immunization prevented systemic spread of EV-A71 in rhesus macaques. High levels of IgM and IgG were detected in VLP-vaccinated animals and these responses were highly specific for EV-A71 particles and capsid proteins. Serum from vaccinated animals also exhibited similar neutralizing activity against different subgenogroups of EV-A71 demonstrating that the VLPs induced cross-neutralizing antibodies. In conclusion, our EV-A71-B5 VLP is safe, highly immunogenic, and prevents systemic EV-A71-B3 infection in nonhuman primates making it a viable attractive vaccine candidate for EV-A71.
Studies of optical defocus on refractive development and ocular growth in animals are presented and discussed in relation to the accommodation hypothesis. None of these studies fully support the accommodation hypothesis. The problems encountered in these studies are also discussed.
Relatively few cases of myocardial infarction associated with coronary artery atherosclerosis have been described previously in macaques. In this study the authors report the prevalence and characteristics of coronary artery atherosclerosis and myocardial infarction in 10 rhesus (Macaca mulatta) and two cynomolgus (Macaca fascicularis) macaques that were fed atherogenic diets for 16 months or longer. Our findings show clearly that myocardial infarction occurs in macaques with diet-induced atherosclerosis. The frequency seems to be related to the species, composition of the atherogenic diet, and length of time fed the atherogenic diet. The myocardial lesions are remarkably similar to those described in human beings in terms of location and gross and microscopic characteristics. The characteristics of coronary artery atherosclerosis, including the occurrence of thrombosis, severe stenosis, mineralization, atheronecrosis, and sterol clefts, especially in animals fed the atherogenic diets for longer periods of time, also closely resemble those of the arterial lesions found in human beings. The greatest prevalence of myocardial infarcts was found in rhesus monkeys fed a cholesterol-containing diet with 40% of calories supplied by peanut oil and in cynomolgus macaques from Malaya that were fed the same amount of cholesterol with 40% of calories from lard. Electrocardiographic abnormalities as well as the occurrence of unexpected and relatively sudden death in several of these nonhuman primates are also consistent with signs frequently observed in human beings.
The dominant cause of malaria in Malaysia is now Plasmodium knowlesi, a zoonotic parasite of cynomolgus macaque monkeys found throughout South East Asia. Comparative genomic analysis of parasites adapted to in vitro growth in either cynomolgus or human RBCs identified a genomic deletion that includes the gene encoding normocyte-binding protein Xa (NBPXa) in parasites growing in cynomolgus RBCs but not in human RBCs. Experimental deletion of the NBPXa gene in parasites adapted to growth in human RBCs (which retain the ability to grow in cynomolgus RBCs) restricted them to cynomolgus RBCs, demonstrating that this gene is selectively required for parasite multiplication and growth in human RBCs. NBPXa-null parasites could bind to human RBCs, but invasion of these cells was severely impaired. Therefore, NBPXa is identified as a key mediator of P. knowlesi human infection and may be a target for vaccine development against this emerging pathogen.
Dengue diseases have an economic as well as social burden worldwide. In this study, the antiviral activity of protegrin-1 (PG-1, RGGRLCYCRRRFCVCVGR) peptide towards dengue NS2B-NS3pro and viral replication in Rhesus monkey kidney (MK2) cells was investigated. The peptide PG-1 was synthesized by solid-phase peptide synthesis, and disulphide bonds formation followed by peptide purification was confirmed by LC-MS and RPHPLC. Dengue NS2B-NS3pro was produced as a single-chain recombinant protein in E. coli. The NS2B-NS3pro assay was carried out by measuring the florescence emission of catalyzed substrate. Real-time PCR was used to evaluate the inhibition potential of PG-1 towards dengue serotype-2 (DENV-2) replication in MK2 cells. The results showed that PG-1 inhibited dengue NS2B-NS3pro at IC(50) of 11.7 μM. The graded concentrations of PG-1 at nontoxic range were able to reduce viral replication significantly (P < 0.001) at 24, 48, and 72 hrs after viral infection. However, the percentage of inhibition was significantly (P < 0.01) higher at 24 hrs compared to 48 and 72 hrs. These data show promising therapeutic potential of PG-1 against dengue infection, hence it warrants further analysis and improvement of the peptide features as a prospective starting point for consideration in designing attractive dengue virus inhibitors.
Several critical factors of an influenza microneutralization assay, utilizing a rapid biotin-streptavidin conjugated system for detecting influenza virus subtypes A and B, are addressed within this manuscript. Factors such as incubation times, amount of virus, cell seeding, sonication, and TPCK trypsin were evaluated for their ability to affect influenza virus neutralization in a microplate-based neutralization assay using Madin-Darby canine kidney (MDCK) cells. It is apparent that the amount of virus used in the assay is the most critical factor to be optimized in an influenza microneutralization assay. Results indicate that 100xTCID(50) of influenza A/Solomon Islands/03/2006 (H1N1) virus overloads the assay and results in no, to low, neutralization, in both ferret and macaque sera, respectively, whereas using 6xTCID(50) resulted in significantly improved neutralization. Conversely, strong neutralization was observed against 100xTCID(50) of B/Malaysia/2506/04 virus. In this manuscript the critical factors described above were optimized and the results indicate that the described biotin-streptavidin conjugated influenza microneutralization assay is a rapid and robust method for detecting the presence of functional, influenza virus-neutralizing antibodies.
Although divergent dengue viruses (DENVs) have been isolated in insects, nonhuman primates, and humans, their relationships to the four canonical serotypes (DENV 1-4) are poorly understood. One virus isolated from a dengue patient, DKE-121, falls between genotype and serotype levels of sequence divergence to DENV-4. To examine its antigenic relationship to DENV-4, we assessed serum neutralizing and protective activity. Whereas DENV-4-immune mouse sera neutralize DKE-121 infection, DKE-121-immune sera inhibit DENV-4 less efficiently. Passive transfer of DENV-4 or DKE-121-immune sera protects mice against homologous, but not heterologous, DENV-4 or DKE-121 challenge. Antigenic cartography suggests that DENV-4 and DKE-121 are related but antigenically distinct. However, DENV-4 vaccination confers protection against DKE-121 in nonhuman primates, and serum from humans immunized with a tetravalent vaccine neutralize DENV-4 and DKE-121 infection equivalently. As divergent DENV strains, such as DKE-121, may meet criteria for serotype distinction, monitoring their capacity to impact dengue disease and vaccine efficacy appears warranted.
Dengue virus (DENV) is considered to be the most important arthropod-borne viral disease and causes more than 100 million human infections annually. To further characterize primary DENV infection in vivo, rhesus macaques were infected with DENV-1, DENV-2, DENV-3, or DENV-4 and clinical parameters, as well as specificity and longevity of serologic responses, were assessed. Overt clinical symptoms were not present after infection. However, abnormalities in blood biochemical parameters consistent with heart, kidney, and liver damage were observed, and changes in plasma fibrinogen, D-dimers, and protein C indicated systemic activation of the blood coagulation pathway. Significant homotypic and heterotypic serum immunoglobulins were present in all animals, and IgG persisted for at least 390 days. Serum neutralizing antibody responses were highly serotype specific by day 120. However, some heterotypic neutralizing activity was noted in infected animals. Identification of serotype-specific host responses may help elucidate mechanisms that mediate severe DENV disease after reinfection.
Four nucleic acid extraction protocols were examined for their suitability for extraction of the ssRNA, dsRNA and dsDNA genomes of gastroenteritis viruses, for PCR detection. Protocol (A), employed specimen lysis with guanidinium thiocyanate, extraction with phenol-chloroform-isoamyl alcohol and nucleic acid purification by size-fractionated silica particles. Protocol (B), utilised specimen lysis with guanidinium thiocyanate and nucleic acid purification by silica, followed by phenol-chloroform-isoamyl alcohol extraction. Protocol (C), employed specimen lysis with guanidinium thiocyanate and nucleic acid purification by RNAID glass powder. Protocol (D), employed specimen lysis with sodium dodecyl sulphate, proteinase K digestion and extraction with phenol-chloroform-isoamyl alcohol. Of the four protocols, (B) appeared to be a suitable candidate 'universal' nucleic acid extraction procedure for PCR detection of different viral agents of gastroenteritis in a single nucleic acid extract of a faecal specimen, irrespective of genome composition. Omission of the phenol-chloroform extraction step did not affect negatively the ability of protocol (B) to allow PCR detection of gastroenteritis viruses in faecal specimens. PCR detection of NLVs, astroviruses, rotaviruses and adenoviruses, in single nucleic acid extracts of faecal specimens obtained from the field, confirmed the universality of the modified protocol (B). We propose the modified protocol (B) as a 'universal' nucleic acid extraction procedure, for monoplex PCR detection of gastroenteritis viruses in single nucleic acid extracts of faecal specimens and for development of multiplex PCR for their simultaneous detection.