Comparative studies with six extenders for sperm cryopreservation in the cynomolgus monkey (Macaca fascicularis) and rhesus monkey (Macaca mulatta)

Ejaculated spermatozoa from cynomolgus monkeys and rhesus monkeys were frozen in straws with six different extenders (TTE, DM, mDM, LG-DM, G-DM, and TCG) containing glycerol. Sperm motility and head membrane and acrosomal integrity were evaluated after freezing and thawing, and the cryoprotective effects were compared among the extenders and the two species studied. The results showed that sperm motility and motility recovery with the six extenders were comparable for the cynomolgus and rhesus monkeys. There was no significant difference in sperm motility and head membrane integrity among the six extenders in either the cynomolgus or rhesus monkeys (P>0.05). However, a slightly but statistically lower percentage of acrosomal integrity was found with TCG in both species compared to the other extenders (P<0.05). These findings demonstrate that TTE, DM, mDM, LG-DM, G-DM, and TCG are equally suitable extenders for the cryopreservation of spermatozoa from cynomolgus and rhesus monkeys.     

Responses of Leishmania (Viannia) braziliensis cutaneous infection to N-methylglucamine antimoniate in the rhesus monkey (Macaca mulatta) model

The antileishmanial efficacy of the reference drug N-methylglucamine antimoniate (Glucantime) was evaluated in groups of rhesus monkeys with acute and chronic Leishmania (Viannia) braziliensis cutaneous infection. The therapeutic responses in experimental animals to either a low dose (5 mg/kg body wt/day for 28 days) or a routine dose (20 mg/kg/day for 28 days) of pentavalent antimony were similar to those reported in the human disease. Primates were cured of their lesions after treatment, but with cryptic parasitism and/or relapse. The rhesus model of L. (V.) braziliensis cutaneous leishmaniasis therefore provides an additional resource for preclinical trials with newer drugs.     

Comparison of experimental Rickettsia tsutsugamushi infections in silvered leaf (Presbytis cristatus) and cynomolgus (Macaca fascicularis) monkeys.

Both silvered leaf and cynomolgus monkeys were infected with the Gilliam, Karp and Kato strains of Rickettsia tsutsugamushi. The two species developed similar clinical syndromes, but the antibody responses were greater in cynomolgus monkeys. In both species of monkeys, the Gilliam strain induced more severe clinical manifestations. At 10 months post-infection, silvered leaf monkeys were immune to homologous intradermal (id) challenge. Cynomolgus monkeys, at 15 months post-infection, were relatively resistant to homologous intravenous challenge, but not to a homologous or heterologous id challenge.     

Aerosol exposure to Zaire ebolavirus in three nonhuman primate species: differences in disease course and clinical pathology

There is little known concerning the disease caused by Zaire ebolavirus (ZEBOV) when inhaled, the likely route of exposure in a biological attack. Cynomolgus macaques, rhesus macaques, and African green monkeys were exposed to aerosolized ZEBOV to determine which species might be the most relevant model of the human disease. A petechial rash was noted on cynomolgus and rhesus macaques after fever onset but not on African green monkeys. Fever duration was shortest in rhesus macaques (62.7 ± 16.3 h) and longest in cynomolgus macaques (82.7 ± 22.3 h) and African green monkeys (88.4 ± 16.7 h). Virus was first detectable in the blood 3 days after challenge; the level of viremia was comparable among all three species. Hematological changes were noted in all three species, including decreases in lymphocyte and platelet counts. Increased blood coagulation times were most pronounced in African green monkeys. Clinical signs and time to death in all three species were comparable to what has been reported previously for each species after parenteral inoculation with ZEBOV. These data will be useful in selection of an animal model for efficacy studies.     

Urinary cyclic AMP excretion is increased in Macaca fascicularis and Macaca mulatta compared to humans

Changes in parathyroid hormone and its second messenger cyclic AMP have been implicated in the pathogenesis of osteoporosis. Nonhuman primate models have been useful in the study of osteoporosis, but the physiology of mineral metabolism in certain species is different than in humans. We investigated parameters of mineral metabolism in 15 normal adult female cynomolgus and 14 normal adult female rhesus monkeys. In both species, urinary cyclic AMP was increased compared with humans, and the nephrogenous cyclic AMP in the cynomolgus monkeys was also elevated. Despite this, there was no evidence for hyperparathyroidism in either species as evaluated by serum or plasma phosphorus and midregion-specific and/or aminoterminal-specific immunoreactive parathyroid hormone. Given the increasing use of nonhuman primates in the study of osteoporosis, understanding basic changes in mineral metabolism is important before pathologic effects of bone loss can be understood.     

Metagenomic comparison of the rectal microbiota between rhesus macaques(Macaca mulatta) and cynomolgus macaques(Macaca fascicularis)

Rhesus macaques(Macaca mulatta) and cynomolgus macaques(Macaca fascicularis) are frequently used in establishing animal models for human diseases. To determine the differences in gut microbiota between these species, rectal swabs from 20 rhesus macaques and 21 cynomolgus macaques were collected, and the microbial composition was examined by deep sequencing of the 16 S rR NA gene. We found that the rectal microbiota of cynomolgus macaques exhibited significantly higher alpha diversity than that of rhesus macaques, although the observed number of operational taxonomic units(OTUs) was almost the same. The dominant taxa at both the phylum and genus levels were similar between the two species, although the relative abundances of these dominant taxa were significantly different between them. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States(PICRUSt) showed significant differences in the functional components between the microbiota of the two species, in particular the lipopolysaccharide(LPS) synthesis proteins. The above data indicated significant differences in microbial composition and function between these two closely related macaque species, which should be taken into consideration in the future selection of these animals for disease models.     

Outbreak of Mycobacterium bovis in a conditioned colony of rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) macaques

We describe a tuberculosis outbreak caused by Mycobacterium bovis in a conditioned colony of rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) macaques. Animals in five rooms were exposed, but most (16/27) infections were confined to the room that housed a mixed population of cynomolgus and rhesus macaques. In this room, rhesus (8/8) and cynomolgus (10/11) macaques naturally exposed to M. bovis were infected at nearly identical rates (Fisher exact test, 2-tailed P = 1). The clinical signs of disease and pathologic lesions in infected macaques, however, were moderately different between the two species. Rhesus macaques were more likely (5/8) to exhibit clinical signs of persistent coughing and inappetance, and had more severe pulmonary lesions. By contrast, clinical signs of disease were seen in only 1 of 19 cynomolgus macaques, and overall, the pulmonary lesions were often focal and less severe, although some still had severe involvement of the lungs similar to that seen in rhesus macaques. These differences should be taken into consideration when developing or evaluating a tuberculosis-screening program. On the basis of observations made during this outbreak, we recommend that alternative screening methods such as the PRIMAGAM test and the ESAT-6 ELISA, be incorporated into the screening program to aid in the identification of infected animals.     

Viremia and antibody response in green monkeys (Chlorocebus aethiops sabaeus) infected with dengue virus type 2: A potential model for vaccine testing

The increasingly limited availability and high cost of the hitherto most commonly used monkey species in dengue vaccine research has augmented the importance of identifying alternative suitable models for these studies. In this study we examined the capacity of green monkeys ( Chlorocebus aethiops sabaeus ) to develop dengue viremia, and thus provide a potential model for dengue vaccine testing. Monkeys were inoculated with two different doses of dengue virus type 2. All animals in both groups became viremic after inoculation of the virus. In the lower dose group, mean viremia duration of 5.66 days was detected, whereas in the group that received the 10⁶ PFU dose, viremia had a mean duration of only 1.66 days. Antibody titers were similar to those obtained in previous experiments with rhesus and cynomolgus macaques. We conclude that green monkeys develop viremia and antibody responses and therefore provide a potential model for the preclinical evaluation of novel candidates for dengue vaccines.     

Biologic data of Macaca mulatta, Macaca fascicularis, and Saimiri sciureus used for research at the Fiocruz primate center

Physiological parameters of laboratory animals used for biomedical research is crucial for following several experimental procedures. With the intent to establish baseline biologic parameters for non-human primates held in closed colonies, hematological and morphometric data of captive monkeys were determined. Data of clinically healthy rhesus macaques (Macaca mulatta), cynomolgus monkeys (Macaca fascicularis), and squirrel monkeys (Saimiri sciureus) were collected over a period of five years. Animals were separated according to sex and divided into five age groups. Hematological data were compared with those in the literature by Student's t test. Discrepancies with significance levels of 0.1, 1 or 5% were found in the hematological studies. Growth curves showed that the sexual dimorphism of rhesus monkeys appeared at an age of four years. In earlier ages, the differences between sexes could not be distinguished (p < 0.05). Sexual dimorphism in both squirrel monkeys and cynomolgus monkeys occurred at an age of about 32 months. Data presented in this paper could be useful for comparative studies using primates under similar conditions.     

Establishment of a B-lymphoblastoid cell line infected with Epstein-Barr-related virus from a cynomolgus monkey (Macaca fascicularis)

A B-lymphoblastoid cell line (Ts-B) was established from lymph nodes of an apparently healthy cynomolgus monkey. The cells were demonstrated to contain Epstein-Barr virus-related antigens. Moreover, herpesvirus particles were found in the cells by electron microscopy. The cell-free culture medium transformed lymphocytes of cynomolgus rhesus monkeys, and of Japanese monkeys.     

A Flexible Restraint Chair for the Cynomolgus Monkey (Macaca fascicularis)1

We have found that commercial restraint chairs suitable for the rhesus monkey (Macaca mulatta) cause severe physical distress to many cynomolgus monkeys (Macaca fascicularis). A new restraint chair has therefore been designed specifically for the cynomolgus. The key features of the chair are an angled neck piece, a large opening to provide free movement for the tail, a soft waist restraint, and a level surface for both the heels and ischial pads. This new chair is not only suitable for the cynomolgus monkey but may also be more comfortable for the rhesus monkey than standard commercial chairs.     

Transmission of Sarcocystis suihominis from humans to swine to nonhuman primates (Pan troglodytes, Macaca mulatta, Macaca irus).

Sporocysts of Sarcocystis suihominis obtained from human feces were used to infect swine. Heart, tongue, and skeletal muscle from experimentally infected and noninfected control swine were fed via stomach tube to nonhuman primates including chimpanzees (Pan troglodytes), rhesus monkeys (Macaca mulatta), and cynomolgus monkeys (Macaca irus). All primates fed infected swine tissues shed sporocysts beginning 13 to 15 days postinfection and were still shedding sporocysts at the conclusion of the experiment, 30 days postinfection. Rhesus and cynomolgus monkeys were fed infected swine tissues a second time and shed sporocysts. All primates remained in good health throughout both experiments and exhibited no unusual clinical signs as a result of infection.     

Enzootic hepatitis A infection in cynomolgus monkeys (Macaca fascicularis)

During a toxicology study in cynomolgus (long-tailed or crab-eating) monkeys (Macaca fascicularis), a randomly distributed incidence of significantly increased hepatic enzyme activity was observed. Premedication hepatic enzyme activity in all monkeys of this study was normal, but increased alanine aminotransferase (ALT) activity was found in 4 of the 24 animals 2 weeks after initiation of the study and in 10 of 24 at 4 weeks. A drug-related effect was considered unlikely initially because the increases were not doserelated, and a 3-year review of 655 cynomolgus monkeys revealed a 15–20% incidence of increased hepatic enzyme activity. Good correlation was subsequently established between increased hepatic enzyme activity, active hepatitis A virus (HAV) infection, and histomorphologic confirmation of hepatitis (chronic periportal inflammation). Follow-up viral serodiagnostic screening of resident macaques revealed an overall incidence of anti-HAV IgG in 80% (155/193) of cynomolgus and in 70% (14/20) of rhesus monkeys. Serial screening demonstrated that several initially negative monkeys became seropositive for anti-HAV IgG, and a few acquired active infection (anti-HAV IgM). Among newly acquired cynomolgus monkeys, 2.5% (2/80) had an acute HAV infection, and 35% (28/80) eventually tested positive for anti-HAV IgG while quarantined in the primate facility. The characterization of an enzootic HAV infection in incoming monkeys posed a significant risk for the primate colony and handlers. Rigorous sanitation, isolation, and quarantine procedures, including personnel training and additional protective clothing for personnel working in the primate colony, reduced tho potential for transmission and arrested the outbreak. Experimenters should be cautious in ascribing toxicity to a test article based solely on increased hepatic enzyme activity associated with chronic periportal inflammation.     

Coxiella burnetii stimulates production of RANTES and MCP-1 by mononuclear cells: modulation by adhesion to endothelial cells and its implication in Q fever

Q fever is an infectious disease caused by Coxiella burnetii, which may become chronic when cytokine network and cell-mediated immune responses are altered. Chemokines, such as Regulated upon Activation, Normal T cell Expressed and Secreted (RANTES, CCL5) and Monocyte Chemoattractant Protein-1 (MCP-1, CCL2), are specialized in the trafficing of peripheral blood mononuclear cells (PBMC), and are associated with T cell polarization that is essential for intracellular survival of C. burnetii. The present study investigated whether or not the infection status (no infection and acute or chronic infection with C. burnetii) of donors, affected the production of the two chemokines by PBMC with or without stimulation with virulent and avirulent C. burnetii. Our findings indicate that in vitro exposure to virulent or avirulent C. burnetii stimulated the production of RANTES and MCP-1 in PBMC obtained from healthy adults. The co-cultivation of endothelial cells and human PBMC resulted in an increased production of MCP-1 and the up-regulation of RANTES, which were contact-dependent. Unstimulated PBMC from patients with acute or chronic Q fever overproduced MCP-1. Interestingly, the addition of C. burnetii resulted in an increased production of RANTES and MCP-1 by PBMC obtained from patients with chronic Q fever, and the co-cultivation of PBMC with endothelial cells amplified increased production of chemokines. Circulating levels of RANTES and MCP-1 were also increased in chronic Q fever. We suggest that the overproduction of RANTES and MCP-1 secondary to the contact of PBMC with endothelium may perpetuate exaggerated inflammatory responses leading to inappropriate PBMC trafficking and to the pathogenesis of Q fever.     

Study of the safety,immunogenicity and efficacy of attenuated and killed Leishmania (Leishmania) major vaccines in a rhesus monkey (Macaca mulatta) model of the human disease

We have compared the efficacy of two Leishmania (Leishmania) major vaccines, one genetically attenuated (DHFR-TS deficient organisms), the other inactivated [autoclaved promastigotes (ALM) with bacillus Calmete-Guérin (BCG)], in protecting rhesus macaques (Macaca mulatta) against infection with virulent L. (L.) major. Positive antigen-specific recall proliferative response was observed in vaccinees (79% in attenuated parasite-vaccinated monkeys, versus 75% in ALM-plus-BCG-vaccinated animals), although none of these animals exhibited either augmented in vitro gamma interferon (IFN-gamma) production or positive delayed-type hypersensitivity (DTH) response to the leishmanin skin test prior to the challenge. Following challenge, there were significant differences in blastogenic responses (p < 0.05) between attenuated-vaccinated monkeys and na?ve controls. In both vaccinated groups very low levels of antibody were found before challenge, which increased after infective challenge. Protective immunity did not follow vaccination, in that monkeys exhibited skin lesion at the site of challenge in all the groups. The most striking result was the lack of pathogenicity of the attenuated parasite, which persisted in infected animals for up to three months, but were incapable of causing disease under the conditions employed. We concluded that both vaccine protocols used in this study are safe in primates, but require further improvement for vaccine application.     

Effects of X-irradiation on the immune response of guinea pigs to Q fever vaccine

The immune response of guinea pigs to Q fever vaccine following 75 to 250 R (60 to 180 rads) of acute whole-body irradiations was investigated. Complement-fixing (CF) antibody titers and protection against febrile response to challenge with virulent Coxiella burnetii were studied. Exposures ranging from 75 to 250 R, 24 hours prior to inoculation, did not detectably alter the CF antibody response. Similar results were observed with 175 R delivered 48 or 72 hours before immunization. Protection against febrile response to challenge with 10(3) median fever doses of C. burnetii was seen in animals irradiated with 175 R, 24 or 72 hours before immunization. Significant protection was detectable at 14, 21, and 42 days after immunization in both irradiated and nonirradiated animals. Acute irradiation of the degree studied increases the mortality in normal animals infected 15 to 17 days later with virulent C. burnetii. The lethal effect could be prevented by use of Q fever vaccine.     

Single‐Nucleotide Polymorphisms Reveal Patterns of Allele Sharing Across the Species Boundary Between Rhesus (Macaca mulatta) and Cynomolgus (M. fascicularis) Macaques

Both phenotypic and genetic evidence for asymmetric hybridization between rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) macaques has been observed in the region of Indochina where both species are sympatric. The large‐scale sharing of major histocompatibility complex (MHC) class II alleles between the two species in this region supports the hypothesis that genes, and especially genes involved in immune response, are being transferred across the species boundary. This differential introgression has important implications for the incorporation of cynomolgus macaques of unknown geographic origin in biomedical research protocols. Our study found that for 2,808 single‐nucleotide polymorphism (SNP) markers, the minor allele frequencies (MAF) and observed heterozygosity calculated from a sample of Vietnamese cynomolgus macaques was significantly different from those calculated from samples of both Chinese rhesus and Indonesian cynomolgus macaques. SNP alleles from Chinese rhesus macaques were overrepresented in a sample of Vietnamese cynomolgus macaques relative to their Indonesian conspecifics and located in genes functionally related to the primary immune system. These results suggest that Indochinese cynomolgus macaques represent a genetically and immunologically distinct entity from Indonesian cynomolgus macaques. Am. J. Primatol. 75:135‐144, 2013. © 2012 Wiley Periodicals, Inc.     

Bone Marrow Depletion as a Coincidental Finding to Hypothermia in Macaca mulatta

Three of 16 juvenile rhesus monkeys (Macaca mulatta) and 1 rhesus of 79 adult rhesus and cynomolgus monkeys (Macaca fascicularis) were found comatose in a state of profound hypothermia after a heating failure occurred in the room in which they were housed. One juvenile monkey died shortly thereafter. The three other monkeys were revived with gradual warming and supportive therapy but later experienced separate acute clinical crises manifesting shock and died at 19, 31, and 51 days after the initial episode. Histopathologic findings of severe bone marrow depletion were observed in each of the three monkeys that died after the initial episode.     

Whole-genome sequencing and analysis of the Malaysian cynomolgus macaque (Macaca fascicularis) genome

ABSTRACT: BACKGROUND: The genetic background of the cynomolgus macaque (Macaca fascicularis) is made complex by the high genetic diversity, population structure, and gene introgression from the closely related rhesus macaque (Macaca mulatta). Herein we report the whole-genome sequence of a Malaysian cynomolgus macaque male with more than 40-fold coverage, which was determined using a resequencing method based on the Indian rhesus macaque genome. RESULTS: We identified approximately 9.7 million single nucleotide variants (SNVs) between the Malaysian cynomolgus and the Indian rhesus macaque genomes. Compared with humans, a smaller nonsynonymous/synonymous SNV ratio in the cynomolgus macaque suggests more effective removal of slightly deleterious mutations. Comparison of two cynomolgus (Malaysian and Vietnamese) and two rhesus (Indian and Chinese) macaque genomes, including previously published macaque genomes, suggests that Indochinese cynomolgus macaques have been more affected by gene introgression from rhesus macaques. We further identified 60 nonsynonymous SNVs that completely differentiated the cynomolgus and rhesus macaque genomes, and that could be important candidate variants for determining species-specific responses to drugs and pathogens. The demographic inference using the genome sequence data revealed that Malaysian cynomolgus macaques have experienced at least three population bottlenecks. CONCLUSIONS: This list of whole-genome SNVs will be useful for many future applications, such as an array-based genotyping system for macaque individuals. High-quality whole-genome sequencing of the cynomolgus macaque genome may aid studies on finding genetic differences that are responsible for phenotypic diversity in macaques and may help control genetic backgrounds among individuals.     

Serum estradiol and progesterone during pregnancy and the status of the corpus luteum at delivery in cynomolgus monkeys (Macaca fascicularis).

Levels of estradiol and progesterone in peripheral serum of seven cynomolgus monkeys (Macaca fascicularis) were measured from about the 30th day of pregnancy until parturition. Although the pattern of each steroid in circulation differed somewhat from the respective patterns in rhesus monkeys (Macaca mulatta), there were basic similarities. On the day of delivery, the corpus luteum was excised and in vitro incubation of dispersed luteal cells was performed. Isolated luteal cells produced progesterone under control conditions and responded to the addition of HCG with enhanced steroidogenesis. Accordingly, "rejuvenation" of the corpus luteum may occur during advanced gestation in cynomolgus monkeys. These findings, along with establishing the efficacy of the Subhuman Primate Pregnancy Test kit to diagnose pregnancy in this macaque, extend previous evidence for utility of cynomolgus monkeys as a primate model for study of steroid hormones in pregnancy.