SNP-based genetic characterization of the Tulane National Primate Research Center's conventional and specific pathogen-free rhesus macaque (Macaca mulatta) populations.

BACKGROUND: The rhesus macaque is an important biomedical model organism, and the Tulane National Primate Research Center (TNPRC) has one of the largest rhesus macaque breeding colonies in the United States. METHODS: SNP profiles from 3266 rhesus macaques were used to examine the TNPRC colony genetic composition over time and across conventional or SPF animals of Chinese and Indian ancestry. RESULTS: Chinese origin animals were the least genetically diverse and the most inbred; however, since their derivation from their conventional forebearers, neither the Chinese nor the Indian SPF animals exhibit any significant loss of genetic diversity or differentiation. CONCLUSIONS: The TNPRC colony managers have successfully minimized loss in genetic variation across generations. Although founder effects and bottlenecks among the Indian animals have been successfully curtailed, the Chinese subpopulation still show some influences from these events.

NHP BurkPx: A multiplex serodiagnostic bead assay to monitor Burkholderia pseudomallei exposures in non-human primates.

BACKGROUND: Melioidosis is a disease caused by the bacterium Burkholderia pseudomallei, infecting humans and non-human primates (NHP) through contaminated soil or water. World-wide there are an estimated 165,000 human melioidosis cases each year, but recordings of NHP cases are sporadic. Clinical detection of melioidosis in humans is primarily by culturing B. pseudomallei, and there are no standardized detection protocols for NHP. NHP are an important animal model for melioidosis research including clinical trials and development of biodefense countermeasures. METHODOLOGY/PRINCIPLE FINDINGS: We evaluated the diagnostic potential of the multiple antigen serological assay, BurkPx, in NHP using two sera sets: (i) 115 B. pseudomallei-challenged serum samples from 80 NHP collected each week post-exposure (n = 52) and at euthanasia (n = 47), and (ii) 126 B. pseudomallei-naïve/negative serum samples. We observed early IgM antibody responses to carbohydrate antigens followed by IgG antibody recognition to multiple B. pseudomallei protein antigens during the second week of infection. B. pseudomallei negative serum samples had low to intermediate antibody cross reactivity to the antigens in this assay. Infection time was predicted as the determining factor in the variation of antibody responses, with 77.67% of variation explained by the first component of the principal component analysis. A multiple antigen model generated a binary prediction metric ([Formula: see text]), which when applied to all data resulted in 100% specificity and 63.48% sensitivity. Removal of week 1 B. pseudomallei challenged serum samples increased the sensitivity of the model to 95%. CONCLUSION/SIGNIFICANCE: We employed a previously standardized assay for humans, the BurkPx assay, and assessed its diagnostic potential for detection of B. pseudomallei exposure in NHP. The assay is expected to be useful for surveillance in NHP colonies, in investigations of suspected accidental releases or exposures, and for identifying vaccine correlates of protection.

Pedigree reconstruction and distant pairwise relatedness estimation from genome sequence data: A demonstration in a population of rhesus macaques (Macaca mulatta).

A primary challenge in the analysis of free-ranging animal populations is the accurate estimation of relatedness among individuals. Many aspects of population analysis rely on knowledge of relatedness patterns, including socioecology, demography, heritability and gene mapping analyses, wildlife conservation and the management of breeding colonies. Methods for determining relatedness using genome-wide data have improved our ability to determine kinship and reconstruct pedigrees in humans. However, methods for reconstructing complex pedigree structures and estimating distant relatedness (beyond third-degree) have not been widely applied to other species. We sequenced the genomes of 150 male rhesus macaques from the Tulane National Primate Research Center colony to estimate pairwise relatedness, reconstruct closely related pedigrees, estimate more distant relationships and augment colony records. Methods for determining relatedness developed for human genetic data were applied and evaluated in the analysis of nonhuman primates, including identity-by-descent-based methods for pedigree reconstruction and shared segment-based inference of more distant relatedness. We compared the genotype-based pedigrees and estimated relationships to available colony pedigree records and found high concordance (95.5% agreement) between expected and identified relationships for close relatives. In addition, we detected distant relationships not captured in colony records, including some as distant as twelfth-degree. Furthermore, while deep sequence coverage is preferable, we show that this approach can also provide valuable information when only low-coverage (5×) sequence data is available. Our findings demonstrate the value of these methods for determination of relatedness in various animal populations, with diverse applications to conservation biology, evolutionary and ecological research and biomedical studies.

Retrospective analysis of wound characteristics and tetanus development in captive macaques.

Traumatic wounds and access to outdoor enclosures containing soil contribute to development of tetanus in nonhuman primates. A retrospective, matched case-control study was conducted at a primate center to evaluate these factors by analysis of medical records of animals sustaining traumatic injuries during a 3-yr study period. Thirty-one macaques with traumatic injuries and a clinical diagnosis of tetanus were selected as cases, and 62 macaques with traumatic injuries and no diagnosis of tetanus were selected as controls. For an animal with injuries to the digits, the odds of developing tetanus were 9.6 times those of a similar animal without injuries to the digits (Odds Ratio [OR] = 9.55, 95% CI = 1.56-58.59); with injuries to the tail, the odds of developing tetanus were 8.0 times those of a similar animal without injuries to the tail (OR = 7.95, 95% CI = 0.82-77.04); and with injuries in more than one location, the odds of developing tetanus were 8.5 times those for a similar animal with injuries in just one location (OR = 8.45, 95% CI = 1.01-70.46). A nonhuman primate with injuries to the leg was less likely to develop tetanus than a similar nonhuman primate without injuries to the leg (OR = 0.19, 95% CI = 0.03-1.2). Results indicated that wound location is associated with development of tetanus infection in rhesus macaques. Identification of high-risk trauma cases will allow better allocation of wound management and tetanus prophylaxis in institutions, especially in those housing nonhuman primates outdoors.

Birth of rhesus macaque (Macaca mulatta) infants after in vitro fertilization and gestation in female rhesus or pigtailed (Macaca nemestrina) macaques.

A study was conducted to assess the possibility of using pigtailed macaques (Macaca nemestrina) as recipients for rhesus macaque (Macaca mulatta) embryos. A total of 250 oocytes were collected from 11 rhesus monkeys during 12 follicular aspirations. We performed 15 embryo transfers with two embryos each into rhesus recipients, which resulted in eight pregnancies, of which two were lost during the second trimester. Among the remaining six pregnant rhesus macaques, two were carrying twins, resulting in the birth of eight infants. Twelve transfers of rhesus embryos into pigtailed macaques resulted in one pregnancy and the birth of one infant. Fetal growth and development were monitored by monthly ultrasound examinations, during which biparietal measurements were taken and compared with those derived from 22 pregnant control monkeys. In vitro fertilization-derived singletons tended to develop faster than did twins and naturally conceived control singletons during the initial months of pregnancy and weighed more at birth than did twins. There were pronounced morphologic changes in the placenta of the rhesus that developed in the female pigtailed macaque. These included an irregular shape, elevated placenta-to-birth-weight ratio, and an abnormal length and diameter of the umbilical cord. Histologic analyses of the rhesus-pigtailed placenta showed evidence of maternal-placental floor infarction and thrombosis of the spiral artery with resulting infarction of the villi. These results demonstrate that pigtailed macaques can carry rhesus fetuses to term, but further studies are necessary to determine the cause of the decreased pregnancy rates and observed placental abnormalities.

Dehydroepiandrosterone-sulfate as a biomarker of senescence in male non-human primates.

Numerous studies have suggested important and varying roles for dehydroepiandrosterone (DHEA) and dehydroepiandrosterone-sulfate (DHEA-S) in primate physiological functions. Despite these numerous claims, specific actions and significance of DHEA and DHEA-S are still equivocal. A decline of these hormones in adult humans may have functional significance, yet there is no clear relationship between functional impairments of aging and the decline in DHEA or DHEA-S levels. This current study attempts to address the natural history of adrenal hormones by presenting non-human primate evidence of the endocrinology of aging; the age-related patterns of adrenal hormone decline in three species of the subfamily Cercopithecinae, Macaca mulatta, Macaca nemestrina, and Papio cynocephalus are compared. It is concluded that DHEA-S and cortisol represent lineage specific markers of senescence among primates and that parallel age-related patterns of DHEA-S and cortisol likely reflect lineage specific effects, or rather, phylogenetic similarities of endocrine senescence. The use of relative adrenal hormone levels to approximate species' life expectancies is discussed.

Correction: Persistence of Borrelia burgdorferi in Rhesus Macaques following Antibiotic Treatment of Disseminated Infection.

[This corrects the article on p. e29914 in vol. 7.].

Excision of femoral head and neck for treatment of coxofemoral degenerative joint disease in a rhesus macaque (Macaca mulatta).

Nonhuman primates are a valuable model for osteoarthritis. Osteoarthritis has been extensively studied in nonhuman primates in both naturally occurring and induced disease states. However, little published information describes naturally occurring osteoarthritis of the coxofemoral joints of nonhuman primates. We report a case of naturally occurring coxofemoral joint osteoarthritis in a rhesus macaque. This case radiographically resembled hip dysplasia reported in other species and demonstrated a rapid progression in severity of lameness, with accompanying loss of muscle mass in the affected limb. We excised the femoral head and neck to alleviate the pain that accompanied the osteoarthritis. Physical therapy was initiated, and dual-energy X-ray absorptiometry and video recordings were performed to evaluate the macaque's response to surgical intervention. By 3 mo postoperatively, the macaque had regained full use of the affected limb.

Low prevalence of Chagas parasite infection in a nonhuman primate colony in Louisiana.

Chagas disease, an important cause of heart disease in Latin America, is caused by the parasite Trypanosoma cruzi, which typically is transmitted to humans by triatomine insects. Although autochthonous transmission of the Chagas parasite to humans is rare in the United States, triatomines are common, and more than 20 species of mammals are infected with the Chagas parasite in the southern United States. Chagas disease has also been detected in colonies of nonhuman primates (NHP) in Georgia and Texas, and heart abnormalities consistent with Chagas disease have occurred at our NHP center in Louisiana. To determine the level of T. cruzi infection, we serologically tested 2157 of the approximately 4200 NHP at the center; 34 of 2157 primates (1.6%) tested positive. Presence of the T. cruzi parasite was confirmed by hemoculture in 4 NHP and PCR of the cultured parasites. These results strongly suggest local transmission of T. cruzi, because most of the infected NHP were born and raised at this site. All 3 species of NHP tested yielded infected animals, with significantly higher infection prevalence in pig-tailed macaques, suggesting possible exploration of this species as a model organism. The local T. cruzi strain isolated during this study would enhance such investigations. The NHP at this center are bred for use in scientific research, and the effects of the Chagas parasite on infected primates could confuse the interpretation of other studies.

Persistence of Borrelia burgdorferi in rhesus macaques following antibiotic treatment of disseminated infection.

The persistence of symptoms in Lyme disease patients following antibiotic therapy, and their causes, continue to be a matter of intense controversy. The studies presented here explore antibiotic efficacy using nonhuman primates. Rhesus macaques were infected with B. burgdorferi and a portion received aggressive antibiotic therapy 4-6 months later. Multiple methods were utilized for detection of residual organisms, including the feeding of lab-reared ticks on monkeys (xenodiagnosis), culture, immunofluorescence and PCR. Antibody responses to the B. burgdorferi-specific C6 diagnostic peptide were measured longitudinally and declined in all treated animals. B. burgdorferi antigen, DNA and RNA were detected in the tissues of treated animals. Finally, small numbers of intact spirochetes were recovered by xenodiagnosis from treated monkeys. These results demonstrate that B. burgdorferi can withstand antibiotic treatment, administered post-dissemination, in a primate host. Though B. burgdorferi is not known to possess resistance mechanisms and is susceptible to the standard antibiotics (doxycycline, ceftriaxone) in vitro, it appears to become tolerant post-dissemination in the primate host. This finding raises important questions about the pathogenicity of antibiotic-tolerant persisters and whether or not they can contribute to symptoms post-treatment.

Modification of a common BAL technique to enhance sample diagnostic value.

Bronchoalveolar lavage (BAL) by means of bronchoscopy is a diagnostic tool frequently used for clinical and research purposes in nonhuman primates. Although many institutions use this procedure, the technique is not standardized. One technical aspect that can vary is the method by which fluid is recovered. The purpose of this study was to evaluate differences between 2 different BAL aspiration techniques. Bronchoscopy and BAL fluid collection were performed on 20 rhesus macaques (Macaca mulatta). Data collected for comparison included heart rate, oxygen saturation levels, rectal temperature, volume of fluid collected, total cell count, cell viability, differential cell count, and flow cytometry. Results showed no significant differences in the heart rate, oxygen saturation, or body temperature between the 2 groups. Likewise, differential cell counts and cell viability studies of the retrieved fluid did not differ between methods. Compared with the conventional technique, the modified aspiration technique led to an 8.3% increase in overall fluid yield and a higher concentration of cells recovered. These differences are statistically significant and likely will be clinically relevant in the context of diagnosis.

Clinical and immunopathologic alterations in rhesus macaques affected with globoid cell leukodystrophy.

Globoid cell leukodystrophy, or Krabbe's disease, is a severe disorder of the central and peripheral nervous system caused by the absence of galactocerebrosidase (GALC) activity. Herein, we describe the clinical, neuropathological, histochemical, and immunohistological features observed in rhesus macaques affected with Krabbe's disease. Clinical signs included pronounced muscle tremors of head and limbs, difficulty ambulating, ataxia, hypermetria, proprioceptive deficits, and respiratory abnormalities. Histopathologically, all animals presented with evidence of demyelination in the peripheral and central nervous systems and accumulation of mononuclear and multinuclear globoid cells in the cerebral and cerebellar white matter associated with severe gliosis. Using immunohistochemistry and multi-label confocal microscopy, it was determined that globoid cells were CD68+, HAM56+, LN5+, CD163+, IBA-1+, and Glut-5+, suggesting that both peripheral blood-derived monocytes/macrophages and resident parenchymal microglia gave rise to globoid cells. Interestingly, many of the globoid cells and parenchymal microglia with a more ameboid morphology expressed HLA-DR, indicating immune activation. Increased expression of iNOS, TNF-alpha, and IL-1 beta were observed in the affected white matter, colocalizing with globoid cells, activated microglia, and astrocytes. Cytokine mRNA levels revealed markedly increased gene expression of CCL2 in the brain of affected macaques. CCL2-expressing cells were detected throughout the affected white matter, colocalizing with GFAP+ cells and astrocytes. Collectively, these data suggest that dysregulation of monocyte/macrophage/microglia and up-regulation of certain cytokines may contribute to the pathogenesis of Krabbe's disease.

A non-human primate model for gluten sensitivity.

BACKGROUND AND AIMS: Gluten sensitivity is widespread among humans. For example, in celiac disease patients, an inflammatory response to dietary gluten leads to enteropathy, malabsorption, circulating antibodies against gluten and transglutaminase 2, and clinical symptoms such as diarrhea. There is a growing need in fundamental and translational research for animal models that exhibit aspects of human gluten sensitivity. METHODS: Using ELISA-based antibody assays, we screened a population of captive rhesus macaques with chronic diarrhea of non-infectious origin to estimate the incidence of gluten sensitivity. A selected animal with elevated anti-gliadin antibodies and a matched control were extensively studied through alternating periods of gluten-free diet and gluten challenge. Blinded clinical and histological evaluations were conducted to seek evidence for gluten sensitivity. RESULTS: When fed with a gluten-containing diet, gluten-sensitive macaques showed signs and symptoms of celiac disease including chronic diarrhea, malabsorptive steatorrhea, intestinal lesions and anti-gliadin antibodies. A gluten-free diet reversed these clinical, histological and serological features, while reintroduction of dietary gluten caused rapid relapse. CONCLUSIONS: Gluten-sensitive rhesus macaques may be an attractive resource for investigating both the pathogenesis and the treatment of celiac disease.

Age as a determinant of reproductive success among captive female rhesus macaques (Macaca mulatta).

A retrospective analysis was performed on fertility outcomes among a colony of captive Indian rhesus monkeys. The analysis covered over 30 years and was based on 1443 females with a total of 11,453 pregnancies. Various determinants of fertility were assessed including birth rates, pregnancy loss, infant survival, interbirth intervals, and interval from last birth to death. Binary variables were analyzed with generalized linear models with random intercepts, while linear mixed models were used for analysis of continuous variables. Age of the dam was a significant factor in determining whether a pregnancy resulted in a birth and whether an infant survived the first 30 days with primiparous or older mothers being less likely to produce an infant surviving to that age. In contrast, sex proved to be the only significant factor in determining whether an infant lived to 1 year, with females being more likely to survive. The interval between births proved to be affected primarily by dam age, while the late death of an infant depressed the likelihood of an extended time interval between her last birth and her death. Overall, these results demonstrate that maternal age contributes significantly to a decline in fertility and older females can live relatively long periods following birth of their last infant.

Complications of gastric catheters implanted in rhesus macaques (Macaca mulatta).

As part of a study addressing chronic alcohol consumption and simian immunodeficiency virus, 31 rhesus macaques (Macaca mulatta) were implanted with gastric catheters used to deliver alcohol or isocaloric sucrose (control). Once implanted, the animals wore jackets and were housed in specialized cages modified with swivels and tethers. During the course of the study, 3 animals developed clinical signs indicating possible instability of the implanted gastric catheter. All 3 animals were found to have a string foreign body wrapped around the distal end of the catheter, with 2 of the catheters perforating the intestinal wall. Gastroscopy was used to screen remaining animals to determine catheter position and the presence of a foreign body attached to the end of the catheter. Results of the screening revealed that of the 28 remaining animals, 9 had malpositioned catheters; string foreign bodies were associated with 3 of the 9 malpositioned catheters. We initially hypothesized that the peristaltic motion of the stomach, combined with the attachment of string, which was probably ingested by subjects after manipulating their jackets, led to eventual catheter displacement. We later concluded that the string may have played a secondary role but was not the primary cause of catheter instability, because several malpositioned catheters had no string attached at the time of diagnosis. Subsequent modifications were instituted, including modifying the surgical technique, altering the type of gastric catheter used, and increasing environmental enrichment for animals with known tendency to manipulate their jackets.

Comparison of efficacy of moxidectin and ivermectin in the treatment of Strongyloides fulleborni infection in rhesus macaques.

BACKGROUND: Strongyloides infection may result in clinical disease or confound experimental protocols that utilize non-human primates. There is presently a Strongyloides fulleborni infection rate of approximately 27% in the Tulane National Primate Research Center's breeding colonies despite the routine therapeutic and prophylactic use of ivermectin. METHODS: A study was conducted to determine if moxidectin treatment offers advantages to the intestinal parasite control program. A total of 150 rhesus macaques (Macaca mulatta) that were removed from the breeding colonies due to illness were selected for the study. The animals were randomly assigned to treatment groups with 75 receiving ivermectin and 75 receiving moxidectin. Egg counts were performed on fecal samples collected pre- and post-treatment. RESULTS: Both treatments resulted in decreases in the number of eggs/g in the post-treatment sample as compared with the pre-treatment sample; however, no significant difference was found between treatment groups. CONCLUSIONS: With the data demonstrating a similar efficacy in both ivermectin and moxidectin treated macaques, the benefit of moxidectin treatment relates to biosafety and topical application.

Infectivity of the highly transformable BBE02- lp56- mutant of Borrelia burgdorferi, the Lyme disease spirochete, via ticks.

Infectious Borrelia burgdorferi strains that have increased transformability with the shuttle vector pBSV2 were recently constructed by inactivating the gene encoding BBE02, a putative restriction-modification gene product expressed by the linear plasmid lp25 (Kawabata et al., Infect. Immun. 72:7147-7154, 2004). The absence of the linear plasmid lp56, which carries another putative restriction-modification gene, further enhanced transformation rates. The infectivity of these mutants was assessed previously in mice that were inoculated with needle and syringe and was found to be equivalent to that of wild-type spirochetes. Here we examined the infectivity of spirochetes to ticks after capillary inoculation of Ixodes scapularis nymphs and the subsequent spirochetal infectivity to mice via ticks by using B. burgdorferi B31 clonal isolates lacking lp56 and/or BBE02. The absence of lp56 (but not BBE02) correlated with a lower number of spirochetes in ticks after feeding on mice; this plasmid thus may play a role, albeit not an essential one, in supporting spirochetal survival in the feeding tick. Importantly, however, the absence of lp56 and BBE02 did not detectably influence infectivity to mice via ticks.

Serial electrophysiologic studies in rhesus monkeys with Krabbe disease.

Krabbe disease is a progressive leukodystrophy that results in demyelination in the central and peripheral nervous systems in humans. It has been described in a number of mammalian species including the rhesus monkey. We performed serial nerve conduction studies beginning within the first 2 months of life in four homozygous, two heterozygous, and two normal rhesus monkeys (Macaca mulatta) to characterize the peripheral neuropathy. Mean conduction velocities of the median, ulnar, and tibial nerves were significantly slower in the affected than unaffected monkeys at all ages (P < 0.0001). The conduction velocity differences became more apparent between the affected and unaffected as the monkeys aged. When compared to the unaffected monkeys, the serial conduction velocities suggested occurrence of dysmyelination followed by demyelination in the affected monkeys. These observations provide further insight into the disease process and suggest an early window of opportunity for treating Krabbe disease.

Four-year study of controlled timed breeding of rhesus monkeys (Macaca mulatta).

As part of the timed breeding colony at Tulane National Primate Research Center, exogenous progesterone administration (5 mg/day for 10 days) has been used to select conception dates by inducing artificial luteal phases in female rhesus monkeys. A retrospective analysis of data obtained during four breeding seasons (1998-2001) revealed that conceptions occurred an average of 18 days after the last administration of progesterone. The age of the female to be bred, previous pregnancy history, and timing of breeding during the breeding season were determined to be critical factors in the success of the procedure. The benefit of this method of timed breeding is that it does not require tracking of menstrual cycles, which can be labor-intensive and requires that animals be monitored several months in advance of breeding to determine each female's individual cycle length. It also provided an efficient use of breeding-age males.

Use of a recombinant envelope protein subunit antigen for specific serological diagnosis of West Nile virus infection.

Serological diagnosis of West Nile virus (WNV) infection is complicated by extensive antigenic cross-reactivity with other closely related flaviviruses, such as St. Louis encephalitis virus. Here we describe a recombinant, bacterially expressed antigen equivalent to structural domain III of the WNV envelope protein that has allowed clear discrimination of antibody responses to WNV from those against other related flaviviruses in indirect enzyme-linked immunosorbent assays using standardized control antisera and field-collected samples.