Colon volvulus in rhesus macaques (Macaca mulatta) at the Oregon National Primate Research Center-Retrospective analysis.

Colonic volvulus is an uncommon, often life-threatening condition, in non-human primates. Twenty-six cases of colonic volvulus in rhesus macaques (Macaca mulatta) were identified in necropsy records spanning 38 years at the Oregon National Primate Research Center (ONPRC). This report represents the largest collection of colonic volvuli in rhesus macaques.

Bardet-Biedl Syndrome in rhesus macaques: A nonhuman primate model of retinitis pigmentosa.

The development of therapies for retinal disorders is hampered by a lack of appropriate animal models. Higher nonhuman primates are the only animals with retinal structure similar to humans, including the presence of a macula and fovea. However, few nonhuman primate models of genetic retinal disease are known. We identified a lineage of rhesus macaques with a frameshift mutation in exon 3 of the BBS7 gene c.160delG (p.Ala54fs) that is predicted to produce a non-functional protein. In humans, mutations in this and other BBS genes cause Bardet-Biedl syndrome, a ciliopathy and a syndromic form of retinitis pigmentosa generally occurring in conjunction with kidney dysfunction, polydactyly, obesity, and/or hypogonadism. Three full- or half-sibling monkeys homozygous for the BBS7 c.160delG variant, at ages 3.5, 4 and 6 years old, displayed a combination of severe photoreceptor degeneration and progressive kidney disease. In vivo retinal imaging revealed features of severe macular degeneration, including absence of photoreceptor layers, degeneration of the retinal pigment epithelium, and retinal vasculature atrophy. Electroretinography in the 3.5-year-old case demonstrated loss of scotopic and photopic a-waves and markedly reduced and delayed b-waves. Histological assessments in the 4- and 6-year-old cases confirmed profound loss of photoreceptors and inner retinal neurons across the posterior retina, with dramatic thinning and disorganization of all cell layers, abundant microglia, absent or displaced RPE cells, and significant gliosis in the subretinal space. Retinal structure, including presence of photoreceptors, was preserved only in the far periphery. Ultrasound imaging of the kidneys revealed deranged architecture, and renal histopathology identified distorted contours with depressed, fibrotic foci and firmly adhered renal capsules; renal failure occurred in the 6-year-old case. Magnetic resonance imaging obtained in one case revealed abnormally low total brain volume and unilateral ventricular enlargement. The one male had abnormally small testes at 4 years of age, but polydactyly and obesity were not observed. Thus, monkeys homozygous for the BBS7 c.160delG variant closely mirrored several key features of the human BBS syndrome. This finding represents the first identification of a naturally-occurring nonhuman primate model of BBS, and more broadly the first such model of retinitis pigmentosa and a ciliopathy with an associated genetic mutation. This important new preclinical model will provide the basis for better understanding of disease progression and for the testing of new therapeutic options, including gene and cell-based therapies, not only for BBS but also for multiple forms of photoreceptor degeneration.

Cerebral cysts of ependymal or ventricular origin in a juvenile rhesus macaque (Macaca mulatta) with neurologic signs.

Naturally occurring neurologic disease in non-human primates may be attributable to a wide-range of causes, including infectious agents, congenital or acquired malformations, degenerative diseases, and, rarely, neoplasia. We report a case of ataxia and paresis in a juvenile rhesus macaque with ependymal-lined cerebral cysts.

Interferon-Gamma test for the detection of Mycobacterium tuberculosis complex infection in Macaca mulatta and other non-human primates.

We have formatted an assay to detect Mycobacterium tuberculosis complex infections of non-human primates. Commercially available reagents were used to elicit a specific immune response that was measured by interferon-gamma release. Initial evaluation using blood samples from Rhesus macaques experimentally infected with M tuberculosis distinguished infected versus uninfected animals.

Standard growth and diarrhea-associated growth faltering in captive infant rhesus macaques (Macaca mulatta).

Reference growth studies of captive rhesus macaque infants have not accounted for diarrhea and the potential for growth stunting or growth faltering. Healthy infants without diarrhea could be used to build a standard growth chart and a tool used to detect growth faltering associated with diarrhea. We hypothesized infants who develop diarrhea during the first year of life would experience decreased linear weight gain compared to healthy infants, and we used healthy infants to establish standard growth of male and female infants. We hypothesized the lower 3rd percentile of standard growth would be cut-off criteria used in screening for diarrhea-associated growth faltering. Using a retrospective cohort of 6,510 infant weight records in a multiple linear regression, daily weight gain through the first year of life was determined by sex, housing type, and health status. Male standard growth was 4.1 g/day (95%CI: 4.0-4.2 g/day) in corrals and 4.7 g/day (95%CI: 4.5-4.8 g/day) in shelter housing. Female standard growth was 4.0 g/day (95%CI: 3.8-4.2 g/day) in corrals and 4.4 g/day (95%CI: 4.0-4.7 g/day) in shelter housing. Diarrhea was significantly associated with decreased linear weight gain by up to 34% during the first year of life. Odds of growth faltering of infants, defined as those falling below the 3rd percentile of standard growth, were at least 8.9 higher given a history of diarrhea compared to healthy. The growth faltering cut-off criteria had a sensitivity of at least 53% for males and females to screen for diarrhea in infants between 6 and 12 months in shelters housing. Interinstitutional collaborations of infant rhesus macaque weight records would refine the standard growth charts and cut-off criteria, and additional morphometric data would provide a more nuanced picture of growth stunting.

Social hair pulling in captive rhesus macaques (Macaca mulatta).

Alopecia is common among captive populations of nonhuman primates. There are many potential causes of alopecia, including physiological conditions such as hormonal imbalance and infection, features of the captive environment such as housing type, ground substrate, and group density, as well as behavioral abnormalities such as self-plucking. A potential behavioral cause of alopecia in group-housed primates is social hair pulling, where one animal pulls hair from a conspecific. While social hair pulling has been conflated with overgrooming in some of the alopecia literature, other authors have categorized it as a form of aggression rather than a form of excessive grooming. In this study, we examined social hair pulling, grooming, and aggression within seven groups of rhesus macaques (Macaca mulatta) (N = 319). We took weekly 30-min behavioral observations on each group for one year to assess the patterns of hair pulling and grooming, which monkeys were receiving and initiating these behaviors, as well as aggression and other behaviors indicating dominance. We also assessed the amount of alopecia on each individual monthly. While grooming tended to be directed "up" the hierarchy (i.e., monkeys were more likely to groom animals of a higher rank than lower rank), most hair pulling was directed "down" the hierarchy. Further, hair pulling seldom co-occurred with aggressive behaviors, suggesting that it was not a form of aggression. Hair pulling also usually resulted in ingestion of the pulled hair. Hair pulling was correlated with alopecia; monkeys who were frequent recipients of hair pulling scored higher on monthly alopecia ratings than those who were less often observed having hair pulled. Our results suggest that social hair pulling is a behavior distinct from either grooming or aggressive behavior, and that it may contribute to alopecia in socially housed macaques.

Infant diarrheal disease in rhesus macaques impedes microbiome maturation and is linked to uncultured Campylobacter species.

Diarrheal diseases remain one of the leading causes of death for children under 5 globally, disproportionately impacting those living in low- and middle-income countries (LMIC). Campylobacter spp., a zoonotic pathogen, is one of the leading causes of food-borne infection in humans. Yet to be cultured Campylobacter spp. contribute to the total burden in diarrheal disease in children living in LMIC thus hampering interventions. We performed microbiome profiling and metagenomic genome assembly on samples collected from over 100 infant rhesus macaques longitudinally and during cases of clinical diarrhea within the first year of life. Acute diarrhea was associated with long-lasting taxonomic and functional shifts of the infant gut microbiome indicative of microbiome immaturity. We constructed 36 Campylobacter metagenomic assembled genomes (MAGs), many of which fell within 4 yet to be cultured species. Finally, we compared the uncultured Campylobacter MAGs assembled from infant macaques with publicly available human metagenomes to show that these uncultured species are also found in human fecal samples from LMIC. These data highlight the importance of unculturable Campylobacter spp. as an important target for reducing disease burden in LMIC children.

Risk factor analysis may provide clues to diarrhea prevention in outdoor-housed rhesus macaques (Macaca mulatta).

Seventy-five percent of rhesus macaques at national primate research centers are housed outside. Annually, 15-39% of these animals experience diarrhea and require veterinary treatment for dehydration, electrolyte imbalance, or weight loss. An estimated 21-33% of these patients will die or be euthanized. Many studies have explored the various infectious etiologies of non-human primate diarrhea. However, there is little published information on diarrhea incidence rates and risk factors in outdoor-housed rhesus macaques. Without this information, it is challenging to determine endemic and epidemic diarrhea levels, or to develop and evaluate mitigation strategies. Using electronic medical records, we conducted a retrospective cohort study to calculate diarrhea incidence rates for rhesus macaques (N = 3,181) housed in three different outdoor housing types (corrals, shelters, and temporary housing) at the Oregon National Primate Research Center between November 1, 2009 and October 31, 2010. With multiple logistic regression analysis, we determined the relative risk of housing type, sex, and age on development of diarrhea. Diarrhea incidence and mortality in our population was lower than many published ranges. Type of outdoor housing, age, and previous diarrhea episode were positively correlated with diarrhea risk. Younger animals in smaller shelters and temporary housing had a greater risk of acquiring diarrhea, with juvenile animals (0.7-3.9 years) having the highest mortality rate. Sex was not a risk factor, but adult females with diarrhea were more likely to develop life-threatening complications than adult males. We also constructed a predictive model for diarrhea-associated mortality using Classification and Regression Tree. Findings from this study will be used to develop and evaluate mitigation strategies in our outdoor-housed population and to provide a foundation for genetic susceptibility and immune function testing.

Campylobacter vaccination reduces diarrheal disease and infant growth stunting among rhesus macaques.

Campylobacter-associated enteric disease is estimated to be responsible for more than 160 million cases of gastroenteritis each year and is linked to growth stunting of infants living under conditions of poor sanitation and hygiene. Here, we examine naturally occurring Campylobacter-associated diarrhea among rhesus macaques as a model to determine if vaccination could reduce severe diarrheal disease and infant growth stunting. Compared to unvaccinated controls, there are no Campylobacter diarrhea-associated deaths observed among vaccinated infant macaques and all-cause diarrhea-associated infant mortality is decreased by 76% (P = 0.03). By 9 months of age, there is a 1.3 cm increase in dorsal length that equaled a significant 1.28 LAZ (Length-for-Age Z score) improvement in linear growth among vaccinated infants compared to their unvaccinated counterparts (P = 0.001). In this work, we show that Campylobacter vaccination not only reduces diarrheal disease but also potentially serves as an effective intervention that improves infant growth trajectories.

Reduced infant rhesus macaque growth rates due to environmental enteric dysfunction and association with histopathology in the large intestine.

Environmental enteric dysfunction is associated with malnutrition as well as infant growth stunting and has been classically defined by villous blunting, decreased crypt-to-villus ratio, and inflammation in the small intestine. Here, we characterized environmental enteric dysfunction among infant rhesus macaques that are naturally exposed to enteric pathogens commonly linked to human growth stunting. Remarkably, despite villous atrophy and histological abnormalities observed in the small intestine, poor growth trajectories and low serum tryptophan levels were correlated with increased histopathology in the large intestine. This work provides insight into the mechanisms underlying this disease and indicates that the large intestine may be an important target for therapeutic intervention.

Growth faltering regardless of chronic diarrhea is associated with mucosal immune dysfunction and microbial dysbiosis in the gut lumen.

Despite the impact of childhood diarrhea on morbidity and mortality, our understanding of its sequelae has been significantly hampered by the lack of studies that examine samples across the entire intestinal tract. Infant rhesus macaques are naturally susceptible to human enteric pathogens and recapitulate the hallmarks of diarrheal disease such as intestinal inflammation and growth faltering. Here, we examined intestinal biopsies, lamina propria leukocytes, luminal contents, and fecal samples from healthy infants and those experiencing growth faltering with distant acute or chronic active diarrhea. We show that growth faltering in the presence or absence of active diarrhea is associated with a heightened systemic and mucosal pro-inflammatory state centered in the colon. Moreover, polyclonal stimulation of colonic lamina propria leukocytes resulted in a dampened cytokine response, indicative of immune exhaustion. We also detected a functional and taxonomic shift in the luminal microbiome across multiple gut sites including the migration of Streptococcus and Prevotella species between the small and large intestine, suggesting a decompartmentalization of gut microbial communities. Our studies provide valuable insight into the outcomes of diarrheal diseases and growth faltering not attainable in humans and lays the groundwork to test interventions in a controlled and reproducible setting.

Tuberculosis detection in nonhuman primates is enhanced by use of testing algorithms that include an interferon-γ release assay.

OBJECTIVE: To develop a testing algorithm that incorporates multiple assays to evaluate host cellular and humoral immunity and antigen detection concerning Mycobacterium tuberculosis complex (MTBC) infection in captive nonhuman primates. ANIMALS: Cohorts of captive-bred and wild-caught macaques from 5 different geographic regions. PROCEDURES: Macaques were tested for MTBC infection by use of a γ interferon tuberculosis (GIFT) assay, an interferon-γ release assay, and other assays. In the first 2 cohorts (n = 15 and 181), initial validation of the GIFT assay was performed by use of experimentally infected and unexposed control macaques. In the next 3 cohorts (n = 59, 42, and 11), results were obtained for opportunistically collected samples from macaques exposed during spontaneous outbreaks. RESULTS: Sensitivity and specificity of the GIFT assay in the control cohorts were 100% and 97%, respectively, and were variable but enhanced by incorporating results from multiple assays in spontaneous outbreaks. CLINICAL RELEVANCE: The detection and management of MTBC infection in captive nonhuman primate populations is an ongoing challenge, especially with animal imports and transfers. Despite standardized practices of initial quarantine with regular intradermal tuberculin skin testing, spontaneous outbreaks continue to be reported. Since infection encompasses a range of disease manifestations over time, a testing algorithm that incorporates multiple assays, such as the GIFT assay, to evaluate host cellular and humoral immunity in addition to agent detection is needed. Testing a combination of samples from controlled studies and spontaneous outbreaks of MTBC infection in nonhuman primates would advance the development and validation of a functional algorithm that incorporates promising tools such as the GIFT assay.

Vaccine-mediated protection against Campylobacter-associated enteric disease.

Campylobacter coli and Campylobacter jejuni are responsible for 400 million to 500 million cases of enteric disease each year and represent the most common cause of bacterial gastroenteritis worldwide. Despite its global importance, Campylobacter vaccine development has been hampered by the lack of animal models that recapitulate human disease pathogenesis. Here, we describe a naturally occurring Campylobacter-associated diarrhea model in outdoor-housed rhesus macaques. Using this model, we developed novel next-generation H2O2-based Campylobacter vaccines that induced strong antibacterial antibodies to multiple Campylobacter proteins including flagellin and provided up to 83% protection against severe C. coli-associated diarrhea. Whole-genome sequencing of circulating Campylobacter strains revealed little to no homology within lipooligosaccharide or capsular polysaccharide loci with the Campylobacter vaccine strains used in these studies, indicating that vaccine-mediated immunity was not restricted to a single homologous serotype. Together, these results demonstrate an important advance in vaccine development and a new approach to reducing Campylobacter-associated enteric disease.

Maturation of the infant rhesus macaque gut microbiome and its role in the development of diarrheal disease.

BACKGROUND: Diarrhea is the second leading cause of death in children under 5 years of age. Enhanced understanding of causal pathways, pathogenesis, and sequelae of diarrhea is urgently needed. Although the gut microbiota is believed to play a role in susceptibility to diarrheal diseases, our understanding of this association remains incomplete. Infant rhesus macaques (Macaca mulatta) are susceptible to diarrhea making them an ideal model to address this question. RESULTS: The maturation of the infant rhesus macaque gut microbiome throughout the first 8 months of life occurs in a similar pattern as that described for human infants. Moreover, the microbiome of the captive reared infant rhesus macaque more closely resembles that of human infants in the developing world than in the western world. Importantly, prior to disease onset, the gut microbiome of infants that later develop diarrhea is enriched in pathways of immunomodulatory metabolite synthesis, while those of infants that remain asymptomatic are enriched in pathways for short-chain fatty acid production. We identify Prevotella strains that are more abundant at 1 month in infants that later develop diarrhea. At 8 months, the microbiomes of animals that experience diarrhea show increased abundance of Campylobacter and a reduction in Helicobacter macacae. CONCLUSION: The composition of the microbial community could provide a phenotypic marker of an infant's susceptibility to diarrheal disease. Given the significant physiological and immunological similarities between human and nonhuman primates, these findings provide potential markers of susceptibility to diarrhea that could be modulated to improve infant health, especially in the developing world.

Modelling disease risk for amyloid A (AA) amyloidosis in non-human primates using machine learning.

Objective: Amyloid A (AA) amyloidosis is found in humans and non-human primates, but quantifying disease risk prior to clinical symptoms is challenging. We applied machine learning to identify the best predictors of amyloidosis in rhesus macaques from available clinical and pathology records. To explore potential biomarkers, we also assessed whether changes in circulating serum amyloid A (SAA) or lipoprotein profiles accompany the disease. Methods: We conducted a retrospective study using 86 cases and 163 controls matched for age and sex. We performed data reduction on 62 clinical, pathological and demographic variables, and applied multivariate modelling and model selection with cross-validation. To test the performance of our final model, we applied it to a replication cohort of 2,775 macaques. Results: The strongest predictors of disease were colitis, gastrointestinal adenocarcinoma, endometriosis, arthritis, trauma, diarrhoea and number of pregnancies. Sensitivity and specificity of the risk model were predicted to be 82%, and were assessed at 79 and 72%, respectively. Total, low density lipoprotein and high density lipoprotein cholesterol levels were significantly lower, and SAA levels and triglyceride-to-HDL ratios were significantly higher in cases versus controls. Conclusion: Machine learning is a powerful approach to identifying macaques at risk of AA amyloidosis, which is accompanied by increased circulating SAA and altered lipoprotein profiles.

High resolution radiographic and fine immunologic definition of TB disease progression in the rhesus macaque.

Mycobacterium tuberculosis infection in non-human primates parallels human tuberculosis, and provides a valuable vaccine evaluation model. However, this model is limited by the availability of real-time, non-invasive information regarding disease progression. Consequently, we have combined computed tomography scanning with enumeration of antigen-specific T cell responses. Four rhesus monkeys were infected with M. tuberculosis strain H37Rv (1000 cfu) in the right lower lobe via a bronchoscope. All uniformly developed progressive tuberculosis, and required euthanasia at 12 weeks. Computed tomography scanning provided detailed real-time imaging of disease progression. At necropsy, computed tomography and pathohistologic findings were tightly correlated, and characteristic of human disease. Immunologic monitoring demonstrated progressive evolution of high frequency M. tuberculosis-specific CD4(+) and CD8(+) T cell responses. Peripheral blood effector cell frequencies were similar to those observed in tissues. In summary, computed tomography scanning in conjunction with immunologic monitoring provides a non-invasive, accurate, and rapid assessment of tuberculosis in the non-human primate.

The value of extended pedigrees for next-generation analysis of complex disease in the rhesus macaque.

Complex diseases (e.g., cardiovascular disease and type 2 diabetes, among many others) pose the biggest threat to human health worldwide and are among the most challenging to investigate. Susceptibility to complex disease may be caused by multiple genetic variants (GVs) and their interaction, by environmental factors, and by interaction between GVs and environment, and large study cohorts with substantial analytical power are typically required to elucidate these individual contributions. Here, we discuss the advantages of both power and feasibility afforded by the use of extended pedigrees of rhesus macaques (Macaca mulatta) for genetic studies of complex human disease based on next-generation sequence data. We present these advantages in the context of previous research conducted in rhesus macaques for several representative complex diseases. We also describe a single, multigeneration pedigree of Indian-origin rhesus macaques and a sample biobank we have developed for genetic analysis of complex disease, including power of this pedigree to detect causal GVs using either genetic linkage or association methods in a variance decomposition approach. Finally, we summarize findings of significant heritability for a number of quantitative traits that demonstrate that genetic contributions to risk factors for complex disease can be detected and measured in this pedigree. We conclude that the development and application of an extended pedigree to analysis of complex disease traits in the rhesus macaque have shown promising early success and that genome-wide genetic and higher order -omics studies in this pedigree are likely to yield useful insights into the architecture of complex human disease.

Dietary soy may not confound acute experimental stroke infarct volume outcomes in ovariectomized female rats.

Oestrogen administration can alter experimental stroke outcomes. Soy as a source of phytoestrogens may therefore modulate responses in 'oestrogen-sensitive' stroke models, thus potentially confounding results. We evaluated the effects of dietary soy on acute infarct volumes in a pilot study using a rat focal stroke model. We hypothesized that ovariectomized (OVX) rats fed a soy-rich diet would have smaller acute infarct volumes than rats fed a soy-free diet. OVX rats were randomly assigned to a soy-free (n = 6) or a soy-rich (n = 6) diet for four weeks and weighed weekly. Following the dietary trial, rats underwent 2 h of middle cerebral artery occlusion (MCAO). Mean arterial blood pressure, rectal and temporalis muscle temperatures, arterial blood gases and blood glucose were recorded peri-ischaemia. Rats were euthanized 22 h following 2 h of MCAO. Brains were stained with 2,3,5-triphenyl tetrazolium chloride for acute infarct volume analysis. Uterine weight and histology were also evaluated as additional internal oestrogen-sensitive controls. Rats on the soy-free diet had greater gains in body weight (259 +/- 6% baseline body weight) than rats on the soy-rich diet (238 +/- 4% baseline body weight). No differences were seen in uterine weight and histology, peri-ischaemic physiological parameters and infarct volumes between the treatment groups. The results of this pilot study suggest that the dietary soy level tested may not alter acute infarct volumes in ischaemic female rat brain. More studies addressing the potential confounding effects of dietary soy in 'oestrogen-sensitive' stroke models are needed if investigators are to make informed choices regarding diets used in experimental stroke research.