Comparative molecular genomic analyses of a spontaneous rhesus macaque model of mismatch repair-deficient colorectal cancer.

Colorectal cancer (CRC) remains the third most common cancer in the US with 15% of cases displaying Microsatellite Instability (MSI) secondary to Lynch Syndrome (LS) or somatic hypermethylation of the MLH1 promoter. A cohort of rhesus macaques from our institution developed spontaneous mismatch repair deficient (MMRd) CRC with a notable fraction harboring a pathogenic germline mutation in MLH1 (c.1029C

Analysis of cell populations in the normal rhesus macaque (Macaca mulatta) lower intestinal tract and diagnostic thresholds for chronic enterocolitis.

Rhesus macaques (Macaca mulatta) are used extensively in biomedical research, often with a focus on the gastrointestinal tract, and yet a full characterization of their normal resident intestinal cell populations has not been published. In addition, chronic enterocolitis (CE), also known as idiopathic chronic diarrhea, affects up to 25% of colony-housed rhesus macaques, often requiring euthanasia for welfare concerns and severely limiting their value as a breeding animal or research subject. We aimed to characterize subjective and objective variables in sections of the ileum, cecum, colon, and rectum in 16 healthy rhesus macaques and compare these results with a cohort of 37 animals euthanized for CE to produce relevant diagnostic thresholds and to improve case definitions for future studies. We found neutrophils to be an infrequent but expected component of the large intestinal leukocyte population. Animals with CE had significantly increased total leukocyte populations between crypts in the cecum, colon, and rectum; variable increases in specific cell populations across all levels of the distal intestinal tract; and significantly increased intraepithelial CD3+ T cells in the colon and rectum. Concentrations of enteroendocrine cells, enterochromaffin cells, and intestinal mast cells were not significantly different between healthy and affected individuals. This study characterizes individual leukocyte populations in the rhesus macaque lower intestinal tract, is the first to evaluate rhesus macaque intestinal mast cells, and provides key diagnostic thresholds for evaluating animals with potential CE.

Vasocutaneous fistula formation and repair following inguinal hernia repair in a rhesus monkey (Macaca mulatta).

A 6-year-old adult male rhesus macaque (Macaca mulatta) developed a vasocutaneous fistula following an anatomic inguinal hernia repair years earlier. The vasocutaneous fistula was surgically repaired, the vas deferens was ligated, and the wound was closed in layers with non-overlapping suture lines with no further adverse sequalae of events.

Specific pathogen free macaque colonies: a review of principles and recent advances for viral testing and colony management.

Specific pathogen free (SPF) macaques provide valuable animal models for biomedical research. In 1989, the National Center for Research Resources [now Office of Research Infrastructure Programs (ORIP)] of the National Institutes of Health initiated experimental research contracts to establish and maintain SPF colonies. The derivation and maintenance of SPF macaque colonies is a complex undertaking requiring knowledge of the biology of the agents for exclusion and normal physiology and behavior of macaques, application of the latest diagnostic technology, facilitiy management, and animal husbandry. This review provides information on the biology of the four viral agents targeted for exclusion in ORIP SPF macaque colonies, describes current state-of-the-art viral diagnostic algorithms, presents data from proficiency testing of diagnostic assays between laboratories at institutions participating in the ORIP SPF program, and outlines management strategies for maintaining the integrity of SPF colonies using results of diagnostic testing as a guide to decision making.

Whole-genome sequences of Campylobacter coli and Campylobacter jejuni isolates from rhesus macaques (Macaca mulatta) with and without intestinal disease.

Campylobacter jejuni or Campylobacter coli infection can lead to post-infectious irritable bowel syndrome in humans and may produce a similar syndrome in rhesus macaques (Macaca mulatta). We report the complete genomes of 8 C. jejuni isolates and 103 C. coli isolates obtained from rhesus macaques with and without intestinal disease.

Diversity of Campylobacter spp. circulating in a rhesus macaque (Macaca mulatta) breeding colony using culture and molecular methods.

Campylobacter jejuni and Campylobacter coli represent the leading causes of bacterial gastroenteritis in humans, and infections can produce post-infectious irritable bowel syndrome (PI-IBS). Rhesus macaques (Macaca mulatta) (RM) are similarly susceptible to acute campylobacteriosis and represent a potential model of PI-IBS. We characterized the Campylobacter species circulating in an RM breeding colony using culture, qPCR, and whole genome sequencing (WGS). We also compared the C. jejuni and C. coli prevalence in RM as detected with qPCR versus culture and identified risk factors for bacteria presence and intestinal disease. Culture of 275 samples yielded C. coli (103) and C. jejuni (8), of which 21.6% were resistant to quinolones and 3.6% were resistant to macrolides. Multidrug-resistant isolates were obtained exclusively from animals exhibiting diarrhea or with histologically confirmed chronic enterocolitis. WGS revealed a non-clonal population of Campylobacter spp. Genotypic predictions of resistance were excellent except for aminoglycosides. All sequenced isolates contained genes for all subunits of cytolethal distending toxin. qPCR detected a prevalence of 45.9% for C. coli and 29.6% for C. jejuni. The quantity of either bacteria was significantly higher (P < 0.05) in animals with intestinal disease compared to healthy animals, though only young age was significantly associated with the presence of Campylobacter sp. or intestinal disease. Significantly more C. jejuni positive animals were detected with qPCR than with culture. These results provide a comprehensive characterization of Campylobacter spp. circulating in a breeding colony of RM in the United States and suggest that qPCR is superior for the detection of C. jejuni in RM. IMPORTANCE: Gastrointestinal disease is one of the most common reasons for hospitalization in non-human primate colonies and accounts for over one-third of non-research related euthanasia. In rhesus macaques, this manifests as both acute diarrhea and chronic enterocolitis (CE), a syndrome of chronic diarrhea resulting in poor weight gain or weight loss which is minimally responsive to treatment. Campylobacter spp. are major causes of acute enterocolitis in rhesus macaques and may predispose individuals to the development of CE, similar to post-infectious irritable bowel syndrome in humans. Despite these concerns, there are few studies characterizing Campylobacter in rhesus macaque colonies, in particular utilizing whole genome sequencing and assessing findings with respect to the health status of the host. Our findings provide insight into Campylobacter strains circulating in rhesus macaque colonies, which can improve clinical monitoring, assist in treatment decisions, and provide new avenues of investigation into campylobacteriosis as a catalyst for CE.

Reproductive Outcomes in Rhesus Macaques (Macaca mulatta) with Naturally-acquired Trypanosoma cruzi Infection.

Chagas disease is a zoonotic vector-borne disease caused by infection with the protozoan parasite Trypanosoma cruzi. T. cruzi is found in Latin America and the Southern United States, where it infects many species, including humans and nonhuman primates (NHPs). NHPs are susceptible to natural infection and can develop clinical symptoms consistent with human disease, including Chagasic cardiomyopathy, gastrointestinal disease and transplacental transmission, leading to congenital infection. Due to evidence of Chagas transmission in Texas, this study hypothesized T. cruzi infection was present in a closed, outdoor-housed breeding colony of rhesus macaques (Macaca mulatta) located at a biomedical research facility in Central Texas. In addition, we questioned whether seropositive female rhesus macaques might experience reproductive complications consistent with maternal-fetal Chagas disease. The seroprevalence of T. cruzi infection in the colony was assessed using an Enzyme Linked Immunosorbant Assay (ELISA) to detect antibodies against Tc24 antigen as a screening assay, and a commercially available immunochromatographic test (Chagas Stat Pak) as a confirmatory assay. Retrospective serologic analysis was performed to confirm the status of all T. cruzi-infected animals between the years 2012 to 2016. The medical history of all seropositive and seronegative breeding females within the colony from 2012 to 2016 was reviewed to determine each animals' level of reproductive fitness. The percentage of T. cruzi-seropositive animals ranged from 6.7% to 9.7% in adult animals and 0% to 0.44% in juveniles or weanling animals, depending on the year. An overall 3.9% seroprevalence of T. cruzi infection was found in the total population. No significant differences in any measure of reproductive outcomes were identified between seropositive and seronegative females from 2012 to 2016. The lack of significant adverse reproductive outcomes reported here may help inform future management decisions regarding seropositive female rhesus macaques within breeding colonies.

Sequence diversity analyses of an improved rhesus macaque genome enhance its biomedical utility.

The rhesus macaque (Macaca mulatta) is the most widely studied nonhuman primate (NHP) in biomedical research. We present an updated reference genome assembly (Mmul_10, contig N50 = 46 Mbp) that increases the sequence contiguity 120-fold and annotate it using 6.5 million full-length transcripts, thus improving our understanding of gene content, isoform diversity, and repeat organization. With the improved assembly of segmental duplications, we discovered new lineage-specific genes and expanded gene families that are potentially informative in studies of evolution and disease susceptibility. Whole-genome sequencing (WGS) data from 853 rhesus macaques identified 85.7 million single-nucleotide variants (SNVs) and 10.5 million indel variants, including potentially damaging variants in genes associated with human autism and developmental delay, providing a framework for developing noninvasive NHP models of human disease.

Trypanosoma cruzi Transmission Among Captive Nonhuman Primates, Wildlife, and Vectors.

Natural infection of captive nonhuman primates (NHPs) with Trypanosoma cruzi (agent of Chagas disease) is an increasingly recognized problem in facilities across the southern USA, with negative consequences for NHP health and biomedical research. We explored a central Texas NHP facility as a nidus of transmission by characterizing parasite discrete typing units (DTU) in seropositive rhesus macaques (Macaca mulatta), identifying the wildlife reservoirs, and characterizing vector infection. In seropositive NHPs, we documented low and intermittent concentrations of circulating T. cruzi DNA, with two DTUs in equal proportions, TcI and TcIV. In contrast, consistently high concentrations of T. cruzi DNA were found in wild mesomammals at the facility, yet rodents were PCR-negative. Strong wildlife host associations were found in which raccoons (Procyon lotor) harbored TcIV and opossums (Didelphis virginiana) harbored TcI, while skunks (Mephitis mephitis) were infected with both DTUs. Active and passive vector surveillance yielded three species of triatomines from the facility and in proximity to the NHP enclosures, with 17% T. cruzi infection prevalence. Interventions to protect NHP and human health must focus on interrupting spillover from the robust sylvatic transmission in the surrounding environment.

Mismatch repair gene mutations lead to lynch syndrome colorectal cancer in rhesus macaques.

Colorectal cancer accounts for a substantial number of deaths each year worldwide. Lynch Syndrome is a genetic form of colorectal cancer (CRC) caused by inherited mutations in DNA mismatch repair (MMR) genes. Although researchers have developed mouse models of Lynch Syndrome through targeted mutagenesis of MMR genes, the tumors that result differ in important ways from those in Lynch Syndrome patients. We identified 60 cases of CRC in rhesus macaques (Macaca mulatta) at our facility since 2001. The tumors occur at the ileocecal junction, cecum and proximal colon and display clinicopathologic features similar to human Lynch Syndrome. We conducted immunohistochemical analysis of CRC tumors from several rhesus macaques, finding they frequently lack expression of MLH1 and PMS2 proteins, both critical MMR proteins involved in Lynch Syndrome. We also found that most macaque cases we tested exhibit microsatellite instability, a defining feature of Lynch Syndrome. Whole genome sequencing of rhesus macaque CRC cases identified mutations in MLH1 and/or MSH6 that are predicted to disrupt protein function. We conclude that this population of rhesus macaques constitutes a spontaneous model of Lynch Syndrome, matching the human disease in several significant characteristics, including genetic risk factors that parallel human Lynch Syndrome.

Disseminated Hemangiosarcoma in a Juvenile Rhesus Macaque (Macaca mulatta).

Hemangiosarcoma is a malignant tumor of vascular endothelial origin that is sporadically reported in rhesus macaques. This report describes the clinicopathologic features of a 1-y-old rhesus macaque with spontaneous disseminated hemangiosarcoma that originally presented as a focal cutaneous mass. Histopathologic examination of multiple tumor foci revealed regions in which the neoplastic cells formed diffuse sheets, as well as the well-defined vascular channels typically associated with hemangiosarcoma. Multiple endothelial cell immunomarkers were used to confirm the diagnosis in this rhesus macaque. The tumor exhibited staining properties consistent with those seen in domestic animals and humans. In addition, to our knowledge, this animal represents the youngest case of any form of spontaneous hemangiosarcoma reported in the rhesus macaque to date.

Single-nucleotide polymorphisms in the TNF gene are associated with obesity-related phenotypes in vervet monkeys.

Tumor necrosis factor (TNF) promoter single-nucleotide polymorphisms (SNPs) have been extensively characterized in humans, with numerous reports of associations with obesity-related phenotypes as well an array of infectious, immune-mediated, and inflammatory disease phenotypes. Controlling for the multitude of environmental risk factors in human studies has been a major confounder of efforts to elucidate the role and relative contribution of TNF promoter SNPs. As part of an ongoing initiative to further genetically and phenotypically characterize the St Kitts-origin vervet monkey (Chlorocebus aethiops ssp.) as an animal model of human obesity, we have conducted association analyses between TNF SNPs and previously defined obesity-related phenotypes in 265 pedigreed vervets. We report eight SNPs (-809G, -756A, -352C, -322A, +1285T, +2133T, +2362A, +2405), all contained within the same haplotype block and comprising a single haplotype, to be significantly associated with BMI, waist circumference, total plasma cholesterol (P < 0.05), and high-density lipoprotein-cholesterol (HDL-C) (P < 0.01). This study provides additional validation of the St Kitts-origin vervet model of obesity by demonstrating genetic associations analogous to that shown in humans.

Epigenetic changes with dietary soy in cynomolgus monkeys.

Nutritional interventions are important alternatives for reducing the prevalence of many chronic diseases. Soy is a good source of protein that contains isoflavones, including genistein and daidzein, and may alter the risk of obesity, Type 2 diabetes, osteoporosis, cardiovascular disease, and reproductive cancers. We have shown previously in nonhuman primates that soy protein containing isoflavones leads to improved body weight, insulin sensitivity, lipid profiles, and atherosclerosis compared to protein without soy isoflavones (casein), and does not increase the risk of cancer. Since genistein has been shown to alter DNA methylation, we compared the methylation profiles of cynomolgus monkeys, from multiple tissues, eating two high-fat, typical American diets (TAD) with similar macronutrient contents, with or without soy protein. DNA methylation status was successfully determined for 80.6% of the probes in at least one tissue using Illumina's HumanMethylation27 BeadChip. Overall methylation increased in liver and muscle tissue when monkeys switched from the TAD-soy to the TAD-casein diets. Genes involved in epigenetic processes, specifically homeobox genes (HOXA5, HOXA11, and HOXB1), and ABCG5 were among those that changed between diets. These data support the use of the HumanMethylation27 BeadChip in cynomolgus monkeys and identify epigenetic changes associated with dietary interventions with soy protein that may potentially affect the etiology of complex diseases.

Comparative analyses of single-nucleotide polymorphisms in the TNF promoter region provide further validation for the vervet monkey model of obesity.

Tumor necrosis factor is a cytokine that plays critical roles in inflammation, the innate immune response, and a variety of other physiologic and pathophysiologic processes. In addition, TNF has recently been shown to mediate an intersection of chronic, low-grade inflammation and concurrent metabolic dysregulation associated with obesity and its comorbidities. As part of an ongoing initiative to further characterize vervet monkeys originating from St Kitts as an animal model of obesity and inflammation, we sequenced and genotyped the human ortholog vervet TNF gene and approximately 1 kb of the flanking 3' and 5' regions from 265 monkeys in a closed, pedigreed colony. This process revealed a total of 11 single-nucleotide polymorphisms (SNPs) and a single 4-bp insertion-deletion, with minor allele frequencies of 0.08 to 0.39. Many of these polymorphisms were in strong or complete linkage disequilibrium with each other, and all but 1 were contained within a single haplotype block, comprising 5 haplotypes with frequencies of 0.075 to 0.298. Using sequences from humans, chimpanzees, vervets, baboons, and rhesus macaques, phylogenetic shadowing of the TNF promoter region revealed that vervet SNPs, like the SNPs in related species, were clustered nonrandomly and nonuniformly around conserved transcription factor binding sites. These data, combined with previously defined heritable phenotypes, permit future association analyses in this nonhuman primate model and have great potential to help dissect the genetic and nongenetic contributions to complex diseases like obesity. More broadly, the sequence data and comparative analyses reported herein facilitates study of the evolution of regulatory sequences of inflammatory and immune-related genes.