Extraintestinal Campylobacteriosis in Rhesus Macaques (Macaca mulatta)

Two cases of clinical disease associated with extraintestinal Campylobacter infection were recently encountered in rhesus macaques (Macaca mulatta). The first case was that of a 3-y-old, male, rhesus macaque experimentally infected with SIV, who presented with abdominal pain and a midabdominal mass and was euthanized. Pathology findings included an abscess within the median liver lobe, fibrinopurulent peritonitis, and intestinal serositis with isolation of Campylobacter fetus from the blood, liver, and the hepatic abscess. The second case was that of a 1-mo-old, female, rhesus macaque who died with no apparent history of illness. Gross pathology findings included thin body condition and diarrheic staining of the perineum; histologically, acute multifocal hepatitis with intralesional bacteria was noted. Campylobacter coli was isolated from the liver and colon. Extraintestinal Campylobacter infection is uncommon in humans, usually occurring in immunocompromised subjects and most commonly manifesting as bacteremia. Extraintestinal Campylobacter infections in animals are rare but have been associated with bacteremia and cholecystitis. The macaques presented here were either immunocompromised due to SIV infection (case 1) or more vulnerable due to young age (case 2). These factors likely contributed to the extraintestinal spread of Campylobacter.Campylobacter spp. are curved or spiral, gram-negative, microaerobic, typically motile bacteria with a single flagellum at one or both ends of the cell.²⁵ Campylobacter is one of the most common bacterial causes of gastroenteritis in humans worldwide.³ The Centers for Disease Control and Prevention estimate it to affect more than 1.3 million people in the United States each year.¹¹ Campylobacter spp. colonize the intestinal tract of primates, other mammals, birds, reptiles, and shellfish, but infection is not always associated with clinical signs of disease.^2,23 Contaminated or undercooked poultry represents the largest potential source of human infection.²² Campylobacteriosis is a zoonosis, and other significant sources of human infection include livestock, wildlife, pets, and contaminated water.²² The species most commonly isolated from humans and nonhuman primates are C. jejuni and C. coli; other species like C. fetus are less commonly found.^2,4,9,23 Extraintestinal campylobacteriosis in humans usually occurs in immunocompromised or elderly persons with underlying medical problems and most commonly manifests as bacteremia.²³ There have been a few reports of extraintestinal Campylobacter infections in animals, including bacteremia and cholecystitis in 2 dogs.^17,30     

Endometrial Decidualization and Deciduosis in Aged Rhesus Macaques (Macaca mulatta)

Superficial decidualization of the endometrial stroma is an essential feature of the implantation stage of pregnancy in rhesus macaques and other primates. Decidualization involves proliferation of the endometrial stromal cells, with differentiation into morphologically distinct decidual cells. Previous reports involving nonpregnant rhesus monkeys have described localized and widespread endometrial decidualization in response to administration of progesterone and synthetic progestogens. Ectopic decidua or ‘deciduosis’ describes the condition in which groups of decidual cells are located outside of the endometrium, most often in the ovaries, uterus and cervix but also in various other organs. In humans, most cases of deciduosis are associated with normal pregnancy, and ectopic decidua can be found in the ovary in nearly all term pregnancies. Here we describe pronounced endometrial decidualization in 2 rhesus macaques. Both macaques had been treated long-term with medroxyprogesterone acetate for presumed endometriosis, which was confirmed in one of the macaques at postmortem examination. In one animal, florid extrauterine and peritoneal serosal decidualization was admixed multifocally with carcinomatosis from a primary colonic adenocarcinoma. Cells constituting endometrial and serosal decidualization reactions were immunopositive for the stromal markers CD10, collagen IV, smooth muscle actin, and vimentin and immunonegative for cytokeratin. In contrast, carcinomatous foci were cytokeratin-positive. To our knowledge, this report describes the first cases of serosal peritoneal decidualization in rhesus macaques. The concurrent presentation of serosal peritoneal decidualization with carcinomatosis is unique.Abbreviations: GnRH, gonadotropin-releasing hormone; PAS, periodic acid–Schiff; SMA, smooth-muscle actinSuperficial decidualization of the endometrial stroma is an essential feature of the implantation stage of pregnancy in rhesus macaques and other primates.^13,27,29,37 This process typically begins, and is most prominent, adjacent to the spiral arteries, eventually expanding to affect the endometrium uniformly.³⁵ The endometrial stroma surrounds and supports the endometrial glands and is composed mainly of endometrial stromal cells and blood vessels.³⁵ Decidualization involves proliferation of the endometrial stromal cells, with differentiation into morphologically distinct decidual cells.^7,27,38 Endometrial stromal cells transform into large, polyhedral, cytoplasm-rich cells with large amounts of stored glycogen and are often binucleated or polyploid in character.^{6,13,27,30,35} Ultrastructurally, decidualized cells have numerous ribosomes, prominent rough endoplasmic reticulum and Golgi complexes, and cytoplasmic accumulation of glycogen and lipid droplets.^13,35 Consistent with their stromal origin, decidualized cells express mesenchymal immunohistochemical markers, such as vimentin, desmin, and muscle-specific actin.^{6,7,14,16,20,22}Initiation of decidualization by attachment of the blastocyst to the uterine epithelium depends on previous sensitization by progesterone secretion, after a brief priming by estrogen.^12,13,27 Estrogen and progesterone regulate a series of complex interactions at the interface between the developing embryo and the cells in the stromal compartment, leading to the formation of a differentiated maternal tissue (decidua) that supports embryo growth and maintains early pregnancy.²⁷ Postovulatory levels of circulating progesterone increase and help maintain the differentiation of decidual cells.^{7,13,33,37,38}Ectopic decidua or ‘deciduosis’ describes the condition in which groups of decidual cells reside outside of the endometrium, most often in the ovaries, uterus, and cervix; the fallopian tubes, peritoneum, omentum, diaphragm, liver, skin, spleen, appendix, abdominal–pelvic lymph nodes, renal pelvis, and lungs of women have also been reported as affected.^{6,14,18,20,22,28,29,38} In humans, most cases of deciduosis are associated with normal pregnancy, and ectopic decidua have been reported in the ovary in 90.5% to 100% of term pregnancies.^{6-8,14,20,22,28-30,38} Occasional cases in nonpregnant or postmenopausal women have been attributed to progesterone-secreting active corpora lutea, progesterone secretion by the adrenal cortex, trophoblastic disease, exogenous progestational agents, and pelvic irradiation.^{6-8,14,18,20,22,28,38} Deciduosis is usually an incidental finding that regresses postpartum within 4 to 6 wk; rarely, florid reactions have been reported to cause peritonitis, adhesions, hydronephrosis and hematuria, acute bowel obstruction or perforation (or both), abdominal pain mimicking appendicitis, massive and occasionally fatal hemoperitoneum, vaginal bleeding, and pneumothorax.^{6,7,14,18,20,22,28,29,31}Previous reports involving nonpregnant rhesus macaques have described localized and widespread endometrial decidualization in response to the administration of progesterone, synthetic progestogens, or progesterone-releasing bioactive intrauterine devices and intravaginal rings and have referred to these changes as ‘pseudodecidualization’ to indicate the absence of pregnancy in these animals.^12,33,35,37 In macaques given low (but superphysiologic) levels of progestogens, decidual changes have been noted in localized regions (around spiral arteries and underneath superficial epithelium), whereas high doses of progesterone or synthetic progestagens can cause a more pronounced and extensive reaction.³⁵In cynomolgus macaques, extrauterine decidual cell plaques are rare histologic findings in the subcoelomic mesenchyme of the ovarian cortex.^8,30 Despite the frequency of the condition in women, deciduosis is postulated to be a rarely documented lesion in primates because it is most often observed in conjunction with pregnancy, and pregnant cynomolgus macaques are seldom used in toxicity studies.⁸ Here we describe the pronounced endometrial decidualization of 2 rhesus macaques, one of which also had florid extrauterine and peritoneal decidualization that was admixed multifocally with carcinomatosis. Both macaques had been treated long-term with medroxyprogesterone acetate for presumed endometriosis, which was confirmed in one of the macaques at postmortem examination. To our knowledge, this report describes the first cases of peritoneal decidualization in rhesus macaques as well as the concurrent occurrence of carcinomatosis, endometriosis and peritoneal decidualization in a macaque. The extensive intermixing of the cell populations presented a diagnostic challenge at pathologic examination, and accurate diagnosis was achieved only through the use of multiple immunohistochemical markers.     

Alternative Activation of Macrophages in Rhesus Macaques (Macaca mulatta) with Endometriosis

Endometriosis is one of the most frequently encountered gynecologic diseases and a common cause of chronic pelvic pain and infertility. The pathophysiology of this syndrome can best be described as the presence of ectopic endometrium and a pelvic inflammatory process with associated immune dysfunction and alteration in the peritoneal environment. Macrophages play an important role in the progression and propagation of endometriosis. Alternative macrophage activation occurs in rodents and women with endometriosis but had not been examined previously in nonhuman primates. This case–control study aimed to characterize macrophage polarization in the ectopic and eutopic endometrial tissue of nonhuman primates with and without endometriosis. In addition, circulating cytokines in endometriosis cases and normal controls were investigated in an effort to identify serum factors that contribute to or result from macrophage polarization. Endometriosis lesions demonstrated increased infiltration by macrophages polarized toward the M2 phenotype when compared with healthy control endometrium. No serum cytokine trends consistent with alternative macrophage activation were identified. However, serum transforming growth factor α was elevated in macaques with endometriosis compared with healthy controls. Findings indicated that the activation state of macrophages in endometriosis tissue in nonhuman primates is weighted toward the M2 phenotype. This important finding enables rhesus macaques to serve as an animal model to investigate the contribution of macrophage polarization to the pathophysiology of endometriosis.Abbreviations: HLA, human leukocyte antigen; Iba1, ionized calcium binding adaptor molecule 1; M1, classically activated macrophage; M2, alternatively activated macrophage; sCD40L, soluble cluster of differentiation 40 ligand; TGF, transforming growth factor; VEGF, vascular endothelial growth factorEndometriosis is a common cause of chronic pelvic pain and infertility and affects more than 5.5 million women in North America alone.⁴¹ Although endometriosis is one of the most frequently encountered gynecologic health problems among women of reproductive age, the pathophysiology of this disease remains elusive due to its complexity and multifactorial etiology. The presence of functional endometrial glands and stroma outside the uterine cavity defines endometriosis. Currently, the most widely accepted theory for the origin of ectopic endometrial tissue is a combined effect of retrograde menstruation and associated implantation of endometrial fragments at an ectopic site. Progression of endometriosis lesions is thought to then be supported by peritoneal factors that allow cell adhesion and growth.⁴⁴ Although endometriosis is not a neoplastic disease, it exhibits aggressive features such as cellular proliferation, invasion, and vascular proliferation.¹² Strong evidence indicates that endometriosis involves a pelvic inflammatory process, with immune dysfunction and alteration in the peritoneal environment.^13,27 Numerous studies have demonstrated marked increases in macrophage populations and activity in the peritoneum of endometriosis patients.^6,54,59 Although macrophages are integral to homeostasis of the peritoneal environment, during endometriosis they mediate inflammation and facilitate the establishment and maintenance of the disease.Macrophages can be classified into 2 main populations: classically activated macrophages (M1), whose activating stimuli include IFNγ and LPS, and alternatively activated macrophages (M2), whose activating stimuli includes IL4, IL13, IL10, and transforming growth factor (TGF) β.⁵⁵ These polar phenotypes are not expressed together, but the activation state of tissue macrophages can change over time. This phenotypic switch is possible because macrophages retain plasticity, resulting in macrophage polarization that is transient and reversible.⁴⁰ A key component in determining the phenotype of the differentially activated macrophage is their response to microenvironmental signals, and this response allows for expression of a spectrum ranging from the M1 to M2 extremes.⁵¹ M1- and M2-activated macrophages perform different functions by producing pro- or antiinflammatory factors. M1 macrophages have enhanced endocytic functions and an enhanced ability to kill intracellular pathogens; they also secrete large amounts of proinflammatory cytokines such as IL1α, IL6, IL12, and TNFα.⁷ In contrast, M2 macrophages are involved in resolution of inflammation and promotion of tissue repair, and they secrete antiinflammatory and immunosuppressive cytokines including IL10 and TGFβ.³² M2 cells also express proangiogenic factors, such as coagulation factor XIII and vascular endothelial growth factor (VEGF) and have been associated with a high degree of vascularization in vivo.¹ The pathogenesis of endometriosis is therefore a likely combination of inappropriate or sustained polarization, leading to tissue damage (increased M1 response) and immune dysfunction (increased M2 response) and allowing for persistence of ectopic endometrial tissue.The use of animal models in endometriosis research is crucial. Work done with rodents involves the study of induced disease.⁵³ Despite this caveat, rodent models have been the basis for important contributions. Global macrophage depletion in a rat model of endometriosis effectively inhibits the initiation and growth of endometriosis implants.¹⁵ Attenuation of endometriosis has recently also been demonstrated in a mouse model of endometriosis.⁴ In that study, systemic depletion of macrophages was associated with failure of endometrial lesion development and defective angiogenesis of established lesions. Further evaluation of specific roles of differentially activated macrophages in that study⁴ showed that adoptive transfer of alternatively activated macrophages (M2) was associated with enhanced endometriosis progression. Conversely, adoptive transfer of inflammatory macrophages (M1) was associated with abrogated progression. In addition to evaluating murine lesions, the authors of the cited study⁴ investigated markers for alternative macrophage activation in women with endometriosis and matched controls which revealed increased expression of CD163 and CD206 (2 markers of M2 polarized macrophages) in endometriosis lesions as compared with disease-free peritoneum. Although many studies have been published about the pivotal role of macrophages in the pathophysiology of endometriosis, only a few have dealt with activation of the M1 and M2 macrophage phenotypes.^4,57 Furthermore, few studies have examined tissue infiltration of macrophages in eutopic endometrium of human subjects with endometriosis.^6,23 An exhaustive literature search failed to identify studies that investigate the role of M1 and M2 macrophage populations in eutopic endometrium.The current study uses rhesus macaques, which have been studied extensively in reproductive medicine.⁵⁸ Because spontaneous development of the disease requires menstrual shedding, endometriosis occurs naturally only in some nonhuman primate species, making development of lesions more comparable to the establishment of disease in humans.¹⁴ Compared with rodents, the nonhuman primate model of endometriosis is advantageous due to a close recapitulation of human disease and physiology. Work characterizing M1 and M2 macrophage activation in a species with spontaneous disease development may reflect a closer immunologic characterization to humans. In the current study, macrophage populations were evaluated in archival tissue collected from rhesus macaques with a diagnosis of endometriosis as confirmed by histologic examination. To characterize the phenotype of endometrial tissue macrophages in ectopic endometriosis lesions and eutopic endometrium of both cases and controls, immunohistochemistry was used to quantify cells expressing M1- and M2-specific markers. We hypothesized that endometriosis lesions and eutopic endometrium of rhesus macaques would be associated with a polarized macrophage infiltration consisting of increased numbers of M2 macrophages. This increase in M2 response may cause reduced immune clearance of ectopic endometrial cells, facilitating their implantation and growth. Further we speculated that M2 polarization would be associated with increased serum cytokines including IL10 and VEGF and decreased production of IL6, IL12, and TNFα. The lack of findings that support our hypotheses may suggest that the micro- or peritoneal environment is more important for lesion development or that another component of the systemic milieu is the determining factor in the development of endometriosis.     

Correlates of Male Consortship Rate in Free-Ranging Rhesus Macaques (Macaca mulatta)

Male–male competition for access to receptive females can take the form of nonrecurring fights and/or a sustained contest over mating opportunities. Male physical condition has been linked to dominance rank and reproductive success in species characterized by intrasexual fights for dominance and access to females. In group-living species characterized by endurance rivalry, however, factors contributing to male reproductive success are less well understood. In such species, particularly seasonally breeding ones with low sexual dimorphism and seniority-based rank, age and social factors other than rank may prove important. In the absence of genetic data, male mate guarding or consortship can serve as an indicator of male reproductive success. To evaluate the contribution of age and intragroup sociality to male consortship rate, I collected behavioral data during one nonmating and one mating season in two social groups of free-ranging rhesus macaques that experience no predation or food scarcity. Higher-ranking males, younger males, or males that exhibited lower rate of intrasexual aggression had higher consortship rates. Male–female dyads that groomed outside consortship did not form consortships as often as dyads that did not engage in nonconsort grooming. This is the first study to identify the significance of male–male aggression and male–female affiliation to male consortship rate in a species characterized by endurance rivalry, high male rank stability, and strong female mate choice. Social behaviors and male age may be particularly important in determining male reproductive success in populations experiencing high food availability and a lack of predation, which are typical of an increasing number of vertebrates in areas densely populated by humans.     

Hematology and Culture Assessment of Cranially Implanted Rhesus Macaques (Macaca mulatta)

The use of percutaneous cranial implants in rhesus macaques (Macaca mulatta) has long been a valuable tool for neuroscience research. However, when treating and assessing these animals, veterinarians are required to make assumptions about diagnostic results due to a lack of research into how these implants affect physiology. Microbial cultures of cranial implant sites show an abundance of colonizing bacteria, but whether these microbes affect animal health and wellbeing is poorly understood. In addition, microbial antibiotic resistance can present significant health concerns for both the animals and the researchers. To help elucidate the relationship between percutaneous cranial implants and blood parameters, complete blood cell counts and serum chemistry results were assessed on 57 nonhuman primates at our institution from September 2001 to March 2017. Generalized estimating equations were used to compare the results before and after an animal''s first implant surgery. This modelling showed that cranial implants were a significant predictor of alterations in the number of neutrophils, lymphocytes, and red blood cells, and in the concentration of hemoglobin, alkaline phosphatase, creatinine, calcium, phosphorus, total protein, albumin, and globulin. Anaerobic and aerobic bacterial cultures were performed to identify bacteria associated with cranial implants. Staphylococcus spp., Streptococcus spp., and Corynebacterium spp. comprised the majority of the aerobic bacterial isolates, while Fusobacterium spp., Peptostreptococcus spp. and Bacterioides fragilis comprised the majority of anaerobic bacterial isolates. Using a Pearson r correlation for statistical analysis, we assessed whether any of these bacterial isolates developed antibiotic resistances over time. Cefazolin, the most frequently used antibiotic in monkeys in this study, was the only antimicrobial out of 41 agents tested to which bacteria developed resistance over time. These results indicate that percutaneous implants are associated with a generalized inflammatory state, multiple bacterial species are present at the implant site, and these bacteria may contribute to the inflammatory response.

Neuroscience research often employs in vivo analysis of neuronal distribution, activity, and function, all of which require visualization and manipulation of the brain in living animals. To achieve this, cortical structures are accessed by the placement of percutaneous implants that are anchored to the skull and outfitted with transcranial ports. Studies of the auditory system,^72,78 visual cortex,^20,22,46,54 motor cortex,^23,44 perception,⁷⁰ and optogenetics^23,80 have all benefitted from the use of these implants in multiple laboratory species. However, few studies have investigated the effects of these implants on the general physiology of research animals.³²Due to human similarities in neuroanatomy, physiology, social behavior, and cognition, rhesus macaques (Macaca mulatta) have proven invaluable in translational studies of cortical structures. This species has long been the recipient of percutaneous cranial implants²⁹ and therefore, has been the subject of many refinements in their construction to improve research outcomes.^{3,11,40,43,60} Traditionally, these implants are constructed out of titanium or titanium alloy hardware that is anchored in place with acrylic or bone cement. Recording chambers provide access points through craniotomy sites where devices are implanted into deeper structures of the brain. Titanium alloys have been shown to provide a good bone/implant interface through the development of titanium oxides;^17,32,59 however, these implants may leach ions into surrounding tissues, with unknown health or research implications.³² Acrylics provide a moldable and easily repairable substrate, but have poor biocompatibility.^5,53 Their addition to implants increases exposed tissue surface area, creating ideal environments for infectious agents to thrive.^2,43Cranial implants can develop complications, including inflammation and infections at skin margins and the bone/implant interface.³² Multiple species of bacteria, including Staphylococcus spp., Corynebacterium spp., Enterococcus spp., and Providencia rettgeri, have been recovered from the chambers and skin edges of cranial implants in rhesus macaques.^10,43,88 These infections may be due to manipulation and contamination of implants by the animals themselves. Chronicity of infections is also likely due to the inability to apply therapeutic agents to deeper regions of tissue shielded by the implant. Chronic pathology is evident in deeper structures at the neural-implant interface.⁶⁷ For example, sustained glial responses and neural degeneration have been identified at sites where electrodes and arrays contact brain tissue.^12,74Chronic inflammation has well-established effects on hematologic values, such as red and white blood cell counts, platelet numbers, and total protein measurements. Few studies are currently available that link chronic percutaneous implants to changes in hematology in rhesus macaques, requiring clinicians to use established reference intervals of nonimplanted animals when analyzing bloodwork. This requires assumptions about effects of implants on overall health; these assumptions may or may not be correct, but are the necessary basis for assessments and therapies. In this study, systemic physiologic changes were identified in clinically healthy, implanted animals to provide a framework for analysis of complete blood cell counts (CBC) and serum chemistry (CHEM) values for clinicians working with rhesus macaques with percutaneous implants. We also assess bacterial cultures taken from these implants to identify common contaminants and their antibiotic resistance profiles. We hypothesize that clinically healthy rhesus macaques with percutaneous implants have significantly different hematology profiles than those without implants, despite a lack of clinical signs. By understanding how implants and associated bacteria affect routine blood test results, observed changes can be better characterized as normal or abnormal, so that veterinary care can be tailored as needed. This important step will allow improved care and welfare of animals used in neuroscience research.     

Coat Color Variation and Pigmentation Gene Expression in Rhesus Macaques (Macaca mulatta)

Light-dark coat color variation is a common aspect of color diversity within and across mammalian taxa. This variation in pelage brightness is associated with aspects of evolutionary ecology, particularly for primates, but little is known about the genetic mechanisms underlying light-dark differences in pelage pigmentation. Previous work, focusing particularly on macaques (Genus Macaca), has found no clear relationship between color variation and coding sequences of key pigmentation genes. This suggests that other loci and/or gene regulatory differences underlie this variation and raises the question of how patterns of gene expression differ in light verses dark hair follicles. Here, we examine relative expression levels of pigmentation genes in hair follicles from free-ranging rhesus macaques (Macaca mulatta) showing stark light-dark coat color variation. We quantified the brightness (reflectance) of plucked hair tufts using a spectrophotometer. We extracted RNA from the follicles and used quantitative RT-PCR to measure the relative amounts of gene product (mRNA) for seven candidate pigmentation genes (MITF, MC1R, MGRN1, ATRN, SLC24A5, TYRP1, and DCT). Expression values were normalized with the house-keeping gene ACTB. All candidate genes were expressed at similar levels in dark, intermediate, and light hair, and thus, light-dark variation in macaque coat color is unlikely to be due to differences in the expression of these key pigmentation genes. This study represents the first examination of gene expression and natural color variation in a non-human primate population. Our results indicate that even in a system, like pigmentation, where a candidate-gene approach is promising, identifying important intra-specific gene regulatory differences remains challenging.     

The Influence of Kinship on Familiar Natal Migrant Rhesus Macaques (Macaca mulatta)

In most primate species, females remain in the natal group with kin while males disperse away from kin around the time of puberty. Philopatric females bias their social behavior toward familiar maternal and paternal kin in several species, but little is known about kin bias in the dispersing sex. Male dispersal is likely to be costly because males encounter an increased risk of predation and death, which might be reduced by dispersing together with kin and/or familiar males (individuals that were born and grew up in same natal group) or into a group containing kin and/or familiar males. Here we studied the influence of kinship on familiar natal migrant rhesus macaques (Macaca mulatta) on Cayo Santiago, Puerto Rico, by combining demographic, behavioral, and genetic data. Our data suggest that kinship influences spatial proximity between recent natal immigrants and males familiar to them. Immigrants were significantly nearer to more closely related familiar males than to more distantly related individuals. Within a familiar subgroup, natal migrants were significantly closer to maternal kin, followed by paternal kin, then non-kin, and finally to males related via both the maternal and paternal line. Spatial proximity between natal immigrants and familiar males did not decrease over time in the new group, suggesting that there is no decline in associations between these individuals within the first months of immigration. Overall, our results might indicate that kinship is important for the dispersing sex, at least during natal dispersal when kin are still available.     

Efficacy of Oral Fecal Bacteriotherapy in Rhesus Macaques (Macaca mulatta) with Chronic Diarrhea

Chronic diarrhea remains the principal burden in providing health care for nonhuman primates in biomedical research facilities. Although the exact etiology continues to puzzle nonhuman primate clinicians, recent research in humans has shown that restoring the indigenous microbial diversity may be successful in resolving cases of chronic diarrhea when other treatment modalities have failed. The process of restoring this microbial balance, known as fecal bacteriotherapy, uses the complete flora from a normal donor as a therapeutic probiotic mixture. In the current study, Indian-origin rhesus macaques were randomized into treatment (n = 7) and control (n = 6) groups to determine whether orally administered fecal bacteriotherapy would reduce the overall incidence of chronic diarrhea during a 60-d follow-up period in the treatment group compared with control macaques, which received a placebo. Although the treatment effect, determined by comparing the baseline fecal scores of the treatment and control groups, did not reach statistical significance, preprocedure and postprocedure fecal scores in the treatment group differed significantly. These findings are encouraging, and we hope that our study will motivate larger studies evaluating the use of fecal bacteriotherapy in nonhuman primates.Chronic diarrhea is perhaps the most daunting clinical challenge of nearly every biomedical research facility that houses large numbers of nonhuman primates. Our facility, The Oregon National Primate Research Center (ONPRC), is no exception. As of 2008, approximately 14.7% of the total population at this center was reported to have diarrhea requiring medical attention each year, which constituted an average of 35.2% of the total clinical caseload.²⁷ Our more recent analysis of the medical records from 2010 has confirmed these statistics: 16.2% of the total population was treated for diarrhea in 2010, which comprised 29.3% of the total clinical caseload for that year. The cost of chronic diarrhea to institutions such as ours in terms of veterinary staff time, diagnostics, and medications is profound. Consequently, colony management personnel and resources are taxed due to the care and maintenance of these patients, given that nonhuman primates with chronic diarrhea generally are in poor body condition, lag behind the growth rate of their peers, and require frequent medical intervention, thereby making them undesirable as research subjects and unproductive members of the breeding colony.⁸Chronic diarrhea is undeniably the largest, most expensive problem in providing health care for nonhuman primate colonies. Nonhuman primates diagnosed with chronic diarrhea typically test negative for known fecal pathogens^19,24,27 and are recalcitrant to common diarrhea treatment modalities. For these reasons, the underlying cause of chronic diarrhea has been elusive and is likely multifactorial. Over the years, numerous researchers and clinicians in this field have attempted to devise an effective treatment regimen for these patients, with little success.Recent research in humans has shown that restoring the indigenous microbial diversity may be useful in resolving cases of chronic diarrhea when other treatment modalities have failed.^10,18 A normal healthy digestive tract contains numerous bacterial inhabitants, which typically act to impede exogenous bacteria from establishing themselves as pathogens. After an episode of gastrointestinal disease that results in diarrhea, the population of indigenous bacteria often is disrupted, subsequently leading to decreased numbers and diversity of these organisms. This imbalance, or dysbiosis, may result from pathogenic diarrhea or may be nosocomial due to prescribed antibiotic therapy.^7,10,18,21 Several publications have explored the idea that dysbiosis can be treated with an infusion of normal flora.^1,4,16 Fecal bacteriotherapy uses the complete flora of a normal donor as a therapeutic probiotic mixture of living organisms.⁵ Because the bacterial components of the normal fecal flora that are the most important for host defense are unknown, reintroducing all flora is currently recommended.²³ In addition to providing the complete bacterial flora from a normal donor, another possible advantage of this therapy is that it halts the cycle of antimicrobial use in these patients.¹ The discontinuance of intestinal flora disruption through the use of antimicrobials, when combined with the probiotic effects of fecal bacteriotherapy, constitutes the philosophy of this therapeutic approach. Several case series in the human literature have demonstrated that this therapy is capable of resolving refractory cases of diarrhea, with very high success rates after single administrations.^2,4,11,15,28 In addition, the transplantation of donor stool can dramatically change the recipient''s intestinal flora in as little as 14 d.¹⁶ Furthermore, fecal bacteriotherapy has been an effective tool in veterinary medicine for the treatment of ruminants and horses with enteric disease.^6,9,12,22 However, whether this treatment modality will be effective in nonhuman primates or whether successful cases will continue to be sporadic and species-specific remains unknown.The goal of the current study was to test a new treatment modality, fecal bacteriotherapy, which if successful, would reduce the overall incidence of chronic diarrhea in rhesus macaques. Because the need for detailed information regarding techniques used to prepare and administer the fecal suspension has been recognized in the human literature,^5,23,28 we here describe in detail the standardized treatment protocol that we developed. Our hypothesis was that the incidence and severity of diarrhea during the 60-d follow-up period would be decreased in macaques that received fecal bacteriotherapy compared with those that received placebo treatment.     

Videotaped Behavior as a Predictor of Clinical Outcome in Rhesus Macaques (Macaca mulatta)

Understanding the behavior of laboratory NHP facilitates health assessment and clinical care. We sought to characterize the behavior of critically ill rhesus macaques (Macaca mulatta) and determine whether specific behaviors or behavioral changes might facilitate the determination of prognosis and clinical endpoints. Twenty-two critically-ill subjects were videorecorded after they were removed from the outdoor breeding colony for diagnostic work-up and treatment. Subjects were categorized as survivors (n = 15) and those that were euthanized according to existing clinical endpoints (n = 7). Behavior before, during, and after cageside examination was compared between these groups with regard to the presence or absence of direct observation. This approach allowed us to determine whether these settings revealed differences between groups or masking of behaviors during direct observation. Before cageside examination, several behaviors (for example, self-grooming and anxiety behaviors) were significantly more common in surviving subjects than in euthanized subjects. Few significant differences in behavior were detectable during or after the examination. Subjects that were eventually euthanized showed more illness-related behaviors; however, not all animals requiring euthanasia showed these signs when an observer was present. Furthermore, euthanized animals spent more time in an alert posture during direct observation than at other times. Therefore, direct observation of critically ill rhesus macaques may not yield the most accurate assessment of illness severity, and using video to assess behavior may be helpful for prognosis.The assessment and recognition of pain and distress in laboratory animals is crucial to ensure welfare and high-quality research.^{6,10,12,20,23,26,28} Difficulty in identifying species-specific signs of pain and distress suggests that cageside assessment of clinical condition by an observer, as a stand-alone method, may not be an optimal method of determining prognosis in critically ill animals of some species.^10,24,26,27 The behavior of NHP, including rhesus macaques (Macaca mulatta), can be especially difficult to interpret due to their relatively stoic nature and tendency to mask clinical signs of illness in the presence of human observers.^{8,10,12,14,17} Two explanations for the tendency to mask pain, which is also observed in other species, are their status as a prey species and the need to hide weaknesses from group members.^3,17,22,23 According to the 2009 Institute for Laboratory Animal Research (ILAR) recommendations for recognizing pain and distress in nonhuman primates, “[v]iewing an animal from a distance or by video can aid in detecting subtle clinical changes.”¹² The goal of the current study was to evaluate the use of videotaped behavioral data to increase accuracy in predicting prognoses for rhesus macaques that are critically ill.A humane endpoint is defined as the earliest time at which an animal may experience unnecessary pain or distress, undue suffering, or impending death.^4,12 Both subjective and objective diagnostic criteria are currently used to define clinical and research endpoints.^{6,7,10,12,21,26,29}A myriad of diagnostics can be used for this purpose, including physical examination, cageside examination, blood assays (CBC and serum biochemistry), imaging (radiographs, ultrasound, CT, and MRI), bacterial culture and sensitivity, urinalysis, and collection of tissue samples (fine-needle aspiration and biopsy). One specific example of using clinical diagnostics to aid in the prediction of mortality in NHP involved using acid-base levels in animals with severe social trauma.¹¹ Behavioral assessment may be an important and underutilized diagnostic method for assessing pain and distress. Recent studies have evaluated the behavior, specifically facial expression, of mice to detect and assess pain.^16,19 Methods for evaluating behavior typically involve cageside examination, but assessment of behavior via videotape in the absence of an observer may also be useful.Endpoints may vary depending on species, nature of the research conducted, and assessment of degree of pain or distress. At our facility, experimental endpoints (those defined as part of a specific study) are differentiated from clinical endpoints (those determined by an animal''s health and quality of life). The veterinarian has the authority to elect euthanasia for an animal that is assessed to have reached an endpoint by either or both classifications. IACUC-approved endpoint policies have been established to aid in this decision. Criteria included in these guidelines include weight loss (excluding postpartum females and intended weight loss for obese animals), anorexia that is not responsive to treatment, diarrhea that is not responsive to treatment, and major organ failure that is not responsive to treatment. Veterinarians use cageside examination in the overall assessment of animals and to aid in the decision to euthanize.To our knowledge, the current study is the first to assess the behavior of critically ill rhesus macaques that controls for the cause of clinical presentation and to use observer presence or absence as an independent variable. To obtain information that may assist in refining clinical endpoints, we compared the behavior of critically ill rhesus macaques while an observer was absent, present, and recently present (labeled as preobservation, observation, and postobservation, respectively, in this text).We hypothesized that in comparison to in-person observation of critically ill rhesus macaques, the use of videotaping in the absence of direct observation would be more accurate in detecting differences in the behavior between those animals that eventually required euthanasia and those that did not. In addition, we hypothesized that behaviors would be suppressed (‘masked’) during direct observation, accounting for the reduced information available to the direct observer. If our hypothesis is supported, then videotaped behavior may be useful as a prognostic indicator that could be incorporated into development of clinical endpoints.     

南京红山森林动物园猕猴的社会玩耍行为

社会玩耍行为是灵长类动物的常见行为类型之一,作为种群数量最大的非人灵长类之一,猕猴(Macaca mulatta)的社会玩耍行为是它们日常生活的一部分。本研究利用红外摄像技术,对南京红山森林动物园猕猴冬季社会玩耍行为的基本特征进行了研究。研究记录了每一回合玩耍行为的发生时间、持续时间、参与对象年龄、参与个体数以及玩耍行为类型等行为参数。在持续10 d的视频中,共记录到猕猴的社会玩耍行为7 496次,其中触摸和抓打4 018次,追逐1 012次,撕咬和摔跤671次,其他53次,以及各种玩耍行为组合1 742次。利用单因素方差分析,发现猕猴的社会玩耍行为节律明显,两个玩耍高峰分别为08:30~09:30时和10:30~11:30时。利用Kruskal-Wallis检验及Mann-Whitney U两两比较法,发现参与个体数和玩耍行为类型对社会玩耍持续时间影响显著,在玩耍过程中,参与个体数越多,社会玩耍持续时间越长;组合类型的持续时间最长,其次是撕咬和摔跤,再次是触摸和抓打,最后是追逐。0~3岁的少年个体在玩耍行为中更常采用触摸和抓打类型,而亚成年及成年个体则倾向于选择追逐类型。该研究量化了猕猴社会玩耍行为的类别和影响因素,为动物园的猕猴管理和动物福利提供了数据支持。     

Sex Ratio, Conflict Dynamics, and Wounding in Rhesus Macaques (Macaca mulatta)

Rhesus macaques, like many other primates, live in stable, multi-male multi-female groups in which adult females typically outnumber adult males. The number of males in multi-male/multi-female groups is most commonly discussed in terms of mate competition, where the sex ratio is a function of an adult male's ability to monopolize a group of females. However, the relationship between sex ratio and group stability is unclear because the presence of many males may either reduce stability by increasing mate competition or may improve stability if adult males are key conflict managers. We investigated the relationship between sex ratio, male intervention behavior, and trauma in seven groups of captive rhesus macaques. Our results show that high-ranking adult males intervene twice as frequently as adult females (P<0.0001) and are about twice as successful (P<0.0001). Furthermore, the type of adult males present in the group affects the relationship between intervention behavior and rate of traumas: males must be unrelated to the highest-ranking matrilines. Groups with a lower ratio of females per male unrelated to the alpha and beta matrilines had better intervention success (P<0.04) and fewer traumas requiring veterinary care (P=0.003). We conclude that conflict management behavior by adult males, particularly those unrelated to the highest-ranking matrilines, is the mechanism by which sex ratio influences the frequency of traumas, and thus group stability. We therefore suggest that monitoring and managing the sex ratio of captive primate groups is one of many measurements to predict group stability.     

An Outbreak of Tularemia in a Colony of Outdoor-Housed Rhesus Macaques (Macaca mulatta)

Since an epizootic and detection of clinical cases of tularemia (Francisella tularensis) in 1996 at the Oregon National Primate Research Center, only 8 cases were identified in the succeeding 13 y. However, within a period of 7 mo, primarily during Winter 2010, 6 rhesus macaques were confirmed positive for Francisella tularensis type B by the Centers for Disease Control and Prevention by culture and fluorescent antibody testing. All cases had similar gross pathologic findings, which included necrotizing splenitis and lymphadenitis. Recent colony management efforts have focused on minimizing nonhuman primate exposure to commonly observed reservoir species and controlling rodent access to corral-style housing. Strategies continue to evolve with regard to managing a large breeding colony of nonhuman primates in the presence of this threat.Abbreviation: ONPRC, Oregon National Primate Research CenterFrancisella tularensis, the causative agent of tularemia, is a small pleomorphic gram-negative coccobacillus.¹¹ Severe disease and potentially death in humans can result from exposure to as few as 10 cfu of this highly infectious organism.^7,10 The disease is also known as rabbit fever and deer-fly fever, reflecting 2 common sources of infection for humans.³ F. tularensis is classified by the United States Department of Health and Human Services as a Category A Select Agent.⁶ It is considered a potential agent of biologic warfare, and in fact, has been weaponized and stockpiled in the past.¹⁰ The 2 biovars that are referenced most frequently in published human and nonhuman primate literature are tularensis (type A) and holarctica (formerly paleartica; type B).^4,12,13,23 An additional biovar, novicida (type C), has been described, but its virulence in humans is decreased due to its lack of a capsule.¹⁰Tularemia is endemic to many parts of the northern hemisphere, which includes the region surrounding the Oregon National Primate Research Center (ONPRC), an AAALAC-accredited facility.²⁰ Tularemia has one of the broadest host ranges of all bacteria, encompassing well over 200 mammalian species primarily, in addition to birds, amphibians, fish, and various arthropods such as fleas, ticks, mosquitoes, and flies.^10,15,19,20 The ONPRC is located in a mixed forest and field environment which is bordered by wetlands and residential neighborhoods outside of Portland. More than 4500 nonhuman primates are housed here, and most live outdoors in breeding groups. Therefore, exposure to this potentially life-threatening and zoonotic pathogen is inevitable, due to its persistence in the environment and the close proximity of several reservoir species. Presumed reservoir species commonly observed at ONPRC include meadow voles (Microtus pennsylvanicus), brown rats (Rattus norvegicus), deer mice (genus Peromyscus), house mice (Mus musculus), and California ground squirrels (Spermophilus beecheyi). Potential arthropod vectors that are monitored regularly at ONPRC include biting flies and mosquitoes. At this time, testing of prospective rodent carriers for tularemia is ongoing; therefore, in the interest of caution, all of the rodent and arthropod species listed are considered potential carriers of the disease.Tularemia was first recognized at the ONPRC in 1996 during an epizootic that resulted in 24 deaths among corral-housed rhesus macaques. Serology results from banked sera and sera collected during and after the outbreak demonstrated a seroconversion rate of approximately 25% in 723 animals. During the succeeding 13 y, only 8 sporadic cases were diagnosed. However, within a period of 3 mo during the winter of 2010, 5 rhesus macaques were diagnosed with Francisella tularensis type B by the Centers for Disease Control and Prevention (Atlanta, GA). Four months later, an additional case was confirmed. All 6 macaques were younger than 1 y and were assigned to a breeding colony protocol approved by the ONPRC Animal Care and Use Committee. The current report describes the clinical signs and gross and histologic findings associated with these cases, as well as methods for prevention and control of future cases of disease.     

Development of Breeding Populations of Rhesus Macaques (Macaca mulatta) That Are Specific Pathogen-free for Rhesus Cytomegalovirus

Development of breeding colonies of rhesus macaques (Macaca mulatta) that are specific pathogen-free (SPF) for rhesus cytomegalovirus (RhCMV) is relatively straightforward and requires few modifications from current SPF programs. Infants separated from the dam at or within a few days of birth and cohoused with similarly treated animals remain RhCMV seronegative indefinitely, provided they are never directly or indirectly exposed to a RhCMV-infected monkey. By systematically cohousing seronegative animals into larger social cohorts, breeding populations of animals SPF for RhCMV can be established. The additional costs involved in expanding the current definition of SPF status to include RhCMV are incremental compared with the money already being spent on existing SPF efforts. Moreover, the large increase in research opportunities available for RhCMV-free animals arguably would far exceed the development costs. Potential new areas of research and further expansion of existing research efforts involving these newly defined SPF animals would have direct implications for improvements in human health.Abbreviations: HCMV, human cytomegalovirus; NHP, nonhuman primate; RhCMV, rhesus cytomegalovirus; SPF, specific pathogen-freeThe impetus for expanding the current SPF definition to include RhCMV is 2-fold. The first is the increasing number of studies involving infection of rhesus macaques with RhCMV as a nonhuman primate (NHP) model of human infection with human cytomegalovirus (HCMV). The second is the recognition that the current SPF protocols result in animals that are also uninfected with RhCMV, such that a relatively minor change in derivation can generate monkeys that meet the current SPF definition and that are uninfected with other endemic viruses, including RhCMV and simian foamy virus.     

Chagas Disease in 2 Geriatric Rhesus Macaques (Macaca mulatta) Housed in the Pacific Northwest

Chagas disease (American trypanosomiasis) is caused by the protozoan parasite Trypanosoma cruzi. It is endemic in Latin America but also is found in the southern United States, particularly Texas and along the Gulf Coast. Typical clinical manifestations of Chagas disease are not well-characterized in rhesus macaques, but conduction abnormalities, myocarditis, and encephalitis and megaesophagus have been described. Here we report 2 cases of Chagas disease in rhesus macaques housed in the northwestern United States. The first case involved a geriatric male macaque with cardiomegaly, diagnosed as dilated cardiomyopathy on ultrasonographic examination. Postmortem findings included myocarditis as well as ganglioneuritis in the esophagus, stomach, and colon. The second case affected a geriatric female macaque experimentally infected with SIV. She was euthanized for a protocol-related time point. Microscopic examination revealed chronic myocarditis with amastigotes present in the cardiomyocytes, ganglioneuritis, and opportunistic infections attributed to her immunocompromised status. Banked serum samples from both macaques had positive titers for T. cruzi. T. cruzi DNA was amplified by conventional PCR from multiple tissues from both animals. Review of their histories revealed that both animals had been obtained from facilities in South Texas more than 12 y earlier. Given the long period of clinical latency, Chagas disease may be more prevalent in rhesus macaques than typically has been reported. T. cruzi infection should be considered for animals with unexplained cardiac or gastrointestinal pathology and that originated from areas known to have a high risk for disease transmission.Abbreviations: DCM, dilated cardiomyopathy; CMV, cytomegalovirus; NHP, nonhuman primateChagas disease is caused by the hemoflagellate protozoan parasite Trypanosoma cruzi. The disease is endemic in many regions of South and Central America, and its range extends to the southern United States. In the United States, there is evidence that the parasite has established a domestic transmission cycle with canine reservoirs,¹⁹ and there are numerous wildlife reservoirs, most importantly armadillos, raccoons, rodents, and opossums.⁶ The main mode of transmission is via arthropod vectors, primarily triatomine species (kissing bugs or cone-nosed bugs), which serve as intermediate hosts. Vector species are present in the southern half of the United States.² Infection has been reported sporadically in domestic nonhuman primate (NHP) colonies.¹² Autochthonous insect vector-mediated transmission in humans in the United States has been reported rarely.³³ Transmission of T. cruzi to NHP is thought to occur mainly through insect vectors, specifically by contamination of the oral mucous membranes with parasite-containing feces during consumption of the bug. The infection may remain subclinical for years and, similar to that in people, affects the nervous system, digestive system, and heart. Clinical findings in NHP are infrequent but can include subcutaneous edema, fever, anorexia, lethargy, heart failure, and sudden death.^4,5 As in humans, the disease in NHP consists of an acute phase, with a paucity of clinical manifestations, and a chronic phase, characterized by irreversible cardiomyopathy leading to cardiac dysfunction and death. Chronically infected NHP in the indeterminate form of the chronic phase can exhibit subclinical conduction and echocardiographic abnormalities.⁸T. cruzi infections have been reported in a number of NHP species housed in Texas, Louisiana, and Georgia. Species affected include rhesus macaques (Macaca mulatta),^12,17 cynomolgus macaques (M. fascicularis),^29,42 yellow baboons (Papio cynocephalus), olive baboons (P. anubis),^12,14,41 pig-tailed macaques (M. nemestrina),^12,35 squirrel monkeys (Saimiri sciureus),¹³ ring-tailed lemurs (Lemur catta),^15,30 black-eyed lemurs (Eulemur macaco flavifrons), black and white ruffed lemurs (Varecia variegata variegate),¹⁵ pileated gibbon (Hylobates pileatus),³⁶ a lion-tailed macaque (M. silenus),³⁰ a Celebes crested black macaque (M. nigra),²⁷ and a chimpanzee (Pan troglodytes).⁴ Here we report on 2 cases of Chagas disease in rhesus macaques housed in the northwestern United States but that originated from South Texas.     

Rhesus Macaques (Macaca mulatta) Use Posture to Assess Level of Threat From Snakes

Once prey animals have detected predators, they must make decisions about how to respond based on a cost‐benefit analysis of their risk level. The threat sensitivity hypothesis predicts that prey animals match their response to the level of risk, with high‐risk predator encounters eliciting stronger evasive responses than low‐risk encounters. Primates are known prey of snakes, yet they vary their responses toward snakes. We predicted that primates match their response to the threat level from snakes by assessing posture, with striking postures indicating greater risk than coiled postures and coiled postures indicating greater risk than extended sinusoidal postures. We tested this prediction in a series of experimental trials in which captive rhesus macaques (Macaca mulatta) were exposed to snake models in those postures. Results supported the predictions: macaques responded more strongly to a snake model in a striking posture than in a coiled posture and more to a snake model in a coiled posture than to an extended sinusoidal snake model. We also examined responses of macaques to a partially exposed snake model to mimic the condition of incomplete information, as snakes are often occluded by vegetation. The occluded snake model evoked a response comparable to that of the striking snake. These findings support the threat sensitivity hypothesis. Rhesus macaques use the posture of snakes as a cue in threat assessment, responding more intensely as threat increases, and they also behave as if risk is elevated when their information about snakes is incomplete.