Positive Reinforcement Training to Enhance the Voluntary Movement of Group-Housed Sooty Mangabeys (Cercocebus atys atys)

Positive reinforcement training (PRT) has successfully been used to train diverse species to execute behaviors helpful in the everyday care and wellbeing of the animals. Because little information is available about training sooty mangabeys (Cercocebus atys atys), we analyzed PRT with a group of 30 adult males as they were trained to shift from 1 side of their enclosure to the other. Over a 4-mo period we conducted 57 training sessions totaling 26.5 h of training and recorded compliance information. During training, compliance increased from 76% of the animals during the first 5 training sessions to 86% of the animals shifting during the last 5 sessions. This result indicated progress but fell short of our goal of 90% compliance. After 25 training sessions, problem-solving techniques were applied to help the consistently noncompliant animals become more proficient. The techniques included reducing social stress by shifting animals so that noncompliant monkeys could shift into an unoccupied space, using more highly preferred foods, and ‘jackpot’-sized reinforcement. To determine whether social rank affected training success, animals were categorized into high, medium, and low dominance groups, based on 7 h of behavioral observations. A Kruskal–Wallis test result indicated a significant difference in compliance according to the category of dominance. Although training a group this large proved challenging, the mangabeys cooperated more than 90% of the time during follow-up sessions. The training program improved efficiency in caring for the mangabeys.Abbreviation: PRT, positive reinforcement trainingPositive reinforcement training (PRT) has successfully been used to train diverse primate species to execute a variety of behaviors helpful in daily husbandry procedures, facilitating research, and promoting the wellbeing of the animals. Over the last 2 decades increasing emphasis has been placed on the use of PRT techniques, as nonhuman primates living in zoos and laboratories have been trained to cooperate with a variety of husbandry, research, and veterinary procedures. For example, PRT has been used to facilitate daily colony management such as moving animals within or between their enclosures³ and into transport cages. Positive reinforcement training facilitates veterinary and research procedures by training animals to cooperate with intramuscular^7,21,23,28 and subcutaneous injections^18,23 and to present for voluntary blood,^4,8,12 semen^17,23 and urine ^12,27 collection. Training has also been used to improve social dynamics in primate groups by reducing competition and aggression over food² and increasing affiliative behaviors between conspecifics.²²At its most basic level, PRT is a form of operant conditioning in which the animal receives a primary reinforcer or reward, such as food, for exhibiting a particular behavior. Receiving the reward increases the likelihood that the animal will exhibit the behavior again in the future. The reward typically is paired with a clicking sound (called a conditioned reinforcer or ‘bridge’), which serves to pinpoint the exact moment the desired behavior occurs. After multiple presentations of the conditioned reinforcer with the primary reinforcer, the clicking sound develops a meaning of “good job”²⁰ to the animal. Positive reinforcement training contrasts with more traditional training techniques such as negative reinforcement. In negative reinforcement, the animal may be coerced into complying by applying a stimulus that an animal wants to avoid and removing that same stimulus when the animal complies.²⁰ For example, directing water from a hose at an animal might be used to get the animal to move from inside to outside or the pressure of a squeeze mechanism in a cage is used to get the animal to come forward and hold a position. Both PRT and negative reinforcement training can be effective, but PRT is believed to have additional benefits such as reducing distress, increasing choice and control for the animal, and therefore improving wellbeing.^7,10,11 The general principles of PRT are species-independent, and the training techniques are used successfully across taxa. Many species of nonhuman primates such as chimpanzees,^{2,3,7,8,12,16-18,21,27,28} macaques,^4,6,19 and tamarins²⁵ have been trained, yet no information has been published on the training of mangabeys (Cercocebus spp.), even though they live in both zoo and laboratory colonies.Several publications have reported training monkeys to move between portions of their enclosures and to enter smaller transport cages or chute systems.^{5,6,13,19,24,29} These articles describe the use of a combination of PRT and negative reinforcement strategies to coerce animals into complying with the requested behavior and then rewarding them. Several of these publications^6,19 describe animal care staff entering the monkeys’ enclosures as a part of the training process. None of these articles relied solely on PRT techniques to achieve their goals.For this study, we wanted to train a group of mangabeys to move between different portions of their enclosure, on cue, to allow for daily cleaning and cage maintenance. To protect personnel, we wanted to conduct the training without having people enter the mangabey enclosure, and we chose to use only PRT procedures for the presumed benefits to the wellbeing of the mangabeys. This approach was similar to that of to an earlier study with chimpanzees,³ in which the animals were taught to shift between enclosures by using only PRT. We recorded the training time and number of sessions needed to train the mangabeys to move throughout their enclosure on cue. During the training process, we suspected that the dominance ranking of individual animals was affecting their success rate and therefore assessed this factor''s contribution. In addition, we found that a few animals were not cooperating with the request to shift even after considerable training. We therefore used a problem-solving method to develop hypotheses about the lack of compliance and to test possible solutions to improve the performance of this subset of our test population.     

Measurement of Fetal Biparietal Diameter in Owl Monkeys (Aotus nancymaae)

Owl monkeys are New World primates frequently used in biomedical research. Despite the historical difficulty of breeding owl monkeys in captivity, several productive owl monkey breeding colonies exist currently. The animals in the colony we describe here are not timed-pregnant, and determination of gestational age is an important factor in prenatal care. Gestational age of human fetuses is often determined by using transabdominal measurements of fetal biparietal diameter. The purpose of this study was to correlate biparietal diameter measurements with gestational age in owl monkeys. We found that biparietal diameter can be used to accurately predict gestational age in owl monkeys.Abbreviation: BPD, biparietal diameter; CNPRR, Center for Neotropical Research and Resources; FHR, fetal heart rateOwl monkeys are small neotropical primates used in biomedical research as valuable models for research addressing malaria, ocular and infectious disease, and other agents. As New World primates, owl monkeys do not harbor cercopithecine herpesvirus 1 (B virus). Therefore, they do not pose the same risk of zoonosis to personnel as do macaques. Adult owl monkeys are approximately 1 kg in body weight and can be easily hand-caught and restrained by using protective leather gloves. Although transabdominal ultrasonography is used routinely for pregnancy monitoring in macaque^{2,15,17,19,20} and baboon^4,7,8 breeding colonies, animals must be anesthetized before imaging to protect personnel from zoonotic diseases and injury.Determination of gestational age is an important tool in primate breeding colonies. Measurements of biparietal diameter (BPD) can be used to determine gestational age.^{3,4,7-9,11,15-17,19,20} The BPD is the diameter of the fetal head made when outer perimeter of the calvarium is clearly visible. In New World primates, timing of pregnancy in squirrel monkeys^11,14 and capuchin monkeys³ through measurements of BPD has been described. The goal of this study was to establish guidelines and standard measurements for the determination of gestational age in owl monkey breeding colonies using ultrasound-generated BPD measurements. This report describes gestation age determination and pregnancy monitoring via ultrasound in the owl monkey.     

Multiple Extrauterine Pregnancy with Early and Near Full-Term Mummified Fetuses in a New Zealand White Rabbit (Oryctolagus cuniculus)

Extrauterine pregnancy (EP) is infrequent in mammalian species and occurs when fertilized ova implant and develop outside the uterus. A common outcome is abdominal pregnancy resulting in mummified fetuses (lithopedia). Here we describe an unusual case of abdominal pregnancy with early and near full-term lithopedia. Macroscopic findings supported the diagnosis of lithopedia with distinct age differences and facilitated further characterization of primary ectopia and risk factors leading to this occurrence.Abbreviation: EP, extrauterine pregnancyExtrauterine pregnancy (EP) occurs infrequently in most mammalian species.¹² The term derives from the Latin prefix meaning ‘outside’ or ‘beyond’ and refers to the implantation of a fertilized ovum outside the uterine cavity. Extrauterine pregnancy was first recognized more than 900 y ago² among other discoveries with a hereditary nature.¹³ Early reports compared EP in women, cats, dogs, and rabbits⁷ and described the presence of mummified fetuses in laboratory rabbits.^16,35EP is a serious obstetric complication that occurs asymptomatically in most cases.¹⁷ There are 4 classifications of EP: tubal, ovarian, abdominal–peritoneal, and cervical. The fallopian tube is the most common location and leads to tubal pregnancy. When gestation occurs in the abdominal–peritoneal cavity, abdominal pregnancy results and is subdivided as primary, when fertilization occurs outside the uterus after an oocyte is accidentally released from the fimbria, and secondary, when an oocyte is released due to direct tubal trauma.⁴⁵ A rare form of EP associated with high maternal morbidity and fetal mortality is called heterotopic (or combined) pregnancy, which occurs when 2 fertilized eggs coexist, one outside the uterus and the other inside.^18,33,44Undetected EP is frequently associated with fatal outcomes to the dam and offspring, including the formation of mummified fetuses, which may eventually become calcified and are called lithopedia (from the Greek: lithos, stone; paidion, child).^11,51,53 The condition is infrequent, and the factors that influence the unexpected outcomes of this pathology are not well understood.^26,34,35 Epizootiologic investigations are few,¹² although a recent report outlined the prevalence of EP in large NZW rabbit breeding colonies.⁵¹Examples of EP have been documented in dogs,¹⁷ cats,^14,39,42,49 rabbits,^20,29,45,51 hamsters,^9,46 rats,²⁶ mice,^8,12 guinea pigs,^3,30 lambs,⁴⁰ nonhuman primates,^10,34,38,50 and other species including humans.^11,12 However, despite the number of documented species, the majority of reports failed to note detailed clinical symptoms that interfered with reproduction even in instances that led to the formation of lithopedions.⁴²Experimentally, mouse embryos have successfully been transferred to a variety of sites including the peritoneal cavity, kidney, spleen, muscles, testis, and the anterior chamber of the eye.^1,6,21,31 The aim of the current report is to describe a rare case of abdominal pregnancy in which lithopedia developed clinically silently and coexisted with multiple pregnancies in a healthy doe rabbit.     

Eliminating Animal Facility Light-at-Night Contamination and Its Effect on Circadian Regulation of Rodent Physiology, Tumor Growth, and Metabolism: A Challenge in the Relocation of a Cancer Research Laboratory

Appropriate laboratory animal facility lighting and lighting protocols are essential for maintaining the health and wellbeing of laboratory animals and ensuring the credible outcome of scientific investigations. Our recent experience in relocating to a new laboratory facility illustrates the importance of these considerations. Previous studies in our laboratory demonstrated that animal room contamination with light-at-night (LAN) of as little as 0.2 lx at rodent eye level during an otherwise normal dark-phase disrupted host circadian rhythms and stimulated the metabolism and proliferation of human cancer xenografts in rats. Here we examined how simple improvements in facility design at our new location completely eliminated dark-phase LAN contamination and restored normal circadian rhythms in nontumor-bearing rats and normal tumor metabolism and growth in host rats bearing tissue-isolated MCF7(SR^–) human breast tumor xenografts or 7288CTC rodent hepatomas. Reducing LAN contamination in the animal quarters from 24.5 ± 2.5 lx to nondetectable levels (complete darkness) restored normal circadian regulation of rodent arterial blood melatonin, glucose, total fatty and linoleic acid concentrations, tumor uptake of O₂, glucose, total fatty acid and CO₂ production and tumor levels of cAMP, triglycerides, free fatty acids, phospholipids, and cholesterol esters, as well as extracellular-signal-regulated kinase, mitogen-activated protein kinase, serine–threonine protein kinase, glycogen synthase kinase 3β, γ-histone 2AX, and proliferating cell nuclear antigen.Abbreviation: 13-HODE, 13-hydroxyoctadecadienoic acid; γH2AX, histone 2AX; AKT, serine–threonine protein kinase; ERK1/2, extracellular signal-regulated kinase p44/46; GSK3β, glycogen synthase kinase 3β: LAN light at night; MEK, mitogen-activated protein kinase kinase, PCNA, proliferating cell nuclear antigen; SR^–, steroid-receptor–negativeRelocating laboratory animal research from one institution to another can be a daunting task for both scientists and animal care personnel with regard to control of lighting and elimination of light-at-night (LAN) contamination. Appropriate facility lighting and lighting protocols, as outlined in the Guide for the Care and Use of Laboratory Animals,³⁰ are essential for maintaining the health and wellbeing of laboratory animals and ensuring the credible outcome of scientific investigations.^16-18,22 The profound effect of light on circadian behavior and physiology is well established.^{2,3,5,9,11,12,16-18,22,29,32,44,46,49,52,55-58,64}Minor alterations in light intensity,¹¹ spectral quality,¹² and duration⁹ at any given time of day can alter or disrupt chronobiologic rhythms markedly in all mammals.^{6,17,26,44,55-59} Light information, which initially is detected by a small group of intrinsically photosensitive retinal ganglion cells containing the blue light-sensitive photopigment melanopsin,^6,26 is transmitted through the retinohypothalamic tract⁵⁹ to a central molecular clock located in the suprachiasmatic nucleus of the hypothalamus.^32,57 The suprachiasmatic nucleus, the activity of which is entrained by the light:dark cycle,^32,57 sends projections over a polysynaptic pathway to the pineal gland driving a series of molecular events leading to the production of the pineal neurohormone melatonin (N-acetyl-5 methoxytryptamine), primarily during the night.^29,46 The daily rhythmic melatonin signal contributes to the temporal coordination of normal behavioral and physiologic functions including the sleep–wake^23,46,66 and reproductive cycles,^51,55 immune function,^38,41,56 hormone levels,^{19,31,45,47,68} temperature regulation,²³ electrolyte balance,⁶⁹ neural protein synthesis,⁶⁵ and redox states.^24,53Dark-phase LAN exposure suppresses endogenous melatonin concentrations and may lead to various disease states,^42,58 including carcinogenesis,^7,8,16,18,33 and metabolic syndrome.^{17,34-37,39,70} Previous in vivo studies in our former laboratory (at the Bassett Research Institute, Cooperstown, NY) demonstrated that animal room LAN of as little as 0.2 lx (0.08 µW/cm²; rodent eye level) during an otherwise normal dark-phase suppressed normal physiologic nighttime melatonin levels, leading to markedly disrupted circadian regulation of physiology and metabolism in nontumor-bearing host animals^16,18 and a stimulation in metabolism and proliferation of both tissue-isolated MCF7 steroid-receptor–negative (SR^–) human breast cancer xenografts and syngeneic grafts of rodent hepatoma 7288CTC in rats.^7,17 This effect was mediated by melatonin receptor-mediated suppression of cAMP, leading to inhibition of tumor linoleic acid uptake and its metabolism to the mitogenic signaling molecule 13-hydroxyoctadecadienoic acid (13-HODE). These events culminated in downregulation of epidermal growth factor and insulin-like growth factor 1 pathways.^7,8,16-19,62Exposure to LAN is likely to exert pervasive and problematic effects on mammalian behavior and physiology in laboratory animal facilities around the world. During the past decade, improved facility design and better adherence to animal room lighting protocols certainly has helped to reduce the problem. In moving to our laboratory animal facility at Tulane University School of Medicine (New Orleans, LA), we discovered considerable preexisting LAN contamination that had to be eliminated before we could resume our human cancer research.The current study was performed to monitor the effects of the elimination of animal room LAN contamination over time on animal health and wellbeing, tumor growth, and metabolic profiles by assessing well-established circadian parameters in physiology and metabolism.^7,8,16-18 We measured light-induced suppression of melatonin, an accepted and sensitive marker of the effects of light on the circadian system in all mammals,^{2,3,5,9,11,12,15,16,18,20,21,29,44,46,49,52,55-58,64} before and after tumor implantation and growth. Tissue-isolated MCF7(SR^–) human breast cancer xenografts and 7288CTC rat hepatomas have been well-characterized over the years in our former light-tight facilities^7,8,16-18 and provided us with unique markers and measures of the extent to which LAN contaminated our new animal quarters. In tumor-bearing animals exposed to even minimal LAN, the latency-to-onset of tumor growth and proliferation rates of these tumors increase markedly in direct proportion to LAN intensity. As improvements were made to eliminate LAN contamination in the new location over the course of more than 20 generations of tumor passages, we measured the changes and reestablishment of normal rat and host–tumor circadian regulation. The information from this study may assist investigators and animal care personnel in addressing this important influence on the health and wellbeing of laboratory animals and consequent effects on the outcome of scientific investigations.     

Nonsurgical Repair of a Pseudoaneurysm in a Cynomolgus Macaque (Macaca fascicularis)

A cynomolgus macaque presented with an ecchymotic and edematous left leg approximately 1 wk after a blood sample had been collected from the left femoral vein. Ecchymosis was noted in the femoral triangle, prepuce, and scrotum. The animal was not febrile or exhibiting signs of pain or distress. Duplex Doppler ultrasound imaging was used to evaluate the area. An arteriovenous fistula between the femoral artery and vein, accompanied by a pseudoaneurysm arising from the femoral artery, was identified. Various invasive and noninvasive treatment options for the pseudoaneurysm, including surgical repair, thrombin injection, stent placement, and ultrasound-guided compression repair (UGCR), were considered. UGCR was chosen as the first option for treatment. After a total of 20 min of UGCR at the neck of the pseudoaneurysm, complete thrombosis was achieved. Subsequent imaging of the lesion revealed resolution of the pseudoaneurysm. Because of the risks involved with invasive management techniques for this vascular lesion, UGCR is a valuable noninvasive treatment option for the repair of pseudoaneurysms.Abbreviation: CDI, color Doppler imaging; UGCR, ultrasound-guided compression repairVascular malformations have been reported to occur in a wide variety of species.^{2,3,5-7,10,11,13,15-22,24,25,27-35} These abnormalities include arteriovenous fistulas, vascular shunts, hemangiomas, vascular atresias, aneurysms, pseudoaneurysms, telangectasia, and lymphangiomas, with the overwhelming majority of reports describing arteriovenous fistulas.^{2,3,5,6,11,15-19,25,27,30,33,35} Aneurysms have occurred in several primates including chimpanzees, gorillas, squirrel monkeys, howler monkeys, owl monkeys, African green monkeys, spider monkeys, patas monkeys, and capuchin monkeys.²⁴ Pseudoaneurysms are reported infrequently. Pseudoaneurysms (or false) aneurysms are the result of leakage of blood from an artery into a defined space. A pseudoaneurysm associated with the femoral artery in a black and white colobus monkey was detected 6 d after manual restraint for routine blood collection from the femoral artery.³² The pseudoaneurysm was excised, and the resulting vascular defect was closed with an autologous graft. Another report¹⁰ describes a pseudoaneurysm associated with the femoral artery in a rhesus monkey. The diagnosis was obtained interoperatively, and excision of the defect was successful. Because surgical treatment of pseudoaneurysm has inherent risks and requires considerable surgical skill for a successful outcome, a noninvasive approach would be a valuable and cost-effective treatment option. This report is the first to describe the clinical presentation, diagnosis, and nonsurgical treatment of a pseudoaneurysm in a cynomolgus macaque.     

Progression of Ulcerative Dermatitis Lesions in C57BL/6Crl Mice and the Development of a Scoring System for Dermatitis Lesions

Ulcerative dermatitis (UD) is a common, spontaneous condition in mice with a C57BL/6 background. Although initial lesions may be mild, UD is a progressive disease that often results in ulcerations or debilitating fibrotic contractures. In addition, lesions typically are unresponsive to treatment. Euthanasia is often warranted in severe cases, thereby affecting study outcomes through the loss of research subjects. Because the clinical assessment of UD can be subjective, a quantitative scoring method and documentation of the likely time-frame of progression may be helpful in predicting when animals that develop dermatitis should be removed from a study. Such a system may also be helpful in quantitatively assessing success of various treatment strategies and be valuable to clinical laboratory animal veterinarians. In this 1.5-y, prospective cohort study, we followed 200 mice to monitor the development and course of UD. Mice were examined every 2 wk. A clinical sign (alopecia, pruritus, or peripheral lymphadenopathy) was not identified that predicted development of UD lesions in the subsequent 2-wk period. Once UD developed, pruritus, the character of the lesion (single or multiple crust, coalescing crust, erosion, or ulceration), and the size of the lesion were the only parameters that changed (increased) over the course of the disease. Pruritus was a factor in the rapid progression of UD lesions. We used these findings to develop a quantitative scoring system for the severity of UD. This enhanced understanding of the progression of UD and the quantitative scoring system will enhance the monitoring of UD.Abbreviation: UD, ulcerative dermatitis; S number, scratching number; COL, character of lesionsUlcerative dermatitis (UD) is an idiopathic, spontaneous, debilitating syndrome of laboratory mice that is typically a disease of aged^1,19,43,46 C57BL/6 mice or genetically engineered mice on a C57BL/6 background.^1,19,43,44 Some reports discuss a similar condition in young, weanling mice that presents initially as alopecia.^24,42,44,45 Prevalence rates of UD between 4.1% to 21% have been reported.^1,6,19 Although no etiology has been identified, environmental factors,^{6,19,41,42,44} diet,^{5,29,41,42,46} season,^19,41,43,44 age at weaning,⁴² alopecia,^24,42,44,45 sex,^19,39,41,43 immune complex vasculitis,¹ follicular dysplasia,⁴⁴ lesion location,²⁰ and deficiencies in vitamin A metabolism⁴⁴ have all been implicated as predisposing factors for disease development. In addition, oronasal pain and chronic inflammation may lead to self-mutilation as a result of, initiating an “itch” response.¹⁰ UD is diagnosed by ruling out other causes of dermatitis in laboratory mice, such as fur mites,⁹ infections, fight wounds,¹⁷ strain phenotype,^15,35,40,49 and experimentally induced dermatitis.^4,50 Other diagnostic criteria are based on professional judgment and may include strain (C57BL/6 background),^1,19,44 lesion location (head and dorsal thorax),^1,19,43,44 intense pruritus,^1,19,44 peripheral lymphadenopathy,^6,19,39 and failure to respond to treatment.¹⁹ The rapid progression of UD lesions results in significant morbidity in laboratory mice.^6,19,44 Typically, the lesions progress to large, irregularly shaped, confluent ulcerations on the dorsal cervical and thoracic region.^1,19,39,44 As the lesions heal, contracted scar tissue forms, which can impair species-typical behaviors and mobility.^39,41,43,44 The presence of large dermal ulcerations or debilitating contractures affect animal welfare and typically necessitate euthanasia of affected mice. Although reports on the later stages of UD have been consistent,^{1,19,39,41,43,44} information on the initiation and progression of UD lesions is conflicting. Pruritus,^1,19,44 pain,¹⁰ and genetic predisposition^1,19,43,44 have been implicated as initiators of the disease. Alopecia, pruritus, erythema, and single or multiple(s) crust have all been reported as early signs of the disease.^{1,19,39,42-44} However, the majority of this information has been collected retrospectively, at timed necropsies, or based on anecdotal reports.Scoring systems are useful tools to evaluate clinical diseases in laboratory animals. For example, scoring systems have been published for tumors,^14,28 body condition,^14,28,47 and neurologic phenotype¹³ in mice to aid in assessment of clinical disease severity. Even though the progressive and severe nature of UD typically warrants eventual euthanasia, determining the severity of disease has typically been based on professional judgment,^1,39 subjective scoring,^{12,15,19,40,43,48,49} or postmortem histology.^40,44 A quantitative scoring system for UD in live mice has not been described and could greatly aid laboratory animal veterinarians and researchers in determining the severity of the disease and response to treatment.The purpose of the current study is to investigate clinical parameters that reflect the progression of UD to facilitate management and veterinary care of mice with UD. We followed 200 mice from 3 wk of age until the development of UD to determine the initial signs and progression of UD lesions. We hypothesized that mice will first develop signs of pruritus prior to any clinical lesion. From there, we predicted that clinical lesions will progress stepwise though the following stages of severity, with or without alopecia: (1) excoriations; (2) a single, small punctuate crust; (3) multiple, small punctuate crusts; (4) coalescing crust; (5) erosion; and (6) ulceration. In addition, we hypothesized that a quantitative, validated, and reliable UD scoring system can be created that is based on physical examination parameters that do, in fact, predict development and progression of UD. Having a more thorough understanding of the initiation and progression of ulcerative lesions likely will enhance our ability to predict the outcome for a given mouse and develop earlier end-points for that mouse. Furthermore, use of this scoring system will enable accurate monitoring of UD lesions.     

Comparison of the Effects of Ketamine, Ketamine�CMedetomidine, and Ketamine�CMidazolam on Physiologic Parameters and Anesthesia-Induced Stress in Rhesus (Macaca mulatta) and Cynomolgus (Macaca fascicularis) Macaques

This study compared the cardiovascular, respiratory, anesthetic, and glucocorticoid effects of ketamine alone with ketamine–medetomidine and ketamine–midazolam in rhesus and cynomolgus macaques. Macaques were given either intramuscular ketamine (10 mg/kg), intramuscular ketamine–medetomidine (3 mg/kg; 0.15 mg/kg), or oral midazolam (1 mg/kg) followed by intramuscular ketamine (8 mg/kg). The addition of medetomidine, but not midazolam, provided muscle relaxation and abolishment of reflexes that was superior to ketamine alone. In addition, medetomidine did not cause clinically relevant effects on cardiovascular and respiratory parameters when compared with ketamine. These 3 protocols did not have significantly different effects on fecal glucocorticoid metabolites. These results suggest that medetomidine can be a valuable addition to ketamine for healthy patients, whereas oral midazolam at the tested dose does not provide additional benefits.Abbreviations: KetMed, ketamine–medetomidine, KetMid, ketamine–midazolam, NHP, nonhuman primates, PaCO₂, arterial partial pressure of carbon dioxide, PaO₂, arterial partial pressure of oxygen, SO₂, oxygen saturation of hemoglobin, SaO₂, arterial oxygen saturation, SpO₂, peripheral oxygen saturation, T, timeKetamine hydrochloride is commonly used as a sole anesthetic agent in nonhuman primates (NHP) but has several drawbacks that could be ameliorated by combining it with other agents. First, animals sedated with dissociative anesthetics retain their reflexes and can exhibit varying amounts of skeletal muscle movement and, rarely, seizure activity.⁴⁶ This propensity raises concerns about personnel safety when working with animals carrying zoonotic infectious agents. In addition, excessive movement creates challenges when performing certain tasks such as tuberculosis testing or when monitoring animals for appropriate anesthetic depth during minor surgical procedures.⁵⁵ Second, the analgesic properties of ketamine in NHP are unknown. Ketamine generally is considered to be satisfactory for somatic analgesia but inadequate for deep or visceral pain. However, analgesic effects can differ widely between species, and the few studies available show variable results even for minor procedures in NHP.^21,28,53 In addition, ketamine is associated with pain on injection and volume-dependent tissue damage in many species^{7,17,18,46,55} and can cause severe psychomimetic side effects such as hallucinations and delirium in people.^29,44 Although we cannot know whether ketamine causes excessive psychological stress in NHP, policies of the Public Health Service and Animal and Plant Health Inspection Services establish a precedent for assuming that procedures affect animals as they do human beings unless there is evidence to the contrary.^3,43Medetomidine and midazolam are 2 agents that are often combined with ketamine to improve anesthesia.^{15,24,27,33,40,49,50,54,58,59} Medetomidine is a selective α2-adrenergic agonist that is a racemic mixture of 2 optical enantiomers, the active dexmedetomidine and the inactive levomedetomidine. Although the primary benefit of α2 agonists is improved muscle relaxation, they also provide documented analgesic effects in many species, including humans and NHP.^{10,11,23,35,46,57} In addition, reversal of the medetomidine with atipamezole can decrease recovery time and reverse sedation if unacceptable side effects occur.⁵⁵ As a benzodiazepine, midazolam can be expected to offer additional sedation and muscle relaxation to ketamine anesthesia.⁴⁵ In addition, the use of medetomidine or midazolam may accommodate a decreased dose of ketamine, thus reducing tissue necrosis and inflammation.⁵⁵In addition to the physical benefits of medetomidine and midazolam, these drugs may help attenuate anesthesia-related stress responses. In dogs, medetomidine decreases the stress-related hormonal changes that occur with ketamine alone.¹ Human physicians often use benzodiazepines to decrease psychotic events, anxiety, and nausea, particularly when ketamine is used.^{8,9,19,22,29,44} In NHP, premedication with midazolam may decrease the stress associated with injection and the possible psychologic effects associated with ketamine. These potential benefits are difficult to measure in animals, but various methods have been used in an effort to quantify the stress associated with anesthesia in NHP.^2,14,51,63 Food intake and urinary cortisol are affected significantly by ketamine anesthesia in macaques.^14,51 Fecal cortisol is gaining popularity as a noninvasive way to evaluate the hormonal impact of a variety of situations, including restraint and sedation.^{20,38,41,48,56,61,63} For example, fecal cortisol increased significantly in chimpanzees 2 d after sedation.⁶³ Although cortisol was the first hormone measured in fecal samples, a fecal corticosterone assay has been developed that measures glucocorticoid metabolites in primate fecal samples with less variable results than those of fecal cortisol assays.^60,62 Although any anesthetic episode may create stress, whether particular drugs or injections in an unsedated animal are exacerbating factors is unclear. We therefore used the glucocorticoid assay to compare 3 different anesthesia protocols.The purpose of this study was to determine whether medetomidine or oral midazolam could provide a deeper level of anesthesia in macaques than ketamine alone with comparable safety. In addition, we sought to determine whether either of these medications, particularly premedication with midazolam, attenuated the glucocorticoid response associated with anesthesia.     

Recurrent Idiopathic Gingival Enlargement in an Olive Baboon (Papio anubis)

We here describe a case of recurrent gingival enlargement in an olive baboon (Papio anubis). This baboon (a male breeder that had not undergone any experimental procedures) also had shown mild gingival enlargement the 2 y prior to the current lesion. Clinical and histopathologic findings confirmed a diagnosis of idiopathic gingival enlargement.The term ‘gingival enlargement’ describes abnormal, excessive growth of the periodontal tissues.¹¹ Although gingival enlargement is seen and studied in nonhuman primates only rarely, it has been extensively evaluated in humans.¹⁵ The etiology in humans is multifactorial and includes age, genetic predisposition, and induction due to drugs (for example, cyclosporine, dihydropyridines, calcium channel blockers, sodium valproate, erythromycin) or plaque.^{6,13,14,22,29}Numerous case reports of gingival overgrowth in humans have been described during the past decade, but case reports from nonhuman primates are rare. For example, papillary gingival enlargement occurred in baboons used to study the physiologic effects of estrogen and progesterone during pregnancy.²⁰ The oral anatomy and radiographic presentation of baboons exhibits a striking similarity to human dentition.¹ Similar to that in humans, gingivitis in baboon is characterized by the presence of plaque, calculus, and proliferation of pocket epithelium.^1,9,18 Baboons also exhibit an age-associated increase in prevalence and severity of periodontitis.⁹ There are also case reports of gingival enlargement from rhesus macaques (Macaca mulatta),³⁰ mustache guenon monkeys (Cercopithecus cephus),²³ stump-tailed macaques (Macaca arctoides),^25,26 baboons (Papio ssp.),⁹ gorillas (Gorilla gorilla),⁵ ferrets (Mustela putorious furo),^27,28 and dogs (Canis familiaris).²¹ Most of these reported cases involved animals that had undergone experimental treatments, which induced the lesions.We present a case report of recurrent idiopathic gingival enlargement in a male olive baboon (Papio anubis) that has not undergone experimental manipulation.     

Noninvasive Temporal Artery Thermometry as an Alternative to Rectal Thermometry in Research Macaques (Macaca spp.)

Obtaining an animal''s body temperature is essential for the assessment of its clinical status. For many species, rectal thermometry is the technique used most often; however, this method in macaques typically requires sedation or considerable physical restraint. A noninvasive and inexpensive temporal artery (TA) thermometer was evaluated as an alternative method for collecting body temperature measurements from macaques used in neuroscience research. Rectal and arterial temperatures were obtained from 86 macaques (mean age, 10.2 y) that had received ketamine (10 mg/kg IM) or Telazol (5 mg/kg IM); the arterial measurements were taken from behind the right ear. In addition, arterial temperatures were measured behind both ears in a cohort of awake, chaired macaques with cephalic restraint pedestals only (n = 8) or with cephalic restraint pedestals and recording chambers (n = 14). Within-subject repeatability for TA thermometry and agreement between rectal and arterial temperature measurements were assessed by using the Bland–Altman method. Temperature measurements indicated that values from TA thermometry were lower than those from rectal thermometry by 1.57 °C with a 95% agreement limit of ± 1.27 °C. Results show satisfactory repeatability with TA thermometry and agreement between arterial and rectal temperatures, demonstrating that TA thermometry can be a valuable tool in conscious, chaired macaques with restrained heads.Abbreviation: TA, temporal arteryThe measurement of an animal''s body temperature is important for evaluating its health. Even though rectal thermometry, considered the clinical ‘gold standard,’ is thought to be minimally invasive, its use in research macaques is suboptimal given the requirement for sedation or extensive physical restraint. The use of anesthetics that can alter body temperature measurements^17,24 and the potential stress associated with manual restraint and intrusive thermometer placement for a rectal measurement both point to the need for an alternative to rectal thermometry in these nonhuman primates.Previous studies using laboratory animals have evaluated various other thermometry methods, including noncontact infrared thermometry,^4,7,25,26,28 telemetry,^24,27 subcutaneous implantable microchip thermometry,^{4,7,8,10,14,15,20,24,25} and tympanic thermometry,^3,9,10,16 against the standard of rectal thermometry. Infrared thermometry was shown to be invalid in rhesus and cynomolgus macaques, presumably because of the relatively large surface area measured and the influences of ambient temperatures, wet fur, and the presence of hair.^4,26 Telemetry and microchip thermometry both require expensive equipment and device implantation but then allow for measurements to be taken without further handling of animals. While temperatures from microchips in rhesus macaques agreed more closely with rectal temperatures than did those measured by infrared thermometry,⁴ microchip readings were inconsistent, and the macaques became stressed after repeated attempts to position them for readings in the cage. Although tympanic thermometry can be performed in conscious, chair-trained nonhuman primates, this method may cause discomfort, even after a period of acclimation. In addition, the accuracy of tympanic thermometry depends on precise placement of the probe.^3,9,10,16The commercial availability of a temporal artery (TA) thermometer has challenged the preeminent use of other thermometry methods, particularly in human newborns, infants, and children.^11,13,22,23 The TA thermometer is designed to accurately measure core body temperature in a completely noninvasive manner by using infrared technology.^18,19 One report on TA thermometry in human infants found that, although the thermometer had limited sensitivity in detecting cases of rectal fever, it was more accurate than tympanic thermometry and better tolerated than rectal thermometry.¹¹ Additional hospital studies noted marked variability between rectal and TA temperature measurements in febrile children and insufficient accuracy to recommend replacing rectal thermometry with TA thermometry.^5,23 Nevertheless, TA thermometry was suggested as a screening tool to rule out rectal fever or as an option in patients where rectal or other invasive techniques, such as pulmonary artery catheter placement, are contraindicated.^5,23The present study evaluated an inexpensive TA thermometer as an alternative for obtaining body temperatures in sedated and chair-restrained macaques. Macaques involved in neuroscience research often have cephalic implants for neuronal recording, and implanted animals are at risk for developing cranial infections. The routine use of a benign but accurate method to follow body temperature trends in these animals potentially could enable early detection of clinical complications.     

Assessment of Routine Procedure Effect on Breathing Parameters in Mice by Using Whole-Body Plethysmography

We used whole-body plethysmography to investigate the effect of restraint, ear marking, tail vein and retroorbital blood sampling, and tail clipping on respiration in Balb/c × TCR-HA^+/– F1 hybrid mice (F1h). Baseline values of breathing parameters were determined. During the experiment, mice experienced a procedure and then plethysmographic recordings were obtained immediately and at 4, 24, and 48 h afterward. Baseline breathing parameters showed significant differences between sexes. Restraint affected minute volume differently than did handling in male mice and to a lesser extent in female mice. Ear marking significantly changed minute volume compared with handling but not restraint in male mice and in the opposite manner in female mice. Tail vein blood sampling changed minute volume in a significant manner compared with restraint but not compared with handling in both sexes. Retroorbital blood sampling significantly changed minute volume compared with values for both handling and restraint in male mice but only compared with handling in female mice. Tail clipping modified minute volume significantly compared with handling in male mice and compared with restraint in both sexes. Analysis of data showed that routine procedures affect minute volume in mice depending on invasiveness of maneuver and in a sex-biased manner for as long as 24 h after the procedure. Our experiment shows that procedures performed on laboratory mice can change respiratory parameters and can be investigated by plethysmography.Abbreviation: F1h, Balb/c × TCR-HA^+/– F1 hybrid miceHandling, restraint, identification methods, and blood or tissue sampling are regarded as routine procedures in animal experiments. This aspect is particularly important when working with transgenic animals, where the need for genotyping demands the frequent use of these procedures, which can readily yield DNA samples.⁵ The effect of routine procedures on physiologic parameters varies depending on method, frequency, and duration, but these procedures generally are considered to be acute stressors.^2,5,26,27 A large body of research has focused on the effect of routine procedures on animals’ physiology. These effects were assessed by studying changes in behavior,^1,23,36,37 body weight,^29,37 food and water intake,^29,37 body temperature³⁵ and heart rate by radiotelemetry,^{5,10,19,26,27} corticosterone^{11,12,24,25,29} and glucose levels,³³ and blood variables.^29,36 Several differences in the response of laboratory animals to stress have been correlated to breed,^2,33 strain,^11,14,37 age,²⁴ and sex.^12,23,24 Some authors²⁴ have noted that female mice show a lower overall stress level due to the modest effect of social competition. Another study¹² showed a different, sex-specific response to hypoxic ventilation in rats, which was higher in male than female rats. In addition, these authors¹² suggested that female ovarian hormones are prime candidates for stress regulation.Whole-body plethysmography is a noninvasive, indirect method of studying respiratory function in conscious, unrestrained animals. This method has been used particularly in pharmacologic and toxicologic studies in diverse animal species including mice,^3,9,14,15,38 rats,^8,20 cats,²² dogs,^16,34 and pigs.¹³ The present study was designed to investigate the extent to which routine procedures affect breathing parameters in a transgenic mouse colony. Male and female mice were exposed to 4 routine procedures that are used for DNA sampling and identification of transgenic mice (ear marking, tail clipping, and tail vein and retroorbital blood sampling). Respiratory parameters were recorded by whole-body plethysmography immediately and at 4, 24, and 48 h after the procedure. Handling and restraint were used as control procedures.     

Modification of a Common BAL Technique to Enhance Sample Diagnostic Value

Bronchoalveolar lavage (BAL) by means of bronchoscopy is a diagnostic tool frequently used for clinical and research purposes in nonhuman primates. Although many institutions use this procedure, the technique is not standardized. One technical aspect that can vary is the method by which fluid is recovered. The purpose of this study was to evaluate differences between 2 different BAL aspiration techniques. Bronchoscopy and BAL fluid collection were performed on 20 rhesus macaques (Macaca mulatta). Data collected for comparison included heart rate, oxygen saturation levels, rectal temperature, volume of fluid collected, total cell count, cell viability, differential cell count, and flow cytometry. Results showed no significant differences in the heart rate, oxygen saturation, or body temperature between the 2 groups. Likewise, differential cell counts and cell viability studies of the retrieved fluid did not differ between methods. Compared with the conventional technique, the modified aspiration technique led to an 8.3% increase in overall fluid yield and a higher concentration of cells recovered. These differences are statistically significant and likely will be clinically relevant in the context of diagnosis.Abbreviation: BAL, bronchoalveolar lavage; BALPF, bronchoalveolar lavage pooled fluid; cBAL, conventional bronchoalveolar lavage; mBAL, modified bronchoalveolar lavageBronchoalveolar lavage (BAL) is a useful and commonly implemented tool in research and clinical medicine.^6,8,14 This valuable diagnostic procedure is used to evaluate the pathomechanisms of airway diseases.¹⁰ The technique involved in performing BAL consists of inserting a flexible videoscope into the trachea under direct visualization and manipulating the instrument to the chosen site.^2,3,4 The bronchoscope then is wedged into a subsegmental bronchus to create a seal, which then allows fluid to be instilled and aspirated from the area of interest.^3,4,7,10Although BAL is used frequently, the procedure is limited by lack of standardization. Aspects of the technique such as amount of instillate used, size of syringes used, amount of ‘dwell’ time of instillate before aspiration, and the suction application technique vary widely.^3,4,12,13 Sources in the literature recommend fluid instillation volume ranging from 10 to 300 ml in human patients and 50 to 600 ml in dogs.^1,3,12,15 Likewise, there are no standard recommendations regarding syringe size, which affects the amount of vacuum applied during aspiration. One randomized trial focusing on syringe size included 30 patients with chronic obstructive pulmonary disease and found that performing BAL with 50-ml syringes acquires more fluid with less oxygen desaturation than can be attained by using 20-ml syringes.⁵ No other studies addressing syringe sizes were identified during literature searches. The time that instillate remains in the airway before aspiration is yet another variable. Most literature sources recommend immediate aspiration of instillate, but some authors advocate leaving lavage fluid in the airway for the period of a normal breath prior to aspiration.^1,3,7,12 Various methods of fluid aspiration including manual suction, drainage by gravity, and mechanical suction at a judicious negative pressure (50 to 100 mm Hg) also have been described. ^2,4,7,11-13 Due to a lack of comparative studies, no method is specifically recommended.Although attempts have been made to achieve some standardization, primarily by way of professional consensus, few hypothesis-driven studies have been completed to optimize BAL technique.¹³ Many aspects of the BAL procedure require further testing to achieve consistency. The aim of this study was to compare a specific technical modification of the aspiration process with a method used more conventionally during manual recovery of BAL fluid. These techniques were evaluated for differences in fluid composition, animal morbidity, and ergonomics.     

Assessment of Immune Activation in Mice before and after Eradication of Mite Infestation

Mite infestation of mice remains a persistent problem for many institutions, leading to numerous health problems and creating unknown and unwanted variables for research. In this study, mice with mite infestation demonstrated significantly higher levels of inflammatory cytokines, both at draining lymph nodes (axillary) and systemically, as compared with mice without mites. In addition, histologic evaluation revealed significant inflammation in mite-infested mice. Inflammatory changes were still present in the skin of mice at 6 to 8 wk after treatment, despite absence of detectable infestation at that time. Because these significant and lasting local and systemic changes have the potential to alter research findings, eradication of mites infestations should be an important goal for all institutions.Abbreviation: KC, keratinocyte-derived chemokine; MIP, macrophage inflammatory proteinLaboratory mice can harbor several species of acarids (fur mites), including Myobia musculi, Radfordia affinis, Myocoptes musculinus, and Psorergates simplex.^{11,14,29,40,45} Fur mites are an excluded pathogen in most research facilities, particularly within barrier suites, and in order to control or avoid mite infestations, many facilities, including those with ongoing infestations, will not accept infested animals from outside sources. Such policies can prevent or halt collaborative research between investigators in different institutions because mite infestation is a sporadic or endemic problem in many facilities that house mice under conventional conditions, despite attempts at eradication.^{12,22,25,43,62,69}Mite infestations cause several health problems in mice, including ulcerative dermatitis, amyloidosis, and other immune system alterations.^{2,12,22,27,29-31,37,44,45,61} For example, mite infestations are associated with increased serum concentrations of IgE and IgG in mice.^30,44,48 Alterations in immune responses could alter research data and thereby perhaps alter the associated conclusions.^36,65,66,70 Mice with mite infections often develop dermatitis, which can lead to bacterial infection and additional changes in immune status.^{15,30,31,45,61} Because any pathogenic infection can cause variability and alter basal measures of immune function, clinical chemistry, and behavior in mice, maintaining laboratory rodents in a disease-free state is crucial to their use for the collection of valid research data.⁵¹The eradication of external parasites is a difficult process. Many reports have been published that attempt mite eradication using various drug treatments,^{5-7,17,18,23,24,35,39,41-43,46,47,49,50,57,59,67} with each method having distinct advantages and disadvantages. Some, but not all, of these treatment regimens have been compared directly.¹⁰ The mite life cycle complicates treatment, because eggs and larvae can be less susceptible to drugs than are adult parasites.^2,19,20,55 In addition, mite eggs can contaminate the environment, providing a source for re-infection of treated animals.^20,63,64 Some drugs (for example, ivermectin) have been associated with toxicity and death in mice, especially among specific transgenic lines.^{8,12,28,53,55,69} Other drugs may require frequent or repeated treatment of the mice. Furthermore, the drugs themselves may have properties that alter physiology or immune function in animals.^2,13,60 The development of new veterinary drugs for treatment of parasites has increased the available therapies for rodent acariasis. Compounds such as fipronil and selamectin provide good efficacy against external parasites with limited side effects in mammals.^9,22,68Our facility housed a large colony of mice that occupied several rooms and were infested with Myocoptes musculinus and Myobia musculi. Although the majority of mite-infested mice had mild or no dermatitis, some infected mice had severe dermatitis. The goal of this study was to evaluate the local and systemic immune response in mice infested with mites. To our knowledge, this study is the first to comprehensively compare cytokine levels and histologic findings in mite-infested, treated, and mite-negative mice. We hypothesized that the immune response would be altered in mite-infested mice as demonstrated by significantly elevated cytokine levels in the draining lymph nodes or spleen as compared with mice that had never been infested with mites. In addition, we hypothesized that significant pathologic changes in the epidermis, dermis, and subcutaneous tissues would be present in response to mite infestation.     

Intestinal monocytes and macrophages are required for T cell polarization in response to Citrobacter rodentium

Dendritic cells (DCs), monocytes, and macrophages are closely related phagocytes that share many phenotypic features and, in some cases, a common developmental origin. Although the requirement for DCs in initiating adaptive immune responses is well appreciated, the role of monocytes and macrophages remains largely undefined, in part because of the lack of genetic tools enabling their specific depletion. Here, we describe a two-gene approach that requires overlapping expression of LysM and Csf1r to define and deplete monocytes and macrophages. The role of monocytes and macrophages in immunity to pathogens was tested by their selective depletion during infection with Citrobacter rodentium. Although neither cell type was required to initiate immunity, monocytes and macrophages contributed to the adaptive immune response by secreting IL-12, which induced Th1 polarization and IFN-γ secretion. Thus, whereas DCs are indispensable for priming naive CD4⁺ T cells, monocytes and macrophages participate in intestinal immunity by producing mediators that direct T cell polarization.Inducing specific immunity and maintaining tolerance requires cells of the mononuclear phagocyte lineage. This lineage is comprised of three closely related cell types: DCs, monocytes, and macrophages (Shortman and Naik, 2007; Geissmann et al., 2010a,b; Liu and Nussenzweig, 2010; Yona and Jung, 2010; Chow et al., 2011). DCs are essential to both immunity and tolerance (Steinman et al., 2003); however, the role monocytes and macrophages play in these processes is not as well defined (Geissmann et al., 2008).In mice, DCs and monocytes arise from the same hematopoietic progenitor, known as the macrophage–DC progenitor (MDP; Fogg et al., 2006). Their development diverges when MDPs become either common DC progenitors (CDPs) that are Flt3L-dependent, or monocytes, which are dependent on CSF1 (M-CSF; Witmer-Pack et al., 1993; McKenna et al., 2000; Fogg et al., 2006; Waskow et al., 2008). CDPs develop into either plasmacytoid DCs or preDCs that leave the bone marrow to seed lymphoid and nonlymphoid tissues, where they further differentiate into conventional DCs (cDCs; Liu et al., 2009). In contrast, monocytes circulate in the blood and through tissues, where they can become activated and develop into several different cell types, including some but not all tissue macrophages (Schulz et al., 2012; Serbina et al., 2008; Yona et al., 2013).Despite their common origin from the MDP, steady-state lymphoid tissue cDCs can be distinguished from monocytes or macrophages by expression of cell surface markers. For example, cDCs in lymphoid tissues express high levels of CD11c and MHCII, but lack the expression of CD115 and F4/80 found in monocytes and macrophages, respectively. However, this distinction is far more difficult in peripheral tissues, like the intestine or lung, or during inflammation when monocytes begin to express many features of DC including high levels of MHCII and CD11c (Serbina et al., 2003; León et al., 2007; Hashimoto et al., 2011).The function of cDCs in immunity and tolerance has been explored extensively using a series of different mutant mice to ablate all or only some subsets of cDCs (Jung et al., 2002; Liu and Nussenzweig, 2010; Chow et al., 2011). In contrast, the methods that are currently available to study the function of monocytes and macrophages in vivo are far more restricted and less specific (Wiktor-Jedrzejczak et al., 1990; Dai et al., 2002; MacDonald et al., 2010; Chow et al., 2011). For example, Ccr2^−/− and Ccr2^DTR mice (Boring et al., 1997; Kuziel et al., 1997; Serbina and Pamer, 2006; Tsou et al., 2007) have been used to study monocytes (Boring et al., 1997; Peters et al., 2004; Hohl et al., 2009; Nakano et al., 2009). However, CCR2 is also expressed on some subsets of cDCs, activated CD4⁺ T cells, and NK cells (Kim et al., 2001; Hohl et al., 2009; Egan et al., 2009; Zhang et al., 2010). Thus, it is challenging to dissect the precise role of monocytes as opposed to other cell types in immune responses in Ccr2^−/− or Ccr2^DTR mice. Inducible DTR expression in CD11c^Cre x CX₃CR1^LsL-DTR mice is far more specific (Diehl et al., 2013), but restricted to a small subset of mononuclear phagocytes.Here, we describe a genetic approach to targeting monocytes and macrophages that spares cDCs and lymphocytes, and we compare the effects of monocyte and macrophage ablation to cDC depletion on the adaptive immune response to intestinal infection with Citrobacter rodentium.     

Spermatozoa capture HIV-1 through heparan sulfate and efficiently transmit the virus to dendritic cells

Semen is the main vector for HIV-1 dissemination worldwide. It contains three major sources of infectious virus: free virions, infected leukocytes, and spermatozoa-associated virions. We focused on the interaction of HIV-1 with human spermatozoa and dendritic cells (DCs). We report that heparan sulfate is expressed in spermatozoa and plays an important role in the capture of HIV-1. Spermatozoa-attached virus is efficiently transmitted to DCs, macrophages, and T cells. Interaction of spermatozoa with DCs not only leads to the transmission of HIV-1 and the internalization of the spermatozoa but also results in the phenotypic maturation of DCs and the production of IL-10 but not IL-12p70. At low values of extracellular pH (∼6.5 pH units), similar to those found in the vaginal mucosa after sexual intercourse, the binding of HIV-1 to the spermatozoa and the consequent transmission of HIV-1 to DCs were strongly enhanced. Our observations support the notion that far from being a passive carrier, spermatozoa acting in concert with DCs might affect the early course of sexual transmission of HIV-1 infection.The UNAIDS/World Health Organization AIDS epidemic update estimated that 33 million people were living with HIV at the end of 2007. New infections have been occurring in the past few years at a rate of 3.2 million per year, and almost 2 million individuals succumbed to AIDS-related diseases in 2007 (UNAIDS, 2007). Most infections are acquired through sexual transmission during vaginal or anal intercourse, with semen being the major transmission vector for HIV-1 (Miller and Shattock, 2003; Pope and Haase, 2003; Haase, 2005; Lederman et al., 2006).Semen contains three major sources of infectious virus: free virions, spermatozoa-associated virions, and infected leukocytes (Miller and Shattock, 2003; Gupta and Klasse, 2006; Lederman et al., 2006; Hladik and McElrath, 2008). The role of each of these sources in sexual transmission of HIV-1 is not well defined. Few studies have addressed this important question. Free virus and seminal infected leukocytes appear to play an important role in sexual transmission of HIV-1 (Miller and Shattock, 2003; Gupta and Klasse, 2006; Lederman et al., 2006; Hladik and McElrath, 2008). The role of spermatozoa, however, has been a matter of debate (Mermin et al., 1991; Dussaix et al., 1993; Quayle et al., 1997; Pudney et al., 1998), in spite of the fact that the presence of viral particles and/or nucleic acids in spermatozoa from HIV-1–infected men has been largely demonstrated using a variety of techniques (Baccetti et al., 1994, 1998; Bagasra et al., 1994; Nuovo et al., 1994; Dulioust et al., 1998; Muciaccia et al., 1998, 2007; Barboza et al., 2004).The identity of the receptor for HIV-1 expressed in the spermatozoa remains unclear. On the basis of its ability to recognize the HIV gp120, it has been proposed that mannose receptors (MRs) might function as HIV-1 receptors on the spermatozoa (Bandivdekar et al., 2003; Cardona-Maya et al., 2006; Fanibunda et al., 2008). It has also been shown that the gp120 can be recognized by galactosyl-alkyl-acylglycerol, a glycolipid expressed on the spermatozoa, suggesting that, as described for keratinocytes and epithelial cells, this molecule also contributes to the attachment of HIV-1 to the spermatozoa (Brogi et al., 1996, 1998).After deposition of HIV-1 on the mucosa, the virus must cross the mucosal epithelium to interact with T CD4⁺ lymphocytes, macrophages, and DCs, which are the most important targets of infection (Mermin et al., 1991; Miller and Shattock, 2003; Haase, 2005; Gupta and Klasse, 2006; Lederman et al., 2006; Hladik and McElrath, 2008). These cells express the HIV-1 receptors CD4 and coreceptors CCR5 or CXCR4 which are required for infection (Haase, 2005; Gupta and Klasse, 2006; Lederman et al., 2006; Hladik and McElrath, 2008). It is now clear that DCs are able to capture HIV-1 at entry sites and transport the virus to draining lymph nodes, where HIV-1 is efficiently transmitted to T CD4⁺ cells, which become the center of viral replication (Geijtenbeek et al., 2000; Gurney et al., 2005; Wilkinson and Cunningham, 2006; Wu and KewalRamani, 2006).The pathways used by HIV-1 to cross the mucosal epithelium are not well defined. The virions may transcytose through the genital epithelium (Gupta and Klasse, 2006; Hladik and McElrath, 2008) or may pass the barrier through genital lesions, either as cell-free or cell-associated virus (Piot and Laga, 1989; Serwadda et al., 2003; Galvin and Cohen, 2004). The latter possibility could account for the association of HIV infections with sexually transmitted diseases (Miller and Shattock, 2003; Haase, 2005; Lederman et al., 2006). It is of note that epithelial microabrasions in the vagina are detected in 60% of healthy women after consensual intercourse (Norvell et al., 1984; Guimarães et al., 1997), suggesting that the presence of genital microlesions represent a frequent scenario for the transmission of HIV-1. Anal intercourse is also often associated with mucosal trauma and, because the rectal epithelium is only one cell layer thick, it provides a low degree of protection against trauma, favoring the access of virus to the underlying target cells (Shattock and Moore, 2003). Moreover, the access of HIV-1 to target cells may be facilitated by other mechanisms such as the binding of HIV-1 to DC projections that extend to the luminal surface (Pope and Haase, 2003; Haase, 2005; Wu and KewalRamani, 2006; Sharkey et al., 2007). Transmission may also be favored by the induction of a local inflammatory response triggered by semen (Pandya and Cohen 1985; Thompson et al., 1992; Berlier et al., 2006; Sharkey et al., 2007).In this paper, we analyze the interaction of HIV-1 with human spermatozoa and the ability of spermatozoa to transmit the virus to immature DCs. We found that spermatozoa capture HIV-1 and efficiently transmit the virus to DCs through a mechanism that requires cell-to-cell contacts. DC–spermatozoa contacts lead to the phenotypic maturation of the DCs and the production of IL-10 (but not IL-12). Acidic values of extracellular pH similar to those found in the vaginal mucosa after sexual intercourse markedly increased both the attachment of HIV-1 to the spermatozoa and the consequent transmission of HIV-1 to DCs. Our results suggest that spermatozoa-associated virus may play a central role in the sexual transmission of HIV-1.     

Soiled Bedding Sentinels for the Detection of Fur Mites in Mice

Identification and eradication of murine fur mite infestations are ongoing challenges faced by many research institutions. Infestations with Myobia musculi and Myocoptes musculinus can lead to animal health problems and may impose unwanted research variables by affecting the immune and physiologic functions of mice. The purpose of this study was to evaluate the utility and efficacy of soiled bedding sentinels in the detection of fur mite infestations in colony mice. Female young-adult CRL:CD1(ICR) mice (n = 140) were exposed over a 12-wk period to various volume percentages of soiled bedding (11%, 20%, 50%, and 100%) from fur-mite–infested animals. Mice were tested every 2 wk with the cellophane tape test to identify the presence of fur mite adults and eggs. At the end of 12 wk, all mice exposed to 11%, 20%, and 50% soiled bedding tested negative for fur mites. One of the 35 mice (3%) receiving 100% soiled bedding tested positive for fur mites at the end of the 12-wk follow-up period. These findings suggest that the use of soiled bedding sentinels for the detection of fur mite infestations in colony mice is unreliable.Ectoparasite infestations present an ongoing threat to barrier facilities. Murine acariasis in laboratory mice frequently is caused by Myobia musculi, Myocoptes musculinus, and Radfordia affinis.^{1,13,17,40,41} These infestations can be challenging to identify and control and often lead to animal health problems and research complications. For this reason, many institutions strive to exclude these parasites from their barrier facilities.^1,17,18,41 Infestations can further compromise ongoing research by disrupting collaboration with institutions affected by sporadic or endemic mite infestations in their facilities.¹⁸Myocoptes musculinus is the most common fur mite identified among laboratory mice, although mixed infections with Myobia musculi are common.¹⁷ The life cycles of Myocoptes and Myobia are 14 and 23 d, respectively.^2,17 Myobia mites most frequently are found to inhabit the head and neck of mice, whereas Myocoptes are reported to have a predilection for the back, ventral abdomen, and inguinal regions.^2,17 Mite infestations in live animals are often diagnosed by using cellophane tape tests.^5,14,25 A clear piece of cellophane tape is pressed against the fur of the mouse, affixed to a slide, and examined microscopically for the presence of eggs or adult mites. Pelage collection and examination and skin scraping are 2 other common diagnostic methods. These tests have been shown to have increased sensitivity when compared with the tape test, but they have the disadvantage of requiring an anesthetized or recently euthanized animal.^2,5,17Fur mites feed on the superficial skin tissues and secretions of the animals they infest.^1,2,17 Mite infestations in mice have been associated with numerous health problems. Common clinical manifestations of acariasis include alopecia, pruritis, and scruffiness.^{1,2,10,15,17-20,22,26,31,42,44} Severe health problems including ulcerative dermatitis, hypersensitivity dermatitis, and pyoderma can develop also.^1,2,10,17,41 Infested mice may also be prone to secondary infections, reduced life span, and decreased body weight.^2,17,42 Several studies have analyzed the potential research complications associated with murine acariasis.^{10,15,18-20,22,26,31,42,44} Mite infestations have been shown to cause elevations in IgE, IgG, and IgA levels; mast cell degranulation; increased levels of inflammatory cytokines; and lymphocytopenia.^{18-20,22,26,31,44} The changes in the immunologic function of affected mice can persist even after mite eradication.¹⁸Multiple chemical treatment modalities have been proposed for the eradication of fur mites in infested animals.^{2,3,5,8,12,14,17,25,29,30,32,36,43} Conflicting information exists regarding the success of many of these treatment regimes. In addition, several of the proposed treatments have been associated with toxicity, adverse health effects in mice, and alterations in the physiologic or immune function of the animals.^{2,3,5,8,12,14,17,25,29,30,32,36,43} The complications associated with identifying an effective treatment for murine acariasis while minimizing toxicity and the introduction of unknown research variables highlight the importance of rapid and effective detection of mite infestations in barrier facilities.Many institutions rely on soiled bedding sentinels for their primary source of information on colony health status.^9,21,33,35 Several studies have demonstrated the efficacy of soiled bedding sentinels to detect common murine pathogens such as mouse hepatitis virus, mouse norovirus, Helicobacter spp., and pinworms.^{4,7,24,28,37,38} However, not all pathogens are easily transmitted through soiled bedding exposure. Agents that are not routinely identified through soiled bedding sentinels include those that are shed in low numbers, are susceptible to environmental factors, or are not easily transmitted through the fecal–oral route.^6,21,33 Examples of pathogens that are not easily transmitted or detected through soiled bedding exposure include mouse Sendai virus, Pasteurella pneumotropica, lymphocytic choriomeningitis virus, and cilia-associated respiratory bacillus.^7,9,11,16,35 In addition, the sensitivity of soiled bedding sentinel programs varies with the number of animals affected within the colony.^27,38In 2008, our institution faced a fur-mite outbreak that affected more than 25 rooms in a single barrier facility. Animals positive for Myobia musculi, Myocoptes musculinus, or both were identified through either health check requests for itching and scratching animals and by testing of animals scheduled for export to other institutions. Despite the extent of this outbreak, the soiled bedding sentinels in all mite-positive rooms consistently tested negative on cellophane tape tests for fur mites.To our knowledge, only one study has specifically examined the efficacy of soiled bedding sentinels in the detection of fur mites in mice.³⁴ A separate study, examining the transmission of mouse hepatitis virus to soiled bedding sentinels,³⁸ demonstrated that 75% of cages (3 of 4) exposed to soiled bedding from colony animals tested positive for fur mites after 19 wk of exposure. That previous study used 8 cages of 12 mice each; 4 cages received soiled bedding from colony animals, whereas the other 4 cages received clean nonsoiled bedding. In that study,³⁸ 56.3% of colony mice were known to be mite-positive. Other literature suggests that spread of mites to naïve animals requires direct contact and that soiled bedding does not serve as an effective mechanism for transmission.^1,17,23,39 However, we were unable to identify any research or experiments that substantiated these conclusions.The purpose of the present study was to evaluate whether CRL:CD1(ICR) mice housed in static microisolation caging on soiled bedding from mice with Myobia and Myocoptes infestations can be used as sentinels for the detection of fur mites and to determine how the efficacy of these soiled bedding sentinels for fur-mite detection varies with the prevalence of fur-mite infestation among colony animals.     

Induction and stability of human Th17 cells require endogenous NOS2 and cGMP-dependent NO signaling

Nitric oxide (NO) is a ubiquitous mediator of inflammation and immunity, involved in the pathogenesis and control of infectious diseases, autoimmunity, and cancer. We observed that the expression of nitric oxide synthase-2 (NOS2/iNOS) positively correlates with Th17 responses in patients with ovarian cancer (OvCa). Although high concentrations of exogenous NO indiscriminately suppress the proliferation and differentiation of Th1, Th2, and Th17 cells, the physiological NO concentrations produced by patients’ myeloid-derived suppressor cells (MDSCs) support the development of RORγt(Rorc)⁺IL-23R⁺IL-17⁺ Th17 cells. Moreover, the development of Th17 cells from naive-, memory-, or tumor-infiltrating CD4⁺ T cells, driven by IL-1β/IL-6/IL-23/NO-producing MDSCs or by recombinant cytokines (IL-1β/IL-6/IL-23), is associated with the induction of endogenous NOS2 and NO production, and critically depends on NOS2 activity and the canonical cyclic guanosine monophosphate (cGMP)–cGMP-dependent protein kinase (cGK) pathway of NO signaling within CD4⁺ T cells. Inhibition of NOS2 or cGMP–cGK signaling abolishes the de novo induction of Th17 cells and selectively suppresses IL-17 production by established Th17 cells isolated from OvCa patients. Our data indicate that, apart from its previously recognized role as an effector mediator of Th17-associated inflammation, NO is also critically required for the induction and stability of human Th17 responses, providing new targets to manipulate Th17 responses in cancer, autoimmunity, and inflammatory diseases.Nitric oxide (NO; a product of nitrite reduction or the NO synthases NOS1, NOS2, and NOS3; Culotta and Koshland, 1992), is a pleiotropic regulator of neurotransmission, inflammation, and autoimmunity (Culotta and Koshland, 1992; Bogdan, 1998, 2001; Kolb and Kolb-Bachofen, 1998) implicated both in cancer progression and its immune-mediated elimination (Culotta and Koshland, 1992; Coussens and Werb, 2002; Hussain et al., 2003; Mantovani et al., 2008). In different mouse models, NO has been paradoxically shown to both promote inflammation (Farrell et al., 1992; Boughton-Smith et al., 1993; McCartney-Francis et al., 1993; Weinberg et al., 1994; Hooper et al., 1997) and to suppress autoimmune tissue damage through nonselective suppression of immune cell activation (Bogdan, 2001; Bogdan, 2011), especially at high concentrations (Mahidhara et al., 2003; Thomas et al., 2004; Niedbala et al., 2011). Although previous studies demonstrated a positive impact of NO on the induction of Th1 cells (Niedbala et al., 2002) and forkhead box P3–positive (FoxP3⁺) regulatory T (T reg) cells (Feng et al., 2008) in murine models, the regulation and function of the NO synthase (NOS)–NO system have shown profound differences between mice and humans (Schneemann and Schoedon, 2002, Schneemann and Schoedon, 2007; Fang, 2004), complicating the translation of these findings from mouse models to human disease.In cancer, NOS2-derived NO plays both cytotoxic and immunoregulatory functions (Bogdan, 2001). It can exert distinct effects on different subsets of tumor-infiltrating T cells (TILs), capable of blocking the development of cytotoxic T lymphocytes (CTLs; Bronte et al., 2003), suppressing Th1 and Th2 cytokine production, and modulating the development of FoxP3⁺ T reg cells (Brahmachari and Pahan, 2010; Lee et al., 2011). NOS2-driven NO production is a prominent feature of cancer-associated myeloid-derived suppressor cells (MDSCs; Mazzoni et al., 2002; Kusmartsev et al., 2004; Vuk-Pavlović et al., 2010; Bronte and Zanovello, 2005), which in the human system are characterized by a CD11b⁺CD33⁺HLA-DR^low/neg phenotype consisting of CD14⁺ monocytic (Serafini et al., 2006; Filipazzi et al., 2007; Hoechst et al., 2008; Obermajer et al., 2011) and CD15⁺ granulocytic (Zea et al., 2005; Mandruzzato et al., 2009; Rodriguez et al., 2009) subsets (Dolcetti et al., 2010; Nagaraj and Gabrilovich, 2010).Production of NO in chronic inflammation is supported by IFN-γ and IL-17 (Mazzoni et al., 2002; Miljkovic and Trajkovic, 2004), the cytokines produced by human Th17 cells (Veldhoen et al., 2006; Acosta-Rodriguez et al., 2007a,b; van Beelen et al., 2007; Wilson et al., 2007). Human Th17 cells secrete varying levels of IFN-γ (Acosta-Rodriguez et al., 2007a; Acosta-Rodriguez et al., 2007b; Kryczek et al., 2009; Miyahara et al., 2008; van Beelen et al., 2007; Wilson et al., 2007) and have been implicated both in tumor surveillance and tumor progression (Miyahara et al., 2008; Kryczek et al., 2009; Martin-Orozco and Dong, 2009). Induction of Th17 cells typically involves IL-1β, IL-6, and IL-23 (Bettelli et al., 2006; Acosta-Rodriguez et al., 2007a,b; Ivanov et al., 2006; van Beelen et al., 2007; Veldhoen et al., 2006; Wilson et al., 2007; Zhou et al., 2007), with the additional involvement of TGF-β in most mouse models (Bettelli et al., 2006; Mangan et al., 2006; Veldhoen et al., 2006; Zhou et al., 2007; Ghoreschi et al., 2010), but not in the human system (Acosta-Rodriguez et al., 2007a; Wilson et al., 2007). IL-1β₁, IL-6, and IL-23 production by monocytes and DCs, and the resulting development of human Th17 cells, can be induced by bacterial products, such as LPS or peptidoglycan (Acosta-Rodriguez et al., 2007a; Acosta-Rodriguez et al., 2007b; van Beelen et al., 2007). However, the mechanisms driving Th17 responses in noninfectious settings, such as autoimmunity or cancer, remain unclear.Here, we report that the development of human Th17 cells from naive, effector, and memory CD4⁺ T cell precursors induced by the previously identified Th17-driving cytokines (IL-1β, IL-6, and IL-23) or by IL-1β/IL-6/IL-23-producing MDSCs, is promoted by exogenous NO (or NO produced by human MDSCs) and critically depends on the induction of endogenous NOS2 in differentiating CD4⁺ T cells.     

Characterization of Cardiac Time Intervals in Healthy Bonnet Macaques (Macaca radiata) by Using an Electronic Stethoscope

Nonhuman primates are used frequently in cardiovascular research. Cardiac time intervals derived by phonocardiography have long been used to assess left ventricular function. Electronic stethoscopes are simple low-cost systems that display heart sound signals. We assessed the use of an electronic stethoscope to measure cardiac time intervals in 48 healthy bonnet macaques (age, 8 ± 5 y) based on recorded heart sounds. Technically adequate recordings were obtained from all animals and required 1.5 ± 1.3 min. The following cardiac time intervals were determined by simultaneously recording acoustic and single-lead electrocardiographic data: electromechanical activation time (QS1), electromechanical systole (QS2), the time interval between the first and second heart sounds (S1S2), and the time interval between the second and first sounds (S2S1). QS2 was correlated with heart rate, mean arterial pressure, diastolic blood pressure, and left ventricular ejection time determined by using echocardiography. S1S2 correlated with heart rate, mean arterial pressure, diastolic blood pressure, left ventricular ejection time, and age. S2S1 correlated with heart rate, mean arterial pressure, diastolic blood pressure, systolic blood pressure, and left ventricular ejection time. QS1 did not correlate with any anthropometric or echocardiographic parameter. The relation S1S2/S2S1 correlated with systolic blood pressure. On multivariate analyses, heart rate was the only independent predictor of QS2, S1S2, and S2S1. In conclusion, determination of cardiac time intervals is feasible and reproducible by using an electrical stethoscope in nonhuman primates. Heart rate is a major determinant of QS2, S1S2, and S2S1 but not QS1; regression equations for reference values for cardiac time intervals in bonnet macaques are provided.Abbreviations: CTI, cardiac time intervals; QS1, electromechanical activation time; QS2, electromechanical systole; S1S2, interval between first and second heart sounds; S2S1, interval between second and first heart soundsNonhuman primates are used widely in biomedical research. Macaques and baboons are the most widely studied species.^{6,12,20,31,38,39,42,45} Macaques are anatomically similar to humans and exhibit similar cardiovascular physiology and metabolism,^{2,4,15-17,22,24,28} which make them useful as a model of left ventricular dysfunction and heart failure.^{2,23,30,32,37,44} Echocardiography has emerged as the most commonly used technique to assess left ventricular function, because it provides a variety of indices pertaining to left ventricular systolic and diastolic function. However, the availability of high-quality ultrasound equipment, transducers, technical expertise in image acquisition, and interpretation are factors that can limit the use of echocardiography in nonhuman primate research.Left ventricular function has long been assessed by using cardiac time intervals (CTI) derived by phonocardiography and carotid pulse tracings. In humans, CTI have been found highly correlated with echocardiographic, angiographic, and hemodynamic measures of left ventricular function^11,13. Low cost, ease of use, high accuracy, and excellent reproducibility have led to the frequent use of CTI in human cardiovascular research. However, electrical bioimpedence and phonocardiography with external carotid wave recordings have in large part been replaced by echocardiography as techniques to measure CTI.³⁵In recent years, electronic advances in the stethoscope have enhanced quantitative assessment of heart sounds.^5,9,33,34,43 Electronic stethoscopes are capable of recording heart sounds that can be digitally processed for display, storage, and analysis on computer. Therefore, electronic stethoscopes provide a portable low-cost alternative means to obtain CTI. Several investigators have incorporated analytical techniques to derive information pertaining to left ventricular function and pulmonary artery pressure from humans by using electronic stethoscopes.^1,5,9,43The results of several studies have led to the use of the electronic stethoscope as an alternative to the phonocardiogram, an older technology for determining CTI.^1,7,27,40 Data pertaining to the uses of CTI and electronic stethoscopes in nonhuman primates is lacking. We are unaware of a single study that has either evaluated left ventricular function by measuring CTI or has used the electronic stethoscope for cardiac assessment in monkeys. The ability to obtain CTI data and reference values may be very useful for the identification of cardiovascular abnormalities in primates. The objective of the current study was to ascertain the feasibility of determining CTI from recorded heart sounds in apparently healthy bonnet macaques (Macaca radiata). Bonnet macaques have a close physical resemblance to rhesus monkeys but are relatively smaller and lighter.²⁶