A One Health Perspective for Defining and Deciphering Escherichia coli Pathogenic Potential in Multiple Hosts

E. coli is one of the most common species of bacteria colonizing humans and animals. The singularity of E. coli’s genus and species underestimates its multifaceted nature, which is represented by different strains, each with different combinations of distinct virulence factors. In fact, several E. coli pathotypes, or hybrid strains, may be associated with both subclinical infection and a range of clinical conditions, including enteric, urinary, and systemic infections. E. coli may also express DNA-damaging toxins that could impact cancer development. This review summarizes the different E. coli pathotypes in the context of their history, hosts, clinical signs, epidemiology, and control. The pathotypic characterization of E. coli in the context of disease in different animals, including humans, provides comparative and One Health perspectives that will guide future clinical and research investigations of E. coli infections.

Escherichia coli (E. coli) is the most common bacterial model used in research and biotechnology. It is an important cause of morbidity and mortality in humans and animals worldwide, and animal hosts can be involved in the epidemiology of infections.^{240,367,373,452,727} The adaptive and versatile nature of E. coli argues that ongoing studies should receive a high priority in the context of One Health involving humans, animals, and the environment.^{240,315,343,727} Two of the 3 E. coli pathogens associated with death in children with moderate-to-severe diarrhea in Asia and Africa are classified into 2 E. coli pathogenic groups (also known as pathotypes or pathovars): enterotoxigenic E. coli (ETEC) and enteropathogenic E. coli (EPEC).³⁶⁷ In global epidemiologic studies, ETEC and EPEC rank among the deadliest causes of foodborne diarrheal illness and are important pathogens for increasing disability adjusted life years.^355,382,570 Furthermore, in humans, E. coli is one of the top-ten organisms involved in coinfections, which generally have deleterious effects on health.²⁷⁰ETEC is also an important etiologic agent of diarrhea in the agricultural setting.¹⁸³ E. coli-associated extraintestinal infections, some of which may be antibiotic-resistant, have a tremendous impact on human and animal health. These infections have a major economic impact on the poultry, swine, and dairy industries.^{70,151,168,681,694,781,797} The pervasive nature of E. coli, and its capacity to induce disease have driven global research efforts to understand, prevent, and treat these devastating diseases. Animal models for the study of E. coli infections have been useful for pathogenesis elucidation and development of intervention strategies; these include zebrafish, rats, mice, Syrian hamsters, guinea pigs, rabbits, pigs, and nonhuman primates.^{27,72,101,232,238,347,476,489,493,566,693,713,744,754} Experiments involving human volunteers have also been important for the study of infectious doses associated with E. coli-induced disease and of the role of virulence determinants in disease causation.^{129,176,365,400,497,702,703} E. coli strains (or their lipopolysaccharide) have also been used for experimental induction of sepsis in animals; the strains used for these studies, considered EPEC, are not typically involved in systemic disease.^{140,205,216,274,575,782}This article provides an overview of selected topics related to E. coli, a common aerobic/facultative anaerobic gastrointestinal organism of humans and animals.^{14,277,432,477,716} In addition, we briefly review: history, definition, pathogenesis, prototype (archetype or reference) strains, and features of the epidemiology and control of specific pathotypes. Furthermore, we describe cases attributed to different E. coli pathotypes in a range of animal hosts. The review of scientific and historical events regarding the discovery and characterization of the different E. coli pathotypes will increase clinical awareness of E. coli, which is too often regarded merely as a commensal organism, as a possible primary or co- etiologic agent during clinical investigations. As Will and Ariel Durant write in The Lessons of History: “The present is the past rolled up for action, and the past is the present unrolled for understanding”.     

Inflammatory Responses with Left Ventricular Compromise after Induction of Myocardial Infarcts in Sheep (Ovis aries)

Ischemic myocardial disease is a major cause of death among humans worldwide; it results in scarring and pallor of the myocardium and triggers an inflammatory response that contributes to impaired left ventricular function. This response includes and is evidenced by the production of several inflammatory cytokines including TNFα, IL1β, IL4, IFNγ, IL10 and IL6. In the current study, myocardial infarcts were induced in 6 mo old male castrated sheep by ligation of the left circumflex obtuse marginal arteries (OM 1 and 2). MRI was used to measure parameters of left ventricular function that include EDV, ESV, EF, SVI, dp/dt max and dp/dt min at baseline and at 4 wk and 3 mo after infarct induction. We also measured serum concentrations of an array of cytokines. Postmortem histologic findings corroborate the existence of left ventricular myocardial injury and deterioration. Our data show a correlation between serum cytokine concentrations and the development of myocardial damage and left ventricular functional compromise.

Heart failure is a globally significant problem in both humans and lower animals.^3,18 The medical literature is replete with predisposing causes of heart disease,¹³ yet the prevalence of heart failure remained high.^4,5,16 Regardless of the cause of myocardial damage and subsequent left ventricular compromise, the literature indicated that the proinflammatory response that occurs after myocardial infarction is an important contributor to the deterioration of the myocardium^{1,9,12,14,17,18,20,21} Sheep and pigs are excellent translational models of human cardiology because their hearts bear many physiologic and anatomic similarities to the human heart.^4,8,15 The primary use of these models in cardiology is primarily to study myocardial infarction^5,13,16 and to a lesser extent, physiologic processes that develop after myocardial insult.Our study measured some of the major proinflammatory cytokines that contribute to myocardial damage. Most of these cytokines, including: TNFα, IL6, and IFNγ, are important correlates of myocardial ischemia that contribute to a decline in left ventricular myocardial function.^1,9,14 In our study, we detected left ventricular compromise as early as 4 wk after the infarction, while the proinflammatory response was recorded at 48 h after the infarct and peaked at 4 wk. Cardiac functional parameters began to decline early in the study consistent with the proinflammatory response. The cardiac functional parameters continued to decline until 3 mo, which was the termination of the study. These findings may support antiinflammatory intervention as an important adjunct of any therapeutic regimen.     

Comparison of Insulins Glargine and Degludec in Diabetic Rhesus Macaques (Macaca mulatta) with CGM Devices

Treating and monitoring type 2 diabetes mellitus (T2DM) in NHP can be challenging. Multiple insulin and hypoglycemic therapies and management tools exist, but few studies demonstrate their benefits in a NHP clinical setting. The insulins glargine and degludec are long-acting insulins; their duration of action in humans exceeds 24 and 42 h, respectively. In the first of this study''s 2 components, we evaluated whether insulin degludec could be dosed daily at equivalent units to glargine to achieve comparable blood glucose (BG) reduction in diabetic rhesus macaques (Macaca mulatta) with continuous glucose monitoring (CGM) devices. The second component assessed the accuracy of CGM devices in rhesus macaques by comparing time-stamped CGM interstitial glucose values, glucometer BG readings, and BG levels measured by using an automated clinical chemistry analyzer from samples that were collected at the beginning and end of each CGM device placement. The CGM devices collected a total of 21,637 glucose data points from 6 diabetic rhesus macaques that received glargine followed by degludec every 24 h for 1 wk each. Ultimately, glucose values averaged 29 mg/dL higher with degludec than with glargine. Glucose values were comparable between the CGM device, glucometer, and chemistry analyzer, thus validating that CGM devices as reliable for measuring BG levels in rhesus macaques. Although glargine was superior to degludec when given at the same dose (units/day), both are safe and effective treatment options. Glucose values from CGM, glucometers, and chemistry analyzers provided results that were analogous to BG values in rhesus macaques. Our report further highlights critical clinical aspects of using glargine as compared with degludec in NHP and the benefits of using CGM devices in macaques.

Diabetes is a group of metabolic diseases that cause hyperglycemia secondary to deficient insulin response, secretion, or both.⁴ Diabetes is categorized by the American Diabetes Association into 4 types: 1) type 1 diabetes mellitus, in which the pancreas is unable to produce insulin for glucose absorption; 2) type 2 diabetes mellitus (T2DM), when the body does not use insulin correctly; 3) gestational diabetes, in which the body is insulin-intolerant during pregnancy (or is first discovered then); and 4) other specific forms of diabetes in which the patient is particularly predisposed to becoming diabetic due to various comorbidities or to inadvertent induction caused by some medications.⁴ In 2018, 34.2 million (10.5%) Americans of all ages were diagnosed with diabetes.^22,23,30 Approximately 90% to 95% of Americans with diabetes have T2DM,²⁴ making T2DM the most common form of diabetes diagnosed in humans.T2DM is a multifactorial disease primarily determined by genetics, behavioral and environmental factors (for example, age, diet, sedentary lifestyle, obesity).^4,46,50,74 As a consequence of these factors, the pancreas increases insulin secretion to maintain normal glucose tolerance.⁷⁴ Over time, the high insulin demand causes pancreatic β-cell destruction, resulting in reduced production of insulin.^39,50,74 As β-cell destruction increases, hyperglycemia and T2DM develop. Insulin resistance and hyperglycemia are tolerated for a period of time^19,82,83 before clinical signs associated with T2DM develop (e.g., polydipsia, polyuria, polyphagia with concurrent weight loss).⁴ Once clinical signs develop, T2DM is most commonly diagnosed as a fasting blood glucose level (FBG) of 126 mg/dL or greater,^2,4 2-h plasma glucose value of 200 mg/d or greater during a 75-g oral glucose tolerance test,^2,4 and/or glycosylated hemoglobin (HbA1c) of 6.5% or greater.^2,4 Depending on the FBG, oral glucose tolerance test, and HbA1c results, various treatment options are recommended by the American Diabetes Association. Most importantly, lifestyle changes, including diet and exercise, are recommended as the first line of treatment, along with oral antihyperglycemic drugs such as metformin.^5,25,46 Treatment efficacy is evaluated with self-monitoring blood glucose or continuous glucose monitoring (CGM) devices.³ Human patients using CGM devices have achieved considerable reductions in HbA1c compared with patients not using them.³ As CGM devices have become more readily available, user friendly, and affordable, they have become an essential tool in managing T2DM.Similar to humans, most NHP affected by diabetes are diagnosed with T2DM.^80,83 NHP are predisposed to similar genetic, behavioral and environmental factors (e.g., age, diet, sedentary lifestyle, obesity);^{6,18,19,37,44,52,82,83} have similar pathophysiology;^38,81-83 are diagnosed via FBG,^39,83 HbA1c,^21,31,49,56 fructosamine,^20,83,87 and weight loss;^49,80,83,86 and are treated with exercise and diet modifications as a first line of treatment.^{11,19,39,53,79} Although the human and NHP conditions are similar, the treatment and management of T2DM is somewhat different, especially when NHP have restricted physical activity due to housing constraints.Previous studies indicate that daily dosing with insulin glargine achieves appropriate glycemic control in NHP.⁴⁸ Therefore, we implemented glargine, along with some diet modification, to improve glycemic control in our diabetic colony. Other noninsulin therapies, such as metformin, had been used, but compliance was low (for example, due to large pill size, unpleasant taste, etc.). However, achieving glycemic control using diet modification, insulin glargine treatment, monthly scheduled FBG, quarterly HbA1c, and regular weight monitoring was challenging in a large colony. Monthly FBG and fructosamine testing were performed due to affordability and practicality for NHP in a research setting. Given that fructosamine levels correlate with BG concentrations for the preceding 2 to 3 wk and HbA1c percentages relate to BG concentration over 1.5 to 3 mo,^49,87 HbA1C was selected over fructosamine for T2DM management in our colony. Determining which T2DM treatment and diagnostics are most effective can be difficult in large colonies of NHP. Therefore, improved treatment and management strategies would help to manage T2DM in NHP more efficiently.Insulin glargine is a long-acting insulin, with a half-life of 12 h and duration of action of 12 to 24 h in humans^40,55 and 12 h in dogs.^34,43,60 Once injected subcutaneously, insulin glargine forms a microprecipitate in the neutral pH environment, which delays and prolongs absorption in subcutaneous tissues.¹² Insulin degludec is a newer form of long-acting insulin, with a half-life of 25 h^41,63,62,77 and duration of action that exceeds 42 h in humans.^40,41,68,77 Insulin degludec forms a soluble and stable dihexamer in the pharmaceutical formulation, which includes phenol and zinc.^63,78 The phenol diffuses away, leading to the formation of a soluble depot in the form of long multihexamer chains in which zinc slowly diffuses from the end of the multihexamers, causing a gradual, continuous, and extended-release of monomers from the depot of the injection site.^63,78 Pharmacodynamic studies in humans, demonstrate that the “glucose-lowering effect” of insulin degluc⁴⁰ is evenly distributed over 24 h, allowing a more stable steady-state and improved wellbeing.⁷⁸ This approach could potentially reduce the number of hypoglycemic events and provide a less rigid daily injection schedule,⁵⁸ thus potentially making insulin degludec—compared with insulin glargine—a safer, alternative diabetes therapy.In addition to medical intervention, glycemic control is achieved through regular management and monitoring of BG. Self-monitoring blood glucose checks in humans^3,5 and glucose curves in animals¹⁰ are some of the management tools used to determine or evaluate therapy for T2DM patients. Telemetry systems like CGM devices are used to monitor interstitial glucose and have been used extensively in humans^3,17,33 and animals^{16,27,36,42,47,84,85} to monitor BG in real-time. Using CGM devices 1) reduces or eliminates the number of blood draws needed to collect FBG,⁶¹ 2) accurately assesses insulin therapy via a real-time glucose curve,^72,84,85 3) allows patients and clinicians to titrate treatment^61,73 as indicated, and 4) obtains continuous glucose data with reduced manipulation and subsequent decreased stress.^72,84,85 Therefore, CGM devices can be a safe and informative tool in monitoring spontaneous T2DM in NHP.Between 2015 and 2030, the prevalence of diabetes is predicted to increase by 54% to more than 54 million Americans affected by diabetes (i.e., diabetes mellitus types 1 and 2).⁷⁰ NHP are an essential model for human T2DM because of their similar pathophysiology, diagnostics, treatment, and management. As more people develop diabetes, novel therapies will continue to be developed. Studying new treatments and management tools in NHP can further human and NHP T2DM research to prevent the progression of T2DM and hopefully diminish projections for the number of future diabetes cases. Human medical literature, American Diabetes Association, and drug manufacturers all recommend giving equal doses (i.e., number of units/day) of long-acting insulins when changing from one long-acting insulin to degludec.^26,63,67 Therefore, we hypothesized that insulin degludec would provide effective glycemic control for rhesus macaques with T2DM when dosed at equivalent doses (that is, the same number of units/day) as insulin glargine. In addition, we hypothesized that CGM devices would provide accurate BG readings as compared with chemistry analyzer and glucometer BG readings, making it a more efficient and effective tool for measurement of BG levels in rhesus macaques with T2DM.     

Indicators of Postoperative Pain in Syrian Hamsters (Mesocricetus auratus)

Despite the use of Syrian hamsters (Mesocricetus auratus) in research, little is known about the evaluation of pain in this species. This study investigated whether the frequency of certain behaviors, a grimace scale, the treat-take-test proxy indicator, body weight, water consumption, and coat appearance could be monitored as signs of postoperative pain in hamsters in a research setting. Animals underwent no manipulation, anesthesia only or laparotomy under anesthesia. An ethogram was constructed and used to determine the frequencies of pain, active and passive behaviors by in-person and remote videorecording observation methods. The Syrian Hamster Grimace Scale (SHGS) was developed for evaluation of facial expressions before and after the surgery. The treat-take-test assessed whether surgery would affect the animals’ motivation to take a high-value food item from a handler. The hypothesis was that behavior frequency, grimace scale, treat-take-test score, body weight, water consumption, and coat appearance would change from baseline in the surgery group but not in the no-intervention and anesthesia-only groups. At several time points, pain and passive behaviors were higher than during baseline in the surgery group but not the anesthesia-only and no-intervention groups. The SHGS score increased from baseline scores in 3 of the 9 animals studied after surgery. The frequency of pain behaviors and SHGS scores were highly specific but poorly sensitive tools to identify animals with pain. Behaviors in the pain category were exhibited by chiefly, but not solely, animals that underwent the laparotomy. Also, many animals that underwent laparotomy did not show behaviors in the pain category. Treat-take-test scores, body weight, water consumption, and coat appearance did not change from baseline in any of the 3 groups. Overall, the methods we tested for identifying Syrian hamsters experiencing postoperative pain were not effective. More research is needed regarding clinically relevant strategies to assess pain in Syrian hamsters.

Pain experienced by laboratory animals can affect both animal welfare and research results. Little is known about the evaluation of pain in Syrian hamsters (Mesocricetus auratus) in the laboratory setting. However, various research models using Syrian hamsters involve surgery and are presumed to cause pain.^16,47,49 In 2018 alone, the USDA reported that 35,695 hamsters were used for research studies involving painful procedures.⁴⁸ Previously published behaviors exhibited by hamsters in response to pain include hunched posture with head down, reluctance to move, increased depression or aggression, extended sleep periods, and weight loss.^7,8,10,16,21 How these behaviors are affected by factors such as the type of painful stimulus, anesthetic protocol, handling procedures, and environmental conditions is unclear. The practicality of observing these signs in the research environment is uncertain and likely complicated by the nocturnal nature of Syrian hamsters and an assumed propensity of this species to mask pain, much like other prey species.^8,14,16A significant need exists for published data investigating whether behavioral observations or other clinical indicators can help recognize, quantify, or monitor pain in hamsters in a research setting. Detailed behavioral observations and well-controlled studies are needed to develop a system to assess postoperative pain in laboratory animals.^8,33 Moreover, little information is available on the efficacy of analgesic agents in hamsters.¹ The few studies of analgesics in hamsters rely on the mitigation of evoked pain responses (such as using a hot plate), which has limited relevance to clinical situations such as postoperative pain.^8,32,36,51 To date, no published literature has evaluated the efficacy or safety of analgesics to treat postoperative pain in hamsters. Validated real-time and practical methods for evaluating pain in Syrian hamsters would support the evaluation of analgesic efficacy in this species.Various assessments have been developed to identify signs of pain in other species. Behavioral ethograms have been used to evaluate pain and analgesic efficacy in mice, rats, rabbits, and guinea pigs in the research environment.^{5,6,20,23,25,34,35,39-41,53} Another tool used to evaluate pain in animals is the grimace scale, which has been developed for mice, rats, rabbits, ferrets, cats, sheep, pigs, horses, and even harbor seals.^{3,4,9,11,13,15,19,22,26,30,37,45,50} The use of a proxy indicator, such as burrowing and time-to-integrate-to-nest in mice and time-to-consume in guinea pigs, can be used as an additional tool for the evaluation of pain.^{5,17,18,35,38}Because none of the previously mentioned assessment techniques were specific to hamsters, we here explored using these approaches to detect pain in Syrian hamsters that underwent laparotomy in a laboratory setting. We developed a species-specific ethogram and the Syrian Hamster Grimace Scale (SHGS). We also devised a novel proxy indicator of pain for use in Syrian hamsters, the treat-take-test (TTT), which is based on hamsters’ natural behavior to hoard food.^16,46,49,52 Although water intake, body weight, and coat appearance are non-specific indicators of pain, we also measured these parameters.^5,19,23,33 Furthermore, we analyzed the effects of the presence of an observer and time of day. We hypothesized that behavior frequency, grimace scale, treat-take-test score, body weight, water consumption, and coat appearance would change from baseline in the surgery group but not in the no-intervention and anesthesia-only groups.     

Effects of Buprenorphine,Chlorhexidine, and Low-level Laser Therapy on Wound Healing in Mice

Systemic buprenorphine and topical antiseptics such as chlorhexidine are frequently used in research animals to aid in pain control and to reduce infection, respectively. These therapeutics are controversial, especially when used in wound healing studies, due to conflicting data suggesting that they delay wound healing. Low-level laser therapy (LLLT) has been used to aid in wound healing without exerting the systemic effects of therapies such as buprenorphine. We conducted 2 studies to investigate the effects of these common treatment modalities on the rate of wound healing in mice. The first study used models of punch biopsy and dermal abrasion to assess whether buprenorphine HCl or 0.12% chlorhexidine delayed wound healing. The second study investigated the effects of sustained-released buprenorphine, 0.05% chlorhexidine, and LLLT on excisional wound healing. The rate of wound healing was assessed by obtaining photographs on days 0, 2, 4, 7, and 9 for the punch biopsy model in study 1, days 0, 1, 2, 4, 6, 8, 11, and 13 for the dermal abrasion model in study 1, and days 0, 3, 6, and 10 for the mice in study 2. Image J software was used to analyze the photographed wounds to determine the wound area. When comparing the wound area on the above days to the original wound area, no significant differences in healing were observed for any of the treatment groups at any time period for either study. Given the results of these studies, we believe that systemic buprenorphine, topical chlorhexidine, and LLLT can be used without impairing or delaying wound healing in mice.

A recent retrospective analysis using a medical insurance dataset estimated that approximately 8.2 million people experienced wounds ranging from acute to chronic conditions within the particular year analyzed, and estimated that the cost of acute and chronic wound treatments ranged from $28.1 to $96.8 billion dollars.⁵² The projected rise in the number of people experiencing wounds and the cost of wound care products⁵² have made wound healing a growing area of interest in both clinical medicine and research. Wound healing is a complex process that involves many overlapping, intricate physiologic processes. Each step can have associated deviations that may lead to enhanced, altered, impaired, or delayed healing. Animal research has been used to develop a better understanding of the basic, physiologic mechanisms of wound healing. Mice are the most commonly used animal in biomedical research, and they are used to model a host of conditions, including wound healing. Despite known anatomic and physiologic differences between murine and human skin,^17,53 this species is commonly used due to their small size, ease of handling, and relatively low cost. In addition, the overlapping phases of the wound healing process are similar in mice and humans, making mice a valuable model.⁶⁵Pain is inherent to the development of wound models. Pain receptors in the skin are sensitized during the actual wounding process and during the inflammatory response that occurs immediately after wounding.¹⁹ Pain can also occur during the cleansing and treatment of wounds.¹⁹ Just as managing wound pain is critical in human patients, The Guide for the Care and Use of Laboratory Animals (the Guide)³⁰ and other federal guidelines and regulations governing the care and use of laboratory animals strongly encourages the use of analgesics for animals that experience pain and/or distress.³⁰ Pain, which can also cause stress, may evoke a persistent catabolic state and may ultimately delay wound healing.^19,28,31,43 Therefore, adequate pain control is necessary to avoid negatively affecting or altering the wound healing process.As in human medicine, opioids are commonly used to provide analgesia to research rodents. Buprenorphine, a mixed agonist-antagonist opioid,^26,54 is a common analgesic that acts as a very weak partial agonist of the mu opioid receptor and an antagonist of the κ opioid receptor.²⁶ Buprenorphine is frequently used in animals as both a pre- and post-operative analgesic. It works by binding to the opioid receptors in the skin and other tissues. This ligand-receptor binding regulates the physiologic responses of nociception and inflammation,⁷ which are key factors in the process of healing and regeneration. Buprenorphine is often used instead of full mu-opioid receptor agonist drugs, such as morphine or hydromorphone, because it has fewer systemic side effects.²⁸ Despite their common use as analgesics, reports are mixed in terms of whether opioids, as a class, delay or impair wound healing.^11,28,35,40In addition to controlling pain, minimizing wound contamination and preventing infection is critical to wound healing. The use of antiseptics is often favored over the use of antibiotics as the former presents less chance for developing antibiotic resistance.⁶ As an antiseptic, chlorhexidine is commonly used to irrigate, cleanse, and treat cutaneous wounds. Chlorhexidine has high antimicrobial activity against gram-positive and gram-negative bacteria and some fungi and viruses.⁴ Although considered to be relatively safe, reports are conflicting with regard to whether chlorhexidine delays or impairs wound healing.^4,9,50,57Laser techniques have been used medically for many years, and their powerful, but precise capabilities have rendered them a unique surgical and therapeutic modality. In brief, when the electrons of atoms move to higher energy levels, these electrons absorb energy. This excited energy state is unstable and temporary. The natural return of electrons to their more stable ground state releases energy in the form of photons or light. Light Amplification by Stimulated Emission of Radiation (LASERS) are characterized by the photon stimulation of an already excited electron. This stimulation causes the emitted light to be amplified, as demonstrated by the intense, bright light that is emitted from lasers.⁶³ The concept of low-level laser therapy (LLLT) has garnered interest as a therapeutic modality in both human and veterinary medicine. Specifically characterized as laser therapy using a low power output and a low power range, LLLT is distinguished from other forms of laser therapies by certain parameters such as wavelength, pulse rate and duration, total irradiation time, and dose.⁴⁴ Although the mechanism of action for LLLT is not completely understood,^46,64 the absorption of red and near infrared light energy may reduce detrimental, inflammatory substances^13,15,24,56 while simultaneously stimulating restorative processes.^15,24,46,64 The reduced photothermal impact of LLLT⁴⁴ is reported to produce beneficial physiologic and biologic effects including analgesia, reduction in inflammation, and acceleration of healing.⁴⁸ The initial report of LLLT as a therapeutic modality found accelerated wound healing and fur regrowth in mice exposed to LLLT.^13,44,46,64 LLLT has since been used as a sole or adjunct therapy for a variety of conditions including tooth root resorption,⁵⁵ traumatic brain injuries,⁵⁸ and tendon, muscle, and bone injuries.^2,3,25,38Studies conducted to assess the effects of LLLT on healing often use parameters of normal wound healing to analyze how LLLT influences those parameters in comparison to healthy, undamaged tissue and damaged tissue not receiving laser therapy. Despite the numerous studies designed to investigate the effects of LLLT on wound healing, conflicting reports exist regarding its efficacy.^{15,17,46,22,23,24,29,34,38,39,55,56,60,64} A recent study in dogs reported accelerated healing and improved cosmetic appearance of a hemilaminectomy surgical site after LLLT,⁶⁰ while other canine studies reported no significant differences in the healing of surgically induced skin wounds between dogs that did and did not receive LLLT.^22,34 Similarly, in an attempt to study the effects of LLLT in pigs, an animal with skin very similar to that of humans, no significant differences were reported in the healing of surgically created skin wounds between swine that did and did not receive LLLT.²⁹ Studies using diabetic rats with excisional cutaneous wounds reported accelerated wound healing,^17,46 and beneficial results were reported in a similar study using diabetic mice.^56,64 While fewer studies have been conducted on the use of LLLT in rodents without concomitant comorbidities, LLLT has been reported to accelerate wound healing in healthy rodents.^15,24 Conversely, some studies found that LLLT does not accelerate or significantly improve wound healing in rodents.^24,39We performed 2 separate studies to investigate the effects of a commonly used opioid, a topical antiseptic solution, and LLLT on excisional wound healing in mice. At the time the initial study (study 1) was conducted, some of our investigators were reluctant to use the recommended analgesic, buprenorphine, due to concern about interference with their study outcomes. Therefore, we conducted study 1 to determine if a single dose of peri-operative buprenorphine would delay healing of a full-thickness excisional wound or a partial-thickness felt wheel dermal abrasion. We also examined the effects of topical chlorhexidine solution on wound healing. The chlorhexidine concentrations used in study 1 were prepared using our standard operating procedure at that time. Study 2 was conducted after study 1, with the design expanded to evaluate a sustained release buprenorphine formulation and LLLT. Study 2 used a full-thickness excisional biopsy to determine the effect of LLLT on excisional wound healing. Commonly used doses of systemic Buprenorphine Sustained Release (SR) and topical chlorhexidine were also included to evaluate their effect on excisional wound healing. The concentration of chlorhexidine in the revised, approved standard operating procedure had been decreased due to literature suggesting that higher concentrations may inhibit healing.^4,49,61 For both studies, we hypothesized that the use of buprenorphine and chlorhexidine would have no effect on the rate of wound healing, and that LLLT would accelerate wound healing in a full-thickness excision as compared with a control.     

Long-Term Effects of Sulfadiazine-Trimethoprim Medicated Diet on Cardiac Function,Hematology, and Weight Gain in Hsd:ICR (CD1) and Tac:SW Mice

With the alarming increase in heart disease and heart failure, the need for appropriate and ethical animal models of cardiac dysfunction continues to grow. Currently, many animal models of cardiomyopathy require either invasive procedures or genetic manipulation, both of which require extensive expertise, time, and cost. Serendipitous findings at our institution revealed a possible correlation between sulfadiazine-trimethoprim (SDZ-TMP) medicated diet and the development of cardiomyopathy in IcrTac:ICR mice. We hypothesized that mice fed SDZ-TMP medicated diet continuously for 3 to 6 mo would develop cardiomyocyte degeneration and fibrosis, eventually leading to dilated cardiomyopathy. A total of 44 mice (22 Hsd:ICR (CD1) and 22 Tac:SW) were enrolled in the study. Half of these 44 mice were fed standard rodent diet and the other half were fed SDZ-TMP medicated diet. Baseline samples, including weights, CBCs, select biochemistry parameters, and echocardiography were performed prior to the start of either diet. Weights were obtained monthly and all other parameters were measured at least once during the study, and again at its conclusion. After 42 wk, mice were euthanized, and heart, lung and bone marrow tissue were submitted for histopathologic evaluation. Histologically, hearts were scored for the degree of degeneration, fibrosis, inflammation, and vacuolation. The data showed that SDZ-TMP did not have a significant effect on cardiac function, RBC parameters, biochemistry parameters (ALT, AST, calcium, magnesium, creatine kinase, and creatinine), hematopoiesis, or histologic heart scores. In addition, mice fed the SDZ-TMP medicated diet gained less weight over time. In summary, we were unable to reproduce the previous findings and thus could not use this approach to develop a novel model of cardiomyopathy. However, these results indicate that SDZ-TMP medicated diet containing 1,365 ppm of SDZ and 275 ppm of TMP does not appear to have long-term detrimental effects in mice.

Heart disease is currently the leading cause of death in the United States. The American Heart Association estimates that the prevalence of heart failure will increase to 46% by 2030,⁴ and the cost associated with heart disease is expected to rise from $55 billion in 2016 to $1.2 trillion (US dollars) by 2030.⁴ The existing animal models of heart disease have enhanced our knowledge of its pathophysiology and has led to the development of numerous therapies.^9,30,47,54 However, many animal models of cardiomyopathy require invasive procedures such as aortic constriction,^16,58 left anterior descending artery ligation,^15,31,52 cryogenic injury,^27,57 or renal ischemia⁶⁸ to induce the cardiac changes associated with heart failure. The chemotherapeutic agent doxorubicin has also been used to induce heart failure in mice,^66,69 but a major disadvantage is the risk it poses to workers as a hazardous agent. Genetically engineered mice can replace invasive surgical models of cardiac disease,¹⁴ but they also have significant limitations including cost, time, and failure to express the anticipated phenotype.^3,38,39 Despite advancements in cardiac research, reproducible and economical animal models of heart disease are still needed to further our understanding and explore novel therapeutic solutions.Researchers at the University of Chicago reported that IcrTac:ICR sentinel mice that were accidently fed sulfadiazine trimethoprim (SDZ-TMP) medicated diet for 3 to 6 mo developed cardiomyopathy and subsequently cardiac dysfunction.⁴⁸ Prior to the onset of cardiomyopathy, these animals showed no signs of clinical disease.⁴⁸ SDZ-TMP is a potentiated sulfonamide, and the synergistic activity of sulfadiazine and trimethoprim allows it to not only inhibit folic acid metabolism in bacteria but also in some protozoa and fungi.²⁵ Potentiated sulfonamides are commonly administered to immunocompromised mice to prevent or treat opportunistic infections such as Pneumocystis murina.²⁰ However, SDZ-TMP may have contributed to the development of cardiomyopathy in these mice,⁴⁸ as potentiated sulfonamides are known to have adverse side effects in other animals, such as keratitis sicca, hypothyroidism, anemia, leukopenia, and hypersensitivity reactions.⁵⁰Based on the previous findings,⁴⁸ the goal of this study was to develop a cost-effective, reproducible, and noninvasive animal model of dilated cardiomyopathy. The prior data suggested that this model would have a slow and progressive onset, mimicking many aspects of heart disease and heart failure in humans.⁴⁸ We hypothesized that mice fed SDZ-TMP medicated diet continuously for 3 to 6 mo would develop cardiomyocyte degeneration and fibrosis, eventually progressing to dilated cardiomyopathy. Initial findings were discovered in the outbred stock IcrTac:ICR, but at the time of the present study, live production of this stock had ceased. Therefore, we elected to investigate our hypothesis in 2 readily available outbred stocks. We first attempted to monitor the progression of cardiomyopathy over time via echocardiography in Hsd:ICR (CD1) and Tac:SW mice fed SDZ-TMP medicated diet. In addition to capturing this progression, other objectives of this study were to investigate the long-term effects of SDZ-TMP medicated diet on other health parameters, including CBCs, a subset of biochemistry parameters to assess kidney and liver function, and weight gain. Thus, our goals were to both create an improved animal model of cardiomyopathy and enhance our understanding of prolonged prophylactic antibiotic use and its potential effects on biomedical research models and results.     

Dose-Finding in the Development of an LPS-Induced Model of Synovitis in Sheep

Models of transient synovitis that can be controlled with antiinflammatory and analgesic drugs have been used to study pain amelioration. To this end, we aimed to determine the dose of intraarticularly administered E. coli LPS that induced signs of synovitis without systemic signs in clinically healthy male castrated sheep (n = 14). In phase 1, a single dose of LPS (0.5, 1.0, 1.5, or 2.0 ng in a total volume of 0.5 mL) was administered into the right stifle joint. In phase 2, a dose of LPS (1.0 or 2.0 μg) in 0.3 mL was administered to 4 naïve sheep. In phase 3, 4 sheep from phase 1 were inoculated after a 60 d washout period with either 0.5 or 1.0 μg of LPS. During the first 48 h after LPS administration, the following were performed: assessment of clinical parameters; scoring for lameness, pain on limb flexion, and local swelling; and ultrasonography of the joints were performed. The doses tested during phase 1 produced subtle signs. During phase 2, mild to moderate lameness with no evidence of systemic signs occurred at both doses. In phase 3, clinical responses were similar between the 0.5- and 1-µg doses. Signs of swelling were not observed at any time. Therefore, we consider the 0.5-µg to be the most appropriate for this model, because it was the lowest dose tested capable of causing lameness without signs of systemic inflammation in all animals.

As an experimental model for the study of arthropathies, the aseptic administration of small doses of endotoxin in the joint induces mild to moderate inflammation and the development of clinical signs similar to those of the naturally occurring disease.⁶ Some studies have used models of transient synovitis to determine whether the associated pain can be controlled with antiinflammatory and analgesic drugs. The use of an LPS-induced model of synovitis to evaluate the analgesic effect of various therapeutic protocols has mainly been reported for horses.^9,16,27,28 However, sheep are an important model species in biomedical research, particularly in orthopedic studies,^15,20,32 due to their similarity in weight, size, and joint and bone structure with humans, and in cardiovascular^7,11 studies, because they are good models of cardiac anatomy and physiology. Consequently, the development of analgesia protocols for acute pain conditions is greatly needed.Animal experiments are under increasing focus regarding their ethical and legal aspects. In vivo studies are permitted when methods consistent with the 3Rs principals (replacement, refinement, and reduction) are considered and implemented.²⁶ This means that experiments have to be performed without animals when possible (replacement) or with as few animals as possible (reduction) and with as little pain and distress as possible (refinement). In this context, species-specific analgesia is considered an important refinement method applicable to the majority of research.²⁵ However, few studies have been conducted to determine analgesia protocols for different pain conditions in sheep. The standardization of animal pain models is necessary for the reliable evaluation of efficient and different drug protocols.^10,19,24To guide standardization of the dose of E. coli LPS for intraarticular administration, with the aim of developing a pain model for studies of analgesia in sheep, we here assessed the ability of various intraarticular doses of LPS to trigger synovitis. Our hypothesis was that the dose established for use in horses (0.5 ng/joint) would trigger similar effects in sheep.     

Comparative Efficacy of Two Types of Antibiotic Mixtures in Gut Flora Depletion in Female C57BL/6 Mice

Over the last decade, interest in the role of the microbiome in health and disease has increased. The use of germ-free animals and depletion of the microbial flora using antimicrobials are 2 methods commonly used to study the microbiome in laboratory mice. Germ-free mice are born, raised, and studied in isolators in the absence of any known microbes; however, the equipment, supplies, and training required for the use of these mice can be costly and time-consuming. The use of antibiotics to decrease the microbial flora does not require special equipment, can be used for any mouse strain, and is relatively inexpensive; however, mice treated in this manner still retain microbes and they do not live in a germ-free environment. One commonly used antibiotic cocktail regimen uses ampicillin, neomycin, metronidazole, and vancomycin in the drinking water for 2 to 4 wk. We found that the palatability of this mixture is low, resulting in weight loss and leading to removal of mice from the study. The addition of sucralose to the medicated water and making wet food (mash) with the medicated water improved intake; however, the low palatability still resulted in a high number of mice requiring removal. The current study evaluated a new combination of antibiotics designed to reduce the gut microbiota while maintaining body weights. C57BL/6NCrl mice were placed on one of the following drinking water regimens: ampicillin/neomycin/metronidazole/vancomycin water (n = 16), enrofloxacin/ampicillin water (n = 12), or standard reverse osmosis deionized water (RODI) (n = 11). During an 8 day regimen, mice were weighed and water consumption was measured. Feces were collected before and after 8 d of treatment. Quantitative real-time PCR (real-time qPCR) for 16S bacterial ribosome was performed on each sample, and values were compared among groups. The combination of enrofloxacin and ampicillin improved water intake, together with a greater reduction in gut flora.

Interest in the intestinal microbiome and its role in human health has increased dramatically over the last decade. The microbiota has been implicated in metabolic, infectious, and inflammatory disease, and its role has been investigated not only in the gut,^2,8,18,38 but also in vasculature,^5,6,19,39 kidney,¹³ liver,²⁸ lung,^9,34,37 and brain.^12,15 Animal models have been important in furthering our understanding of the microbiota. Two approaches to studying microbiota in mice are the use of germ-free mice^22,35,42 and depletion of the flora with oral administration of antibiotics.^12,17,18 Both approaches have advantages and disadvantages. Germ-free mice are bred in isolators and are free of microorganisms from birth, allowing studies in mice with no microbes present; mice can then be used to generate gnotobiotic mice in which only known microbes are present. However, to remain germ-free, mice must be maintained in isolators under aseptic housing conditions, which is both costly and labor intensive. In addition, alterations of microbiota in early life may cause sustained effects on body composition¹⁰ and lasting negative consequences on the host immune system.³¹ A more economic approach has been to deplete mouse gut microflora using a combination of broad-spectrum antibiotics given either by oral gavage or in the drinking water. The primary limitation with antibiotic treatment of mice is that not all microbes are eliminated; which can potentially make reproducibility in certain types of studies such as those involving microbial transplantation²⁹ very difficult. However, antibiotic-induced gut dysbiosis can be used on conventionally raised mice without the limitations imposed by maintaining a sterile living environment. Direct handling of the mice is possible, allowing behavioral and imaging assessments, which are not be feasible for mice housed in isolators. Several broad-spectrum antibiotic treatment regimens in the drinking water have been used for gut microbe depletion.^{7,16,20,25-27,41} One of the more commonly used combinations is comprised of 4 antibiotics (ampicillin, neomycin, metronidazole, and vancomycin) added to the drinking water for periods ranging from 1 to 4 wk.^{5,9,13,19,23,30,32,34} This cocktail is effective at depleting gut microbes; however, a previous study in our laboratory found it to be highly unpalatable. Dehydration and weight loss can occur in mice receiving antibiotics in the drinking water, and the magnitude of the effect can be significant, depending on the mouse strain.^21,30,33 The weight loss can result in a substantial number of mice being removed from studies due to animal welfare concerns as reported in a previous study in which 5 of 5 mice given ampicillin, neomycin, metronidazole, and vancomycin reached eighty percent of baseline body weight and were subsequently removed.³³ A reduction in water consumption is also likely to interfere with effective antibiotic treatment and may prolong the time necessary to achieve adequate microbial depletion. Palatability enhancers such as glucose,^5,13 sucrose,⁴¹ and flavored water²³ are sometimes combined with the antibiotics in drinking water. The aim of the current study was to determine whether 8 days of treatment with an alternative mixture comprised of 2 antibiotics (enrofloxacin and ampicillin) was sufficient to deplete the gut flora as compared with the widely used combination of ampicillin, neomycin, metronidazole, and vancomycin. We hypothesized that the combination of 2 antibiotics would be at least equivalent to the combination of 4 antibiotics in reducing the gut flora while causing less weight loss.     

Alpha-1 Acid Glycoprotein as a Biomarker for Subclinical Illness and Altered Drug Binding in Rats

Alpha-1 acid glycoprotein (AGP) is a significant drug binding acute phase protein that is present in rats. AGP levels are known to increase during tissue injury, cancer and infection. Accordingly, when determining effective drug ranges and toxicity limits, consideration of drug binding to AGP is essential. However, AGP levels have not been well established during subclinical infections. The goal of this study was to establish a subclinical infection model in rats using AGP as a biomarker. This information could enhance health surveillance, aid in outlier identification, and provide more informed characterization of drug candidates. An initial study (n = 57) was conducted to evaluate AGP in response to various concentrations of Staphylococcus aureus (S. aureus) in Sprague–Dawley rats with or without implants of catheter material. A model validation study (n = 16) was then conducted using propranolol. Rats received vehicle control or S. aureus and when indicated, received oral propranolol (10 mg/kg). Health assessment and blood collection for measurement of plasma AGP or propranolol were performed over time (days). A dose response study showed that plasma AGP was elevated on day 2 in rats inoculated with S. aureus at 10⁶, 10⁷ or, 10⁸ CFU regardless of implant status. Furthermore, AGP levels remained elevated on day 4 in rats inoculated with 10⁷ or 10⁸ CFUs of S. aureus. In contrast, significant increases in AGP were not detected in rats treated with vehicle or 10³ CFU S. aureus. In the validation study, robust elevations in plasma AGP were detected on days 2 and 4 in S. aureus infected rats with or without propranolol. The AUC levels for propranolol on days 2 and 4 were 493 ± 44 h × ng/mL and 334 ± 54 h × ng/mL, respectively), whereas in noninfected rats that received only propranolol, levels were 38 ± 11 h × ng/mL and 76 ± 16.h × ng/mL, respectively. The high correlation between plasma propranolol and AGP demonstrated a direct impact of AGP on drug pharmacokinetics and pharmacodynamics. The results indicate that AGP is a reliable biomarker in this model of subclinical infection and should be considered for accurate data interpretation.

Protein binding is an important component of pharmacokinetic/pharmacodynamic (PK/PD) research. In vitro measurement of protein drug binding is an essential component of the research and development of novel drugs. However, in vitro studies often poorly mirror the in vivo condition.^9,42 Pharmacokinetic studies early in drug development provide a means to assess the time course of drug effects in the body and drug distribution and availability.⁴² From a PK/PD modeling perspective, protein binding is an important factor in the kinetics and dynamics of drug availability in vivo.^21,35,36,40 These complex relationships are used to project efficacious doses in humans and take into consideration differences in plasma protein binding between preclinical species and humans.^8,44A variety of acute phase proteins (APP) exist across all species and increase in response to inflammatory, infectious and traumatic events.^{5,9,12,13,19,21,22,29,45,53} APPs are potential biomarkers for detection and monitoring of various disease states including cancer.^{2,18,24,34,39,40,47,50,52} Because of this, enhanced understanding of drug binding characteristics to APPs early in the development phase will promote the design of more efficacious therapeutics. Alpha-1 acid glycoprotein (AGP), a ubiquitous major APP that is present in rats,^9,46 has significant drug binding properties and binds to many basic and neutral compounds. Normal AGP levels in plasma of naïve rats range from 0.1 to 0.32 mg/mL.⁴⁴ The importance of AGP as related to drug discovery and development will be bolstered by greater understanding of the sources of AGP stimulation in established animal models. For example, AGP modulates the immune response in a rodent shock model in which it is thought to maintain normal capillary permeability to ensure perfusion of vital organs.^30,33 In addition, elevated AGP levels are present in animal models of infection and inflammation.^{11,20,27,32,41,48}In surgically modified animals, AGP levels may be elevated after surgical manipulation, which unavoidably induces local transient inflammatory responses.^8,25,51 In addition, infections may develop postoperatively leading to increased AGP levels. Chronic catheterization has been linked to increased incidence of infection.^3,8,37 Surgically modified animals should not be placed on study if aseptic technique was not adhered to during surgical preparation and instrumentation.^6,37 Contamination may occur within or at the external portion of a catheter, usually resulting in more obvious signs of infection. Routine PK studies in rats involve implantation of vascular catheters through which drugs are administered and blood samples are taken over time. Catheterized animals are typically perceived as being healthy and thus are enrolled in and remain on study unless they develop obvious clinical signs of infection or illness. However, an occult infection may be present even with a patent catheter. As such, understanding the direct effect of subclinical infection in modulating AGP levels and drug binding is critical, as AGP levels may affect drug levels in study animals with persistent subclinical infection. In this event, the PK data generated may be altered due to selective binding to AGP, thus confounding data interpretation.A possible application of AGP is its potential utility as a biomarker for evaluating health status animals in drug development. The use of AGP as a select biomarker for monitoring and identifying sick animals and/or predicting the potential impact of subclinical infection on drug PK/PD is highly desirable. A screening tool such as this could help to optimize animal selection by reliably identifying healthy animals. Improved intra-study health monitoring would promote confidence in PK/PD data and its predictive value.The focus of this research was to develop a sensitive, reliable and reproducible model of subclinical infection in the rat using the ubiquitous skin contaminant, S. aureus. We selected AGP as a biomarker that would promote health status screening and enhance PK/PD characterization of AGP binding drugs (that is basic and neutral) in the presence or absence of subclinical infection. The model was validated by evaluating the impact of increased AGP levels on propranolol, a drug known to have high binding affinity to AGP.^{4,7,10,26,28,31,49} Ultimately, establishing this model will provide heightened visibility of the protein binding characteristics of drugs and yield more informed data interpretation.     

Fibrous Osteodystrophy,Chronic Renal Disease,and Uterine Adenocarcinoma in Aged Gray Mouse Lemurs (Microcebus murinus)

The gray mouse lemur (Microcebus murinus, GML) is a nocturnal, arboreal, prosimian primate that is native to Madagascar. Captive breeding colonies of GMLs have been established primarily for noninvasive studies on questions related to circadian rhythms and metabolism. GMLs are increasingly considered to be a strong translational model for neurocognitive aging due to overlapping histopathologic features shared with aged humans. However, little information is available describing the clinical presentations, naturally occurring diseases, and histopathology of aged GMLs. In our colony, a 9 y-old, male, GML was euthanized after sudden onset of weakness, lethargy, and tibial fracture. Evaluation of this animal revealed widespread fibrous osteodystrophy (FOD) of the mandible, maxilla, cranium, appendicular, and vertebral bones. FOD and systemic metastatic mineralization were attributed to underlying chronic renal disease. Findings in this GML prompted periodic colony-wide serum biochemical screenings for azotemia and electrolyte abnormalities. Subsequently, 3 additional GMLs (2 females and 1 male) were euthanized due to varying clinical and serum biochemical presentations. Common to all 4 animals were FOD, chronic renal disease, uterine adenocarcinoma (females only), cataracts, and osteoarthritis. This case study highlights the concurrent clinical and histopathologic abnormalities that are relevant to use of GMLs in the expanding field of aging research.

Within the past 5 y, recognition of the translational utility of the gray mouse lemur (Microcebus murinus, GML) has greatly expanded, in part due to the sequencing of its genome.²⁷ GMLs have been proposed as an animal model in the context of aging research,^14,35 most notably within the fields of Alzheimer disease and dementia^33,39 and circadian rhythms.^15,20 GMLs are nocturnal, arboreal, prosimian primates (family Cheirogaleidae) that are endemic to Madagascar. They are among the smallest primates, with a body weight of 49 to 80 g in the wild³⁷ (60 to 110 g in captivity) and have a life expectancy of approximately 8 to 10 y in captivity.¹⁴ A small number of captive breeding colonies have been established throughout Europe and the United States, many of which have arisen from a closed captive breeding colony at the Muséum National d''Histoire Naturelle (MNHN) in Brunoy, France.Despite an ever-growing interest in the GML as a model organism, clinical and pathologic case reports focusing on naturally occurring disease are rare for this species.^{1,4,10,16,17,20,28,31,34,38} Reports of spontaneous disease often focus on neoplasia^28,31,34 or on ocular abnormalities, which are accessible without invasive interventions.^1,4,12 Apart from age-related neurodegenerative disease and cognitive impairment,^{5,23,25,26,32,36} little is known about the natural disease predilection and histologic aging phenotypes of GMLs.In June 2017, a 9 y-old male GML was euthanized after the sudden onset of weakness, lethargy, and tibial fracture. Necropsy and histopathology revealed chronic renal disease, widespread fibrous osteodystrophy (FOD), and systemic metastatic mineralization. These findings prompted colony-wide serum biochemical screenings for potential underlying renal disease and subsequent metabolic bone disease within the population.Herein, we report the clinical, gross, and histologic multisystemic pathology of 4 aged GMLs. This is the first documentation of FOD secondary to chronic renal disease in GMLs in a captive research colony. In addition, we corroborate previous reports^31,34 of uterine adenocarcinoma in aged female GMLs. Together, these findings aid in providing appropriate clinical care to GMLs as their use in the field of aging research continues to expand.     

Hemochromatosis in Two Female Olive Baboons (Papio anubis)

This report describes hemochromatosis associated with chronic parenteral iron dextran administration in 2 female olive baboons (Papio anubis). These baboons were enrolled on an experimental protocol that induced and maintained anemia by periodic phlebotomy for use in studying potential treatments for sickle cell anemia. The 2 baboons both presented with clinical signs consistent with iron overload, including decreased appetite, weight loss, elevated liver enzymes, and hepatosplenomegaly. Histopathologic findings supported a morphologic diagnosis of systemic hemosiderosis, as evidenced by the overwhelming presence of iron in the reticuloendothelial system and liver after the application of Prussian blue stain. This finding, combined with the clinical presentation, lead to a final diagnosis of hemochromatosis. This case report suggests that providing anemic patients with chronic parenteral iron supplementation in the absence of iron deficiency can result in iatrogenic iron overload and subsequent systemic toxicity. Furthermore, these subjects may present with hemochromatosis and its associated clinical signs many years after cessation of iron supplementation.

Iron is an essential micronutrient that plays an important role in cellular proliferation, oxygen transport, and cellular energy generation.^13,26,31 The highest levels of iron in the body is found in the erythrocytes, followed by the liver, reticuloendothelial system, and skeletal muscle.⁹ Three main mechanisms regulate iron: 1) dietary absorption through the proximal duodenum; 2) recycling of senescent red blood cells by macrophages; and 3) storage in the liver. The liver produces the hormone hepcidin, which is the primary negative regulator of systemic iron metabolism.³⁸ Hepcidin controls the release of iron from enterocytes and macrophages into the circulation by binding to and degrading ferroportin, the only mammalian iron exporter.³⁵ When plasma iron levels are high, hepatocytes increase hepcidin synthesis. The increased hepcidin subsequently suppresses gastrointestinal absorption of exogenous iron and iron release from macrophages into circulation.³¹Approximately 1 to 2 mg of iron is lost per day through enterocyte and skin sloughing.³⁸ Iron can also be lost by hemorrhage, menstruation, and parasitic infestation.³⁸ Other than these, the body has no active mechanism for iron excretion. Iron overload can result from acute iron toxicity or chronic accumulation of iron over time.³⁵ Iron is primarily stored in the liver in the form of ferritin, and excess iron is transformed into hemosiderin, an oxidized form of ferritin. Hemosiderin is an iron-containing pigment found primarily in macrophages and hepatocytes.³⁵Hemosiderosis occurs when iron accumulates in tissues, but causes no subsequent organ injury or dysfunction. It is not typically pathologic and can be reversed.⁹ In contrast, hemochromatosis occurs when iron accumulation results in organ injury and dysfunction.³⁵ The 2 types of hemochromatosis are primary and secondary. Primary hemochromatosis, also known as hereditary hemochromatosis, is the result of inherited mutations in genes that are important for iron homeostasis. The most common gene involved in primary hemochromatosis is HFE, an autosomal recessive trait.¹¹ Almost all forms of primary hemochromatosis involve low levels of hepcidin expression.¹¹ Secondary hemochromatosis can occur due to iron-loading anemias such as thalassemia and sideroblastic anemia, chronic liver disease (for example, hepatitis C), and iatrogenic causes, such as excess iron in the diet or parenteral administration.¹⁶ Hemolytic anemia and repeated blood transfusions can also result in secondary hemochromatosis.³⁵ In primary hemochromatosis, iron typically accumulates in the liver, pancreas, heart, and endocrine glands for many years.^16,24 In contrast, secondary hemochromatosis patients often accumulate iron in the reticuloendothelial system, bone marrow, and lymph nodes¹⁶ over a shorter time period,²⁴ with excess iron accumulating in the hepatocytes after the reticuloendothelial system has become saturated with iron.¹⁶ Symptoms of iron overload can vary among individuals due to the number of organ systems affected. These symptoms may include lethargy, arthralgia, skin hyperpigmentation, abdominal pain, abnormal liver chemistry tests, and hepatomegaly.¹⁵ In the current report, we describe 2 cases of hemochromatosis in female baboons after chronic parenteral administration of iron dextran as part of an anemia maintenance protocol used to study sickle cell anemia treatments.     

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.     

Cotton Rat Placenta Anatomy and Fc Receptor Expression and Their Roles in Maternal Antibody Transfer

Respiratory syncytial virus (RSV) is the leading cause of bronchiolitis and viral pneumonia in infants and young children worldwide. Currently no vaccine is available to prevent RSV infection, but virus-neutralizing monoclonal antibodies can be given prophylactically, emphasizing the protective potential of antibodies. One concept of RSV vaccinology is mothers’ immunization to induce high antibody titers, leading to passive transfer of high levels of maternal antibody to the fetus through the placenta and to the neonate through colostrum. Cotton rats are an excellent small animal model for RSV infection and have been used to test maternal immunization. To mechanistically understand antibody transfer in the cotton rat model, we characterized the cotton rat placenta and Fc receptor localization. Placentas from cotton rats at midgestation (approximately day 14) and at late gestation (approximately day 25) and neonatal (younger than 1 wk) gastrointestinal tracts were collected for light microscopy, immunohistochemistry, and transmission electron microscopy. The cotton rat placenta is hemotrichorial and has 5 distinct layers: decidua, junctional zone, labyrinth, chorionic plate, and yolk sac. Consistent with the transfer of maternal antibodies, the majority of the Fc receptors are present in the yolk sac endoderm and fetal capillary endothelium of the chorionic plate, involving 10% of the cells within the labyrinth. In addition, Fc receptors are present on duodenal and jejunal enterocytes in cotton rats, similar to humans, mice, and rats. These findings provide the structural basis for the pre- and postnatal transfer of maternal antibodies described in cotton rats.

Cotton rats (Sigmodon hispidus) have been used to study the pathogenesis of human respiratory syncytial virus (RSV) infection.^6,15,29 This species is considered the best small animal model for RSV pathogenesis, because RSV replicates similarly in cotton rats as in infants and induces an adaptive immune response, as is seen in infants with moderately severe RSV infection.^4,5,33,38 Cotton rats have been used to accurately predict the efficacy of the only prophylactic treatment for RSV, a monoclonal antibody against the fusion protein.^5,17,31 In addition, cotton rats are the ‘gold standard’ for RSV vaccine development.^6,15,29,39A proposed method to protect infants from RSV-induced severe respiratory disease is to vaccinate pregnant mothers to increase the level of maternal antibodies transferred to fetuses and infants.² Human gestation has a duration of approximately 270 d, with midgestation corresponding to approximately day 135 and late gestation to approximately day 225. The human placenta is hemomonochorial in that trophoblasts are in direct contact with the maternal blood supply. A single continuous laver of trophoblasts separates fetal capillaries from the maternal blood.^23,34 Maternal antibodies are predominantly transferred in utero through the placenta. Maternal IgG is endocytosed by syncytiotrophoblasts, where IgG₁ and IgG₄ bind to the neonatal Fc receptor (FnRn) at an acidic pH in early endosomes.^11,25 The endosomal IgG-FcRn complex undergoes transcytosis and fuses with the basal membrane where IgG disassociates from the FcRn. The FcRn is then recycled back to the maternal blood surface of the syncytiotrophoblasts.^11,25,28,30 After birth, the FcRn in the intestine binds maternal IgG in the milk at the acidic apical surface of the epithelium and transports it in an endosome to the basolateral surface, where the maternal IgG disassociates and enters the neonatal blood stream.^20,21In cotton rats, gestation is 28 d in duration, with midgestation at approximately day 15 and late gestation at about day 25. The average litter size is 6 pups, with a range of 4 to 8 pups. Reportedly, maternal antibodies to RSV are transferred to cotton rat pups both prenatally and postnatally.³² Maternal immunization with RSV vaccine candidates in cotton rats has demonstrated that maternal antibodies are transferred to pups and confer protection, but this protection is only short-lived.² In contrast, maternal antibodies after measles immunization provide long-term protection in cotton rats.²⁸ Further investigation of the mechanisms of prenatal and postnatal antibody transfer in cotton rats is necessary to understand these differences and evaluate RSV vaccine candidates. As a basis for these physiologic studies, the placental anatomy and the expression of FcRn in cotton rat must be characterized. In this study, we report the histologic and ultrastructural characterization of the cotton rat placenta at mid- and late gestation and the localization of FcRn in the placenta and neonatal intestine.     

Effects of Chronic Viral Infection on Lymphocyte Populations in Middle‑aged Baboons (Papio anubis)

Aging of the immune system is characterized by the loss of naïve T-cells, increased inflammation, and immune function impairment. Chronic infection with cytomegalovirus is thought to play a role in age-related changes in immunity. Therefore, to assess the effect of pathogens such as cytomegalovirus on the immune system, we determined lymphocyte populations and inflammatory markers over a 3-y period in captive, middle-age baboons, with various exposure to pathogens and shedding pressure. Groups included SPF (i.e., pathogen-negative; n = 14); large-group, conventionally housed (CONV LG; pathogen-positive; n = 14), and small-group, conventionally housed (CONV SM; pathogen-positive; n = 7). All baboon groups showed a decrease in CD45RA+ CD28+ (i.e., naive) cells over time during middle age, but the rate of decline appeared faster in CONV LG baboons than in the other groups. In addition, the reduction in CD45RA+ CD28+ cells in the CONV LG baboons coincided with higher IgG levels against baboon cytomegalovirus, increased serum cortisol concentration, and a greater inflammatory phenotype. The results of this project support a role for cytomegalovirus infection in immune system alterations in middle-aged baboons.

In 2010, an estimated 8% of the world''s population was 65 y or older, and by 2050 the world''s aged population is estimated to reach 16%.²⁹ As people live longer, age-related diseases and conditions place an increased burden on public health and may place tremendous hardship on individual families. Aging is the number one risk factor for numerous diseases, including Alzheimer disease, cardiovascular disease, cancer, and diabetes.³⁰ Practices that improve wellbeing and allow for extended periods of productivity in the aged population will help lower the costs of long-term care for individual families and to society as a whole.²⁹ Further complicating matters, many older adults have multiple, concurrent age-related diseases, making specific disease-targeted treatment approaches less efficient. Treatments targeting the deficiencies underlying aging diseases would therefore be preferable. The increased susceptibility to disease in the elderly is due, in part, to a progressive weakening of the immune system, a process known as immunosenescence. Understanding how age affects immunity may offer more broad-reaching therapeutic targets to reduce the cost of long-term care while it helps to improve health and wellbeing among aged populations.Aging is associated with an altered immune system that includes loss of naïve T lymphocytes, accumulation of T lymphocytes negative for the costimulatory receptor CD28, altered cytokine expression, and increased inflammatory markers.^7,16,28,39 Evidence indicates that immunosenescence reduces overall health and contributes to a rise of disease in the elderly.^7,8,10 Infection with chronic pathogens has been implicated in the aging of immune function.^32,39 Antigenic stress from chronic viruses, particularly cytomegalovirus, is thought to contribute to age-related changes in the immune system.¹ Cytomegalovirus is a β-herpesvirus that rarely causes symptoms in healthy individuals despite establishing a lifelong infection in its host.⁶ Starting in middle age and accumulating over a lifetime, the constant immune response to cytomegalovirus viral antigens is thought to significantly reduce the overall efficiency of the immune system.¹³Chronic infection with pathogens like cytomegalovirus has been suggested to contribute to immunosenescence by causing decreases in naïve cells and increases in late-differentiated memory lymphocytes.^1,6,32 Naive lymphocytes are an essential defense against infection by new pathogens. Some direct evidence in other species suggests that decreases in the naive T-cell pool could limit the ability of the elderly to respond to a novel immune stimulus appropriately.³⁶ Naive lymphocytes are activated through a process that involves the costimulatory receptor, CD28.²⁴ High numbers of lymphocytes negative for CD28 are linked to a reduced response to influenza vaccines and increased inflammatory diseases in the elderly.^35,38,41 Although correlations between chronic viruses and aging of the immune system have been identified in several species, critical questions regarding the direct effects of chronic viruses on the immune system changes remain unanswered.To better determine the role of chronic viruses in age-related changes in the immune system, appropriate animal models are necessary. The genetics and physiology of NHP are similar to those in humans and, therefore, these species have distinct advantages over other animal models. In addition, NHP carry viruses that are closely related to viruses naturally infecting humans. However, little research examining the general effects of age on immunocompetence in NHP is available. Previous studies by our laboratory found that olive baboons (Papio anubis) show age-associated alterations in the immune system.^26,46 In conventionally raised baboons 6 to 26 y old, we discovered that T cells aged in a manner like what has been described in people.⁴⁶ In addition, aged baboons were found to exhibit a proinflammatory state.²⁶ Although the cited study²⁶ supports the idea that aging causes impairment of cell-mediated immunity and a proinflammatory phenotype, the cause of these changes was not addressed. Therefore, in the current study, we sought to test the hypothesis that baboons with the most exposure to chronic pathogens, particularly baboon cytomegalovirus (BaCMV), will show the most profound changes in the immune system, whereas animals lacking exposure to chronic pathogens will have better preservation of immune function over time.     

Pathology of Ground Squirrels in a Research Colony

This paper presents a retrospective review of the postmortem findings in a colony of wild-caught ground squirrels used in medical research. The species included in this study were Richardson''s ground squirrel Urocitellus richardsonii, Columbian ground squirrel Urocitellus columbianus and golden-mantled ground squirrel Callospermophilus lateralis. The pathologic findings in 160 ground squirrels from this colony demonstrated a wide variety of conditions, with chronic nephritis and hepatic adenomas being the most frequent overall. All animals with gross lesions of chronic interstitial nephritis had both glomerular and tubulointerstitial disease upon microscopic examination. As the first review of pathology in a research colony of ground squirrels. this study provides data for use in comparative studies about rodent diseases and important information for those who maintain such animals for research.

Ground squirrels are commonly but incorrectly referred to as gophers and are terrestrial members of the Order Rodentia, family Sciuridae. True gophers are members of the family Geomyidae. In the Canadian province of Alberta, the 3 most common species of ground squirrels are Richardson ground squirrel (Urocitellus richardsonii),^4,13 the Columbian ground squirrel (Urocitellus columbianus),⁵ and the golden-mantled ground squirrel (Callospermophilus lateralis).⁷ Thirteen lined (Ictidomys tridecemlineatus) and Franklin (Poliocitellus franklinii)⁶ ground squirrels are also present but less numerous in Alberta.The 3 species in this study vary in lifespan, litter size, population density,⁵ annual mortality rate and timing of reproduction,¹³ but have several common attributes. An overview of these ground squirrels in the wild is as follows. In a natural habitat, which includes open sagebrush, grasslands,⁴ and mountain meadows, ground squirrels live in complex burrows on stable,⁴ sandy, well-drained soils^4,5 at elevations from 1828 to over 3658 meters in the western 2-thirds of North America.¹ Litters are born in late April to early May and consist of 6 to 8 pups. Ground squirrels are social animals, with densities ranging from 5 to 60 per hectare. In the wild, mortality among male Richardson ground squirrels is as much as 80% per year, partly due to heavy predation.¹⁴ Females have a slightly lower annual mortality rate and are considered to have reached old age at 3 to 4 y of age.¹⁴ One study in southern Alberta found 5 and 6 y old females were present but only a single male that had survived to 4 y.¹³ Ground squirrel burrows can damage to agricultural land because their holes are a hazard to livestock, their mounds can damage equipment, and burrowing can damage crops.¹¹ For these reasons, they are often considered a pest species. On the other hand, ground squirrels till the soil and can serve as a food source for predators.¹¹ As such, they are an integral part of the prairie and mountain ecosystem and food chain.Ground squirrels are most often used to study hibernation and metabolism, as their basal metabolic rate varies according to environmental conditions. They have large amounts of brown fat and are deep seasonal hibernators.³ Unlike most rodents, they are strictly diurnal with peak activities in the morning and late afternoon. Hibernation in ground squirrels consists of periods of torpor interspersed with brief thermogenic arousals that last less than 24 h. Torpor bouts increase in length with decreasing soil temperature. During periods of inactivity, body temperature drops to near ambient soil temperature, falling as low as 2 to 3 °C in late winter.¹² Despite the metabolic costs of thermogenesis and euthermy during intertorpor arousals, hibernating ground squirrels can achieve an overall energy saving over the hibernating season of 88% compared with euthermic animals.¹⁹ Complex regulation of body temperature makes ground squirrels ideal subjects for studies of basal metabolism,⁹ body composition during euthermic and hibernating states, and gene expression during hibernation.⁸ Other research applications for which ground squirrels are useful include the study of liver neoplasia,^10,15,18 cholesterol gallstones,¹⁷ obesity, and diabetes mellitus.¹⁵Ground squirrels seldom produce fertile matings in captivity, and research colonies must be maintained by capture from the wild, posing biosecurity challenges for such colonies. Despite the need to capture of wild ground squirrels for research, these animals habituate easily and adapt well to captivityKnowledge of the pathology of captive ground squirrels is important to investigators and veterinarians who maintain colonies for research purposes. Although wild ground squirrels are used as a laboratory animal in some research institutions, little information is available in the scientific literature about the naturally occurring diseases and pathology of ground squirrels, with the exception of hepatic tumors.¹⁸ The purpose of the current study was to describe conditions that may occur in a research colony of wild-caught ground squirrels, potentially resulting in death or the need for humane euthanasia. The species included in this study were primarily U. richardsonii and U. columbianus, with 6 C. lateralis.     

The Effect of Body Region on Hair Cortisol Concentration in Common Marmosets (Callithrix jacchus)

Common marmosets (Callithrix jacchus) are a valuable research model for the study of neuroscience and the biologic impact of aging due to their adaptivity, physiologic characteristics, and ease of handling for experimental manipulations. Quantification of cortisol in hair provides a noninvasive, retrospective biomarker of hypothalamics-pituitary-adrenal (HPA) axis activity and information on animal wellbeing, including responses to environmental and social stimuli. To obtain valid and reliable measurements of long-term HPA activity, we investigated the variability of cortisol concentration in the hair depending on the body region of marmosets. Hair was collected from the back and tail of 9 adult common marmosets during annual health screenings (male n = 3; female n = 6) and these samples were analyzed for cortisol via methanol extraction and enzyme immunoassay. We found that hair cortisol concentration differed between the tail and back regions, with the tail samples having a significantly higher cortisol concentration. These results indicate intraindividual and interindividual comparisons of hair cortisol concentration should use hair obtained from the same body region in marmosets.

Common marmosets (Callithrix jacchus) are small, New World Primates endemic to Northeast Brazil. Marmosets are similar to humans in their physiology, neuroanatomy, cognition, and sociality.^11,16 They have become an important model in behavioral and biomedical research, including studies of the physiologic effects of aging and neurologic disease. Furthermore, their use in studies of aging or development includes their frequent use in longitudinal studies. Therefore, the ability to frequently and noninvasively measure glucocorticoid hormones as a marker of the response to environmental or social stimuli is an important aspect of longitudinal research in this species.Glucocorticoids are regulated by the hypothalamic-pituitary-adrenal (HPA) axis, a complex neural negative-feedback system that perceives environmental stimuli and regulates hormones accordingly. Cortisol is the dominant glucocorticoid hormone found in most mammals²⁵ and is involved in several physiologic processes, such as metabolizing sugar, fat, and protein stores into usable energy and the inhibition of swelling and inflammation.²¹ Quantification of cortisol concentration can therefore provide valuable insight into how an animal is responding to its environment or to an experimental manipulation and can also provide a valid, retrospective biomarker of HPA axis activity and individual health.^4,5,14,26Cortisol can be measured in blood, saliva, urine, feces, and hair. These tissue types differ in terms of the time frame reflected in the sample and the invasiveness of sample collection.²² Different tissues reflect different intervals with regard to when the cortisol was secreted from the adrenal cortex. Blood and saliva are considered to provide point measures because they indicate a time frame of minutes since the cortisol was secreted.²² Urine and feces are considered to be state measures; they reflect a time frame from hours to a day.²² Finally, hair incorporates a chronic time frame of weeks or months.²² Long-term HPA axis activity can be quantified retrospectively from hair, which accumulates its cortisol concentration over weeks or months, depending upon the length of hair analyzed²² and the growth dynamics of the hair.²⁰ Blood-born substances, such as cortisol, diffuse from blood capillaries into hair follicle cells.^7,27 Only cortisol molecules that are not bound to proteins diffuse into blood capillaries; therefore, hair cortisol concentration (HCC) represents unbound cortisol molecules.^7,27 Once cortisol molecules enter the follicle cells, they are deposited into the hair shaft.⁷ The cortisol deposition process only occurs at a specific time during hair growth, and cortisol deposition into the hair shaft over time is directly proportional to blood cortisol concentration.⁷The stage of growth of a hair follicle influences the cortisol deposition process. All hair has 3 stages of growth: the anagen, catagen, and telogen. The anagen is the active stage when growth occurs; in the catagen or intermediate stage, old hair shafts break down and newer shafts are produced in preparation for the next round of growth; lastly, the telogen stage is the resting phase of hair growth. Most cut or plucked hair samples are expected to be in the anagen phase, while shed hair is likely in the telogen phase.¹³ Cortisol deposition occurs during the anagen stage as active hair follicles require nutrients via the bloodstream to grow. This slow rate of deposition provides information on HPA activity over weeks or months.²²While hair cortisol concentration is increasingly used to assess retrospective HPA axis activity in captive and wild animals, a standard body region from which to obtain hair samples has not been determined. Studies report sampling from various regions, including the back of the neck, back, shoulder, chest, the base of tail, thigh, deltoid, and interscapular regions.^1,7-9 In this investigation, we sought to determine if HCC was affected by the body region from which the samples were taken in common marmosets. We compared HCC from 2 common and readily accessible locations of sample collection in common marmosets, the back and the tail.