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Suggested Citation:"7 Marmoset Care and Management." National Academies of Sciences, Engineering, and Medicine. 2019. Care, Use, and Welfare of Marmosets as Animal Models for Gene Editing-Based Biomedical Research: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25356.
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Suggested Citation:"7 Marmoset Care and Management." National Academies of Sciences, Engineering, and Medicine. 2019. Care, Use, and Welfare of Marmosets as Animal Models for Gene Editing-Based Biomedical Research: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25356.
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Suggested Citation:"7 Marmoset Care and Management." National Academies of Sciences, Engineering, and Medicine. 2019. Care, Use, and Welfare of Marmosets as Animal Models for Gene Editing-Based Biomedical Research: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25356.
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Page 43
Suggested Citation:"7 Marmoset Care and Management." National Academies of Sciences, Engineering, and Medicine. 2019. Care, Use, and Welfare of Marmosets as Animal Models for Gene Editing-Based Biomedical Research: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25356.
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Page 44
Suggested Citation:"7 Marmoset Care and Management." National Academies of Sciences, Engineering, and Medicine. 2019. Care, Use, and Welfare of Marmosets as Animal Models for Gene Editing-Based Biomedical Research: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25356.
×
Page 45
Suggested Citation:"7 Marmoset Care and Management." National Academies of Sciences, Engineering, and Medicine. 2019. Care, Use, and Welfare of Marmosets as Animal Models for Gene Editing-Based Biomedical Research: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25356.
×
Page 46
Suggested Citation:"7 Marmoset Care and Management." National Academies of Sciences, Engineering, and Medicine. 2019. Care, Use, and Welfare of Marmosets as Animal Models for Gene Editing-Based Biomedical Research: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25356.
×
Page 47
Suggested Citation:"7 Marmoset Care and Management." National Academies of Sciences, Engineering, and Medicine. 2019. Care, Use, and Welfare of Marmosets as Animal Models for Gene Editing-Based Biomedical Research: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25356.
×
Page 48
Suggested Citation:"7 Marmoset Care and Management." National Academies of Sciences, Engineering, and Medicine. 2019. Care, Use, and Welfare of Marmosets as Animal Models for Gene Editing-Based Biomedical Research: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25356.
×
Page 49
Suggested Citation:"7 Marmoset Care and Management." National Academies of Sciences, Engineering, and Medicine. 2019. Care, Use, and Welfare of Marmosets as Animal Models for Gene Editing-Based Biomedical Research: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25356.
×
Page 50
Suggested Citation:"7 Marmoset Care and Management." National Academies of Sciences, Engineering, and Medicine. 2019. Care, Use, and Welfare of Marmosets as Animal Models for Gene Editing-Based Biomedical Research: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25356.
×
Page 51
Suggested Citation:"7 Marmoset Care and Management." National Academies of Sciences, Engineering, and Medicine. 2019. Care, Use, and Welfare of Marmosets as Animal Models for Gene Editing-Based Biomedical Research: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25356.
×
Page 52
Suggested Citation:"7 Marmoset Care and Management." National Academies of Sciences, Engineering, and Medicine. 2019. Care, Use, and Welfare of Marmosets as Animal Models for Gene Editing-Based Biomedical Research: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25356.
×
Page 53
Suggested Citation:"7 Marmoset Care and Management." National Academies of Sciences, Engineering, and Medicine. 2019. Care, Use, and Welfare of Marmosets as Animal Models for Gene Editing-Based Biomedical Research: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25356.
×
Page 54
Suggested Citation:"7 Marmoset Care and Management." National Academies of Sciences, Engineering, and Medicine. 2019. Care, Use, and Welfare of Marmosets as Animal Models for Gene Editing-Based Biomedical Research: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/25356.
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Page 55

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  7 Marmoset Care and Management Approaches to the care and management of captive marmosets for research vary widely between in- stitutions. In advance of the workshop, Anna Goodroe, Monica Burns, Casey Fitz, Jessica Izzi, and Keith Mansfield surveyed a broad range of marmoset facilities around the globe for their insights on key veteri- nary and clinical care practices in successful colonies. The data showed a number of similarities but also some differences. Presenters detailed their facilities’ practices and lessons learned related to routine animal care, diet, environment, reproduction, disease, and pain management. This chapter summarizes the main issues addressed; full details and references can be accessed in the presentation slides on the workshop website.11 ROUTINE CARE Marmoset facilities around the world manage the health of their colonies in various ways. In his presentation, Casey Fitz, an assistant research animal veterinarian at WPNRC, summarized operational differences at various marmoset facilities, including staff selection and animal care and offered recommen- dations on these practices. Jaco Bakker, a veterinarian at the Biomedical Primate Research Centre in the Netherlands, continued this discussion, elaborating on ways to harmonize care practices across facilities. Facility Operations The successful operation of a marmoset colony relies on a diverse array of personnel roles and exper- tise. The specific roles and division of responsibility vary across facilities. Generally, a colony manager controls resource allocation, oversees the husbandry staff, manages the process of maintaining ample breed- ing pairs and selecting animals for breeding or research. The breeding manager is generally responsible for hands-on breeding, including pairing, monitoring pregnancies, administering ultrasounds, and drawing blood, as necessary. Many facilities maintain a husbandry staff to manage daily observations, administer medications, and train animals. Often, veterinary technicians assist with daily care as well, though it is important that these individuals have substantial specialized training on working with marmosets, even if they had previous experience with other primates such as macaques. Working in conjunction with all of these team members veterinarians also oversee the animal program and pertinent staff. Health Checks Fitz underscored the need for observing marmoset colonies closely through monthly weigh-ins and once or twice annual physical exams, including dental care. In the WPNRC marmoset facility the animals are trained to go into a detachable nest box attached to the cage for easy transport for small procedures, e.g., weighing, observation, brief clinical examination. Staff members at this facility also use a marmoset tube restraint as an alternative to sedation for brief procedures like blood draws. This restraint also makes it possible for a staff member to complete a test or procedure singlehandedly without support personnel. 11 See http://nas-sites.org/ilar-roundtable/roundtable-activities/care-use-and-welfare-of-marmosets-as-animal-models- for-gene-editing-based-biomedical-research/abstracts-and-speakers. Prepublication Copy 41

Care, Use, and Welfare of Marmosets as Animal Models for Gene Editing–Based Biomedical Research During the discussion, attendees deliberated on protocols for drawing blood from marmosets. Re- searchers described using a transport box high up in the cage to first attract the animals into a gauntlet through which they can be transported out of the cage. Then, handlers lift the marmoset onto a pipe of appropriate diameter, where the animal is secured with Velcro at the waist and legs. Blood samples are taken alternately from the femoral veins in the left or right leg and then the animals are rewarded with a small treat. One researcher noted the possibility of displaying videos to keep the marmoset distracted during the blood draw. Between comprehensive health exams and research procedures, daily observations are important to monitoring marmoset health and well-being and rapid identification of any problems. Any abnormalities observed during these regular checks should be recorded in a standardized way, either on paper or electron- ically, to ensure correct animal identification and available notes for veterinarian review. Facilities use a variety of methods for identifying individual animals. Permanent measures like tattoos and subcutaneous microchips, or more temporary markers such as ear dye or shaving fur, are options for identifying young marmosets. For fully grown animals, neck tags can be used. Veterinary Care When new animals are added to a colony, they are commonly quarantined for 1-3 months to monitor their health and avoid the introduction of disease. During this period, many facilities test for tuberculosis, a particular concern for marmosets. Quarantining is also crucial for avoiding the spread of measles, bacterial pathogens like Klebsiella, and intestinal parasites. To reduce the burden on veterinary staff, some facilities use video monitoring to track the health of animals suspected of having an infectious disease or other health problems. However, when done effec- tively this still requires a great deal of staff time because the video must be actively monitored. For routine care and especially for research procedures such as imaging, several researchers recom- mended the use of a small, prepared box of emergency medical supplies. This “crash cart” could include an external positive pressure ventilation device, warming device, catheter, and fluids. If opioids are used, it is important to have naloxone. To reverse significant respiratory depression, one researcher reported success poking the GB26 acupuncture point with a 25- or 22-gauge needle to reinstate regular breathing. Harmonizing Practices Most facilities house marmosets in traditional indoor enclosures while some facilities, like the Bio- medical Primate Research Center in the Netherlands, provide large, outdoor enclosures with access to sun- light and limited or no use of chemical cleaning products. While these conditions offer many benefits, they also introduce variables that make it hard to compare research results across facilities, such as increased vitamin D levels from access to sunlight or increased cortisol level from the stress of capture in a larger space. Although recommendations exist to standardize housing practices, many of the standards have min- imum but not maximum requirements. Such discrepancies underscore the need to communicate protocols between facilities and harmonize practices where feasible. Harmonization of diet and nutritional guidelines is equally important. In an investigation of how changes in diet can affect susceptibility to EAE, twin marmosets were given diets supplemented with either yogurt or water. Animals consuming yogurt had less multiple sclerosis-like demyelination of the spinal cord and a smaller pro-inflammatory immune response, i.e., reduced T-cell proliferation. The yogurt diet was also related to a decreased likelihood of developing EAE as well as increased levels of Bifidobacteria in the gut microbiota (Kap et al. 2018). These findings emphasize the potential impact of diet on research outcomes and the need for commu- nication about feeding practices among facilities. Furthermore, factors such as age, bedding, cleaning pro- cedures, gut microbiome, catching, fixation, and sedation need to be taken into consideration, particularly when studying the immune system or related diseases. For example, a study comparing the effects of chlo- 42 Prepublication Copy

Marmoset Care and Management  rine-based disinfectants on captive marmosets found that their use resulted in elevated levels of chromoso- mal disorders (Delimitreva et al. 2013). Despite their impact on research outcomes, these factors are not harmonized or standardized across facilities or laboratories. With regard to animal health, mortality rates, biomarkers like glucose levels, and the subclinical and transient presence of Klebsiella pneumonia differ across research colonies. It may also be the case that different colonies show different patterns of aging. Given this, some attendees suggested establishing what “normal” means for each colony rather than attempt to develop global guidelines. It would also be helpful to have more evidence regarding the use of different glucose tests and assessment tools for other biomarkers in marmosets. Comparing notes on health issues between colonies can help colony managers stay abreast of the evidence and proactively monitor for early signs of health problems. DIET Michael Power, an animal scientist at the Smithsonian Conservation Biology Institute, presented on several nutrition-related challenges in marmoset care, including not-well established nutrient requirements; variation in diets between colonies and dietary husbandry informed by practice or anecdotes; the role of vitamin D; factors contributing to Marmoset Wasting Syndrome and metabolic bone disease.; and obesity in captive marmosets. Addressing these concerns requires study of marmosets’ ability to digest and receive appropriate nutrition. He provided a few standard references regarding NHP and marmoset diets: the 2003 National Research Council publication is focused on NHP nutritional requirements, but very few facts are marmoset-specific; chapter 10 (Power et al. 2012) in the first volume of Nonhuman Primates in Biomedical Research (Abee et al. 2012) similarly takes a general approach to dietary requirements for NHPs, including some marmoset-related facts; and the just-released marmoset-specific book edited by Fox et al. (2018) that includes a chapter on nutrition and dietary husbandry (Power and Koutsos 2018). Overall Approaches to Feeding Marmosets can be fed single-item diets that meet all of the animals’ nutritional needs. Such a diet does not require gluten. Gluten sensitivity may be present in subsets of marmosets, but previous experience providing gluten-free diets does not support removing gluten as an effective way to prevent vitamin D deficiency and other digestive difficulties. Additionally, nutrient-rich food will be a more successful train- ing treat if the animal’s base diet is simpler. Importantly, food used as enrichment, training or rewards must be accounted for as part of the animal’s diet and should not compromise nutrition, Power said. In the same context, food is not a good enrichment item unless it is used in creative ways, e.g., puzzle boxes, to reinforce marmosets’ natural behavior. As noted during discussion sessions, researchers have experimented with different types of treats for training and rewards. Some use mini marshmallows, others use drops of Ensure or sugar water, and others use gum. Several attendees reiterated that it is ideal for treats to be aligned with an overall healthy diet and, crucially, that any treats be counted as part of the animal’s diet. To enhance the evidence-base for dietary guidelines, it will be important for researchers to share in- formation openly and in detail, reporting not only what foods were offered but a full nutrient profile break- down of each food, detailing what was offered versus what was consumed, and indicating whether the choice of food varies depending on the time of day or other factors. It also would be helpful to report and compare levels of nutritional components such as vitamin D in the blood, as this data could affect research results and comparisons across laboratories. Examining Differences Between Colonies Although recommendations for nutrient requirements for healthy marmosets exist, experimental test- ing has exposed some unexpected results. For example, digestibility of vitamin D tends to be unusual in Prepublication Copy 43

Care, Use, and Welfare of Marmosets as Animal Models for Gene Editing–Based Biomedical Research marmosets. When examining a single-item diet at three separate colony locations, each sample demon- strated a large amount of variability in diet digestibility. Although one would expect healthy animals to process a single-item diet similarly, some animals presented with high levels of vitamin D while others were deficient. Over time, vitamin D deficiency can affect healthy bone density (Jarcho et al. 2013). Indi- vidual differences like these complicate the task of defining nutritional needs of marmosets and suggest that underlying problems, disease, or other dysfunction may contribute to biological unpredictability. Furthermore, the fecal fat content in these animals was much higher than anticipated, suggesting po- tential problems with fat-soluble vitamin (A, D, E, and K) absorption. Animals that have difficulty digesting nutrients typically adapt by ingesting more food, but even when they do so, they are unlikely to absorb enough nutrients, specifically fat-soluble vitamins. Animal behavior further complicates this picture; ad- justments to diet in an attempt to help an obese animal lose weight may be met with the animal decreasing energy output, thereby nullifying the effort. Despite these complications, further research into these differ- ences and their impact on metabolic state and health is imperative. For instance, animals with vitamin D deficiency likely exhibit different immunological functions than seen in those without this deficiency. These differences could contribute to significant unintended variation in research results. Gum A large debate surrounds the role of gum in marmoset diets. Gum-eating has played an important role in marmoset evolutionary biology; it is central to the natural diet of marmosets, has nutritional value, and provides a fermentable substrate. On the other hand, although gum is a source of energy and minerals, it provides only half of the dietary calcium recommended for marmosets. Power cautioned that despite gum’s importance to marmosets’ evolutionary biology, it is an incom- plete food; if a marmoset eats nothing but gum, it will die. Gum has a far lower energy density and nutri- tional value than other foods in the marmoset diet, like insects and fruits. Another participant noted that the role of gum in the diet also appears to vary from species to species, playing a more central role in the diets of C. jacchus and C. penicillata. The role of gums in intestinal health and gut microbiome requires further study. Moreover, if research indicates positive effects from the inclusion of gum in the marmoset diet, the question follows of whether the benefit is specific to gum or if a saccharide, agar, or other fermentable carbohydrate could serve the same purpose. Vitamin D and Iron During the discussion, participants considered the wide variation in vitamin D supplementation prac- tices between laboratories. One attendee noted that the picture is further complicated by the fact that some laboratories allow marmosets access to outdoor enclosures, where exposure to sunlight can increase endog- enous vitamin D. Several research groups are exploring ways to measure vitamin D precursors to untangle the beneficial or detrimental impacts on health of dietary vitamin D. Indoor lighting can also influence vitamin D and other factors, and researchers are working to deter- mine which lighting approaches are best. The challenge with marmosets is that they can be anywhere in the enclosure and the level of vitamin D an animal gets is highly dependent on how close the animal is to the light source. Compensating for this by using lights that are too strong can risk eye damage both for animals and their caretakers. Labs have reported different downstream effects of high vitamin D supplementation, including soft tissue calcification, though it is unclear whether this is actually related to vitamin D. It has previously been hypothesized that vitamin D metabolism differences may be related to vitamin D receptor resistance, but studies have not supported this, suggesting there is another factor that interferes with vitamin D absorption before the receptor comes into play. Determining appropriate levels of vitamin D in marmosets (and, in- deed, in humans) is an area ripe for further research, many participants agreed. 44 Prepublication Copy

Marmoset Care and Management  Iron presents another nutritional concern raised in the discussion. While increased iron levels have been reported in tissues, particularly in the liver and in older animals, it does not seem to be a cause of death. Anemia has also been reported in some marmosets. One researcher noted that it could be important to select enrichment foods that are low in iron. Obesity Obesity is a common and complex concern in captive marmoset colonies, reported in labs around the world. According to an attendee, adult animals are considered obese if they weigh 500 grams or more. Noting that marmosets typically present with around 10 percent body fat and that obesity often develops early in life, one researcher said marmosets less than a year old were considered obese if their percentage of body fat was 14 percent or higher. Animals that grow faster and have higher fat mass have been shown to reach developmental milestones earlier. Several researchers noted that the number of obese animals tends to increase within a research colony over time. This trend is seen not only in adults but also in the first few months of life and perhaps even prenatally. The pattern is accompanied by a shift in the ratios between litter sizes and birth weights; while larger litters historically have meant smaller infants, that is less and less the case. The factors that drive obesity in research colonies and in younger animals in particular remain unknown. Researchers are inves- tigating genetic factors such as the role of the RNA complex. HABITS AND ENVIRONMENT Behavioral needs of animals in the wild and in semi-natural conditions have been extensively re- searched and are used to guide care recommendations for captive animals. For marmosets in particular, these types of behaviors fall into several categories that include: social behaviors, feeding and foraging, and sleeping site selection. Aspects of each of these activities can inform recommendations for husbandry and care of marmosets in captivity. Nancy Caine, professor of psychology at California State University, San Marco, addressed multiple aspects of marmoset habits and environments. Social Life Marmosets are incredibly social creatures, living in groups of up to 15 close relatives. Although they are not usually aggressive, they can be so when facing territorial or breeding competition. In captivity, groups are created to provide these social conditions and are monitored to ensure assimilation. Additionally, these animals are generally easy to match for breeding, which they do monogamously and cooperatively, meaning that males and females are both responsible for rearing litters (Schiel and Souto 2017). Eating Behavior Food availability presents a major difference between captivity and the wild where marmosets have to invest time to collect and prepare food, including adapting their behaviors to match the demands of their living environments (e.g., resting more and foraging less in high temperatures; de la Fuente et al. 2014). They are adept in extracting gums from tree trunks (Francisco et al. 2014), which is observed in their be- havior both in and out of captivity. Beyond gums, marmosets eat bugs, small lizards, and nesting birds and eggs. In captivity, it is beneficial to provide food in ways that are as naturalistic as possible, for example, forcing the animal to peel or bite into whole fruits instead of providing prepared food, so that it also becomes a source of enrichment. Moreover, captive marmoset habitats can be adjusted to help change behavior and even solve problems such as obesity. Including ropes and nets to encourage running, jumping, and swinging can help replicate behaviors of wild marmosets in captive environments. Prepublication Copy 45

Care, Use, and Welfare of Marmosets as Animal Models for Gene Editing–Based Biomedical Research Sleeping Behavior Marmosets are very deliberate in their selection of sleeping location. Sleep is the time when they are most vulnerable to predation by raptors, hawks, cats, and snakes. In the wild, after selecting a sleeping spot, marmosets become stealthy to not attract attention. The group then gathers together in a tight ball, where they remain until the morning. In captivity, marmosets require sleeping boxes and will select the most secure, covered box. When an ideal box is not available they will select one that offers the most cover or is in the highest position. This exemplifies the highly adaptable nature of marmoset behavior, contradicting the outdated assumption that marmosets are primitive, cognitively simple, or fragile (Warren 1965). Density and Cage Design One common thread among all of these behaviors is the critical nature of social interaction; guarding against potential threats, sleeping, raising young, gathering food are all done in groups. In the context of research, the effect of this is that keeping marmosets in isolation can substantially alter the resulting model. In general, there is a dearth of evidence regarding optimal cage sizes. Several researchers noted that overall cage size may not be as important as the density of marmosets within an area. Higher densities can lead to territorial behaviors and aggression, and also increase the risk of infectious and respiratory diseases, and other health problems, though there is mixed evidence on whether density also affects wasting syn- drome. Density is an issue not only within each cage but between cages; it is important to consider, from a given marmoset’s perspective, how many other marmosets can be seen, smelled, or heard, and how many of those marmosets are within and outside of that individual’s family group. Stress and other problems tend to increase when marmosets are housed very close to animals outside of their family group. REPRODUCTION Female marmosets have the specialized endocrinology typical of New World primates, i.e., pituitary gonadotropin release and action and higher levels of steroid binding hormones. David Abbott, a professor of obstetrics and gynecology at WPNRC, discussed his research aimed at understanding and controlling the marmoset reproductive cycle. Monika Burns presented the breeding practices from mate matching to care of newborn marmosets that she implements as a senior research/clinical veterinarian at the Massachusetts Institute of Technology. Reproductive Biology Although puberty in marmosets begins at 8-10 months of age, the reproductive cycle is irregular until 2-6 years of age. As they age, marmosets do not experience menopause, but have decreased litter sizes as they near the end of their lifespan. Ovarian cycles of adult female marmosets are typical for an anthropoid primate, ranging from 28-30 days. The cycle includes several phases. The first is the follicular phase, an approximately 7-day period when progesterone levels are low in the corpus luteum. Unlike humans who only exhibit one follicle stimulating hormone peak during the follicular phase, marmosets have two (Gilchrist et al. 2001), which coincide with ovarian follicle growth and development, followed by ovulation and the luteal phase, the second half of the cycle (Adams et al. 2011; Kutteyil et al. 2017). Following pregnancy, the marmoset cycle returns to normal, leaving no post-partum control on immediate pregnancy (Abbott 1992). Social cues and conditioning can play an important role in reproductive behaviors and controlling reproductive cycles in marmosets. Socially subordinate female marmosets show suppressed ovulation cy- cles and sexual behavior when around a dominant female. Moreover, babies born to socially subordinate females are often killed and eaten by the dominant female. In groups with related marmosets, inbreeding avoidance further inhibits sexual behavior (Abbott et al. 2009). 46 Prepublication Copy

Marmoset Care and Management  Abbott and other researchers have developed methods to control the timing of marmoset ovarian cycle onset to increase its predictability. While social cues can be powerful, pharmacological interventions can predictably control reproductive cycles and end the post-ovulatory phase or early pregnancies. To control the timing of ovarian cycles, pharmacological mechanisms induce luteolysis, the degradation of the corpus luteum. Prostaglandin F2alpha analogues and gonadotropin-releasing hormone antagonists are established methods and 90 percent effective at controlling the cycle (Summers et al. 1985; Webley et al. 1991, 2010; Fraser et al. 1995; Alper and Fauser 2017; Kraynak et al. 2017; Kropp et al. 2017). Breeding Practices Managing breeding colonies of marmosets requires careful attention to selecting mates, choosing and administering contraception as needed, monitoring pregnancies, handling difficult births, and caring for infants. With a range of standardized and acceptable options available, in many cases colony managers are able to choose the best approaches for their unique colony. Although aggression between pairs of males and females is rare, the introduction of partners to create new pairs of marmosets should be done gradually. At MIT, this entails using two cages with a modifiable window to regulate interaction, while limiting visual access to the previous family group. If the two animals initially seem compatible, the likelihood that the relationship will turn aggressive is low. This gradual in- troduction method, providing first visual then tactile access to the new mate, has a high success rate for forming pairs. It is important to use genetic information to drive pair selection for captive marmosets. Com- puter programs with pedigree trackers help facilitate this. Once two animals form a pair, colony managers may choose from various methods of contraception depending on the needs of the colony and the position in the female’s cycle. Of the available injections, one needs to be administered 3 weeks postpartum and then monthly (Cloprostenol) and another must be injected within 10 days postpartum or it will not be effective (Medroxyprogesterone). Other more permanent options include subcutaneous implants (Etonogestrel and Melengestrol acetate) or surgical remedies, such as vas- ectomy or tubal ligation. Monitoring Pregnancy and Birth If no contraceptive methods are being utilized, a female marmoset typically becomes pregnant within a few months of being matched with a mate. If not, the colony manager will need to identify the root cause, for example performing a thorough physical exam to look for abnormalities in external genitalia, measure- ment of male and female hormone levels, and the use of ultrasound to look for internal maladies. Diagnosing pregnancy can be done by an expert trained in uterine palpitation or ultrasonography to obtain uterine and fetal measurements. Measurement of the length from crown to rump gives an indication of when the fetus will be born. Generally, marmosets experience a 150- to 170-day inter-birth interval and litter size ranges from one to five with twins as the default litter size. Early pregnancy loss is common in marmosets and often goes unnoticed, except when examining inter-birth intervals. The majority of marmoset births occur in the evening after the lights have been turned off. In some locations, lights will be manipulated to encourage births during hours when staff are present. Most births are uncomplicated but dystocia can occur and may require surgical intervention. Other reproductive issues that can require surgery are uterine prolapse, uterine rupture, and several congenital anomalies. Researchers have not established the threshold of gestational length beyond which caesarian is rec- ommended, though one facility reported using a threshold of four days past the expected due date in preg- nancies when the date of embryo implantation is known. If a pregnancy seems to be extending well beyond the expected delivery date but there may be some uncertainty about the timing of implantation, the first step would be an ultrasound to confirm normal fetal heart rates (although more work is needed to determine what is a normal fetal heart rate and what variation can be considered within the normal range). If fetal heart rates appear normal and the mother does not show signs of dystocia, cesarean is likely unnecessary. Prepublication Copy 47

Care, Use, and Welfare of Marmosets as Animal Models for Gene Editing–Based Biomedical Research Some participants suggested a consortium of researchers could examine available records on gestation and birth to elucidate possible differences in outcomes and offer practice guidance on this issue. Infant Care Once born, infants’ weights vary based on litter size and they are carried for the first month of life on the backs of parents or older siblings. Infant viability can be assessed by a checklist of reflexes and meas- urements, including tail curl, position on parent, grip strength, temperature, weight, and others. Triplet litters present one of the most significant areas of concern for breeding marmosets. In these instances, which currently account for more than one-third of marmoset births in captivity, the mother does not increase resources and care to accommodate the additional infant, thus spreading the same amount of resources more thinly among three instead of two infants. In this scenario, colony managers can take any or some combination of these actions: (1) no intervention, (2) remove one infant, (3) foster, (4) supple- mental feeding and assisted rearing, or (5) full nursery rearing. At MIT, Burns implements a protocol that supplements natural feeding on a rotational schedule. Different practices for handling triplet litters likely have some impact on immunological response, microbiome, and other factors. The selection of milk replacement is also likely important, and varies from facility to facility. These differences and their impacts are worthy of further investigation. The choice of milk replacement may also have implications for obesity, because marmoset milk, human breastmilk, and various artificial formulas all have different densities of protein and calories. DISEASE PREVENTION AND MANAGEMENT Along with variation in husbandry practices and diets, each marmoset colony has unique genetic and infectious disease profiles. These characteristics lead to significant variation in patterns of morbidity and mortality between colonies. Of specific concern is the recent upward trend in mortality and morbidity in juvenile and young adults. Keith Mansfield, Takashi Inoue, and Jessica Izzi presented data on some of the chronic and infectious diseases that affect marmoset health and the role of microbiological control in pro- tecting colonies. Mansfield is the executive director and global lead of molecular pathology at Novartis Institutes for Biomedical Research in Cambridge, Massachusetts. Inoue is the head of the Disease Model Animal Labor- atory in the Department of Marmoset Research at the Central Institute for Experimental Animals, Japan. Izzi is an assistant professor and the director of large animal medicine and surgery at Johns Hopkins Uni- versity. Disease Pathology Understanding the diseases that affect each colony is important but complicated due to the numerous underlying causes of mortality in marmosets: infectious and inflammatory diseases, cardiovascular and neoplastic events. Moreover, causes of mortality in marmoset populations vary between age groups. Mans- field presented data from a common marmoset colony showing that adult animals over 5.78 years old (median age) exhibited increased rates of amyloidosis, diabetes, cancer, and renal diseases while younger animals were more prone to inflammatory processes, such as inflammatory bowel disease (IBD) and infec- tious diseases. Even within these categories of disease, colonies vary in their disease profiles, as survey results from common marmoset colonies showed (Mansfield presentation). Gastrointestinal pathologies, specifically in- flammatory bowel diseases, were distinct from one colony to another. Determining the etiology and patho- genesis of these diseases, in particular the reason they manifest in certain colonies and not in others, requires thorough study of colony management variables, including genetics, diet, housing, stress, infectious agents and others. 48 Prepublication Copy

Marmoset Care and Management  Chronic lymphocytic enteritis is a distinct form of IBD characterized by intermittent diarrhea accom- panied by wasting, leading to maldigestion/malabsorption syndrome. Mansfield encouraged researchers to stop using the term “Marmoset Wasting Syndrome” as it lacks diagnostic specificity. Instead, he recom- mended relying on the pathological assessment of tissues for a specific diagnosis. Diagnostic criteria for chronic lymphocytic enteritis including infiltrates of inflammatory cells in the lamina propria and epithe- lium, villous atrophy, and crypt hyperplasia. Similar evaluations can be done to distinguish other types of IBD. Despite the morphologic similarity between chronic lymphocytic enteritis in marmosets and celiac disease in humans, removal of gluten from the diet did not alleviate the underlying symptoms, a fundamen- tal difference from celiac disease. Other initial studies of this disease developed a multi-factorial hypothe- sis: environmental, viral or bacterial triggers cause inflammation worsened by T-cells recognizing self- antigens; inflammation disrupts the mucosal barrier, damages the villous architecture and subsequently causes malabsorption, diarrhea, and weight loss. Animals with chronic lymphocytic enteritis should be re- moved from the colony to isolate the disease and attempt to stop its transmission. Another chronic disease characterized by diarrhea and weight loss is systemic Amyloid A amyloido- sis, which can be diagnosed using histological stains and observing the characteristic liver damage. Though transmission through oral exposure has not been experimentally demonstrated in marmosets, it has been in other species. There is no treatment for this disease; as such, affected animals are either euthanized or isolated to prevent its spread. Marmosets are also very susceptible to bacterial and viral infections. Although it is important to in- troduce new animals to achieve the desired genetic diversity in a colony, this practice does raise the risk of introducing infections to a healthy, established colony. Enteropathogenic E. coli causes acute hemorrhagic diarrhea in marmosets, resulting in hypovolemia and cardiovascular shock. Enrofloxacin and supportive therapy are the treatments of choice for this infection. Campylobacter species are frequent causes of diar- rhea in marmosets. Environmental waterborne organisms such as mycobacterial strains can cause atypical mycobacteriosis in new world primates, including marmosets. While exposure to atypical mycobacteria is common, the disease itself is infrequent and most often recognized by a positive intradermal skin test. Animals with atypical mycobacteriosis may experience weight loss or anorexia. If contracted, the disease should be differentiated from Mycobacterium tuberculosis infection Some researchers have explored the use of probiotics for gastrointestinal issues, though not with for- mal studies. Most agreed that it is unlikely to hurt, and may help, suggesting it may be worth investigating further. Less is known about viral infections in marmosets. The most commonly encountered and studied are infections from herpes simplex 1 and herpesvirus tamarinus. These are rapidly progressive and fatal viral diseases that are quickly transmitted through a colony and associated with high morbidity and mortality rates. Infected animals must be isolated and facility staff should use personal protective equipment. Another viral infection caused by callitrichid herpesvirus 3 is encountered in more than 60 percent of animals over 3 years of age in North American marmoset colonies. Animals with this common infection are generally subclinical, but preliminary studies at WNPRC suggest that the infection may be linked to gastrointestinal lymphomas. Mansfield discussed the likelihood that genetic or environmental cofactors could affect callitrichid herpesvirus 3 disease outcomes in marmosets. Finally, GB virus A causes a common subclinical infection in marmosets with unknown disease associations. In humans, GB virus C infects lym- phocytes and modulates HIV infection. Mansfield recommended conducting autopsies on all deceased colony and research animals to under- stand which disease processes are present in a facility because many present with no or difficult-to-diagnose clinical symptoms. Even in subclinical cases, the presence of disease may influence research outcomes. Comparative analysis of colony morbidity and mortality is needed to help study disease etiology and pub- lication of disease entities and outbreaks would also help elucidate the impact of diet and infectious diseases on animal health. Prepublication Copy 49

Care, Use, and Welfare of Marmosets as Animal Models for Gene Editing–Based Biomedical Research Microbiological Control Microbiological control is an important aspect of marmoset husbandry and critical to regulating the impact of pathogens on the health of colonies and research standards. Most marmoset colonies are not specific-pathogen-free; nonetheless, it is important to take precautions when introducing new animals into an established colony. For example, new animals can be quarantined for a period of time and checked for zoonotic pathogens that pose a high risk in both marmosets and humans. Inoue described experiments examining several protozoa, bacteria, and viruses responsible for diar- rhea, pseudomembranous colitis, sepsis, and possibly even lymphoma. Clostridioides difficile is a naturally- occurring bacterium found in the environment, as well as in humans and animals. In humans, it is believed to be the cause of nosocomial and antibiotic-related diarrhea but in severe cases it can also cause pseudo- membranous colitis. In cotton-top tamarins C. difficile has been associated with colitis. The bacterium has recently been found in Japanese marmoset colonies. To study this situation further, Inoue tested 153 mar- mosets from his colony for the presence of fecal C. difficile toxin and occurrence of various types of diarrhea using a rapid cassette assay that detects both A and B toxins of C. difficile as well as glutamate dehydro- genase antigen in fecal samples. The results indicated the presence of toxins together with a high incidence of mucous, bloody, and other types of diarrhea. Treating this infection is possible with antibiotic therapy using metronidazole or vancomycin; how- ever, the majority of cases recur. Fecal microbiota transplants are used to treat recurring cases of C. difficile in humans and Inoue tested this alternative treatment in marmosets. Preliminary data from his study suggest that fecal transplant can be effective in rebalancing marmoset intestinal microbiota. Immunodeficient and germ-free marmosets may have special research applications but require strict microbiological control. One study used genome editing techniques to produce immunodeficient marmosets (see Chapter 4, Emerging Gene Editing Techniques). Although the results were promising, the extra costs of time and resources needed for the microbiologic control of such a colony are significant. Animals in an SPF colony must be delivered via Caesarean section; immunodeficient newborn marmosets are hand-fed milk in a clean room to prevent exposure to contaminants. Researchers have been able to maintain marmo- sets in such conditions for up to four years. Yet, despite these extensive precautions, C. difficile infection affects even carefully quarantined populations. Germ-free marmosets, which are free of all microorganisms, can play an important role in microbiome research, but require similarly complicated rearing strategies. Although strides have been made in under- standing the underlying husbandry needed to care for these animals, further investigation is required to maximize the efficacy of using these special populations in research applications. Common Marmoset Diseases and Treatments Based on surveys conducted in several marmoset facilities, Izzi discussed a number of common mar- moset diseases. Five types of infectious gastrointestinal disease commonly occur in marmoset colonies. Two are caused by enteropathogenic E. coli and Giardia. Both diseases are prevalent enough to be part of the stand- ard differential diagnosis list, especially when symptoms align. Three other common pathogens are Klebsiella pneumoniae, Salmonella, and C. difficile. Klebsiella pneumoniae primarily causes enterocolitis that often results in rapid animal death some- times even before any clinical symptoms are exhibited. Virulent strains can also result in meningitis, pneu- monia, or septicemia. Once diagnosed animals can be treated with antibiotics such as sulfamethoxazone/tri- methoprim and enrofloxacin. Colonies can be protected against Klebsiella by an autogenous vaccine developed from bacteria isolates taken from affected animals within the colony or other carriers of Klebsiella such as macaques. The vaccination sites may react poorly or develop abscesses but this risk should be weighed against a possible colony infection. 50 Prepublication Copy

Marmoset Care and Management  There are no published reports of outbreaks of Salmonella enterica in captive marmosets, although reports do exist for outbreaks in wild marmosets or other primate species. Izzi noted an outbreak at her marmoset colony at Johns Hopkins University. Clinical presentations of salmonellosis include diarrhea, weight loss, and lethargy, but since these are not pathognomonic a culture is the gold-standard diagnostic tool. When using culture to diagnose specific pathogens, selecting the culture media will impact the test result’s error rate. This is easiest to address by specifying the suspected pathogens to the bacteriologist performing the culture tests so the appropriate media is selected, which is selenite broth for Salmonella. Diagnosed cases can similarly be treated with enrofloxacin. Though not many reported cases of C. difficile colitis are found in the literature, several surveyed marmoset facilities reported finding C. difficile in their colonies. In Johns Hopkin’s first case of C. difficile, the animal experienced mucoid diarrhea and was diagnosed using a fecal smear, doing a PCR analysis for toxins, and an antigen test. The disease was successfully treated with metronidazole. Not all diseases threatening marmoset health are infectious. The most frequently mentioned is Marmoset Wasting Disease, a non-infectious gastrointestinal disease that is difficult to compare across in- stitutions as facilities define it in different ways (see previous discussion by Keith Mansfield). Chronic lymphoplasmacytic enterocolitis is a defining feature of the disease, as is weight loss without diarrhea. All marmosets diagnosed with wasting disease at Johns Hopkins have been characterized by low serum albumin and low body weight, as well as metabolic bone disease, likely a result of vitamin D malabsorption. Marmosets are also affected by bone disorders including metabolic bone disease and traumatic and idiopathic bone disease. Traumatic wounds and fractures can be caused by fighting among the animals or iatrogenic factors like improper catching or restraint. If a marmoset does suffer a fracture, amputation or splints are viable treatment options and euthanasia is not necessarily required. Idiopathic bone disease generally, but not exclusively, affects long bones unilaterally. Radiograph- ically the most specific indicator of idiopathic bone disease is radiolucency, and histologic findings include marked osteoclastic bone resorption. One specific type of this disease is fibrous dysplasia, a hereditary disease characterized by increased bone diameter from expansive lesions and fibrous connective tissue that has replaced the bone marrow. This can cause bone pain, swelling, and physical impairment in marmosets. Other bone diseases are metabolic. For instance, rickets is a metabolic bone disease that affects the growth plates of juvenile animals. Induced by prolonged vitamin D deficiency, often caused by gastroin- testinal disease, rickets leads to stunted growth and bowed legs. Fibrous osteodystrophy is another type of metabolic bone disease that has been reported at the Johns Hopkins colony (Olson et al. 2015). This disease affects the long bones, the mandible and maxilla, and vertebrae, and is characterized by multifocal areas of radiolucency and increased number of osteoclasts. Numerous other issues can affect marmoset health in captivity, including complications from cranial implants, viral infections as previously discussed, chronic renal disease, type 2 diabetes, lymphoma, and dental disease. PAIN PREVENTION AND MANAGEMENT Anesthesia and analgesia are necessary in the routine clinical and research procedures conducted in marmosets. Although the practice of using these types of drugs is ubiquitous in captive animal colonies, the regimens and procedures used differ between species. In this case, marmosets’ small size and elevated metabolic rate affect dose amount, fasting time, and post-procedural nutrition. Anna Goodroe, a veterinar- ian with Animal Care Operations at the University of Houston, presented data from a survey of marmoset facilities on the common regimens used and suggestions for optimizing anesthesia and analgesia in mar- mosets. Anesthesia Anesthesia provides a temporary period during which the animal loses sensation and/or awareness. Sedation is milder than general anesthesia, in that it depresses the central nervous system but does not result Prepublication Copy 51

Care, Use, and Welfare of Marmosets as Animal Models for Gene Editing–Based Biomedical Research in loss of consciousness. Anesthesia can be induced using injectable or inhalant drugs. Injectable anesthetic regimens can be based on single drugs, such as ketamine or alfaxalone, or on combinations of those with others. Manipulating their dose amounts can shift the length or depth of anesthesia. Isoflurane and sevoflu- rane, inhalant anesthetics, have very reliable onset of action and duration. This predictability enables precise manipulation of anesthetic depth. Inhalant anesthetics can be delivered either through a mask or endotracheal tube. Intubation requires unobstructed visualization of the larynx through the oral cavity, which can be achieved by suspending the animal from a tilt table or having an assistant hold the top jaw open for the insertion of the laryngoscope. A local anesthetic may also help minimize laryngeal spasms and facilitate intubation. In either case, while the animal is under anesthesia and connected to a non-rebreathing gas delivery system, intravenous access for fluid administration is established through the saphenous or lateral tail veins. Once the procedure is underway, the animal must be constantly monitored for physiological signs relating to the expected depth of anesthesia. A few critical factors to monitor in an animal during any anes- thetic state are heart rate and rhythm, hemoglobin oxygen saturation, blood pressure, end tidal carbon di- oxide, and blood gases. While under anesthesia marmosets are particularly susceptible to hypothermia and dehydration, therefore they should be regularly checked by the anesthetist. This physiological monitoring continues after the procedure is completed to ensure a quick recovery to the animal’s normal behavior and physiological function. A small contained recovery environment may be suitable to decrease the likelihood of accidental injury and facilitate re-catching the animal should it become necessary. However, many institutions return animals within line of sight and close to their family group, which has been reported to reduce recovery time. Marmosets’ high metabolic rate necessitates their return to regular feeding as soon as possible after a procedure. Supplementing a complete diet with a sweet liquid or preferred foods may be needed to ensure the animal is consuming enough calories and is successfully transitioning back to a normal diet. Supportive therapies, including many common medications to lessen nausea, decrease gastric acid, and treat ulcers are also useful in this post-procedural transition period. It is unknown whether administering anesthesia to a single animal frequently or over extended periods of time can result in neural damage, but may be a risk worth considering in experimental designs. Some monitoring and anesthetic equipment designed for rats or other small laboratory animals can be successfully used for marmosets; examples include electrophysiologic suites for rats and transreflectant probes with Masimo pulse oximetry units. One researcher reported an attempt to use the Kent Scientific sono-suite but found it to be prone to errors in the end tidal carbon dioxide module. Analgesia Analgesia provides pain relief. Local anesthetics, non-steroidal anti-inflammatory drugs (NSAIDs), opioids, among other options, are used as analgesics in marmosets. Determining which of these treatments is the most appropriate therapy depends on a thorough pain evaluation. Local anesthetics are able to block pain signals from reaching the central nervous system. They can be used before surgical incisions or topical application for less invasive procedures such as tattoo placement. Furthermore, NSAIDs are able to reduce inflammation while also providing analgesia. Several NSAIDs op- tions are available. One published study demonstrated that sustained release of meloxicam can provide ap- proximately 72 hours of analgesia in macaques (Bauer et al. 2014). Similar applications in marmosets have shown equally promising results. Moreover, opioids can be used for managing both acute and chronic pain in marmosets. However, multiple reports have linked buprenorphine, one such opioid, with respiratory depres- sion and delayed recovery when used in conjunction with anesthesia. Other opioid options, such as fentanyl, can bridge the intra-surgical period when buprenorphine may be unsafe to administer. Goodroe emphasized the importance of adapting anesthetic and analgesic therapies to the marmosets’ unique physiological features. Although a few studies have been published on anesthetic doses, even less evidence exists regarding effective analgesia in marmosets. Across the board, clinical practices can greatly 52 Prepublication Copy

Marmoset Care and Management  benefit from open communication between institutions, including sharing protocols for anesthesia and an- algesia in marmosets. Assessing Stress During the discussion, attendees considered how to appropriately characterize the stress level imposed on marmosets during various types of research activities. For example, imaging studies sometimes require awake animals to be restrained for several hours. When the restraint is conditioned and the session can be terminated if the animal shows signs of distress, one participant suggested it would not necessarily warrant a high stress classification. Another attendee suggested looking for signs of assent—indications that the marmoset is going willingly into a situation in which it knows what will likely happen. While marmosets cannot consent to a procedure the way humans can, studies can be designed in such a way that the animal receives sufficient training to associate the early stages of a procedure with what will happen next and has the opportunity to show an adverse response versus calmly accepting the procedure. Interactions between facility personnel and marmosets can be important in managing the animals’ stress levels. Based on studies of cortisol levels and ovulation cycles, one attendee reported developing protocols that emphasize a great deal of contact between marmosets and human handlers, including specific practices for keeping stress levels low when new personnel are introduced. Approaches for routine handling and interaction with marmosets can affect how stressful certain procedures are, therefore different routine practices between facilities can induce varying amounts of stress that could affect outcomes of the same research activities. It is important to account for the animals’ holistic experience at the facility when clas- sifying the stress level associated with a research procedure. DISCUSSION Many attendees agreed that better methods are needed to objectively evaluate marmosets’ stress levels to guide care and management practices and inform ethics reviews. Other participants noted that while cortisol levels can be informative, single measurements are not reliable because cortisol levels can fluctuate as a result of daily cycles and many other factors. Unlike other primate species, scratching behavior is not a reliable indicator of stress or arousal in marmosets. A few participants added that when developing mark- ers for stress it is important to distinguish between stress and distress. REFERENCES Abee CR, Mansfield K, Tardif SD, eds. 2012. Nonhuman Primates in Biomedical Research. Volume I: Biology and Management, 2nd ed. San Diego, CA: Elsevier. Abbott D. 1992. Reproduction in female marmoset monkeys, Callithrix Jacchus. In Reproductive Biology of South American Vertebrates, Hamlett WC, ed. New York, NY: Springer-Verlag. Abbott D, Digby L, Saltzman W. 2009. Reproductive skew in female common marmosets: Contributions of infanticide and subordinate self-restraint. In Reproductive Skew in Vertebrates: Proximate and Ultimate Causes, Hager R, Jones C, eds. Cambridge, England: Cambridge University Press. Adams C, Henke A, Gromoll J. 2011. A novel two-promoter-one-gene system of the chorionic gonadotropin b gene enables tissue-specific expression. J Mol Endocrinol 47:285-298. Alper MM, Fauser BC. 2017. Ovarian stimulation protocols for IVF: Is more better than less? Reprod Biomed Online 34(4):345-353. Available at: https://www.rbmojournal.com/article/S1472-6483(17)30038-X/fulltext. Ansel TV, Nour AK, Benavente-Perez A. 2016. The effect of anesthesia on blood pressure measured noninvasively by using the tail-cuff method in marmosets (Callithrix jacchus). J Am Assoc Lab Anim Sci 55(5):594-600. Bakker J, Uilenreef JJ, Pelt ERJ, et al. 2013. Comparison of three different sedative-anaesthetic protocols (ketamine, ketamine-medetomidine and alphaxalone) in common marmosets (Callithrix jacchus). BMC Vet Res 9:113. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3686586. Baskerville M. 1984. Canine tooth root infection as a cause of facial abscess in the common marmoset (Callithrix jacchus). Lab Anim 18:115-118. Prepublication Copy 53

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Care, Use, and Welfare of Marmosets as Animal Models for Gene Editing-Based Biomedical Research: Proceedings of a Workshop Get This Book
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The marmoset, a type of small monkey native to South America, is a research model of increasing importance for biomedical research in the United States and globally. Marmosets offer a range of advantages as animal models in neuroscience, aging, infectious diseases, and other fields of study. They may be particularly useful for the development of new disease models using genetic engineering and assisted reproductive technologies. However, concerns have been voiced with respect to the development of new marmoset-based models of disease, ethical considerations for their use, the supply of marmosets available for research, and gaps in guidance for their care and management.

To explore and address these concerns, the Roundtable on Science and Welfare in Laboratory Animal Use hosted a public workshop on October 22-23, 2018, in Washington, DC. The workshop focused on the availability of marmosets in the United States and abroad; animal welfare and ethical considerations stemming from the use of wildtype and genetically modified marmosets; and standards of housing and care, dietary needs, and feeding requirements for marmosets in captivity. This publication summarizes the presentations and discussions from the workshop.

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