Genetic characterization of a captive marmoset (Callithrix jacchus) colony using genotype-by-sequencing.

The marmoset is a fundamental nonhuman primate model for the study of aging, neurobiology, and many other topics. Genetic management of captive marmoset colonies is complicated by frequent chimerism in the blood and other tissues, a lack of tools to enable cost-effective, genome-wide interrogation of variation, and historic mergers and migrations of animals between colonies. We implemented genotype-by-sequencing (GBS) of hair follicle derived DNA (a minimally chimeric DNA source) of 82 marmosets housed at the Southwest National Primate Research Center (SNPRC). Our primary goals were the genetic characterization of our marmoset population for pedigree verification and colony management and to inform the scientific community of the functional genetic makeup of this valuable resource. We used the GBS data to reconstruct the genetic legacy of recent mergers between colonies, to identify genetically related animals whose relationships were previously unknown due to incomplete pedigree information, and to show that animals in the SNPRC colony appear to exhibit low levels of inbreeding. Of the >99,000 single-nucleotide variants (SNVs) that we characterized, >9800 are located within gene regions known to harbor pathogenic variants of clinical significance in humans. Overall, we show the combination of low-resolution (sparse) genotyping using hair follicle DNA is a powerful strategy for the genetic management of captive marmoset colonies and for identifying potential SNVs for the development of biomedical research models.

Discovery of PSMA in the prostate of the common marmoset (Callithrix jacchus).

BACKGROUND: Prostate-specific membrane antigen (PSMA) is a biomarker and therapeutic target of high relevance in prostate cancer. Although upregulated PSMA expression is a well-documented feature of prostatic neoplasia in both humans and canids, to date humans are the only species known to express PSMA basally in the prostate. Thus, traditional laboratory animal species have limited utility for studying PSMA biology in the prostate or for predicting efficacy or toxicity of PSMA-targeted agents. METHODS: PSMA expression in human, macaque, and marmoset prostates was determined by immunohistochemistry, employing an antibody with validated cross-species reactivity in a PSMA-positive control tissue; kidney. RESULTS: We newly discover that the common marmoset endogenously expresses PSMA in non-diseased prostate, similar to humans, and thus may be a valuable preclinical model for researchers studying PSMA.

Protective efficacy of a Zika purified inactivated virus vaccine candidate during pregnancy in marmosets.

Zika virus (ZIKV) infection during pregnancy poses significant threats to maternal and fetal health, leading to intrauterine fetal demise and severe developmental malformations that constitute congenital Zika syndrome (CZS). As such, the development of a safe and effective ZIKV vaccine is a critical public health priority. However, the safety and efficacy of such a vaccine during pregnancy remain uncertain. Historically, the conduct of clinical trials in pregnant women has been challenging. Therefore, clinically relevant animal pregnancy models are in high demand for testing vaccine efficacy. We previously reported that a marmoset pregnancy model of ZIKV infection consistently demonstrated vertical transmission from mother to fetus during pregnancy. Using this marmoset model, we also showed that vertical transmission could be prevented by pre-pregnancy vaccination with Zika purified inactivated virus (ZPIV) vaccine. Here, we further examined the efficacy of ZPIV vaccination during pregnancy. Vaccination during pregnancy elicited virus neutralizing antibody responses that were comparable to those elicited by pre-pregnancy vaccination. Vaccination also reduced placental pathology, viral burden and vertical transmission of ZIKV during pregnancy, without causing adverse effects. These results provide key insights into the safety and efficacy of ZPIV vaccination during pregnancy and demonstrate positive effects of vaccination on the reduction of ZIKV infection, an important advance in preparedness for future ZIKV outbreaks.

Impact of prior dengue virus infection on Zika virus infection during pregnancy in marmosets.

Zika virus (ZIKV) infection during pregnancy causes severe developmental defects in newborns, termed congenital Zika syndrome (CZS). Factors contributing to a surge in ZIKV-associated CZS are poorly understood. One possibility is that ZIKV may exploit the antibody-dependent enhancement of infection mechanism, mediated by cross-reactive antibodies from prior dengue virus (DENV) infection, which may exacerbate ZIKV infection during pregnancy. In this study, we investigated the impact of prior DENV infection or no DENV infection on ZIKV pathogenesis during pregnancy in a total of four female common marmosets with five or six fetuses per group. The results showed that negative-sense viral RNA copies increased in the placental and fetal tissues of DENV-immune dams but not in DENV-naïve dams. In addition, viral proteins were prevalent in endothelial cells, macrophages, and neonatal Fc receptor-expressing cells in the placental trabeculae and in neuronal cells in the brains of fetuses from DENV-immune dams. DENV-immune marmosets maintained high titers of cross-reactive ZIKV-binding antibodies that were poorly neutralizing, raising the possibility that these antibodies might be involved in the exacerbation of ZIKV infection. These findings need to be verified in a larger study, and the mechanism involved in the exacerbation of ZIKV infection in DENV-immune marmosets needs further investigation. However, the results suggest a potential negative impact of preexisting DENV immunity on subsequent ZIKV infection during pregnancy in vivo.

Efficacy of an inactivated Zika vaccine against virus infection during pregnancy in mice and marmosets.

Zika virus (ZIKV) is a mosquito-borne arbovirus that can cause severe congenital birth defects. The utmost goal of ZIKV vaccines is to prevent both maternal-fetal infection and congenital Zika syndrome. A Zika purified inactivated virus (ZPIV) was previously shown to be protective in non-pregnant mice and rhesus macaques. In this study, we further examined the efficacy of ZPIV against ZIKV infection during pregnancy in immunocompetent C57BL6 mice and common marmoset monkeys (Callithrix jacchus). We showed that, in C57BL/6 mice, ZPIV significantly reduced ZIKV-induced fetal malformations. Protection of fetuses was positively correlated with virus-neutralizing antibody levels. In marmosets, the vaccine prevented vertical transmission of ZIKV and elicited neutralizing antibodies that remained above a previously determined threshold of protection for up to 18 months. These proof-of-concept studies demonstrate ZPIV's protective efficacy is both potent and durable and has the potential to prevent the harmful consequence of ZIKV infection during pregnancy.

Advancing Autism Research From Mice to Marmosets: Behavioral Development of Offspring Following Prenatal Maternal Immune Activation.

Understanding the mechanism(s) by which maternal immune activation (MIA) during gestation may disrupt neurodevelopment and increase the susceptibility for disorders such as autism spectrum disorder (ASD) or schizophrenia is a critical step in the development of better treatments and preventive measures. A large body of literature has investigated the pathophysiology of MIA in rodents. However, a translatability gap plagues pre-clinical research of complex behavioral/developmental diseases and those diseases requiring clinical diagnosis, such as ASD. While ideal for their genetic flexibility, vast reagent toolkit, and practicality, rodent models often lack important elements of ethological validity. Hence, our study aimed to develop and characterize the prenatal MIA model in marmosets. Here, we adapted the well-characterized murine maternal immune activation model. Pregnant dams were administered 5 mg/kg poly-L-lysine stabilized polyinosinic-polycytidylic acid (Poly ICLC) subcutaneously three times during gestation (gestational day 63, 65, and 67). Dams were allowed to deliver naturally with no further experimental treatments. After parturition, offspring were screened for general health and vigor, and individual assessment of communication development and social behavior was measured during neonatal or adolescent periods. Similar to rodent models, offspring subjected to MIA exhibited a disruption in patterns of communication during early development. Assessment of social behavior in a marmoset-modified 3-chamber test at 3 and 9 months of age revealed alterations in social behavior that, in some instances, was sex-dependent. Together, our data indicate that marmosets are an excellent non-human primate model for investigating the neurodevelopmental and behavioral consequences of exposure to prenatal challenges, like MIA. Additional studies are necessary to more completely characterize the effect of prenatal inflammation on marmoset development and explore therapeutic intervention strategies that may be applicable in a clinical setting.

Womb to womb: Maternal litter size and birth weight but not adult characteristics predict early neonatal death of offspring in the common marmoset monkey.

A singular focus on maternal health at the time of a pregnancy leaves much about perinatal mortality unexplained, especially when there is growing evidence for maternal early life effects. Further, lumping stillbirth and early neonatal death into a single category of perinatal mortality may obscure different causes and thus different avenues of screening and prevention. The common marmoset monkey (Callithrix jacchus), a litter-bearing nonhuman primate, is an ideal species in which to study the independent effects of a mother's early life and adult phenotypes on pregnancy outcomes. We tested two hypotheses in 59 marmoset pregnancies at the Southwest National Primate Research Center and the Barshop Institute for Longevity and Aging Studies. We explored 1) whether pregnancy outcomes were predicted independently by maternal adult weight versus maternal litter size and birth weight, and 2) whether stillbirth and early neonatal death were differentially predicted by maternal variables. No maternal characteristics predicted stillbirth and no maternal adult characteristics predicted early neonatal death. In univariate Poisson models, triplet-born females had a significantly increased rate of early neonatal death (IRR[se] = 3.00[1.29], p = 0.011), while higher birth weight females had a decreased rate (IRR[se] = 0.89[0.05], p = 0.039). In multivariate Poisson models, maternal litter size remained an independent predictor, explaining 13% of the variance in early neonatal death. We found that the later in the first week those neonates died, the more weight they lost. Together these findings suggest that triplet-born and low birth weight females have distinct developmental trajectories underlying greater rates of infant loss, losses that we suggest may be attributable to developmental disruption of infant feeding and carrying. Our findings of early life contributions to adult pregnancy outcomes in the common marmoset disrupt mother-blaming narratives of pregnancy outcomes in humans. These narratives hold that the pregnant person is solely responsible for pregnancy outcomes and the health of their children, independent of socioecological factors, a moralistic framing that has shaped clinical pregnancy management. It is necessary to differentiate temporal trajectories and causes of perinatal loss and view them as embedded in external processes to develop screening, diagnostic, and treatment tools that consider the full arc of a mother's lived experience, from womb to womb and beyond.

Current practices in nutrition management and disease incidence of common marmosets (Callithrix jacchus).

BACKGROUND: A survey was developed to characterize disease incidence, common pathology lesions, environmental characteristics, and nutrition programs within captive research marmoset colonies. METHODS: Seventeen research facilities completed the electronic survey. RESULTS: Nutritional management programs varied amongst research institutions housing marmosets; eight primary base diets were reported. The most common clinical syndromes reported were gastrointestinal disease (i.e. inflammatory bowel disease like disease, chronic lymphocytic enteritis, chronic malabsorption, chronic diarrhea), metabolic bone disease or fracture, infectious diarrhea, and oral disease (tooth root abscesses, gingivitis, tooth root resorption). The five most common pathology morphologic diagnoses were colitis, nephropathy/nephritis, enteritis, chronic lymphoplasmacytic enteritis, and cholecystitis. Obesity was more common (average 20% of a reporting institution's population) than thin body condition (average 5%). CONCLUSIONS: Through review of current practices, we aim to inspire development of evidence-based practices to standardize husbandry and nutrition practices for marmoset research colonies.

Publisher Correction: Experimental Zika Virus Infection in the Pregnant Common Marmoset Induces Spontaneous Fetal Loss and Neurodevelopmental Abnormalities.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Experimental Zika Virus Infection in the Pregnant Common Marmoset Induces Spontaneous Fetal Loss and Neurodevelopmental Abnormalities.

During its most recent outbreak across the Americas, Zika virus (ZIKV) was surprisingly shown to cause fetal loss and congenital malformations in acutely and chronically infected pregnant women. However, understanding the underlying pathogenesis of ZIKV congenital disease has been hampered by a lack of relevant in vivo experimental models. Here we present a candidate New World monkey model of ZIKV infection in pregnant marmosets that faithfully recapitulates human disease. ZIKV inoculation at the human-equivalent of early gestation caused an asymptomatic seroconversion, induction of type I/II interferon-associated genes and proinflammatory cytokines, and persistent viremia and viruria. Spontaneous pregnancy loss was observed 16-18 days post-infection, with extensive active placental viral replication and fetal neurocellular disorganization similar to that seen in humans. These findings underscore the key role of the placenta as a conduit for fetal infection, and demonstrate the utility of marmosets as a highly relevant model for studying congenital ZIKV disease and pregnancy loss.

Developmental origins of pregnancy loss in the adult female common marmoset monkey (Callithrix jacchus).

BACKGROUND: The impact of the intrauterine environment on the developmental programming of adult female reproductive success is still poorly understood and potentially underestimated. Litter size variation in a nonhuman primate, the common marmoset monkey (Callithrix jacchus), allows us to model the effects of varying intrauterine environments (e.g. nutrient restriction, exposure to male womb-mates) on the risk of losing fetuses in adulthood. Our previous work has characterized the fetuses of triplet pregnancies as experiencing intrauterine nutritional restriction. METHODOLOGY/PRINCIPAL FINDINGS: We used over a decade of demographic data from the Southwest National Primate Research Center common marmoset colony. We evaluated differences between twin and triplet females in the number of pregnancies they produce and the proportion of those pregnancies that ended in fetal loss. We found that triplet females produced the same number of total offspring as twin females, but lost offspring during pregnancy at a significantly higher rate than did twins (38% vs. 13%, p = 0.02). Regardless of their own birth weight or the sex ratio of the litter the experienced as fetuses, triplet females lost more fetuses than did twins. Females with a male littermate experienced a significant increase in the proportion of stillbirths. CONCLUSIONS/SIGNIFICANCE: These striking findings anchor pregnancy loss in the mother's own fetal environment and development, underscoring a "Womb to Womb" view of the lifecourse and the intergenerational consequences of development. This has important translational implications for understanding the large proportion of human stillbirths that are unexplained. Our findings provide strong evidence that a full understanding of mammalian life history and reproductive biology requires a developmental foundation.

Digestive efficiency mediated by serum calcium predicts bone mineral density in the common marmoset (Callithrix jacchus).

Two health problems have plagued captive common marmoset (Callithrix jacchus) colonies for nearly as long as those colonies have existed: marmoset wasting syndrome and metabolic bone disease. While marmoset wasting syndrome is explicitly linked to nutrient malabsorption, we propose metabolic bone disease is also linked to nutrient malabsorption, although indirectly. If animals experience negative nutrient balance chronically, critical nutrients may be taken from mineral stores such as the skeleton, thus leaving those stores depleted. We indirectly tested this prediction through an initial investigation of digestive efficiency, as measured by apparent energy digestibility, and serum parameters known to play a part in metabolic bone mineral density of captive common marmoset monkeys. In our initial study on 12 clinically healthy animals, we found a wide range of digestive efficiencies, and subjects with lower digestive efficiency had lower serum vitamin D despite having higher food intakes. A second experiment on 23 subjects including several with suspected bone disease was undertaken to measure digestive and serum parameters, with the addition of a measure of bone mineral density by dual-energy X-ray absorptiometry (DEXA). Bone mineral density was positively associated with apparent digestibility of energy, vitamin D, and serum calcium. Further, digestive efficiency was found to predict bone mineral density when mediated by serum calcium. These data indicate that a poor ability to digest and absorb nutrients leads to calcium and vitamin D insufficiency. Vitamin D absorption may be particularly critical for indoor-housed animals, as opposed to animals in a more natural setting, because vitamin D that would otherwise be synthesized via exposure to sunlight must be absorbed from their diet. If malabsorption persists, metabolic bone disease is a possible consequence in common marmosets. These findings support our hypothesis that both wasting syndrome and metabolic bone disease in captive common marmosets are consequences of inefficient nutrient absorption.

Characterization of obese phenotypes in a small nonhuman primate, the common marmoset (Callithrix jacchus).

This report explores aspects of developing obesity in two captive populations of common marmosets (Callithrix jacchus), a small primate with a short lifespan that may be of value in modeling chronic aspects of obesity acquisition and its lifetime effects. Two populations were examined. In study 1, body composition, lipid parameters, and glucose metabolic parameters were measured in a population of 64 adult animals. Animals classified as obese (>80th percentile relative fat based on sex) displayed both dyslipidemia (higher triglyceride and very low-density lipoprotein (VLDL)) and altered glucose metabolism (higher fasting glucose and HbA(1c)). Using operational definitions of atypical values for factors associated with metabolic syndrome in humans, five subjects (7.8%) had at least three atypical factors and five others had two atypical factors. A previously unreported finding in these normally sexually monomorphic primates was higher body weight, fat weights, and percent fat in females compared to males. In a second study, longitudinal weight data for a larger population (n = 210) were analyzed to evaluate the development of high weight animals. Differences in weights for animals that would exceed the 90th percentile in early adulthood were evident from infancy, with a 15% difference in weight between future-large weight vs. their future-normal weight litter mates as early as 4-6 months of age. The marmoset, therefore, demonstrates similar suites of obesity-related alterations to those seen in other primates, including humans, suggesting that this species is worthy of consideration for obesity studies in which its fast maturity, high fertility, relatively short lifespan, and small size may be of advantage.