Subjects

Subjects included 170 healthy infant rhesus macaques (90 female, 80 male). Subjects were either raised by their biological mothers (BIO, n = 132) or cross-fostered to a new unrelated female in a new social group (FOSTER, n = 38). These subjects represent a subset of subjects from a larger longitudinal study of 206 mother-infant pairs. All mother-offspring pairs were housed in the outdoor breeding colony at the California National Primate Research Center (CNPRC). Animals were housed in one of sixteen established social groups (30–160 members). These groups were comprised of similar social demographics, including 6–13 distinct matrilines with extended kin networks and animals of all age/sex classes. All social groups included 5–10 reproductively mature males, 23–105 reproductively mature females, and 25–75 subadult, juvenile or infant monkeys. Enclosures were 0.2 hectares with chain link fencing to allow visual access. Animals were fed monkey chow (LabDiet 5405) twice per day, once in the morning and once in the afternoon. All procedures were approved by the UC Davis Animal Care and Use Committee and were in compliance with the National Institutes of Health guide for the care and use of Laboratory animals.

Cross-fostering procedures

As infants were born, they were assigned to BIO or FOSTER conditions. BIO dyads remained in large social groups for the first twelve weeks of life. Pairs of dyads to be cross-fostered were removed from outdoor enclosures on the day of infant birth. During indoor FOSTER procedures, infants were removed from the sedated (10 mg/kg ketamine) biological mothers by trained CNPRC staff and then placed on the ventrum of the sedated FOSTER mother that had recently given birth, FOSTER pairs were observed indoors for 18–24 hours by CNPRC staff for successful fostering/reunion. Within 24 hours of FOSTER manipulations, FOSTER dyads were returned to field enclosures until infants were 3–4 months of age. Cross fostering was conducted on only one pair per day.

Social hierarchy rank determination

In nature and captivity, group living rhesus macaques form social hierarchies, in which each social group member occupies a rank-ordered place compared with all other group members. Hierarchy rank of the biological and foster mothers was measured by trained CNPRC Behavioral Management staff. Infant subjects are not considered to have social rank, as they are not yet independent from mothers. Hierarchy data were collected in all outdoor field corrals for at least 30 minutes on a bi-weekly basis, totaling at least one hour of data collection per month. Data were collected with scan sampling to record dyadic displacement interactions between individuals. Displacement interactions include aggression (chasing, biting, threats) from one individual, and subordination (fear grimace, moving out of proximity) from the other. The number of “wins” (when an individual receives subordination in response to an aggression) and losses (the same individuals subordinates in response to an aggression) are plotted against all other adult females in the group. Animals with the most wins and fewest losses are considered the highest ranked, and the animals with the most losses and fewest wins are considered the lowest ranked. We calculated maternal social rank as the absolute rank at the time of infant birth divided by the number of adult females in the social group (continuous variable). Ranks ranged from the 2% percentile for higher-ranked animals (i.e., 98% of female adults and adolescents are lower-ranked than mother) to the 100th percentile (i.e., lower ranked animals: 0% of adults and adolescents are lower ranked than mother) The average rank was 47.4%, such that 52.6% of animals were lower-ranked than our subjects’ mothers.

Mother-infant observations

Mother-infant interactions were assessed and we present the detailed methods as we have described previously18,40. Dyads were observed in their social groups for five minutes per observation using focal dyadic sampling conducted one to four times weekly. Observations were conducted between 0700 h and 1300 h, during postnatal weeks 1–12. Observation order was rotated daily so dyads were observed at different times of day across the proscribed observation period.

Mother-infant interactions were coded using a transactional coding system, describing the overall theme of an interaction from the perspectives of the initiator and the recipient18,40. A transaction was defined as a change from one state of association that lasted three seconds or more, to a new state that was maintained for at least three seconds. Themes are defined in Table 1, and include protection, affiliation, neutral, rejection and aggression. Each dyadic interaction was characterized from the perspective of the mother and of the infant, and a theme assigned for each based on their behavior. The initiator of the transaction is considered to be the individual who changed the state of interaction between the pair, which the receiver then responds to. For example, common types of transaction types include: (1) infant approaches mother and initiates contact, mothers initiates physical contact (which would be scored as Affiliative-Affiliative, initiated by the infant), (2) Mother retrieves infant and infant grabs mother’s ventrum or back (Protective-Affiliative), initiated by the mother, (3) Infant jumped on mother, mother swats the infant to the ground (Affiliative-aggressive, initiated by the infant).

Table 1 Transaction Theme Definitions. Full size table

Male and female adult raters were trained by a primatologist with experience in mother-infant interactions for at least eight two-hour training sessions. Rater reliability was determined at the end of this period by calculating whether the rater was correct in recording each aspect of the transaction (maternal theme, infant theme, and recipient) for ten sequential five-minute trials. These trials were required to include with a wide variety of possible transaction calls or they were excluded from reliability calculations. The criterion for inter-rater reliability was 90% or better, and averaged 94% across all observers. Rates of all transaction themes by the mother (Protective, Affiliative, Accommodating, Neutral, Rejecting or Aggressive) were calculated per observation period for analysis.

Maternal sensitivity scale

Our maternal sensitivity measure was characterized from factor analysis of 206 mother-infant dyads that were part of a longitudinal ongoing study. We present the methods here as we have described previously18,40.Transactions in which the infant initiated social overtures toward that mother were considered in this analysis. Rates of each transaction response type by the mother (continuous variables) were entered into a principal components analysis with Promax rotation. The analysis yielded five factors. We identified a factor that explained a high proportion of the total variance explained in maternal responses (17% out of 67%). This factor was novel in that it did not fit into the remaining factor structure which described factors that specifically overlapped with our other maternal behavior themes (ie, protectiveness, affiliation, aggressiveness and neutral; Kinnally et al.18,40). This factor was considered to be a naturally occurring aspect of macaque maternal care that shares similarities with Ainsworth’s MS scale: high MS mothers are more accepting and symmetrical in response to infants than low MS mothers, as in humans18,27,28,29 Maternal sensitivity scores were generated based on all factor loadings using the regression method. Higher MS scores reflect higher rates of affiliative or neutral responses to infant neutral and affiliative overtures (See Table 2 for factor loadings, bolded loadings over 0.4 are considered to represent significant contributors to factors).

Table 2 Factor Analysis for Maternal Sensitivity Score. Full size table

Social climate measures

Social climate was recorded at the end of every mother-infant observation. The social climate scale included five descriptors of the activity status of proportions of the social group, where activity was defined as locomotion, eating, drinking, or other activities in which the animal’s body was in motion. “Torpor” was recorded when less than 10% of the social groups members were engaged in activity, “Calm” was recorded when 10–25% of the group was active, “Moderately Active” was recorded for 25–50%, and “Active was recorded when 50–100% of groups members were engaged in activity. “Conflict” was recorded if two or more members of the social group engaged in contact aggression at any time during the observation. Another requirement was that 10% or more of the group must attend to conflict or change their behavior in response to conflict for conflict to be scored. The criterion for observer reliability for this measure was 90% or better, and across all observers there was an average of 95% reliability. For each dyad, a proportion of observations in which each social climate state was recorded was generated. Rate of social conflict ranged from 0% per observations to 39% per total observations per subject, with a mean of 11.4%.

Cytokine and protein assays

At 3–4 months (90–120 days) of age, infants underwent a 25-hr social and maternal separation as part of a standardized biobehavioral assessment at the CNPRC. Inflammatory stress response was measured by assaying plasma cytokines and proteins (Interleukin 8 [IL-8], monocyte chemoattractant protein 1 [MCP-1] and C-reactive protein [CRP]) collected 2 hours after a mother-infant separation. Samples were assayed using the MILLIPLEX MAP Non-Human Primate Cytokine Magnetic Bead Panel Array Kit (Millipore, Billerica, MA, USA). Samples were assayed following manufacturer’s instructions. While the panel included a larger number of possible analytes (including IL-6 and TNFα), pilot testing revealed that reliability standards met our stringent criteria only for IL-8 and MCP-1 (ie, >99% standard curve fit and ~20% intra-assay coefficient of variance). Plates were read using a Bio-Plex HTF System with Luminex xMAP Technology (Bio-Rad, Hercules, CA, USA), and values were calculated using Bio-Plex Manager Software, version 4.1. Samples were run in duplicate, and duplicates with coefficients of variance less than 30% were included for analysis (average intra-assay CVs: IL-8 = 7.15% and MCP-1 = 8.16%). To control for inter-assay variation, a pooled sample was run on each plate. Interassay coefficients of variance were: MCP1 average = 18.05%; IL-8 average = 22.19%). Assay sensitivity for MCP-1 was 3.1 pg/ml and for IL-8 1.1 pg/ml. Observed ranges and means were: IL-8 (0.00–5131.06; 437.98 pg/ml) and MCP-1 (30.14–632–0.44; 213.28 pg/ml). A total of 36 out of our original 206 subjects were removed from the study because of missing or incomplete data.

C-reactive protein was measured using a latex agglutination method43. Latex particles are coated with rabbit anti-CRP antibodies and incubated with plasma from each subject. Displacement reactions are read using a Beckman Coulter AU 480 chemistry system (Beckman Coulter, Brea, CA). Observed CRP ranged from 0–33 pg/ml, with an average of 1.81 pg/ml.

Health follow-up

Approximately 145 months (up to 12 years) later, we extracted health data from our subjects’ lifetime veterinary health records as we have described previously39. We recorded presence or absence of the most common infectious disease in captive macaques, colitis/diarrhea, at any time in the intervening years between assessment and the present, up to 12 years. These were defined as (1) having been hospitalized for one of these conditions, and (2) testing positive for one or more enteric pathogens that cause diarrhea including Shigella, Campylobacter lari, Campylobacter coli, Campylobacter jejuni, orTrichomonas. Of our subjects, 47% fit these criteria.

Data analysis

All data analysis was conducted using SPSS version 26. The effects of MS (independent variable) on three plasma pro-inflammatory measures (dependent variables) was assessed using multivariate analysis of variance (MANOVA). This method was selected because it allows us to assess the effect of our predictors on correlated outcome measures (interclass correlation coefficient among cytokines = 0.432, p < 0.0001) and enhances power. Maternal sensitivity groups were trichotomized as one standard deviation or more above (high MS, n = 24), within one standard deviation (medium MS, n = 123), and one standard deviation or more below (low MS, n = 23) the population mean. MS group and foster condition (FOSTER or BIO rearing) were independent variables. Maternal social hierarchy rank (continuous variable), average proportion of observations containing social conflict across the dyad observation period (continuous variable), foster status (dichotomous variable), and infant sex (dichotomous variable), were entered as covariates. Earlier models included group level covariates (group identity and group size), but these were non-significant and therefore removed from the analysis. To test the specificity of association with MS, we included our other maternal behavior factors (protectiveness, affiliation, neutrality and aggression,18) in earlier models, but they were non-significant and therefore removed from the analysis. Interactions between infant sex, foster condition, maternal rank and social group conflict and MS were tested in separate MANOVAs. We used this stepwise approach to testing interactions because our study was not powered to test all interactions in the same model (ie, some cell sizes were too small to permit analysis). Two-tailed statistical significance was set at p < 0.05.

The effects of MS, foster status, maternal social hierarchy rank and social conflict on risk for lifetime inflammatory disease was analyzed using multinomial logistic regression with likelihood ratio testing. Non-significant contributors were manually removed from the model. For this analysis, one-tailed statistical significance was set at p < 0.05. One-tailed testing was used because our hypothesis was directional based on our previous study showing a link between poor maternal care and greater rates of colitis/diarrhea40. Finally, we determined whether inflammation in infancy predicted lifetime disease risk using independent samples t-tests.