Macaca nemestrina

Ten chronically catheterized pregnant monkeys (Macaca nemestrina) at 118–125 days gestation (term = 172 days) received one of two experimental treatments: 1) choriodecidual and intra-amniotic saline infusions (n = 5), or 2) GBS choriodecidual inoculation (n = 5). Saline infusions were performed separately in the choriodecidual and amniotic fluid to confirm that saline would not stimulate cytokine production in either compartment. In two saline controls, fetal samples were not collected due to either an inability to place the fetal catheter during initial surgery or clotting of the fetal catheter. This resulted in three fetal cytokine analyses in the saline group. In one GBS case, technical problems led to only intermittent data collection and so the remaining uterine activity data was excluded for this animal.
In our model, pregnant pigtail macaques were time-mated and fetal age determined using early ultrasound. The tethered chronic catheter preparation was used for all in vivo experiments and is a major breakthrough in studying maternal-fetal immunologic responses [53] (link), [54] (link). The animal was first conditioned to a nylon jacket/tether system for several weeks before surgery, which allowed free movement within the cage, but protected the catheters. On day 118–125 of pregnancy (term = 172 days) catheters were implanted into the maternal femoral artery and vein, fetal internal jugular vein, amniotic cavity, and choriodecidual interface in the lower uterine segment (between uterine muscle and fetal membranes, external to amniotic fluid). Fetal ECG electrodes and a maternal temperature probe were also implanted. After surgery, the catheters/electrodes were tracked through the tether system and cefazolin and terbutaline sulfate were administered to reduce postoperative infection risk and uterine activity. Both cefazolin and terbutaline were stopped at least 72 hours before experimental start (∼5 half-lives for terbutaline, 40 half-lives for cefazolin, >97% of both drugs eliminated). Cefazolin 1 gram was administered intravenously each day in saline controls to minimize chances of a catheter-related infection. Experiments began approximately two weeks after catheterization surgery to allow recovery (∼30–31 weeks human gestation). At our center, term gestation in the non-instrumented pigtail macaque population averages 172 days.

Three-fold (pigtail macaque) or four-fold (human) serial dilutions of heat inactivated serum and 600 plaque-forming units (PFU)/ml solution of SARS-CoV-2/WA/20 (BEI resources) were mixed 1:1 in DPBS (Fisher Scientific) + 0.3% gelatin (Sigma G7041) and incubated for 30 min at 37°C. Serum/virus mixtures were added in duplicate, along with virus only and mock controls, to Vero E6 cells (ATCC) in a 12-well plate and incubated for 1hr at 37°C. Following adsorption, plates were washed once with DPBS and overlayed with a 1:1 mixture of Avicel RC-591 (FMC) + 2 x MEM (ThermoFisher) supplemented with 4% heat-inactivated FBS and Penicillin/Streptomycin (Fisher Scientific). Plates were then incubated for 2 days at 37°C. Following incubation, overlay was removed and plates were washed once with DPBS and then 10% formaldehyde (Sigma-Aldrich) in DPBS was added to cells and incubated for 30 min at room temp. Plates were washed again with DPBS and stained with 1% crystal violet (Sigma-Aldrich) in 20% EtOH (Fisher Scientific). Plaques were enumerated and percent neutralization was calculated relative to the virus-only control.

We used 128 S. fuelleborni genotypes compiled as part of previous work (Barratt et al., 2019a (link); Barratt and Sapp, 2020 (link)), plus 18 from an undefined Strongyloides species identified in Bornean slow lorises (Frias et al., 2018 (link)). Our dataset included 24 cox1 sequences and HVR-IV sequences from Thai isolates of S. fuelleborni: one from a human and 23 from pig-tailed macaques (Macaca nemestrina) (Janwan et al., 2020 (link)). We also included 39 S. fuelleborni cox1 sequences generated by Ko et al. (2023) (link) from a range of Asian non-human primates including rhesus macaques from Myanmar (Macaca mulatta), Japanese macaques (Macaca fuscata), and sequences from four primate species housed in zoological parks in Japan; siamang (Symphalangus syndactylus), red-shanked douc (Pygathrix nemaeus), Francois’ langur (Trachypithecus francoisi), and Bornean Orangutan (Pongo pygmaeus) (Ko et al., 2023 (link)). To serve as an outgroup for our phylogenetic analysis, we sequenced our S. stercoralis PCR positive control amplicons in the same Illumina library as the vervet samples, and included 28 previously published S. stercoralis genotypes (Barratt and Sapp, 2020 (link)), plus a cox1 sequence extracted from the mitochondrial genome of S. stercoralis reference strain PV001 (GenBank [GB] - accession: NC_028624.1). A detailed overview of all specimens within this dataset, including information on hosts, genotype, and GenBank accession numbers, is provided in File S1, Tab A.

We used two approaches to test for evidence of introgression and if detected, to permit us to evaluate hypotheses regarding introgression of N_interact and non-N_interact windows that are articulated in the results. The first approach uses a hidden Markov model to infer introgression in 100 kb windows as implemented by the software admixfrog (Peter 2020 ). Because the data produced errors under some single-nucleotide polymorphism densities, for each chromosome, we thinned the data to differing degrees, removing variant sites that were within 200–2,000 positions from other variable sites. Because the extent of introgression that was detected in M. arctoides and M. f. aurea was low, no statistical analyses were performed on these introgression blocks.
The second approach we used to study introgression used Patterson's D statistic (Patterson et al. 2012 (link)). This analysis was performed using 30 and 100 kb windows and using taxon settings that matched established evolutionary relationships among nuclear genes or genomes (Fan et al. 2018 (link); Matsudaira et al. 2018 ), namely for the M. f. aurea data set (((M. fascicularis, M. f. aurea), M. assamensis), Macaca nemestrina) and for the M. arctoides data set (((M. arctoides, M. assamensis), M. mulatta), M. nemestrina). For both data sets, we also analyzed Patterson's D after substituting the M. thibetana genome for the M. assamensis genomes. For the M. f. aurea data set, we used previously published genomic data from an M. nemestrina individual (PM1206) as an outgroup; genotypes for this individual were called along with the other individuals in this data set for this analysis only using the pipeline described above. Following Evans et al. (2021) , permutation tests were used to evaluate expectations based on evolutionary relationships among mitochondrial genomes of these samples that are described in the results.