Effects of relocation on Immunological and physiological measures in squirrel monkeys (Saimiri boliviensis boliviensis)

In the present study, we have quantified the effects of transport, relocation and acclimate/adapt to their new surroundings on squirrel monkey. These responses are measured in blood samples obtained from squirrel monkeys, at different time points relative to their relocation from their old home to their new home. A variety of immunological assays are performed on the phenotype and function of peripheral blood mononuclear cells (PBMCs) in a group of squirrel monkeys that were transported by road for approximately 10 hours from one facility to another. Using a panel of human antibodies and a set of standardized human immune assays, we evaluated the phenotype of lymphocyte subsets by flow, mitogen-specific immune responses of PBMCs in vitro, and levels of cytokines at various time points including immediately before transport, immediately upon arrival, and after approximately 150 days of acclimation. We observed significant changes in T cells and subsets, NK and B cells (CD4+, CD8+, CD4+/CD8+, CD16+, and CD20+). Mitogen specific (e.g. PHA, PWM and LPS) proliferation responses, IFN-g by ELISPOT assay, and cytokines (IL-2, IL-4 and VEGF) significant changes were observed. Changes seen in the serum chemistry measurements mostly complement those seen in the hematology data. The specific goal was to empirically assess the effects of relocation stress in squirrel monkeys in terms of changes in the numbers and functions of various leukocyte subsets in the blood and the amount of time require for acclimating to their new environment. Such data will help to determine when newly arrived animals become available for use in research studies.


Introduction
grouping, each kennel was opened, and the animals were released into their new housing. At 126 no point were the animals sedated. The entire colony of 500 animals was moved over a five 127 weeks period following the same routine procedures. 129 Ten animals from three of separate shipments, for a total of 30, were sampled for this study. 130 Baseline blood samples were obtained one day prior to the relocation as part of the animals' 131 "pre-shipment" physical exam. The next blood samples "Day 1" were obtained within the first 132 few hours upon arrival at the Keeling Center as part of the animals' first "quarantine" physical 133 exam. The final used for this study was collected at "Day150" post-arrival to monitor the 134 animals' adaptation to their assigned housing conditions at the Keeling Center. Blood 135 samples (3 mL) were collected, 1.5mL in EDTA anti-coagulant tubes and 1.5 mL in 136 coagulation CBC tubes, from the femoral vein at the different time points. The animals were 137 manually restrained for each of the three blood draws, and no sedatives or other chemical 138 restraints were utilized. All blood sample collections occurred in the morning (8-10AM) before 139 the animals were fed. Before the separation of peripheral blood mononuclear cells (PBMC) 140 from the blood samples, plasma was collected and stored immediately at -80ºC until 141 analyzed. The PBMC prepared from the blood samples by the standard ficoll-hypaque 142 density-gradient centrifugation were used for various immune assays [23,26]. All blood 143 samples were processed at the Keeling Center following domestic overnight shipment (for the 144 pre-travel blood collections) or within 2-4 hours of collection at the Keeling Center (for arrival 145 and day 150 post-arrival samples). The PBMCs, freshly prepared from whole blood collected 146 in EDTA tubes, were more than 90% viable as determined by the trypan blue exclusion 147 method. For each immune assay, 10 5 cells /well were used for various immune assay.

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Complete blood count and blood chemistry analysis. 149 EDTA whole blood samples were analyzed for a complete blood count on Siemens Advia 120 150 Hematology Analyzer, Tarrytown, NY. The Parameters analyzed included: total WBC, total 151 RBC, hemoglobin, hematocrit, RBC indices, WBC differential counts, and platelet count. 152 Serum Chemistry was analyzed on Chemistry Analyzer (Beckman Coulter AU680® 153 Chemistry Analyzer). The different parameters analyzed were for example, Glucose, Na, K, 154 Cl, CO2, Cholesterol, Triglycerides, Iron, BUN, Creatinine etc.).

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Flow cytometry: 156 A series of commercially available human monoclonal antibodies that cross react with 157 nonhuman primate mononuclear cells were used in flow cytometry analyses as described 158 previously [10,[26][27][28]. Briefly, 100μl of whole blood from each sample was added to each Jose, CA, USA). All samples acquired in this study were compensated using the single-color 169 stained cells. Lymphocytes that were gated on forward scatter versus side scatter dot plot 170 were used to analyze CD3 + , CD4 + , and CD8 + T cell and CD20 + B cell lymphocyte subsets 171 using FlowJo software (Tree Star, Inc., Ashland, OR, USA).

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For analysis of NK cells, a separate tube with 100ul of blood was stained with separate 173 cocktail of consisting of -CD3 PE (clone SP-34 and CD16 APC (clone 3G8), (all from BD 174 Pharmingen, San Jose, USA) antibodies, as described above. The gating scheme for T-cell, 175 B and NK markers in peripheral blood from a representative cynomolgus macaques has been 176 identified previously [10]. The absolute number of lymphocytes and monocytes, as obtained 177 from hematologic analysis, was used to convert the percentages identified through FACS 178 analysis into absolute numbers for each of the lymphocyte and monocyte subset populations. The PBMCs, freshly prepared from whole blood collected in EDTA tubes, were more than 181 90% viable as determined by the trypan blue exclusion method. For each immune assay, 10 5 182 cells /well were used for various immune assay. Briefly, aliquots of PBMCs (10 5 /well) were  atmosphere. During the last 16-18 hr., 1 µCr of 3 H thymidine was added. Cells were 195 harvested onto filter strips for estimating 3 H-incoporation and counted using a liquid 196 scintillation counter. The proliferative response in terms of stimulation index (SI) was 197 calculated as fold-increase in the radioactivity over that of the cells cultured in medium alone.

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The responses to antigens were considered positive when the SI values were ≤ 2.0 [29,30].

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ELISA for IFN-α cytokine were performed according to the manufacturer's instructions.

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The minimum IFN-a detectable concentration was 2.9 pg/mL.

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NK Assay: 224 The natural killer activity (NK) was measured as previously described [33]. Briefly, PBMCs 225 from blood were purified by centrifugation on a Ficoll-Hypaque density gradient as described min) and 25 mL of aliquots were used in assay. The 96-well filter plate was blocked with 244 assay buffer for 10 min at room temperature, washed, and 25 mL of standard or control 245 samples were dispersed into appropriate wells. After adding 25 mL of beads to each well, the 246 plate was incubated on a shaker overnight at 40C. The next day, after washing two times with 247 wash buffer, the plate was incubated with detection antibody for 1 h at room temperature and 248 again incubated with 25 mL of Streptavidin-Phycoerythin for 30 min at room temperature.

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After washing two times with wash buffer, 150 mL of sheath fluid was added into each well   Table 1.   Figure 1 (B). Relocation-dependent differences in lymphocytes in squirrel monkeys.

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Proliferative responses 317 Since, we found significant differences in expression of T, and B cells, we investigated 318 functional hallmark of proliferation of PBMCs samples from the squirrel monkeys. We 319 measured proliferation in 3 H thymidine incorporation assay (Fig 2A). The proliferative

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Freshly isolated PBMCs were either unstimulated (medium) or stimulated with PHA, PWM 337 and LPS (1ug/mL) for 24 hr. and supernatant was collected to measure IFN-α (Fig.3A)  PWM, and LPS assays (Fig 3A). Day 1 arrival levels (Fig 4). The blood samples collected at pre shipment Day 1 and Day150 was subjected hematology 359 and analysis is shown in Fig 5A and   natural killer cell activity [15,40].

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The data from the present study provide some insight into the time that it takes squirrel to 457 acclimatize to their new surroundings. Some standard clinical chemistry and hematologic 458 values appeared to return to Pre levels by about 6 weeks after arrival, while others did not.

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Some of the cell-mediated immune responses that were affected by transport and relocation 460 also did not return to Pre levels. The squirrel monkeys were still affected by the transport 461 process weeks after transport and relocation, and probably should not be considered