Overall Abstract The CNPRC has maintained a pedigreed colony of Indian-origin rhesus macaques for over 22 years. The SPF Pedigreed Indian origin rhesus colony includes both an SPF level 1 colony negative for Simian Retrovirus (SRV), Simian Immmunodeficiency Virus (SIV), Simian T Cell Lymphotrophic Virus (STLV) , and Herpes B virus (B virus) and an SPF Level 2 colony free of three additional viruses including rhesus cytomegalovirus (RhCMV), simian foamy virus (SFV), and rhesus rhadinovirus (RRV). This application will expand the percentage of SPF Level 2 rhesus while maintaining the current overall colony size. We will continue pedigree determination for all colony animals and characterize the Major Histocompatibility Complex (MHC) Class I (Mamu-A and -B) and Class II (Mamu-DRB, -DQA, -DQB, -DPA, and -DPB) loci in our breeding SPF rhesus macaques. This application will continue to collect fecal microbiome and peripheral blood lymphoid cells as a biorepository for use by HIV/AIDS investigators. The four principal goals of this application are: Continue to expand the SPF Level 2 population relative to Level 1 while maintaining overall colony size. Develop improved and expanded screening and confirmatory tests for the detection of SPF agents to facilitate the maintenance of the SPF colony. We will initiate annual surveillance for Trypanosoma cruzi in recognition of the expansion of the geographic range of this parasitic infection. This work includes new assays for agents such as tuberculosis, which represents the first SPF agent. Maintain genetic analysis to determine the pedigree of Indian rhesus born into the colony and test all adult breeders to characterize their Major Histocompatibility Complex (MHC) Class I (Mamu-A and -B) and Class II (Mamu-DRB, -DQA, -DQB, -DPA, and -DPB) loci. Continue the collection and storage of intestinal microbiome and peripheral blood lymphocyte samples in a unique SPF Level 2 biorepository to facilitate future research projects investigating the interaction of HIV with the host microbiome and immune system.