The TNPRC maintains a variety of resources to enable research requiring nonhuman primates. These research resources are maintained within existing research divisions and are described below.
DIVISION OF BACTERIOLOGY & PARASITOLOGY
The Division of Bacteriology & Parasitology has a significant service commitment to the Center. These service functions can be divided into four major areas: 1) Diagnostic Parasitology Core, 2) Vector-borne Diseases Core, 3) DNA Microarray and Expression Core, and, 4) Enzootic Pathogens Survey.
This Core provides diagnostic support to investigators and clinical veterinarians whenever monkeys are suspected of harboring parasites. All animals entering quarantine from outside sources are examined for blood and intestinal parasites at monthly intervals before they are allowed to enter the colony. Additionally, mice in the rodent colony are periodically checked for mites and pinworms prospectively. The Core also collaborates with researchers outside the TNPRC when projects involving nonhuman primates require diagnostic services. In addition, the laboratory is examining blood and fecal samples in direct support of a survey of enzootic pathogens from the breeding colony.
Vector-Borne Diseases Core
This Core maintains insects and arthropods that are important to reproduce the natural mode of transmission of vector-borne diseases that are studied at the TNPRC, e.g. Lyme disease and malaria.
The DNA Microarray and Expression Core
This core provides both bioinformatics expertise to investigators who already have obtained DNA microarray results, and technical support to perform the microarray experiments de novo.
The Enzootic Pathogens Survey
This survey centers on the collection and examination of blood and stool samples from each animal in the TNPRC breeding colony. This survey permits the early detection of microbes that may affect the general health status of the monkey colony.
DIVISION OF COMPARATIVE PATHOLOGY
The Division of Comparative Pathology has a significant service commitment to the Center, and these service functions can be divided into four major areas: 1) necropsy and biopsy service, 2) clinical pathology, 3) molecular pathology, and 4) confocal microscopy and image analysis.
Pathology service support is provided to the Center’s clinical veterinary staff as part of our colony surveillance program. The Division also furnishes support in each of these areas to staff scientists within other divisions at the Center as well as to collaborating scientists from around the world.
Necropsy and Biopsy Service Core
The necropsy and biopsy service is the core of the Division’s service functions. The necropsy and biopsy service provides investigators and collaborators at the Center with gross and histopathologic evaluations of organs and tissues for the purpose of understanding pathologic changes either for diseases that spontaneously arise in the colony or in relation to experimental protocols. In addition, the service assists the clinical veterinarians with colony health and management. The necropsy and biopsy service is the Center’s primary means to identify and investigate new conditions in nonhuman primates. These investigations serve not only to provide further understanding of disease processes in general, but also have the potential to identify new models for the study of human disease.
The Clinical Pathology Laboratory Core provides clinical data for animals involved in specific research projects as well as colony animals. The laboratory furnishes hematology, chemistry, fecal analysis, fluid analysis, urinalysis, and cytology on all Center animals. The Clinical Pathology Laboratory Core is currently staffed by two Medical Research Specialists and a Laboratory Supervisor, all three of whom are ASCP registered Medical Technologists. The primary responsibility of this unit is to perform hematology, clinical chemistry and bacteriology for the medical care of the animal colony and to support numerous research projects from scientists here at the TNPRC and around the world. The service also provides important diagnostic support for the veterinarians in managing the health and well being of the animals in the colony.
The Molecular Pathology Core facility provides investigators at Tulane and around the world with an array of tools and techniques to investigate the molecular mechanisms involved in diseases. The Core facility provides multilabel immunohistochemistry, PCR testing, the generation of labeled RNA and DNA probes (for cytokines, EGFP, and SIV) via in vitro transcription or random primed labeling, in situ hybridization on paraffin and frozen tissue sections, and molecular manipulation of genetic and plasmid material to provide the necessary templates. This section of the Pathology Division also provides a significant amount of consulting regarding molecular techniques to scientists, technicians, and postdoctoral fellows in molecular biology techniques.
Confocal Microscopy and Image Analysis
This Core provides state-of-the-art confocal microscopy, multilabel fluorescent labeling and detection, and image analysis support to every Division in the TNPRC and to numerous affiliate research scientists at several institutions around the world. The Core has a Leica TCS SP2 laser scanning confocal microscope system equipped with three lasers, with six laser lines available, capable of simultaneously collecting information in four channels (three fluorescent and one for differential interference contrast). The system is attached to two microscopes, upright (DMRE) and inverted (DMIRE2), that allow for confocal microscopy of fixed preparations and also living cells. The confocal system offers such benefits as: a) multi-dimensional imaging, as it is possible to obtain images in four dimensions, length (x axis), width (y axis), depth (z axis) and time (t); b) resolution improvement; c) contrast improvement; and e) multicolor imaging. Several fluorochromes can be imaged at the same time.
DIVISION OF REGENERATIVE MEDICINE
The Regenerative Medicine Division has a significant service commitment to the Center. The current service function is mesenchymal stem cells.
Mesenchymal stem cells (MSC) are a subset of adult stem cells from bone marrow or adipose tissue. These cells are of medical and therapeutic interest because they have been shown to differentiate into osteoblasts, adipocytes, chondrocytes, myocytes, astrocytes, oligodendrocytes and neurons. Due to their inherent plasticity, these cells have the potential to be useful for the treatment of a large number of genetic diseases. The Mesenchymal Stem Cell Production Core Facility (SCPC) focuses on generation, maintenance and distribution of nonhuman primate MSCs. We routinely prepare MSCs from rhesus macaque bone marrow and adipose tissue samples.
The goals of the Core are:
1. Prepare a continuous supply of quality-tested nonhuman primate MSCs for distribution to other investigators.
2. Develop improved methods for isolating and characterizing nonhuman primate MSCs.
3. Prepare MSCs engineered to express various reporter genes enhanced green fluorescent protein (EGFP), beta –galactosidase (b-gal), luciferase, etc.)
4. Consult with investigators on design of research studies using MSCs.
DIVISION OF IMMUNOLOGY
The Division of Immunology has a significant service commitment to the Center. These service functions can be divided into two major cores: 1) Flow cytometry core, 2) Immunology core.
Flow Cytometry Core (FCC)
The FCC is equipped with two FACS-Calibur analytic flow cytometers capable of 4-color analysis, one FACS Aria high-speed cell sorter capable of 9-color analysis and simultaneous 4-way cell sorting and a LSRII analytic flow cytometer capable of 12-color analysis. The FCC offers assistance ranging from study design through data acquisition and analysis.
Immunology Core (IM)
The IC provides immunology services to specific research projects of in-house and outside investigators, as requested. Current services include sample preparation under standardized procedures for optimal assay analysis, planning and performance of ELISPOT assays, data processing and presentation, and intracellular cytokine staining.
DIVISION OF MICROBIOLOGY
The Division of Microbiology maintains two service cores to support the clinical staff and investigators that use the resources of the TNPRC. These are:
Viral Diagnostic Core (VDC)
A healthy nonhuman primate (NHP) colony that is free of specific pathogens is vital to the success of a primate center. The VDC, established in 2003, is a key resource in this regard. It has two components:
- The viral diagnostic component provides serological testing for the NHP colony. For the “Specific Pathogen Free” colony, each monkey is tested for SIV, STLV, SRV and herpes B virus infection. For SIV specific antibody detection, a peptide ELISA against SIVmac/SIVsm was developed. Confirmation of SIV antibody is provided by a Western blot (WB) assay. Herpes B virus serodiagnosis is done using a commercial Herpes simplex virus type1 ELISA. Commercial ELISA kits and a second generation WB assay that allows discrimination between STLV-1 and STLV-2 are being used for diagnosis of STLV infections. The methodology for SRV diagnosis is currently under development. The VDC processed approximately 3,000 samples in the past year and is growing to meet future needs.
- A real-time PCR component is also part of the Viral Diagnostic Core. Currently SIVmac and SIVagm quantification is available. Other services, such as cytokine and chemokine quantification, are also available. Additionally, the Core will assist investigators with custom-designed real-time PCR assays.
The VDC also provides other serological assays upon the investigator’s request. Examples are TB Primagam and SIV p27 antigenemia quantification.
Retrovirus Challenge Stock Production and SIV/SHIV Isolation Core
This Core provides research investigators with primate lentiviruses for inoculation of nonhuman primates (NHP). The Core fills this need because many investigators who use NHPs are not able to produce and maintain their own virus challenge stocks. Therefore, the Core produces and maintains titered viral stocks for testing the efficacy of candidate AIDS vaccines, for testing new anti-HIV drugs and for viral pathogenesis studies. The inventory consists of 65 separate stocks of SHIVs and SIVs. These viruses were grown in human cell lines and in primary cell cultures of human and NHP origin. The Core has also prepared specially requested stocks for use by investigators.
DIVISION OF VETERINARY MEDICINE
The Division of Veterinary Medicine has a significant service commitment to the Center, and these service functions can be divided into four major areas: 1) Animal colony management and care, 2) Clinical imaging, 3) Surgical support and 4) Assisted reproductive technologies.
Animal Colony Management and Care
Animal colony management and care is provided through the cooperation of several different units within the Division of Veterinary Medicine including Clinical and Research Medicine, Environmental Enrichment, Research Resources, and Animal Resources. The TNPRC animal colonies include the research colony and the breeding colonies. The breeding colonies are further separated into the conventional breeding colony and the specific pathogen free (SPF) colonies. The specific pathogen free colonies are comprised of animals that are free of targeted viruses that have the potential to affect health or could confound infectious disease research.
Nine species of nonhuman primates are represented at the TNPRC for a total population of approximately 5000 nonhuman primates. The represented African species are sooty mangabey (Cercocebus torquatus atys), white crowned mangabey (Cercocebus torquatus lunulatus), African green monkey (Chlorocebus aethiops), patas monkey (Erythrocebus patas), and baboon (Papio spp). Macaque species consist of rhesus monkey (Macaca mulatta), pigtail macaque (Macaca nemestrina), and cynomolgus macaque (Macaca fascicularis). The TNPRC has both Chinese origin and Indian origin M. mulatta. These regional variants have been maintained as separate populations in the breeding colony. The Center also houses a small number of squirrel monkeys (Saimiri sciureus) maintained for malaria research.
The Unit of Animal Resources is a service unit that provides routine husbandry care for the animal colonies at the Center. The Unit also has responsibility to provide support to the Units of Clinical and Research Medicine, Environmental Enrichment and Research Resources, and to core and affiliate scientists. The Unit assists the Center in complying with relevant regulatory requirements including, but not limited to, those of the USDA, Public Health Service (PHS), and the U.S. Fish and Wildlife Service.
Routine husbandry practices include the reporting of any abnormal clinical sign or activity by animals to the appropriate veterinary medical staff and faculty. Animal Care Technicians provide support during diagnostic and therapeutic procedures and the administration of the preventive medicine program. The Unit closely coordinates its activities with research personnel to provide assistance, equipment and support for their work. The Unit of Animal Resources provides after-hours care, which includes administration of treatments, collection of biologic samples for research activities and observation of animals. The TNPRC facilities are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC).
The Unit’s staff consists of a Vivarium Manager, a Breeding Colony Manager, Resource Manager, two Quality Assurance Specialists, Animal Care Supervisors, and Animal Care Technicians.
All nonhuman primates are acquired from established USDA registered nonhuman primate importers. A veterinarian examines all animals entering the TNPRC after arrival in the quarantine facility. During the quarantine period, a minimum of three TB tests are performed in addition to Primagam testing, fecal examination for parasites, rectal swab bacterial culture, serum chemistry, complete blood count, thin and thick blood smears for parasites, and testing for type D simian retrovirus. At each sample collection time point, a veterinarian performs a body weight measurement and physical examination. Prior to release from quarantine, all animals must have a normal thoracic radiograph, negative TB skin test and negative Primagam test.
Surveillance, Diagnosis, Treatment and Control of Animal Diseases
Animals are housed and separated based on species and infections they have encountered. Prior to assignment to research protocols, all nonhuman primates have a physical examination performed by the veterinarian assigned to the research project to determine fitness for the particular study. A specific veterinarian is assigned to each research project at the time of IACUC review. This procedure allows continuity in the provision of care to individual animals.
Breeding colony animals are housed in outdoor social groups. A minimum of twice yearly, all animals in a social group receive tuberculin testing, reproductive evaluations, and examination for pregnancy. Body weights, physiological samples, and demographic data are collected, and genealogical records are updated. Data collection and entry into the computerized animal records database allows the careful analysis of breeding colony production and clinical data.
The Unit of Environmental Enrichment is dedicated to improving nonhuman primate well-being through collaboration with the units of Clinical and Research Medicine and Animal Resources. The Tulane Environmental Enhancement Plan involves a number of strategies that are implemented according to animal needs and research requirements. The Plan is dynamic, permitting modification of techniques in accordance with in-house assessments and the scientific literature. New items are added to the program through an approval system including veterinary staff, animal care supervisory staff, and the Enrichment Coordinator. Conspecific social contact is the most critical element of the enrichment program, in recognition of the social nature of nonhuman primates. The socialization program places nonhuman primates into social groupings when compatible with research protocols, and dedicated staff monitors social introductions, ongoing compatibility, and social group dynamics. Other elements of the enrichment program include nonhuman primate/human positive interaction, feeding enrichment, structural enhancements, manipulable objects, and devices permitting foraging, grooming, problem-solving, and sensory enrichment. Several enrichment techniques are utilized concurrently with each individual nonhuman primate, scaled to the number and intensity of other feasible elements. Daily enrichment is implemented by Animal Care Technicians and Environmental Enrichment personnel.
Animal Resource Allocation
The Tulane Resource Allocation Committee (TRAC) was created to evaluate all proposed research projects that request utilization of the resources of the Center. The Committee, composed of ten members, includes research scientists, veterinarians, program coordinators and the animal colony epidemiologist. Several members represent facilities and programs from outside the Center. Requests are reviewed after both IACUC approval and funding is in place. Once TRAC approval is in place, the Division of Veterinary Medicine assigns the animal or space resource as it becomes available. Since the inception of the TRAC in 2001, animal allocations to affiliate (outside) investigators has been approximately 60% of the total, with the remainder allocated to core (inside) investigators.
The TNPRC Rhesus Monkey Serum Bank is maintained by the Division of Veterinary Medicine and stores serum and plasma samples collected during routine veterinary care procedures for the breeding colony. The serum bank also includes samples collected during routine monitoring of viral status for the SPF colony. The purpose of the serum bank is to provide samples for retrospective analysis of the colony and for investigator use, if required, to minimize the need to access animals from the colony for serum samples. The samples have been catalogued and entered into the Center’s database.
Breeding Colony Management
The Breeding Colonies of the TNPRC provide nonhuman primates to core investigators and affiliate investigators for research. The breeding colonies make up the largest population of nonhuman primates at the TNPRC. All animals in the colony are tracked via a centralized, computerized animal records system. With exception of the animals housed for treatment of illness, the animals assigned to the breeding colony are housed in outdoor enclosures in social groups. Social groups are housed in large fenced corrals and field cages that allow for the establishment of a normal social dynamics similar to that found in feral troops. The breeding colony management program is designed and administered by veterinarians, the breeding colony manager, environmental enrichment coordinator, and the breeding colony epidemiologist through the Breeding Colony Management Committee. In addition, the Tulane Resource Allocation Committee (TRAC) facilitates breeding colony management by determining appropriate allocation of animals for assignment to research protocols based on statistical analysis of colony demographics.
The demand for animals from the SPF and conventional colonies has increased dramatically over the past five years. Requests from investigators reflect the need for more thoroughly characterized nonhuman primates, with regard to viral status and genetic background. Our long-range goal is to expand the breeding colonies so that all animals are SPF.
Radiology support is provided with a Continental 150KV, 300 mA fluoroscopy unit with image intensification. A direct digital radiography system is utilized to capture images to a picture archiving system (PACS) server. Approximately 700 radiographs are taken each year.
Ultrasonographic examinations and procedures are performed using one of three Toshiba or a portable GE Logicbook ultrasound machine. Color doppler capability is present on all of the machines.
MRI is performed on the TNPRC campus on a contract basis utilizing a private imaging company.
Surgery is performed in either of two fully-equipped operating rooms. Surgical facilities are under the direct supervision of a veterinarian, who is assisted by the surgery supervisor and two surgery technicians. Procedures performed are those approved by the IACUC and/or administered for the medical management of non-research animals.
Assisted Reproductive Technologies (ART) Core
The ART Core is administered through the Unit of Reproductive Biology in the Division of Veterinary Medicine. The program is focused on several areas of embryo production and embryo manipulation. The significance of ART in rhesus monkeys for biomedical research is growing with rapid advances in production, preservation and manipulation of embryos. These advances may ultimately facilitate genetic modification of embryos, the identification of embryos with specific genotypes, or the production of identical twins by embryo splitting.
Timed Breeding Program
This program is used to provide timed bred rhesus monkeys for investigator use in approved research protocols.