Necropsy is literally defined as "examination of the dead". UAC Pathology Services personnel routinely perform necropsy on animals within the contexts of diagnostic investigations, routine rodent health monitoring, and comparative pathology support. A systematic examination of the entire body at the gross visual level is performed, with collection of samples needed for additional analyses as indicated.top
UAC Pathology Services tests for numerous infectious diseases which may be detrimental to the animal research studies performed at the University of Arizona. It is very important to detect diseases early and accurately to prevent disease transmission and to expedite appropriate disease management procedures. We utilize the Multiplexed Fluorometric Immunoassay (MFI) for serologic detection of a comprehensive panel of viral and bacterial agents with a very small amount of sera (20 microliters).
MFI technology is a solid-phase immunoassay based upon flow cytometry principles. It is also known as xMAP technology, originally developed by the Luminex Corporation. Each purified antigen is covalently linked or "labeled" onto one of 100 different types of polystyrene beads, which vary slightly in the intensity of their internal dye. If IgG antibody to a particular antigen is present, it will bind to the antigen on a specific bead and will then be detected by subsequent binding of species-specific antibody conjugated to a fluorochrome, R-phycoerythrin. The reader channels single beads through a dual laser detector which simultaneously determines both the bead type by the internal dye combination and the fluorescent intensity associated with each individual bead. The fluorescent intensity associated with 50 individual beads of each type is used to determine the median fluorescent intensity value.
The antigens used for the immunoassay are produced in our laboratory. Each viral or bacterial organism is grown to high titers in culture and taken through various purification procedures in order to remove any cellular or contaminating proteins. Purification protocols are based upon the physical properties of each antigen. For example, the purification protocol for viruses takes into account the density of the virus, whether it is enveloped or non-enveloped, and if it is secreted or membrane associated. After purification, the antigens are inactivated by chemical and/or mechanical means to ensure they are non-infectious before use. Each preparation produces enough purified antigen to meet our demand for multiple years.
For a few antigens that grow poorly in culture, we produce recombinant protein using the Invitrogen Bac-to-Bac Baculovirus Expression System. The advantage of this approach is that milligram quantities of viral protein can be produced with no contaminating mammalian proteins. In some cases viral capsid proteins also self-assemble into virus-like particles (VLPs), thereby providing conformational epitopes for improved immunogenicity. With the expression system, the DNA sequence of the protein to be produced is cloned into a bacterial plasmid. This plasmid is used to transpose the DNA sequence into a bacmid that can transfect insect cells. The insect cells produce recombinant baculovirus particles which are used to infect insect cells to make large amounts of recombinant protein. Protein is then purified similarly to non-enveloped viral antigens and capsid formation is verified by electron microscopy. VLP's are non-infectious so inactivation steps are unnecessary. Once the purified antigens are produced, they are quantitated by a bicinchoninic acid protein assay and labeled onto beads in different buffers and protein concentrations for signal (noise optimization). After optimization, every antigen preparation goes through a validation process using known positive and negative serum samples. Baseline, borderline, and positive ranges are determined for each antigen on the basis of statistical analysis.
This lab uses the Qiagen LiquiChip system to perform MFI. The antigen-bound beads are processed in a similar way to the measurement of fluorescently labeled cells in a fluorescence-activated cell sorter (FACS). Several thousand beads are read in 30 seconds. A computer algorithm processes the signals from each bead and calculates the signal average and standard deviation for each bead set. Sera that generate positive, borderline, and unexpected results are retested by an extramural reference laboratory to confirm results. Multiple controls are employed in every assay to ensure accuracy and reliability.
The MFI requires a small sample volume and is highly sensitive, specific, and fast. In combination with intramural antigen production, this technology is helping our laboratory efficiently meet the increasing demand for rodent health monitoring as the rodent populations within our facilities continue to expand.
View the Pathology Services Price List for information on available serology testing.top
Whenever possible, specimens should be obtained before antimicrobial agents have been administered. Use aseptic technique when collecting specimens. When transport systems (culturettes) are used, always cap the system and ensure tube tops are firmly secure. Provide an adequate amount of specimen. The specimen must be properly labeled with the identification number or the name of the animal, source, and date. Specimens should be refrigerated until pick up and transported with a cold pack. Results are only as good as the specimen submitted. Please make every effort to submit good quality specimens and properly filled out submission forms. Make sure that you include the source of the culture and antibiotics the animal is on or may be given when filling out the submission form.
Antibiotic Susceptibility Testing:
Susceptibility testing is performed on pathogenic bacteria isolated.
ANTIBIOTIC SENSITIVITY PANEL Amoxicillin-Clavulinic Acid
Oxacillian (Staph only)
Sampling for endoparasites in research animals is done in a variety of ways including: cellophane tape tests, examinations of direct wet mounts of intestinal contents or feces, microscopic examination of flotation preparations of fecal material, direct inspection of the gastrointestinal tract, and histopathological examination of intestinal tissues.
Cellophane tape impressions detect ova from parasites that deposit their eggs around the perianal area of an animal. Tape is pressed to the perianal area and applied to a labeled glass microscope slide. Direct wet mounts are taken directly from feces or intestines and examined grossly or placed on labeled glass slides, cover slipped and examined for ova, trophozoites, and cysts. Flotation preparations allow the detection of parasite ova in a solution that is heavier than the ova, causing the ova to float to the surface. Ova can then be recovered from the surface of the solution and placed on a microscope slide. Microscopic examination of prepared slides is performed systematically under a light microscope to identify parasites.
Collection of intestinal tissues for histological examination is similar to techniques used during routine necropsies. Tissue should be collected as quickly as possible to prevent post mortem autolysis which could hinder detection. Injecting 10 percent buffered formalin into the lumen of the intestinal tissue will hasten fixation of both the tissue and the parasites. Be careful not to flush too vigorously so as to not dislodge attached or embedded parasites.
Detection of ectoparasites is accomplished through direct examination of the pelt. The ear and face regions or scalp in small rodents are collected since fur mites tend to congregate in this are. Pelt samples are placed in a Petri dish and examined using a dissecting microscope. Ectoparasites are removed from the Petri dish using an oil-tipped applicator stick or probe and immersed in an oil droplet on a glass microscope slide. A cover slip is applied prior to viewing under a light microscope. Skin scrapings may also be immersed in oil and examined under a light microscope for parasite identification.top
Proper tissue fixation is essential for accurate histopathologic evaluation. The tissue fixative of choice for routine H&E staining is 10 percent neutral buffered formalin, though alcohol or formalin substitutes are also acceptable. Tissues should be placed in a 10:1 volume of fixative to tissue immediately after dissection. Ideally, solid tissues should be no thicker than 5 mm, with larger tissues incised to allow penetration of fixative. Formalin infusion or flushing is recommended for lung and intestinal samples; especially for rodents.
Samples must be submitted in containers that are adequately sized, leak proof, and properly labeled. Multiple tissues from the same animal may be placed in one container. Submission of tissues from control animals is strongly recommended to ensure accurate interpretation of experimentally induced lesions. Histology slides prepared by other histotechnology laboratories may also be submitted for histopathologic interpretation.
A concise, complete history is vital for the pathologist to provide the most meaningful interpretation of the submitted specimens. Relevant clinical signs, manipulations, procedures, experiments, etc. should be indicated in the "Comments/History/Special Instructions" section of the submission form. Identification of control animals is suggested, although "blind" interpretation will be performed if requested.
UAC Pathology Services uses molecular diagnostics as one way to monitor rodent health. A primary molecular diagnostic assay that Pathology Services uses is quantitative real-time PCR (qPCR), a method of PCR which efficiently amplifies and detects a region of DNA belonging to a specific pathogen in a given sample. Unlike traditional PCR, which requires products to be run on a gel to detect positive samples, qPCR makes use of a fluorescently labeled probe specifically matched to the target pathogen, allowing real time results to be displayed via computer during amplification. The computer algorithm tells us about how many starting copies of the pathogen were in the sample, with a minimum detection range of as few as 1-10 copies.
Our laboratory employs two Stratagene mx3000p machines, each allowing up to 96 samples per plate per machine to be run at once. These machines can detect up to four differently colored probes in a single reaction well, essentially allowing up to four different assays to be run in a single well. For example, each assay for detecting a different species of Helicobacter uses a different colored probe allowing for the simultaneous detection and quantization of each species in just one well, saving time and resources.
Rodent health is routinely monitored through our quarterly serology testing panel, however if a rodent room is found to be positive for a specific pathogen, that room can then be monitored monthly through follow up qPCR testing. Samples that are tested through qPCR can range from tissues to feces to cell culture aliquots. In addition to routine rodent health monitoring via qPCR, our lab offers detection of a whole range of pathogens through PCR-based IMPACT testing.
The Infectious Microbe PCR AmplifiCation Test, or IMPACT, is a panel of qPCR assays that detects murine pathogens in biological samples as an alternative to the Mouse Antibody Production (MAP) test; the traditional method for testing biological specimens for murine pathogens. The major disadvantage of MAP testing is that it requires 6 to 8 weeks to get results. Turnaround time for our molecular diagnostic results including IMPACT results is usually 10 business days. Contamination of biological specimens, such as cell lines, hybridomas and tumor cells, with rodent pathogens can result in outbreaks of disease in laboratory animals implanted with these materials and deleterious effects on tissue culture-based experiments.top