Abstract

Zoo veterinarians and pathologists have an opportunity to collect a wide variety of tissue and body fluid samples for archiving. Serum samples are among the most common, but fixed and frozen postmortem tissues are also frequently collected. Unfortunately, many samples are not collected unless specific outside requests are on file, particularly those from SSPs. Zoos all too often do not take full advantage of the opportunity to create biomaterial archives for their own use, or to contribute to centralized archives, such as SSP archives and the San Diego Zoo’s Frozen Zoo. Furthermore, when samples are collected for archiving, sample collection or handling errors can limit the usefulness of the specimen. With ongoing advances in molecular diagnostics and research, such biomaterial archives represent invaluable resources that are well worth the effort to establish.

Creating an archive is not difficult, but requires appropriate storage capability and a little preparation. Common problems with archiving include use of household frost-free freezers that thaw samples repeatedly, lack of back-up power supply, lack of accurate inventory, and incompletely labeled specimens. Samples must at least have a unique identifier that enables you to determine the nature of the sample, animal identification, and date of collection. It is also helpful to note any significant sample handling issues, such as previous thaws, special fixatives, etc., in the inventory or catalogue record.

Sending samples off site for processing or archiving creates additional problems, since samples can get lost or be mishandled during transfer. In addition, most pathology service providers discard their paraffin blocks after 3–5 yr. You might think you have an extensive archive of tissue blocks at your local university or private diagnostic lab, when in fact you might only have 3–5 yr worth of samples. Another issue to consider is that once your samples are in the hands of outside laboratories or investigators, it can be difficult to retrieve them for your own use.

With considerably more effort, zoos can create accredited museum archives. The pathology department at the San Diego Zoo is currently in the process of having its specimen archives accredited. Requirements for museum accreditation include having a collection plan with policies and procedures for specimen acquisition, accessioning, cataloguing, inventory tracking, conservation/preservation, deaccessioning, disposal, loans, and availability for education or research. It also requires that the institution commit the staff and financial resources necessary to maintain the accredited archive. Accreditation is of limited value to most institutions, but has the added benefits of facilitating transfers of materials with special CITES permits, and providing the political leverage sometimes needed to marshal the resources necessary to maintain the archive. It also puts proper emphasis on the value of the archive, the need for upkeep and preservation, and facilitates use by investigators, both internal and external.

The kinds of investigations that can be done with archived biomaterials depend on the nature of the sample and method of preservation. We can broadly categorize the types of investigations into genetics, infectious diseases, nutrition, toxicology, and diseases of unknown etiology. The following are examples of how we use our own biomaterials archives for each of these types of investigations.

For genetic studies, living cells in a “frozen zoo” yield the most, followed by tissue snap frozen in liquid nitrogen and stored at –80°C, tissues frozen and stored at –80°C, tissues frozen and stored at –20°C, tissues fixed and stored in absolute ethanol, and finally formalin-fixed, paraffin-embedded tissues. The progressively lower yields with each of the above preservation methods result from incremental increases in degradation of DNA and RNA. Recovery of viable cells, messenger RNA, or very long segments of DNA generally requires snap freezing in liquid nitrogen and storage at –80°C. Because ethanol fixes tissue by coagulation, while formalin causes cross-linking, ethanol-fixed tissues yield much longer lengths of DNA than formalin-fixed tissues.

Examples of genetic studies we have undertaken with archived samples include albinism in koalas, and chondrodysplasia in California condors. Molecular characterization of the koala tyrosinase gene has involved PCR amplification of genomic DNA from individual exons of the gene and amplification of full-length transcripts of cDNA derived from mRNA. Genomic DNA has been obtained from frozen peripheral blood leukocytes and kidney, while pigmented skin, colored hair and hair follicles, footpad skin, nose skin, and eye were sources of mRNA. Since the defect in California condors might involve one or several genes, a combination of PCR and a cDNA library could be used to screen candidate genes. Frozen genomic DNA samples could be assembled into a phage or cosmid library and screened with condor gene fragments obtained by PCR for candidate genes.

For most infectious diseases, the best samples are those snap frozen in liquid nitrogen and stored at –80°C, since this preserves viability of many infectious agents and preserves RNA of RNA viruses. Otherwise, the preferred preservation methods follow the pattern described above for genetic studies. We regularly utilize archived samples for infectious disease investigations. Two of the larger studies have involved avian adenoviruses and Atoxoplasma spp. For these studies, tissues frozen at –80°C have been used successfully for PCR, while formalin-fixed paraffin-embedded tissues have been used successfully for both PCR and in situ hybridization. PCR from paraffin tissues utilized smaller genetic targets (approximately 300 bp for Atoxoplasma and 400 bp for the adenoviruses) and both have worked with some success—but with lower sensitivity than frozen tissues. In situ hybridization on formalin-fixed tissues utilized oligonucleotide probes (20–30 bp) to circumvent any problem with DNA degradation and worked well on falcon tissues but only on sexual stages of Atoxoplasma spp. in the intestine.

Generally, samples frozen at –20°C are adequate for nutritional or toxicologic investigations. Samples we routinely collect include liver, kidney, and gastrointestinal content. In selected cases, we also collect brain, urine, blood, bone, and hair or feathers. We have successfully used our archive to investigate cases of metabolic bone disease, copper deficiency in ruminants, rodenticide toxicity, and lead toxicity in California condors.

Diseases of unknown etiology present special problems in terms of sample collection, but can best be covered by collecting a routine set of tissues, such as liver, kidney, spleen, lung, gastrointestinal tract, and brain, with additional samples as appropriate to the condition being investigated (e.g., vomitus, feces, whole blood, etc.). We are currently using our archives to investigate chronic gastrointestinal problems of uncertain etiology in douc langurs. By using routinely archived samples, complemented by more directed sampling, our molecular diagnostics lab has identified as yet unspeciated Helicobacter and Campylobacter bacteria. We are now saving biopsy, necropsy, vomit, and fecal samples from douc langurs in 20% glycerol brucella broth to facilitate culture of these fastidious organisms.

Conclusion and Recommendations

Biomaterials archives are one of the most important repositories of information we can create for our own use and are a valuable resource we can provide for the conservation and research communities. In order to create the most useful archives, we recommend the following:

• Freeze samples at –80°C whenever possible.
• Follow specimen handling and sample labeling guidelines.
• Always use an adequate volume of formalin for fixed tissues, and limit fixation time to less than 72 h.
• Consider collecting selected duplicate samples in absolute ethanol for molecular diagnostics when freezing is not practical.
• Find out where your tissue blocks are stored and make sure older blocks are not being discarded.
• Whenever possible, send duplicate samples to central repositories, such as SSP archives and the San Diego Zoo’s frozen zoo.
• For health problems of unknown etiology, consider archiving unconventional samples relevant to the problem. Examples include ocular fluid, postmortem lung smears, whole blood, yolk samples from eggs or chicks, stomach and intestinal content (especially from wild or released animals), urine, hair, feather, bone, food and water samples.
• Maintain an inventory of your archive in a computerized database, with appropriate backup.