USGS National Wildlife Health Center Diagnostic Laboratories
Microbiology | Virology | Parasitology | Chemistry | Pathology | Diagnostic Tests
The DML routinely performs a variety of procedures to isolate and identify bacterial and fungal agents from wildlife. Microbes are identified based upon morphological characteristics, DNA sequence analysis, and biochemical properties using bioMerieux API, the Biolog, and BBL Crystal systems.
- Routine culture and identification of aerobic and microaerophilic bacteria using a variety of culture media. Isolates are identified using API and Biolog systems; PCR amplification/DNA sequencing of the 16S rRNA gene is also used.
- Culture and identification of anaerobic bacteria using nonselective isolation culture media. Isolates are identified using API and BBL Crystal anaerobic identification systems; PCR amplification/DNA sequencing of the 16S rRNA gene is also used.
- Mycobacteria. Impression smears of suspected mycobacterial containing samples are evaluated by acid-fast staining. Specimens are forwarded to a reference laboratory for histology, culture, and identification.
- Routine culture and identification of fungi and yeasts using a variety of culture media. Isolates are identified by morphology, with the Biolog system, and/or by PCR amplification/DNA sequencing of the rRNA gene ITS region.
- Culture and identification of psychrophilic fungi, such as Geomyces destructans. Isolates are identified based on morphological characteristics, isolate-specific PCR, and by PCR amplification/DNA sequencing of the rRNA gene ITS region.
Mycoplasma, Chlamydia/Chlamidophila, and Rickettsia
- Routine Mycoplasma culture is conducted using SP4 medium, and isolates are identified by morphology and/or PCR amplification/DNA sequencing of the 16S rRNA gene. Potential Mycoplasma-containing materials may also be submitted to a reference laboratory for culture and identification.
- Tissues suspect for Chlamydia/Chlamidophila and Rickettsia are submitted to a reference laboratory.
- Identification of Clostridium botulinum neurotoxins types C and E using the mouse protection assay.
Additional Characterizations of Isolates
- Non-capsular serotyping of Pasteurella multocida.
- Serotype of isolates of Salmonella is determined by a reference laboratory.
- Serotype screening for Escherichia coli O157:H7 with confirmation by a reference laboratory.
- Identification of Streptococcal antigen groups A, B, C, D, F, & G.
- Serotype determination for Vibrio cholerae 01.
- Identification of Clostridium perfringens toxin types by multiplex PCR ? not done routinely.
The DVL performs isolation and identification of common and novel viruses from diagnostic and research samples. Isolation procedures used are specific to the host animal and suspected pathogen. The DVL has expertise in recognizing morphological changes in cell culture and effects on embryonated avian eggs caused by viral infection. Some of the identification techniques used include PCR, RT-PCR, serum neutralization, serology, electron microscopy and DNA sequencing. The DVL participates in the Select Agent Program and is a member of the USDA National Animal Health Laboratory Network (NAHLN).
Virus isolation and propagation
- Culture in cells lines appropriate to host animal.
- Cell culture lines maintained and utilized at NWHC include Muscovy duck embryo fibroblasts (MSDEF), Madin Darby canine kidney (MDCK), African green monkey kidney (VERO) and fat head minnow (FHM) cells.
- Microscopically detect typical cytopathic effect in cell culture for known viruses and atypical cytopathic effect for possible novel viruses.
- Inoculation of embryonating chicken eggs
- Allantoic route of inoculation [example: avian influenza virus (AIV) and avian paramyxovirus (APMV)].
- Chorioallantoic membrane (CAM) route of inoculation [example: avian pox virus].
Identification of virus isolates
- Electron microscopy grid preparation for viewing virus particles.
- Virus-neutralizing tests using constant-serum varying-virus (example: duck plague).
- Virus-neutralizing tests using constant-virus varying-serum (example: APMV).
- Determination of viral nucleic acid and presence of lipid coat.
- ELISA kits (example: Influenza types A and B, canine parvovirus).
- Nucleic acid extraction and PCR (see molecular testing below).
Characterization of novel virus isolates
- Determine optimal cell culture necessary and number of virus particles present per ml (TCID57/ml).
- Describe morphology by electron microscopy.
- Determine pathogenicity by mean death time and/or EID57 in embryonating chicken eggs.
- Characterize viral nucleic acid as RNA or DNA and as enveloped with lipid coat or non-enveloped.
Serologic tests (antibody detection)
- Microtiter neutralization and plaque reduction assays to determine serum antibody titers
- Inclusion body disease in cranes.
- AMPV 1, 2, 3, 4, 6, 7, 8, 9.
- Novel whooping crane herpes virus.
- Novel common eider orthomyxovirus (Wellfleet Bay Virus).
- West Nile virus (WNV).
- St. Louis encephalitis (SLE) & Japanese encephalitis.
- Avian adenovirus.
- Eastern equine encephalitis (EEE) and others.
Nucleic acid extraction, screening and sequencing
- Perform multiple procedures for RNA and DNA extraction on tissue, swab, and environmental samples.
- Molecular screening (PCR) for known viral genes for AIV, APMV-1, WNV, Ranavirus, Wellfleet Bay virus, Duck Plague, Buggy Creek virus, avian pox, flavivirus.
- Sequencing viral genome segments in AIV sub typing, virulent Newcastle disease virus identification and anything that needs sequencing for further analysis such as complete viral genomes and phylogenic comparison.
The DPL supports the NWHC’s diagnostic surveillance as related to eukaryotic parasites, and is based on dissection and identification of parasites. Identifications are reliant primarily upon parasite morphology, host affiliation, tissue location and geographic location of the host. A broad knowledge of the parasitological literature is required to make accurate identifications. Knowledge of the host’s ecology, age, and food habits are also necessary for identification and assessing the potential impact of the infection.
Laboratory diagnostic procedures
- Staining processing of protozoal, helminth or ectoparasites of birds, mammals, amphibians, reptiles, and fish for identification.
- Fecal and blood tests for identification of parasite stages.
- Collection and examination of intermediate hosts for identification of larval stages of parasites.
- Identification of parasites in H&E tissue sections.
- Archiving parasite specimens for reference and study.
The Chemistry Laboratory technical staff provides toxicology support for diagnostic cases, including metal analysis (primarily lead), screening for organophosphate and carbamate pesticide exposure, and other toxicants. The Chemistry Laboratory is also the main conduit for submission of toxicology to external laboratories.
Chemistry Laboratory Capabilities
Toxicology support for diagnostic cases
- Analysis of tissues for metals toxicity (i.e., lead, sodium, copper, zinc, cadmium, thallium) by microwave digestion/ flame atomic absorption spectrophotometry and graphite furnace atomic absorption spectrophotometry.
- Organophosphate/carbamate pesticide exposure testing by use of brain cholinesterase assay.
- Other toxicant/poison exposure (e.g., strychnine and cyanide analysis by UV/Vis spectrophotometry; aflatoxin analysis in feed/UGI by thin layer chromatography).
- Sourcing of appropriate analyses for which in-house capabilities are not present through a network of state veterinary diagnostic laboratories and/or other fee-for-service laboratories.
The Pathology Section includes diagnostic veterinary pathologists and necropsy technical staff whose principal role is to determine the cause of death for animals submitted to the NWHC. Section staff examines carcasses to verify species and condition, collect and process photographic and radiographic images, conduct detailed necropsies, collect appropriate samples for histological preparation, and collect and submit appropriate tissue samples for diagnostic laboratory evaluation. Histology, laboratory assay results, and necropsy findings are interpreted by pathologists to infer cause of death.
- Infectious disease: viral, bacterial, fungal, parasitic.
- Toxicity: natural, anthropogenic (pesticides, contaminants, heavy metals), malicious, incidental.
- Trauma (electrocution, gunshot, vehicular trauma, intraspecific aggression, etc.).
- Degenerative, nutritional and metabolic diseases
- Detecting emerging diseases and novel trends.
- Forensic investigations for potential legal cases
- Assistance with natural resource damage assessments.
Specialties and specific interests
- Wildlife, birds, mammals (e.g., ungulates and sea otters), amphibians, reptiles, fish .
- Emerging diseases and diseases that cause significant wildlife populations declines or ecological disturbance, including harmful algal blooms and associated affects, amphibian declines, white-nose syndrome, corals, parasitic, infectious and novel disease agents.
USGS National Wildlife Health Center Diagnostic Test Menu
This document is a reference guide to the NWHC Diagnostic Laboratories commonly requested tests. It is not a full list of testing capabilities of the laboratories. Determination of which test will be run on any given submission will be based on case history, gross findings at necropsy, and the scientific judgment of the case pathologist.