SAMPLE SELECTION, PRESERVATION AND SUBMISSION (FOR HISTOPATHOLOGY AND PCR/RT-PCR) Dr.

Donald Lightner Aquaculture Pathology Laboratory Department of Veterinary Science and Microbiology University of Arizona 1117 E. Lowell St., Bldg. 90, Rm 106 Tucson, Arizona 85721 USA phone: 1-520-621-4438 or 621-8414; fax: 1-520-621-4899 e-mail: ritar@u.arizona.edu or solangel@u.arizona.edu http://microvet.arizona.edu/research/aquapath/index.htm These guidelines are provided to assist you with selection and preparation of samples for submission to the University of Arizona Aquaculture Pathology Laboratory for disease diagnosis or health status confirmation by methods that include: 1. 2. 3. 4. Routine histology. In situ hybridization with DNA probes. Immunohistochemistry with labeled antibodies. DNA/RNA amplification tests by PCR/RT-PCR.

If you have questions concerning sample selection, sample size, method(s) of preservation, packing and shipping requirements, etc. please contact us for instructions by phone, fax or e-mail at the numbers given in the letterhead. If your government or shipping company requires copies of our U.S. Import Permits to facilitate shipment of samples from your country to the USA, please contact us to obtain copies. Please provide the following information with each sample submission: 1. The correct invoicing address and the address where the report is to be sent. 2. A brief written account or history why the samples are being submitted. This should include a description of any abnormalities that were observed (i.e. mortalities, poor growth, abnormal behavior, obvious lesions) or specify that the samples are being submitted for a routine health check. 3. The species of the samples. In the case of larval samples, the life stage (i.e. nauplii, zoea, mysis, PL) of each sample set should also be noted. 4. The origin of each group of samples (country, name of farm or hatchery, corresponding pond/tank or, if applicable, your companys specimen identification number). 5. Date of collection and fixation or preservation (if not the same). 6. The name of the test(s) that you are requesting to be run on the sample(s). 7. The method of preservation and/or the name of fixative, if used (e.g. Davidsons, 90%95% ethyl alcohol (PCR), glutaraldehyde, 10% buffered formalin, frozen in dry-ice, etc.).

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This general information is requested to facilitate rapid processing of the samples, provide background information to the diagnostician who will analyze the samples, and for use in documenting the epidemiology and geographic distribution of pathogens within specific penaeid species. The main purpose for requesting this information is to give us some idea of the disease problem, which will assist us in our final diagnosis and in identification of the causative agent(s). If the findings of a particular case should be used as part of an epizootiological report concerning a given pathogen, no reference to the submitting company will be made. Failure to provide the above mentioned information may result in delayed processing until the information is obtained. For those companies using brokers or governmental agencies, please inform the appropriate customs or agriculture inspection officials to leave all the case submission information, regarding the samples, within or securely attached to the box being shipped. If the official requires a copy, please include two copies - one for them and one for the UAZ laboratory.

SAMPLES FOR HISTOLOGY Shrimp for Histological microscopy to be fixed in Davidsons fixative should be fixed live by the injection/immersion method. 1. Larvae and early postlarvae - fix by immersion in fixative with fixative volume to shrimp volume exceeding 10 to 1. Fix for 12 to 24 hours; transfer to 50% alcohol for storage or shipment in screw-cap glass or plastic vials. 2. Larger postlarvae, juveniles and adults: Inject fixative into hepatopancreas, stomach, and midgut region in ~2-4th abdominal segment; then on small shrimp open shell longitudinally for the length of the animal or bisect or trisect larger shrimp as well as opening the shell. When opening the shell cut only through the cuticle as to not harm the underlying organs. Special precautions should be made when cutting the cephalothorax area to only cut just through the carapace and not into the underlying organs. The fixative should be made up as follows: Davidsons Fixative (for 1 liter): 95% ethyl alcohol* 37% formaldehyde (technical grade) Glacial Acetic Acid** Tap Water

330 ml 220 ml 115 ml 335 ml

DO NOT USE10% FORMALIN

Notes: * Isopropyl alcohol may be used as a substitute for ethyl alcohol, but the results may be less than optimal. ** DO NOT USE OTHER ACIDS SUCH AS HCl (Hydrochloric acid) AND H2S04 (Sulfuric acid) AS THESE ACIDS INTERFERE WITH TISSUE PROCESSING, STAINING, AND MOLECULAR OR ANTIBODY TESTS.

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Detailed fixation procedure for Davidsons fixative (injection and immersion method): 1. Select (if possible) moribund or otherwise compromised shrimp (dead shrimp are almost always useless as specimens, especially for histology) and kill shrimp by fixation: 2. Immerse live larvae and early postlarvae (<PL5) directly into fixative (without the injection step of fixative into the hepatopancreas). 3. All larger shrimp must be injected with fixative while still alive. This is accomplished by injection of 0.1 to 5 ml of fixative into the hepatopancreas and other body regions. The amount injected depends on the size of the specimen; inject a volume equal to ~10% of the body weight as a guideline (i.e., a 1g shrimp is injected with 0.1 ml of fixative, a 10g shrimp with 1ml, etc.). 4. Then open the cuticle over the cephalothorax and abdomen just lateral to the dorsal midline using dissecting scissors; bisect or trisect large shrimp (i.e. 12 g or larger); 5. Then immerse shrimp in fixative with volume of fixative to tissue ratio of at least 10 to 1. 6. Fix for 12 (larvae and PLs), 24 hr (juveniles), or 48 to 72 hours (sub-adults to adults) (depending on size, longer for larger shrimp to insure adequate decalcification of exoskeleton). 7. Transfer samples to 70% ethyl alcohol. 8. Specimens (juveniles to adults) may be shipped by wrapping in paper towels saturated with 50% alcohol and packed in double plastic zip-lock bags. Pack and ship larvae and postlarvae in small glass or plastic vials, that are in turn well packed in double plastic bags that are placed in a well padded, crush-proof container for shipping/mailing. 9. Label each specimen container carefully using a #2 soft pencil on water resistant white paper. DO NOT use markers or ink pens! Ink and marker will dissolve in the alcohol resulting in a blank or unreadable label. FOR PCR SAMPLES The above listed information, about the submitted samples, should be included with the PCR samples. It is also important to specify which viruses are to be tested since each virus uses a different set of reagents for detection by PCR. As for histology, sample only live or moribund shrimp for analysis (dead shrimp are useless). PCR samples are sent to the UAZ diagnostic lab in several ways. 1. Larvae, postlarvae, small juvenile shrimp (< 2 g) and clipped pleopods should be submersed into 95% ethanol. This is the most convenient method for preserving and shipping samples to the laboratory for PCR analysis. Larger shrimp should be injected with 95% ethanol first and then immersed in it for 24-48 hours prior to shipping. Pack and ship small PCR samples preserved in 95% ethanol in small glass or plastic vials, that are in turn packed in double plastic bags. Label each specimen container carefully in #2 soft pencil on water resistant white paper. Do Not use markers or ink pens! Ink and marker will dissolve in the alcohol.
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2. Larger specimens (juveniles to adults) should be shipped by wrapping in paper towels moistened with 95% alcohol and packed in double plastic bags. Do not send large specimens in jars with alcohol. 3. Hemolymph samples may also be fixed in 95% ethanol. We require at least 200 :l of hemolymph to perform DNA and RNA extractions for PCR. Expel the hemolymph sample into a sterile 1.5 ml microcentrifuge tube and add one volume of 95% ethanol for shipment. Be sure to use a new syringe and needle for each sample to avoid crosscontamination of samples or animals. 4. Other tissues such as gill tissue, pleopods, abdominal tissue or stomach which are biopsied or excised from moribund shrimp should be submersed into 95% ethanol for PCR analysis.

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