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USERS GUIDE Veterinary Diagnostic Laboratory TABLE OF CONTENTS VDL USER INFORMATION .................................................................................................................. 5 LABORATORY BUSINESS HOURS (EXCEPT HOLIDAYS) .......................................................................... 5 CONTACT INFORMATION ........................................................................................................................ 5 LOCATION OF THE LABORATORY............................................................................................................ 5 AFTER BUSINESS HOURS.......................................................................................................................... 6 CONTACT INFORMATION.................................................................................................................. 7 VDPAM ADMINISTRATION .................................................................................................................... 7 VETERINARY DIAGNOSTIC LABORATORY .............................................................................................. 7 VETERINARY MEDICINE EXTENSION ...................................................................................................... 7 FOOD SUPPLY AND VETERINARY MEDICINE.......................................................................................... 7 VETERINARY FIELD SERVICES.................................................................................................................. 7 ADMINISTRATIVE ORGANIZATION .............................................................................................. 8 MISSION OF THE VETERINARY DIAGNOSTIC LABORATORY .................................................................. 9 OBJECTIVES ............................................................................................................................................... 9 PURPOSE OF THIS GUIDE.......................................................................................................................... 9 LABORATORY GUIDELINES AND POLICIES .............................................................................. 10 SPECIMEN SUBMISSION .......................................................................................................................... 10 SUBMISSION FORMS ............................................................................................................................... 10 GUIDELINES FOR PACKAGING SPECIMENS ........................................................................................... 11 SERUM SUBMISSION AND SHIPPING BOXES (BRUCELLOSIS BOXES) .................................................... 11 DELIVERY OF SPECIMENS....................................................................................................................... 12 TESTING SCHEDULE ............................................................................................................................... 12 BIOSECURITY AND DISEASE CONTROL AT THE VDL ........................................................................... 12 REPORTING RESULTS ............................................................................................................................. 12 REPORTABLE AND QUARANTINABLE DISEASES POLICY ..................................................................... 13 CONSULTATION AND INTERPRETATION OF RESULTS .......................................................................... 14 FEES......................................................................................................................................................... 14 INVOICES/PAYMENT OF FEES ............................................................................................................... 14 RETURN OF BIOLOGICAL AGENTS TO SUBMITTER ............................................................................... 15 BACTERIOLOGY ................................................................................................................................... 16 TESTING SCHEDULE ............................................................................................................................... 16 SUBMISSION GUIDELINES ...................................................................................................................... 16 ANTIMICROBIAL SUSCEPTIBILITY TESTING........................................................................................... 18 CLINICAL IMMUNOLOGY TESTS ............................................................................................................ 19 TABLE OF BACTERIOLOGY TESTS .......................................................................................................... 19 MOLECULAR AND VIRAL DIAGNOSTICS ............................................................................................ 23 LIST OF MOLECULAR ASSAYS................................................................................................................ 23 TESTING SCHEDULE ............................................................................................................................... 23 SUBMISSION GUIDELINES ...................................................................................................................... 23 MOLECULAR DIAGNOSTIC METHODS .................................................................................................. 23 MOLECULAR ASSAYS FOR DIFFERENTIATION AND GENETIC CHARACTERIZATION ......................... 25 ORAL FLUID PROGNOSTIC PROFILING OF SWINE POPULATIONS ....................................................... 25 2 PATHOLOGY .......................................................................................................................................... 28 TESTING SCHEDULE ............................................................................................................................... 28 NECROPSY SERVICES .............................................................................................................................. 28 GENERAL GUIDELINES FOR FIELD NECROPSY ..................................................................................... 29 SUBMISSION GUIDELINES ...................................................................................................................... 31 HISTOCHEMISTRY AND IMMUNOHISTOCHEMISTRY ............................................................................ 32 SPECIAL TESTS ........................................................................................................................................ 33 CLINICAL PATHOLOGY .......................................................................................................................... 33 QUICK GUIDES TO SAMPLE SELECTION FOR DIAGNOSIS OF COMMON CONDITIONS ....................... 34 BOVINE ABORTION ................................................................................................................................ 35 BOVINE CENTRAL NERVOUS SYSTEM DISORDERS ............................................................................... 36 BOVINE ENTERITIS – CALVES < 2 MONTHS OF AGE ............................................................................ 37 BOVINE ENTERITIS - CALVES > 2 MONTHS OF AGE, FEEDLOT CATTLE, ADULTS .............................. 38 BOVINE PNEUMONIA ............................................................................................................................. 39 PORCINE ABORTION .............................................................................................................................. 40 PORCINE CENTRAL NERVOUS SYSTEM DISORDERS ............................................................................. 41 PORCINE ENTERITIS – NURSING PIGS ................................................................................................... 42 PORCINE ENTERITIS - WEANED PIGS .................................................................................................... 43 PORCINE MULTISYSTEMIC DISEASE INVESTIGATIONS ......................................................................... 44 PORCINE PNEUMONIA / RHINITIS........................................................................................................ 45 OVINE ABORTION .................................................................................................................................. 46 ABORTION SEROLOGY - GENERAL COMMENTS ................................................................................... 47 SPECIFIC DISEASES ................................................................................................................................. 47 SEROLOGY.............................................................................................................................................. 49 LIST OF SEROLOGY ASSAYS ................................................................................................................... 49 TESTING SCHEDULE ............................................................................................................................... 49 SUBMISSION GUIDELINES ...................................................................................................................... 49 SEROLOGY FOR EXPORT CASES ............................................................................................................. 50 REPORTING SEROLOGY RESULTS .......................................................................................................... 50 INTERPRETATION OF SELECTED SEROLOGIC TESTS ............................................................................. 51 TOXICOLOGY/NUTRITION................................................................................................................... 53 LIST OF TOXICOLOGY ASSAYS .............................................................................................................. 53 SUBMISSION GUIDELINES ...................................................................................................................... 53 MAINTAINING PACKAGE INTEGRITY ................................................................................................... 54 TISSUE SPECIMENS ................................................................................................................................. 54 FEEDS AND ENVIRONMENTAL SAMPLES .............................................................................................. 55 RESULTS AND INTERPRETATION ........................................................................................................... 56 VIROLOGY .............................................................................................................................................. 57 LIST OF VIROLOGY ASSAYS ................................................................................................................... 57 TESTING SCHEDULE ............................................................................................................................... 57 SUBMISSION GUIDELINES ...................................................................................................................... 57 CONVENTIONAL VIROLOGICAL ASSAYS .............................................................................................. 59 EXPORT CASES........................................................................................................................................ 60 SUBMISSIONS FOR RABIES EXAMINATION ............................................................................................ 60 3 RABIES SPECIMENS ................................................................................................................................. 60 RABIES TEST PROCEDURES .................................................................................................................... 61 RABIES RESULTS AND REPORTING ........................................................................................................ 61 ABBREVIATIONS USED IN THE VDL USER’S GUIDE ............................................................................. 62 INDEX OF ENTRIES .............................................................................................................................. 63 Doc 9.2.5 9/2014 “Iowa State University does not discriminate on the basis of race, color, age, religion, national origin, sexual orientation, gender identity, genetic information, sex, marital status, disability, or status as a U.S. veteran. Inquiries can be "directed to the Director of Equal Opportunity and Compliance, 3280 Beardshear Hall, (515) 294-7612." 4 VDL USER INFORMATION LABORATORY BUSINESS HOURS (EXCEPT HOLIDAYS) Monday – Friday CONTACT INFORMATION 8:00 a.m. – 5:00 p.m. After hours emergencies see below Mailing Address Veterinary Diagnostic Laboratory ISU College of Veterinary Medicine 1600 South 16th St Ames, IA 50011 Telephone number 515-294-1950 After hours telephone 515-290-1969 (VDL after hours technicians) or 515-294-1500 (Veterinary Teaching Hospital) FAX numbers 515-294-3564 (Main Office) 515-294-6961 (FAX submission forms to this number) E-mail [email protected] Web site www.vdpam.iastate.edu Additional consulting on animal health issues is available through Veterinary Extension (515-294-8790) and Food Supply Veterinary Medicine (515-294-3837). LOCATION OF THE LABORATORY The College of Veterinary Medicine is located southeast of Jack Trice Stadium and north of U.S. Highway 30 at 1600 South 16th Street. From I-35, take the Ames-Nevada exit and drive west on Highway 30 to exit 146. Go north on University Blvd. to South 16th Street, then turn east on South 16th Street. As shown in the map below, the College of Veterinary Medicine is located immediately to the south. Enter via entrance B and follow VDL signs to south side of complex. VDL clients will find parking on the southeast side of the building and the loading dock for unloading large specimens immediately northeast of the walk-in entrance. See the VDL website www.vdpam.iastate.edu for further instructions and maps. 5 AFTER BUSINESS HOURS After-hours submissions to the ISU Vet Diagnostic Lab can now be placed directly in a refrigerator located inside the VDL submission door foyer. After entering the foyer, follow the procedure below for unlocking the refrigerator and leaving samples for submission: • Paperwork MUST accompany the submission. If you do not have paperwork completed, please do so with the forms provided and place the submission form with the sample(s). • Place the sample into the refrigerator in the foyer. • Log your submission on the log sheet provided. Please fill out as much information as possible, so we can contact the submitting veterinarian if there are questions. See the VDL website www.vdpam.iastate.edu for further instructions and maps. 6 CONTACT INFORMATION VDPAM ADMINISTRATION 2412 Lloyd Vet Med Center Iowa State University Ames, Iowa 50011 Phone 515-294-8791 VETERINARY DIAGNOSTIC LABORATORY ISU College of Veterinary Medicine 1600 South 16th Street Ames, Iowa 50011 Phone 515-294-1950 VETERINARY MEDICINE EXTENSION 2412 Lloyd Vet Med Center Iowa State University Ames, Iowa 50011 Phone 515-294-3837 FOOD SUPPLY AND VETERINARY MEDICINE 2412 Lloyd Vet Med Center Iowa State University Ames, Iowa 50011 Phone 515-294-3837 VETERINARY FIELD SERVICES 2412 Lloyd Vet Med Center Iowa State University Ames, Iowa 50011 Phone 515-294-7595 Fax 515-294-1072 E-mail: [email protected] Fax 515-294-3564 Fax 515-294-1072 Fax 515-294-1072 Fax 515-294-1072 Consult our website at www.vdpam.iastate.edu for a complete Faculty and Staff listing including phone numbers and email addresses. 7 ADMINISTRATIVE ORGANIZATION The VDL is administered within the Department of Veterinary Diagnostic and Food Supply Veterinary Medicine. The VDL itself is structured into several sections of specialization for timely and efficient delivery of service and to provide higher levels of expertise. Additional information is available on our website - www.vdpam.iastate.edu DEPARTMENT OF VETERINARY DIAGNOSTIC AND PRODUCTION ANIMAL MEDICINE PG Halbur (Chair of VDPAM and Executive Director of VDL) VETERINARY DIAGNOSTIC LABORATORY (VDL) VETERINARY MEDICINE EXTENSION (VME) RG Main (Director) P Gorden Food Supply and Veterinary MEDICINE (FSVM) P Gorden ADMINISTRATIVE AND INFORMATION SECTIONS CLERICAL INFORMATICS QUALITY ASSURANCE J Holdredge R Berghefer K Boesenberg GENERAL DIAGNOSTIC SECTIONS BACT & CLINICAL MICRO TS Frana TOXICOLOGY/ NUTRITION S Ensley PHaST (Pharmacology Anayltical Support Team) H Coetzee 8 PATHOLOGY SEROLOGY D Madson B Baum MOLECULAR & VIRAL DIAGNOSTICS P Gauger The Iowa State University Veterinary Diagnostic Laboratory (VDL) is accredited as a full service laboratory by the American Association of Veterinary Laboratory Diagnosticians (AAVLD). Services offered include bacteriology, molecular diagnostics, pathology, serology, toxicology, and virology. MISSION OF THE VETERINARY DIAGNOSTIC LABORATORY It is the mission of the Iowa State University Veterinary Diagnostic Laboratory to provide comprehensive and cutting edge diagnostic services to veterinarians, producers, and animal owners in Iowa and nationally. The lab is responsible for delivering accessible, timely, accurate, valid, and consistent test results to aid in the protection of animal and human health. Other services include a wide range of surveillance testing for early detection and identification of foreign animal and emerging domestic disease agents, as well as acts of bioterrorism directed at human and livestock populations and agricultural food supplies. The VDL also provides educational opportunities to professional and graduate students, as well as local, national and international scientists, diagnosticians and practitioners. Research is an important component of the tripartite mission as faculty and staffdevelop state-of-the-art diagnostic tools and techniques and also direct studies which provide new insights and deeper understanding of pathogenesis, transmission, and immunomodulation of infectious diseases. OBJECTIVES 1. To provide accessible, accountable, timely, and accurate diagnostic service to veterinarians and animal owners. 2. To conduct diagnostic examinations, record results, report information, and assist in the interpretation of results to submitting veterinarians. 3. To monitor and report the incidence and threat of animal diseases, as well as diseases that are transmissible from animals to humans. 4. To provide support for teaching and research programs at Iowa State University. These objectives are accomplished by the development and application of diagnostic assays, interpretation of diagnostic procedures, consultation with animal health professionals, diagnostically oriented research, and the training and continuing education of persons responsible for delivering animal health care services. PURPOSE OF THIS GUIDE The purpose of the User’s Guide is to aid veterinary practitioners and their assistants in selection, preservation, and delivery of specimens to the VDL for more timely and accurate evaluations and tests. Please refer to this Guide, telephone the laboratory, or consult our website if you have questions concerning sampling and submission. 9 LABORATORY GUIDELINES AND POLICIES SPECIMEN SUBMISSION The VDL only accepts specimens referred by a licensed veterinarian. The appropriate submission form must accompany all specimens. See individual sections (Pathology, Bacteriology, Virology, Serology, and Toxicology) for complete instructions on packaging and shipping specimens. SUBMISSION FORMS Submission forms are legal documents designed to be concise, yet complete. The information requested is needed to determine which laboratory tests are appropriate, to minimize your laboratory testing charges, and to produce your results as quickly as possible. Please be as specific as possible in your testing requests. • Diagnostic Examination Submission Form – use for all submissions except serologic assays and requests for rabies examinations. • Serum Submission Form – use for routine serum assay requests. NOTE: Serum samples submitted to comply with EIA, Brucellosis, and PRV Disease Programs require special submission forms. Call 515-284-4140 (APHIS) to request EIA and Brucellosis Disease Program forms. Call 515-281-8601 (Iowa State Veterinarian’s Office) to request PRV Forms 1 and 2. • Rabies Examination Form – NOTE: If the specimen tests negative for rabies and additional tests are desired, it is necessary to attach a completed Diagnostic Examination Submission form, as well. Electronic versions of the submission forms are available as fillable PDF files on the VDL website - www.vdpam.iastate.edu. Electronic versions may be saved, downloaded and/or printed for your convenience. Submission forms may also be ordered from the lab. Requests for specialized testing will be managed on an individual basis in consultation between the referring veterinarian and the receiving diagnostician, and with the approval of the laboratory director. Please call the VDL at 515-294-1950 for information if you have questions concerning a particular submission. 10 GUIDELINES FOR PACKAGING SPECIMENS The goals of packaging are to protect the specimens from temperature extremes (freezing and heating) and to protect persons who may come into contact with the package from exposure to infectious agents. For this reason, it is extremely important to prevent leakage of specimens. Neither commercial carriers nor the U.S. Postal Service will deliver containers that leak. Additional fees will be assessed if the VDL is required to pick up leaking packages that carriers will not deliver. Leak-proof specimen containers, abundant icepacks with chilled fresh specimens, and an insulated leak-proof transport container lined with a plastic bag are required. 1. Label all samples with the owner's name in waterproof marker. Please do not use stickon labels, as they often come off. Be sure the contents of the box, as well as the outside of the box, are identified. 2. If multiple cases are submitted in one box, package each case separately to ensure that all samples are assigned to the proper case. This is especially important if the package includes multiple serology submissions. 3. Place tubes in serum shipping boxes (i.e., brucellosis boxes) or similar boxes with dividers to separate tubes from one another. Do NOT package loose blood tubes in crumpled paper or styrofoam bits. 4. Enclose sufficient ice packs to preserve the quality of fresh tissues. This should be at least a 2:1 ratio of ice to tissues. If available, insulated styrofoam-lined containers should be used. 5. Plastic leak-proof jars can be used for formalin-fixed tissues. Whirl-Pak® bags are excellent for holding fresh tissues. Squeeze the air out of the bag, then fold the end over several times before bending over the tabs. Double-bagging tissues improves the biosecurity of the specimen. 6. If inside packages have the potential to leak, line the box with sealed plastic bags to prevent leakage. Pack with absorbent materials to soak up spills should they occur. All specimens should be packed in a manner to avoid leakage or breakage, and to withstand the trauma of mailing. Shipping agencies may choose not to deliver leaking packages because of new Department of Transport regulations on shipment of potentially biohazardous materials. In addition, a special permit is required for interstate transportation of selected veterinary agents within the United States. Practitioners are referred to published federal guidelines and regulations for details pertaining to packaging, labeling, and interstate shipping of infectious agents (Title 42 CFR Part 72; Title 49 CRF Part 173.386-388). Regulations and guidelines for packaging and shipping diagnostic specimens and infectious agents have been summarized by Iowa State University Environmental Health and Safety. SERUM SUBMISSION AND SHIPPING BOXES (BRUCELLOSIS BOXES) Serum samples should be submitted according to diagnostic specimen guidelines (see above). The primary container should be snap-cap tubes. For Iowa pseudorabies continuing surveillance, clients may obtain brucellosis boxes by contacting the VDL (515-294-1950). At the time of this writing, clients who are not residents of the State of Iowa are charged $15.00 for 16 11 brucellosis boxes and 16 shipping containers. Boxes are recycled from submissions to the VDL and are not to be considered sterile containers. DELIVERY OF SPECIMENS Choose a method of transportation that will ensure timely delivery to the laboratory. We receive U.S. Mail and UPS deliveries daily. Other commercial carriers deliver as needed. Upon request, shipping cartons will be returned at the submitter's expense. To be sure that samples receive prompt and proper care, schedule their arrival during standard business hours. Always consider the potential impact of holidays and weekends on shipping schedules before sending packages by commercial carriers. Personal delivery by the owner is often appropriate. A map of the location of the VDL can be copied for the owner's convenience on our website at www.vdpam.iastate.edu TESTING SCHEDULE The VDL website (www.vdpam.iastate.edu) contains a complete list of molecular tests currently offered. Our goal is to provide accurate results and timely service. However, some diagnostic assays are not performed daily and/or testing schedules may change. If your case involves testing animals for export, testing show animals, testing animals to be sold, and/or testing a large number of samples, please contact the VDL (515-294-1950) prior to submission to verify the availability and turnaround time of specific assays. A phone call will often shorten turnaround time by providing us time to prepare for testing prior to receipt of samples. BIOSECURITY AND DISEASE CONTROL AT THE VDL Admission of unauthorized persons to the post mortem room is prohibited. Diseased animals with a multitude of infectious agents are handled on a daily basis by the laboratory and we wish to avoid transmission of disease to your clients' animals. Diagnosticians will discuss case histories outside the post mortem area. REPORTING RESULTS All submissions received by the VDL are assigned an accession number (case number). The referring veterinarian will receive an email or FAX listing the species received, the owner, the VDL accession number, and diagnostician(s) in charge of the case. 12 Results are reported only to the referring veterinarian. When calling to inquire about a case, please be prepared to provide the VDL accession number. If requested, preliminary and/or final results may be emailed, FAXed, or telephoned to the referring veterinarian. The charge for FAX reporting of results is $1 per fax. Final reports are sent to the referring veterinarian via the clients preferred reporting method (web, email, FAX, or mail). The VDL laboratory information management system (ISULIMS) provides Internet access to laboratory results. A user name and password may be obtained that will allow access to reports and to a running total of charges for each case as soon as each test is completed and approved. This allows continuous access to diagnostic results, which can be printed for your files or saved as an Excel spreadsheet. To sign up for this service, please call the lab at 515-294-1950 or complete and fax the form found on our website at www.vdpam.iastate.edu. Click on Web Report, then click on Sign Up. You may also request that results be reported to your email address. REPORTABLE AND QUARANTINABLE DISEASES POLICY Several infectious or contagious diseases are considered reportable and/or quarantinable under current regulations. As required by law, these diseases are reported to the responsible state or federal agencies. If you are uncertain about the reporting status of a disease, please contact the office of the State Veterinarian. Notifiable diseases include any livestock or poultry disease designated as a ‘Foreign Animal Disease’ by the United States Department of Agriculture, Animal and Plant Health Inspection Services, Veterinary Services (USDA, APHIS, VS) and also the following: REPORTABLE DISEASES Bovine spongiform encephalopathy Brucellosis Chronic Wasting Disease Encephalomyelitis (horses) Equine infectious anemia Johne's disease Pseudorabies (any species) Rabies Scabies (cattle or sheep) Scrapie Swine dysentery Tuberculosis Trichomoniasis Vesicular stomatitis West Nile Virus REPORTABLE DISEASES OF POULTRY: Avian influenza Infectious encephalomyelitis Infectious laryngotracheitis Newcastle disease Paramyxovirus infection Psittacosis – ornithosis 13 CONSULTATION AND INTERPRETATION OF RESULTS The VDL faculty and staff are available for assistance with sample and test selection, as well as to offer tips on preservation and transportation of specimens. The VDL can assist in interpretation of results, but the final diagnosis and plan of action are the responsibility of the referring veterinarian. Additional animal health expertise and information is available through Veterinary Medicine Extension (515-294-8790) and faculty in Food Supply Veterinary Medicine (515-294-3837). Refer to Contact Information for additional information. FEES The VDL is authorized to charge fees to users of laboratory services. User fees support the wide range of diagnostic services offered and supplement state funding. All fees are billed to the submitting veterinarian. Please consult the VDL website for a list of current tests and charges. The submission form should be used to indicate specific tests or procedures requested by the referring veterinarian. If multiple tests are requested and/or the diagnostician is instructed to use their own judgement in requesting tests, then an indication of monetary limitations should be provided by the referring veterinarian. Requests for specialized or non-routine testing will be assessed on an individual basis in consultation between the referring veterinarian and the receiving diagnostician, and with the approval of the laboratory director. INVOICES/PAYMENT OF FEES Along with the final report, you will receive an invoice showing the case charges. At the end of the month, a statement is sent from the University Accounts Receivable Office showing each invoice number and the charge. You are encouraged to keep the invoice received from our laboratory for cross-reference on the billing from Accounts Receivable. Payment is due on the 15th of the month. A 1.0% finance charge is added after 30 days. VISA, MasterCard and Discover credit cards are now accepted as another form of payment. These transactions can be made in person or by telephone during normal business hours. RETURNING OF BODIES AND COSMETIC POSTING No animal carcasses, body parts or ashes will be returned/released to veterinarians or animal owners. Arrangement for pickup by authorized crematory service is an option. No cosmetic necropsy will be performed on animals/bodies submitted to the laboratory for necropsy/testing. Please call the Laboratory Director with questions and additional details relating to these policies. 14 RETURN OF BIOLOGICAL AGENTS TO SUBMITTER Rapid changes in livestock production and disease management coupled with the fast pace of technology in medicine and diagnostics have resulted in increased requests for the return of biological agents. The VDL will return isolates either to the submitting veterinarian or to a third party laboratory, e.g., for use in the manufacture of immunizing agents. Because of issues of safety, efficacy, and ownership, the VDL is required by the University to obtain a signed release form prior to providing agents to the submitter. Release forms can be faxed to you and the VDL will accept a faxed, signed copy in return. There is a fee for providing isolates (see fee schedule). Following a request for a virus isolate, 7-10 days is required to propagate sufficient virus for transfer. If you anticipate multiple requests, we can issue a Memorandum of Understanding (MOU) that will cover all agents requested within a specified time period. Then with each request you will receive a statement confirming transfer for the specific agent to which the transfer agreement will apply. You can access this form on our web site or request it from the VDL. 15 BACTERIOLOGY TESTING SCHEDULE Our goal is to provide accurate results and timely service. However, many diagnostic assays are not performed daily and/or testing schedules may change. If your case involves testing animals for export, testing show animals, testing animals to be sold, and/or testing a large number of samples, please contact the VDL (515-294-1950) prior to submission to verify the availability and turnaround time of specific assays. A phone call will often shorten turnaround time by allowing us time to prepare for testing prior to receipt of samples. SUBMISSION GUIDELINES General guidelines for submission are found in the Pathology section. If you are requesting bacteriology tests, remember to keep samples moist, cool, and send by overnight transport. The following are important points to keep in mind: 1. 2. 3. 4. 5. Tissues should be VERY fresh and collected aseptically. Collect samples prior to antibiotic treatment. Submit generous portions of tissue or several milliliters of pus, exudate, or feces. Avoid swab submissions whenever possible. Submit samples individually in separate bags or jars with correct and clear identification. 6. Maintain samples at refrigeration temperature (40ºF/4ºC) and send with ice packs. Freezing is generally not recommended. Anaerobic Cultures Care in collection is essential. Do not contaminate the samples with surfaces which have resident anaerobic bacteria. Exposure to air for more than 20 minutes can be detrimental. Samples from animals that have been dead longer than 4 hours are usually unsuitable. Tissues and liquid exudates are recommended (ship in anaerobic pouches or tubes). Swabs are not acceptable unless shipped in proper containers. Special collection devices and transport tubes with reduced oxygen environment are available. Blood Not normally used for recovery of animal pathogens because bacteremia is intermittent. Call the laboratory for recommendations. Milk Collect milk in sterile snap cap or screw cap tubes. Do not ask owner to collect milk samples without first providing training in proper sampling technique. 16 Cool samples before submitting to the laboratory, and mail with ice packs. Samples may also be frozen without altering recoverability of pathogens. Urine Collect by cystocentesis (best), catheter, or mid-stream catch. Submit a 3 ml sample. Skin Lesions To collect from pustules or vesicles, disinfect surface with alcohol, allow to dry, and aspirate material with syringe and needle. Pluck hair from lesion and scrape edge of lesion when ringworm in suspected. Submit hair, skin scrapings, scab material, and toe nails. 17 ANTIMICROBIAL SUSCEPTIBILITY TESTING Antimicrobial susceptibility testing is provided for bacterial pathogens recovered in the VDL or submitted by veterinarians. The system utilized at this time is a semi-automated broth dilution technique known as a modified MIC (minimum inhibitory concentration). The panels contain different antimicrobials, depending upon the animal from which the organism was recovered. Panels are as follows: food animal (porcine, bovine, ovine), companion animal/equine, poultry, and mastitis. Antibiograms for organisms requiring special techniques are also available. The antimicrobials selected for all panels are consistent with CVM regulatory policies. Additional antimicrobial disks are available upon request for Kirby-Bauer (disk diffusion) tests. The disclaimer on food animal/equine susceptibility reports states: ‘This report contains only in vitro antimicrobial susceptibility results and does not represent a therapeutic recommendation. Some antimicrobial use is limited or prohibited in food animals by FDA-CVM regulations. Inappropriate extra-label drug use in food producing animals may lead to violative residues and/or enforcement actions.’ FOOD ANIMAL COMPANION ANIMAL / EQUINE MASTITIS SIR SIR SIR SIR SIR SIR SIR SIR SIR SIR SIR SIR SIR SIR SIR SIR SIR SIR SIR SIR SIR SIR SIR SIR SIR SIR SIR SIR SIR SIR SIR SIR SIR SIR ampicillin apramycin ceftiofur chlortetracycline clindamycin enrofloxacin erythromycin florfenicol gentamicin neomycin oxytetracycline penicillin amikacin amoxicillin/clavulanic acid ampicillin cefazolin cefoxitin ceftiofur cephalothin chloramphenicol clindamycin enrofloxacin erythromycin gentamicin S I R spectinomycin S I R sulfachlorpyridazine S I R sulfadimethoxine S I R sulfathiazole S I R tiamulin S I R trimethoprim/ sulfamethoxazole S I R tylosin tartrate S I R *tilmicosin. (in swine approved for feed grade only) S I R imipenem S I R orbifloxacin S I R oxacillin S I R penicillin S I R rifampin S I R spectinomycin S I R sulfadimethoxine S I R tetracycline S I R ticarcillin S I R ticarcillin/clavulanic acid S I R trimethoprim/ sulfamethoxazole BRACHYSPIRA SPECIES ANAEROBES - E STRIPS SIR SIR SIR SIR SIR SIR SIR SIR carbadox gentamicin lincomycin tiamulin ceftiofur clindamycin penicillin metronidazole S = Susceptible I = Intermediate R = Resistant 18 ampicillin ceftiofur cephalothin erythromycin oxacillin penicillin penicillin/novobiocin pirlimycin sulfadimethoxine tetracycline POULTRY SIR SIR SIR SIR SIR SIR SIR SIR SIR SIR SIR SIR SIR SIR amoxicillin ceftiofur clindamycin enrofloxacin erythromycin gentamicin neomycin novobiocin oxytetracycline penicillin sarafloxacin spectinomycin streptomycin sulfadimethoxine S I R sulfathiazole S I R tetracycline S I R trimethoprim/ sulfamethoxazole S I R tylosin CLINICAL IMMUNOLOGY TESTS SPECIMEN(S) ASSAY Antinuclear antibody titer (ANA) IgG level, single radial immunodiffusion Rheumatoid Factor test serum serum serum TABLE OF BACTERIOLOGY TESTS CONDITION Abortion, bacterial Abortion, mycotic Actinomycosis, bovine ORGANISM(S) Brucella species, Campylobacter species, Leptospira species, Listeria monocytogenes Aspergillus species, etc. Actinomyces bovis TISSUE AND/OR SAMPLES TO SUBMIT Fresh whole fetus. Placenta with cotyledons, lung and liver. Fetal stomach contents for darkfield examination. Uterine discharge. Placenta with cotyledons. Fetal stomach contents. Material from abscess on swab (kept moist) or in syringe. Large piece of tissue or exudate collected in sterile syringe with plugged needle. Anaerobic transport system helps maintain viability. Blood sample taken from superficial vein, such as jugular. Spleen, if necropsy has been performed. Swine – lymph nodes, spleen, kidney, and peritoneal fluid. State on submission form that case involves an anthrax suspect. Take care in sampling! Clean external nares with alcohol, then swab deep into nasal cavity. Swabs should be kept moist during transport to lab. Use mini swabs on small pigs. Send entire snout if animal has died. Entire joint from smaller animals. Joint swab in transport media or joint fluid in sterile syringe. Anthrax Bacteroides species, Fusobacterium species, Eubacterium species Bacillus anthracis Atrophic rhinitis of swine Bordetella bronchiseptica, Pasteurella multocida Arthritis Black leg, gangrene disease Streptococcus suis, Arcanobacterium pyogenes, Haemophilus species, Erysipelothrix rhusiopathiae, Mycoplasma species, Chlamydia species Clostridium chauvoei, novyi, septicum, sordelli Botulism Clostridium botulinum Bovine respiratory disease Histophilus somnus, Pasteurella multocida, Mannheimia hemolytica, Mycoplasma species, other bacteria Anaerobic infections Fresh piece of muscle with lesion. Impression smear for fluorescent antibody test. 19 Food suspected of containing toxin. Section of fresh intestine, tied off. Large section of liver. Serum. Samples may be forwarded to NVSL. Nasal swab in transport medium. Transtracheal wash. Portion of affected lung. CONDITION ORGANISM(S) TISSUE AND/OR SAMPLES TO SUBMIT Brucellosis Brucella ovis, etc. Campylobacteriosis, bovine and ovine Campylobacter fetus, Campylobacter jejuni Campylobacteriosis, canine and equine Chlamydial infections Campylobacter jejuni Colibacillosis Corynebacterial pneumonia of foals Cystitis Dermatophilosis Escherichia coli Rhodococcus (Corynebacterium) equi Escherichia coli, Proteus species, Enterococcus species, Staphylococcus aureus Microsporum and Trichophyton species Dermatophilus congolensis Edema disease Enterotoxemia Escherichia coli Clostridium perfringens Erysipelas Erysipelothrix rhusiopathiae Exudative epidermitis, Greasy pig disease Staphylococcus hyicus, Streptococcus species Glasser's disease Haemophilus parasuis Johne's disease Mycobacterium paratuberculosis Keratoconjunctivitis, bovine Leptospirosis Moraxella bovis, Branhamella ovis Leptospira species Dermatomycosis Aborted fetus stomach contents, placenta. Mammary lymph nodes and milk sample. Fetus, cervical mucus, preputial wash, or semen. Deliver fresh within 5-6 hours of collection or freeze specimens and transport on dry ice. Transport medium can be ordered from lab. Fresh rectal/fecal swabs, feces. Chlamydia psittaci 20 Abortions - affected cotyledon, vaginal swabs, fetal lung and liver. Arthritis - aspirated synovial fluid. Birds - lung, cloaca, and feces. Feline tracheal wash, nasal and conjuctival swabs. Live, sick animal. Intestines and feces. Transtracheal aspirate/wash. Nasal discharge swab. Fresh lung and lymph nodes with lesions. Fresh urine in sterile tube. Plucked hair and skin scrapings sent dry in envelope. Fresh and formalized sections of skin. Scabs, crust and plucked hair. Skin biopsy after scab removal, fresh and fixed. Live, sick pig. Several ounces of fresh intestinal contents in tube or bag. Cool and transport as soon as possible to the VDL. If interested in preserving toxin, freezing of ileal contents is preferred. Acute form - heart blood, kidney, spleen, liver. Arthritic and cardiac form – joints and heart valves (swabs in transport medium). Clean skin with soap, water and alcohol, scrape lesions and send to lab on swab in transport medium or in sterile tube. Live, sick pig. Brain, heart, lung, and fluids from joints, etc. Mailed in swabs are NOT acceptable. Ileocecal valve, mesenteric lymph nodes, mucosal scrapings. Rectally-collected fecal samples in separate screw cap tubes or Whirl-Pak® bags. Contact the lab one week prior to submission if the case involves >20 samples. Allow 16 weeks for culture. Conjuctival swabs from ‘wet faced’ calves (sample 3-6 calves). Send in transport medium. Fluids and urine for darkfield examination. Kidney tissue for fluorescent antibody test. CONDITION ORGANISM(S) Listeriosis Listeria monocytogenes Lymphadenitis Corynebacterium pseudotuberculosis, Streptococcus species Staphylococcus species, Streptococcus species, Mycoplasma species Mastitis Mycoplasma infections Streptococcus species, Streptococcus suis, Histophilus somnus Cryptococcus neoformans Mycoplasma species Nocardiosis Nocardia asteroides Otitis externa Proteus species, Pseudomonas species, Staphylococcus species, Streptococcus species, Yeasts (Malessezia pachydermatis) Actinobacillus pleuropneumoniae Lawsonia intracellularis Meningitis Pleuropneumonia of swine Porcine proliferative enteritis Pyelonephritis, bovine TISSUE AND/OR SAMPLES TO SUBMIT Neural form - brain stem, fresh and fixed. Visceral form - liver, fresh and fixed. Abortion form – fetus and placenta or fetal stomach contents. Feed sample. Abscessed tissue and lymph nodes. Pus in syringe or sterile tube. Avoid bacterial contamination of samples. Clean and dry udders, strip several streams of milk before starting collection. Collect 1-2 streams from each quarter into sterile snap cap or screwcap tubes held at angle. A veterinarian should collect the samples. Refrigerate immediately and maintain at 4º C until cultured. If culture will not be performed within 24 hours, samples may be frozen up to 2 weeks. Cerebrospinal fluid and brain. May include mucosal scrapings, tracheal exudates, and aspirates; lung tissue with bronchi, joint fluids and milk. Submit in tubes placed within Whirl-Pak® bags. Submit swabs in Amies with charcoal. Refrigerate and deliver within 24 hours of collection. Alternatively, samples can be frozen and shipped frozen. Exudate from lesion. Excision may be necessary. Thoracocentesis or tracheal wash may be used. Culturette with transport medium. Portion of affected lung with lesion. Ileum and feces. Corynebacterium renale Salmonellosis Salmonella species Strangles Streptococcus equi Swine dysentery and spirochetal colitis Brachyspira hyodysenteriae, Brachyspira species Midstream sample of urine. Portion of affected kidney, ureter, bladder and urethra. Intestine, liver, spleen, lung and lymph nodes. Feces or fecal swabs from live animals w/ signs. Pus or fluid from abscess on swab in transport medium or in syringe. Live, sick pig. Spiral colon, colonic scrapings, feces, or rectal swabs. 21 CONDITION ORGANISM(S) Trichomoniasis Trichomonas species Ureaplasma Ureaplasma species Water quality analysis Coliforms TISSUE AND/OR SAMPLES TO SUBMIT Preputial wash, semen sample, or vaginal mucous sample sent in TM pouch. (Contact Biomed Diagnostics 800-677-2855 for Transport Media pouch). Vaginal swabs from cow. Respiratory samples. Submit swabs in Amies medium. Minimum of 100 ml of water in a sterile container. 22 MOLECULAR AND VIRAL DIAGNOSTICS LIST OF MOLECULAR ASSAYS The VDL website (www.vdpam.iastate.edu) contains a complete list of molecular tests currently offered, testing schedule and fee for each. TESTING SCHEDULE Our goal is to provide accurate results and timely service. However, many diagnostic assays are not performed daily and/or testing schedules may change. If your case involves testing animals for export, testing show animals, testing animals to be sold, and/or testing a large number of samples, please contact the VDL (515-294-1950) prior to submission to verify the availability and turnaround time of specific assays. A phone call will often shorten turnaround time by allowing us time to prepare for testing prior to receipt of samples. SUBMISSION GUIDELINES In general, samples appropriate for conventional microbiological evaluation (i.e., detection of viruses) are also suitable for molecular diagnostic tests. This includes excretions and secretions, feces, blood, serum, and biopsies from infected, live animals (i.e., ante mortem), and relevant tissues/organs and washings (e.g., bronchial lavage) from necropsied animals (i.e., post mortem). Refer to the Sample Collection and Submission Guidelines (above) for comments on sample handling and shipment. For best results, clinical specimens should be collected aseptically from individual animals. Care should be taken to avoid cross contamination. Samples from different animals can be pooled for molecular assays if cost is a concern, but it is recommended that clients consult with VDL staff prior to pooling samples. Alternatively, samples can be pooled in the lab. MOLECULAR DIAGNOSTIC METHODS Several molecular diagnostic methods are available for detection or differentiation of viruses, including polymerase chain reaction (PCR), in situ hybridization (ISH), fingerprinting, and sequencing. Some, but not all, molecular techniques first require isolation of the target agent. Polymerase chain reaction (PCR) PCR is a process by which a portion of viral nucleic acid (DNA or RNA) is replicated a million times or more. Detection of this amplified product (amplicon) indicates that the sample is positive for the target virus. Proper amplification relies on a set of two short synthetic oligonucleotides (primers). Good test performance requires that primers bind only to the corresponding nucleotide sequences of the viral genome and nothing else. Thus, the specificity of the assay and accuracy of the results depend upon the design of PCR primers. 23 Theoretically, a PCR-based assay is capable of detecting one copy of the viral genome. However, depending upon the target agent, type of sample, and condition of sample, diagnostic sensitivity often is not as sensitive as anticipated and, depending on the circumstances, conventional assays may provide equivalent test performance. Also, PCR is expensive relative to most other diagnostic tests and positive results do not always have biological significance, i.e., PCR reacts with inactivated viruses, as well as infectious viruses. There are several types of PCR assays. RT-PCR is used to detect target RNA from clinical specimens. Reverse transcription (RT) of RNA is required to make complementary DNA for further amplification. This assay is most frequently used for specific detection of RNA viruses. Nested PCR is a PCR done in two steps, a primary PCR reaction and a nested reaction. The primary (or first) reaction uses a set of primers to generate a product that serves as the template for the nested (or second) reaction. The nested reaction uses a set of PCR primers specific for a region within the amplified product from the first reaction. Therefore, the nested reaction often serves as a confirmation for the specificity of the PCR products amplified in the primary reaction. Multiplex PCR is a PCR designed to detect more than one target sequence in a single PCR reaction. The assay uses two or more sets of primers. Each set of primers is specific for a different target sequence. The assay is most commonly used for simultaneous detection of multiple viral genes and differentiation of genotypes or subtypes of related microorganisms. Real-time PCR combines PCR amplification and detection into a single step. The basic principle of real-time quantitative PCR is the detection of target sequences using a fluorogenic 5’ nuclease assay (often called ‘TaqMan’). The advantages of this system include high reproducibility, the capability of handling large numbers of samples, the potential for quantitative results, and decreased turnaround time. The disadvantages include high instrument cost and the requirement for technical proficiency. Hybridization Hybridization procedures rely on base-pairing between a labeled DNA or RNA oligonucleotide (probe) and the complementary nucleotide sequences of the target genomic DNA or RNA. Since there is no amplication step, hybridization-based assays are generally less sensitive than PCR-based assays. Although most constituents for hybridization are commercially available, specific probes for each microorganism have to be generated by the lab performing the test. In situ hybridization is used for the detection of specific microorganisms in tissues. Depending on the protocol, ISH utilizes a labeled DNA, cDNA, or RNA probe that corresponds to a specific portion of pathogen’s genome. Various methods are used to detect the binding of the probe to the agent. In the VDL, the presence of the target microorganism is detected by a colorimetric reaction that can be seen under a light microscope. The advantange of this method is that the location of the pathogen can be assessed in the context of the histological lesions. 24 MOLECULAR ASSAYS FOR DIFFERENTIATION AND GENETIC CHARACTERIZATION Differential PCR Differential PCR can sometimes be used to distinguish closely related targets. Differential PCR is done either in a multiplex format using two or more sets of primers or by running two separate PCR assays. Restriction fragment length polymorphism (RFLP) RFLP analysis is a molecular differential technique that, to a limited extent, can distinguish between two viruses at the genomic level. RFLP is based on the fact that restriction enzymes recognize specific nucleotide sequences and cut the genome at that location. In general, the RFLP procedure consists of isolating the target microorganism from a clinical specimen, extracting DNA or RNA, digesting the nucleic acid material with restriction enzymes, and gel electrophoresis of the resulting products. The pattern of fragments observed on the gel is used to characterize or compare isolates. In general, RFLP requires virus isolates; however, RFLP using PCR products instead of native nucleic acid has recently been developed. This method provides faster turnaround since it is not necessary to isolate and propagate virus. RFLP is useful for differentiating minor differences at the strain level that normally cannot be detected by any antigenic assays, such as tissue immunoassays, serology, and enzyme immunoassays. RFLP analysis is rapid and less expensive than sequence analysis, but RFLP will miss many of the genetic differences that are revealed by sequencing. Sequence analysis Sequence analysis is a molecular tool for use in characterizing the genetic information of microorganism in detail. Sequencing provides a list of the nucleotides in the viral genome in the order in which they appear. Comparison of the genetic information makes complete differentiation between viruses possible. Either partial or whole genomic sequencing can be done, depending upon the size of genome and purpose of the analysis. In addition, RFLP patterns and amino acid composition can be predicted from sequence data. One disadvantage of sequencing is the expense of conducting the analysis. ORAL FLUID PROGNOSTIC PROFILING OF SWINE POPULATIONS What is oral fluid prognostic profiling? Prognostic profiling is a process for monitoring the circulation of pathogens in swine populations. The process is “prognostic” because the goal is to forecast the health and productivity of the population in the immediate future. The process uses oral fluids because they are quick, easy, and inexpensive to collect. Prognostic profiling is not a diagnostic procedure. Pigs showing clinical signs should be evaluated using conventional diagnostic methods. 25 What is “oral fluid”? Oral fluid is the liquid present in the oral cavity. Oral fluid is a mixture of saliva and “oral mucosal transudate.” Saliva is produced by the salivary glands. Oral mucosal transudate enters the mouth by crossing the buccal mucosa from the capillaries. Oral fluids contain both pathogens and antibodies. In humans, oral fluids are used to test for a variety of infections, e.g., HIV, measles, etc. In pigs, research has shown that oral fluids can be used to detect PRRSV, PCV2, SIV, and M hyopneumoniae infections. How to collect oral fluid samples (refer to Figures 1-4) Fig 1. Suspend a length of cotton rope in a location accessible to the pigs. Ropes should be placed in a clean area of the pen and not in close proximity to water or feed. Cotton rope is recommended because it is highly absorbent. Use 1/2” (1.3 cm) rope for nursery pigs; 5/8” (1.6 cm) rope for grow-finish pigs. The figure (above) shows a bracket with a 1” (2.5 cm) hole in the horizontal plate to hold the rope. A knot in the top of the rope secures it in place during collection. Fig 2. Hang rope shoulder high to the pigs (hang the rope, then cut to length). The pigs deposit oral fluids as they chew the rope. In active pens, 20-30 minutes is sufficient sampling time. Fig 3. Extract oral fluids from the rope. Insert the bottom (wet) end of the rope into a clean plastic bag or single-use plastic boot. Squeeze the rope so that the fluid accumulates in one corner. Fig 4. Cut a corner of the plastic and drain the contents into a tube (Falcon 2054 or equivalent). A 4 ml sample is ideal for testing. If samples are clean, no further processing is necessary. If samples contain particulates, centrifuge for 10 minutes and then pour into a clean tube. Handling oral fluid samples • Freeze samples promptly to optimize quality. Oral fluid samples for “same day” submission may be chilled and submitted on wet ice. Maintain the cold chain to preserve sample integrity. • Prepare and handle samples for shipment to the testing laboratory as is required for serum samples, i.e., pack samples with ice packs and absorbent material (to capture spills) in an insulated leak-proof transport container lined with a plastic bag. 26 Sampling strategy • Prognostic profiling relies on testing oral fluid samples at ±2-week intervals for specific pathogens of interest. To optimize detection of pathogens, sample pens spaced throughout the facility. Sampling the same pens repeatedly over time provides longitudinal data on the population. • Sample size calculations for specific pathogens have not been established. Field studies showed that sampling 6 pens in 1,100 head wean-finish barns at 2-week intervals detected circulation of PRRSV, PCV2, and SIV. Interpretation of lab results • PCR-positive results reflect active circulation of pathogens in the population. Caution: to avoid false negative results, PCR procedures optimized for serum must be validated for oral fluid samples. Check with the laboratory prior to submission. • A PRRSV ELISA for the detection of antibodies in oral fluids is available through the IISU VDL. Trouble-shooting oral fluid collection • Do not pool oral fluid samples. • Cotton rope is usually unavailable at local stores, but can be purchased through the Internet. Other materials, especially synthetic materials, do not have the absorbency of cotton. • Pigs are occasionally reluctant to approach the rope for the first collection. Reluctant pigs can be trained by throwing a length of rope in the pen for them to play with or by flavoring “practice” ropes with sugar solution. Once accustomed to the rope, pigs look forward to the next collection. • Pigs are more active in the morning. Afternoon collections may require more time. • Dirty samples are often an indication that the rope was too long. Hang rope at pig shoulder height. • Pigs are attracted to the rope because it is something new. Remove all ropes after each sampling to keep their interest in future collections. Discard rope after use; never re-use. • The presence of other hanging “toys”, e.g., chains or hoses, will reduce the likelihood that pigs will interact with the rope. 27 PATHOLOGY The VDL Pathology Section offers necropsy services, gross examination and histopathological evaluation (histochemistry, and immunohistochemistry) of tissue samples. Telephone consultation with a VDL pathologist prior to the submission may be useful and is particularly encouraged if a case is complex or the diagnostic investigation is an ongoing series of submissions. Please call the laboratory (515-294-1950) and talk to one of the diagnostic pathologists at any time if you have questions concerning specimen selection or preservation. TESTING SCHEDULE Our goal is to provide accurate results and timely service. However, many diagnostic assays are not performed daily and/or testing schedules may change. If your case involves testing animals for export, testing show animals, testing animals to be sold, and/or testing a large number of samples, please contact the VDL (515-294-1950) prior to submission to verify the availability and turnaround time of specific assays. A phone call will often shorten turnaround time by allowing us time to prepare for testing prior to receipt of samples. NECROPSY SERVICES Veterinary pathologists necropsy carcasses, evaluate fresh tissues grossly, and evaluate formalin-fixed tissues microscopically for the presence of lesions. Live animals and intact carcasses may be submitted to the laboratory for examination. Live, untreated animals with signs typical of the acute disease, or dead animals without postmortem decomposition are the submissions of choice. Tests will NOT be performed on specimens deemed unsuitable (autolyzed or incorrect samples). A complete history should be included with all submissions. Special procedures or circumstances (legal issues or insurance cases) should be designated as such at the time of submission. Returning of bodies, carcasses, body parts No animal carcasses, body parts or ashes will be returned/released to veterinarians or animal owners. Arrangement for pickup by authorized crematory service is an option. No cosmetic necropsy will be performed on animals/bodies submitted to the laboratory for necropsy/testing. Please call the Laboratory Director with questions and additional details relating to these policies. 28 GENERAL GUIDELINES FOR FIELD NECROPSY When collecting tissues from necropsied animals, the history, clinical signs, and gross lesions should determine which tissues are collected. The following sampling recommendations and reminders may be helpful in optimizing the diagnostic information that can be obtained. Brain Sever the cranial cervical spinal cord as far caudally as possible when removing the head. Sagittally section brain and submit one-half in formalin and one-half chilled. Be sure to include cerebellum and brainstem in addition to cerebrum. Brain should always be submitted from animals found dead without gross lesions in the thoracic or abdominal viscera. Gross lesions ALWAYS submit representative samples of ANY gross lesions. For example, oral erosions/ulcers are often described on submission forms but less often submitted, especially in suspect BVD-mucosal disease cases. Fixed ulcers are an excellent sample for detecting BVD virus by immunohistochemistry (IHC). Heart Open all chambers and examine all valves. If lesions are identified, submit formaliin-fixed and fresh (if infectious agents are suspected) samples. If no lesions or evidence of heart failure are identified, submit a fixed portion of papillary muscle from the left ventricle. Intestines When sampling the intestinal tract, always begin at the ileocecal junction and work toward the stomach. Examination of multiple areas is often required, since lesions and/or agents are usually not uniformly distributed throughout the intestinal tract. Place 3 widely spaced 1-2 cm segments of ileum, 3 segments of jejunum, and one segment of duodenum in formalin, making sure that the formalin contacts the mucosal surface. Cecal contents should be collected for virology, and a portion of cecum should be fixed. Samples of several areas of spiral colon should be placed in formalin so that both ascending and descending loops will be included. As noted above, also submit any gross lesions. If the animal is of small size, the entire remaining intestinal tract may be submitted chilled. Otherwise, representative loops of ileum, jejunum, and colon should be submitted. Also, include fixed and fresh enlarged lymph nodes. Joints Joint fluids need to be sampled using aseptic techniques. Synovium should be submitted on ice and in formalin. Submission of intact joints on ice is an alternative. Liver, kidney, spleen Since these organs have many potential uses for the diagnosis of many different infectious agents and diseases, please submit formalin-fixed and fresh samples in all cases. 29 Lung Submit both formalin-fixed and fresh tissue samples from at least four different areas: cranial, hilar, middle, and caudal lung. More extensive sampling greatly increases the probability of diagnosis. Fresh portions of lung should be as large as possible. The entire lung (less the parts collected for formalin fixation) should be submitted, if possible. If lung lavage for PRRS virus isolation is desired, submit one entire (unsampled) lung. Also, include both fixed and fresh enlarged lymph nodes. Lymph nodes Lymph nodes, especially those enlarged or hemorrhagic, are useful for detection of viral infections, bacterial infections or neoplasms. Please include fresh and formalin-fixed lymph nodes (and tonsil) when appropriate. Skin Skin lesions should be submitted fixed and fresh. Nonlesional skin from dead bovines is useful for determining BVD virus persistent infection status. Ear (pinna) or coronary band are preferred sites (see discussion under ‘Special Tests’). Spinal Cord When clinical signs suggest spinal cord involvement, it is essential to submit at least parts of spinal cord. A Barnes dehorning tool works well to gouge away bone to access segments of cord. A cleaver, ax, hatchet or saw is also effective. Segments representing cervical, thoracic and lumbar regions in formalin and on ice are appropriate specimens. Alternatively, submit vertebral column or vertebrae on ice. Stomach Submit any lesions in formalin. In ruminants, submit one piece of fixed abomasum even if no gross lesions are present. Thymus Fixed and fresh thymus are useful for detecting BVD virus in cattle and PRRS virus in pigs. Tongue Formalin-fixed tongue is useful for detecting generalized myopathies, especially in fetuses (Neospora caninum) and neonates. Muscular diaphragm is also useful for this purpose. Trachea In general, formalin-fixed and fresh trachea is only useful if gross lesions are present. In cattle, hemorrhages are common but nonspecific in animals with respiratory disease. Pooled exudate from lung is easily wiped away to reveal a smooth, glistening (normal) mucosal surface. This feature distinguishes pooled exudate from an adherent fibrinonecrotic membrane, such as is typical of IBR virus. 30 SUBMISSION GUIDELINES Submission of tissue specimen(s) is usually the best method of obtaining a diagnosis of a clinical disease. However, proper selection and preservation of samples is essential to make the most efficient and economical use of the laboratory. The two conditions that most frequently interfere with diagnosis are: (1) post mortem autolysis, and (2) sample collection too late in the course of disease. Fresh specimens should be large enough to demonstrate the lesion yet small enough to allow for rapid chilling (tennis-ball sized). In some cases (e.g., bronchoalveolar lavage for PRRS virus isolation) submission of an entire organ may be preferred. Ideally, fresh samples should be packaged individually to prevent cross-contamination. Please do NOT to package fresh intestine with other tissues, as this results in fecal contamination of other organs. At a minimum, intestinal samples should be submitted separate from all other tissues. Samples should be shipped in leak-proof containers with artificial freeze packs. Please line shipping containers with additional sealed plastic bags to avoid leakage in transit. Carriers will not deliver leaky packages resulting in delays in processing. A fee of $25 may be assessed for packages that leak. Specimens for histopathology should include thin (0.5-1.0 cm or 1/2 inch) slices of the appropriate organs, including the lesion, transitional zones, and adjacent grossly normal tissue, in 10% buffered formalin in a leak-proof, wide-mouthed solid container. Do NOT use GLASS Containers. When in doubt, collect specimens from multiple organs, including brain. The pathologist can then select the most appropriate specimens for complete microscopic examination. Unless it is important that individual animals be examined and reported separately, specimens from each individual animal can be pooled in a single container provided the specimens are small enough to maintain a 10:1 formalin : tissue ratio. The ratio of formalin : tissue should be at least 10:1 to allow optimal fixation. A minimum of 48 hours is required for histopathology, 24 hours for fixation, and 24 hours for processing. Consequently, specimens for histopathological examination should be collected and placed in formalin at the time of necropsy in order to minimize autolysis and generally allow results to be available the day after the specimen is received. General guidelines for histopathologic examination/submission of formalin-fixed specimens are as follows: 1. Solid organs: 0.5 - 1 cm slices to include lesion and transitional areas between lesions and normal tissue. 2. Intestine: 1 - 2 cm lengths held open as they are immersed in formalin or flushed with formalin prior to immersion. Do NOT tie the ends. 3. Brain (including brain stem): immerse sagittal half of small brain in formalin. One-half of the brain should also be submitted from larger animals. These larger brains should be sliced for better fixation. The preferred procedure is to make transverse slices, 1.0 – 1.5 cm apart that extend to within 1.0 cm of the ventrum of the brain. This allows for more 31 rapid fixation while retaining the architecture of the brain so that specific areas can be sampled. Nerves can be pinned to a tongue depressor and immersed in formalin. 4. Tumors: immerse in 10% formalin. If greater than 1 cm, incise, leaving a base attachment intact. 5. Impression smears and needle aspirates of tumors are helpful in a few situations. However, actual biopsies are usually required for definitive diagnosis. Stain and examine smears and aspirates in your practice laboratory and, if a diagnosis is not obvious (i.e., mastocytoma, abscess, etc.), biopsy the mass and forward both the biopsy and the smears to the reference laboratory. Preparation of 10% neutral buffered formalin Buffered formalin concentrates are available from several suppliers. These are ready for use after the addition of water. Non-buffered 10% formalin (1:9 of 37% formaldehyde : water) can be substituted for neutral buffered formalin in emergency situations. Do NOT submit samples in undiluted 37% formaldehyde. 10% neutral buffered formalin is prepared as follows: 900 ml distilled water 100 ml 37% formaldehyde solution 8.0 g NaCl 4.0 g potassium phosphate monobasic 6.5 g potassium phosphate dibasic HISTOCHEMISTRY AND IMMUNOHISTOCHEMISTRY Various histochemical techniques are available to demonstrate agents/structures within histologic sections. These include the following: Histochemical staining Histochemistry demonstrates bacteria, fungi, cellular components, etc., by use of differential stains, e.g. a Gram stain for demonstrating bacteria or Giemsa stain for mast cell granules. Immunohistochemistry (IHC) IHC is used to demonstrate specific antigens within tissue sections. The location of agents (bacteria, viruses) can be visualized within the tissue in association with lesions. This technique is performed on formalin-fixed specimens. Results are generally available 24 - 48 hours after receipt, providing the proper specimens are submitted in formalin. See the VDL website (www.vdpam.iastate.edu) for a list of current immunohistochemical tests (e.g. IHC, immunoperoxidase, IPT). 32 SPECIAL TESTS Diagnosis of BVD virus persistent infection (PI) Ear notch biopsy is a method to detect cattle persistently infected with BVD virus. BVD virus is present in epidermis and hair follicles throughout the skin of PI cattle. The ear is a convenient site and can be sampled with a small swine ear notcher. Ear notch specimens (at least 1.0 x 0.5 cm) submitted in 10% neutral buffered formalin are processed for immunohistochemistry (IHC) to detect BVD viral antigen. This test offers several advantages: 1. Testing can be conducted on animals of any age. 2. Maternal antibody does not interfere with detection of BVD virus in skin. 3. Identifying PI’s as early as possible allows them to be removed from the herd prior to the next breeding season. 4. Samples may be collected by producers. 5. Samples do not require refrigeration. Samples should be submitted within 7 days after being placed in formalin. 6. Six specimens can be processed on one slide; therefore, submissions in multiples of 6 are preferred. Please submit specimens in numbered (1, 2, 3, . . . ) snap-cap tubes (not in plastic bags) halffilled with 10% neutral buffered formalin. Use a VDL Serum Submission form to write the calf ID number next to the tube number, and write ‘BVD virus IHC‘ on the form. Producers should be instructed to disinfect notchers between calves and to avoid inhalation of and skin contact with formalin. Questions regarding this test should be addressed to Dr. V.L. Cooper. Positive animals may be retested in 10 days to confirm PI status. They should be isolated until testing is complete. Evidence indicates that ear notch IHC positive calves are PI, or CI (congenitally infected), both of which can have negative impact on herd health. Diagnosis of anaplasmosis using blood smears Please submit air-dried smears of blood collected in EDTA. Smears should be made immediately after blood is collected. Parasites disassociate from RBCs over time and, for that reason, we prefer receiving smears rather than whole blood in EDTA. If serum is available, serology is probably more reliable than examination of blood smears for diagnosis of anaplasmosis. CLINICAL PATHOLOGY Clinical pathology (urinalysis, hematology, serum chemistry, cytology, endocrinology, etc.) is generally not offered through the VDL. Specimens that only require routine clinical pathological evaluation should be submitted to commercial reference laboratories or arrangements made directly with the Clinical Pathology Laboratory in the Department of Veterinary Pathology (515-294-3282). 33 QUICK GUIDES TO SAMPLE SELECTION FOR DIAGNOSIS OF COMMON CONDITIONS The following ‘Quick Guides’ have been prepared to assist you in sample selection when a particular system is affected. You may wish to provide copies to your technicians and keep copies at strategic locations for reference. Specific topics are listed in the table of contents. 34 BOVINE ABORTION Specimens to submit: Tissues tissues should include: Brain Dam's serum Heart Ileum Kidney Liver Lung Placenta (very important) are received in best condition if removed at necropsy in the field. Fetal Formalin-fixed (1/2 cm slice) 3 - 5 ml from affected cows. Optional, see notes on abortion serology. Formalin-fixed (1/2 cm slice) Formalin-fixed Fresh/chilled (1 entire kidney), formalin-fixed (1/2 cm slice) Fresh/chilled (1/8- 1/4 of organ), formalin-fixed (1/2 cm slice) Fresh/chilled (1/8- 1/4 of organ), formalin-fixed (1/2 cm slice) 3 cotyledons, fresh/chilled; 2 cotyledons, formalin-fixed (please submit placenta when possible - this increases the diagnostic success rate) Skeletal Muscle Tongue and diaphragm formalin-fixed (1/2 cm slice) Skin (lesions/ear notch) Formalin-fixed (1/2 cm slice) Spleen Fresh/chilled (1/2 of organ), formalin-fixed (1/2 cm slice) Stomach contents 1-3 ml in sterile syringe or tube, fresh/chilled Thoracic fluid 1-3 ml in sterile syringe, fresh/chilled Thymus Fresh/chilled, formalin-fixed (1/2 cm slice) Alternatively, the entire fetus and placenta can be submitted. SAMPLING TECHNIQUES 1. 2. 3. Do NOT freeze fresh tissues. Always submit placenta if possible! Failure to submit placenta severely diminishes the diagnostic success rate of bovine abortion cases. It may be useful to submit serum from affected and unaffected dams. AGENTS DETECTED BY ROUTINE EXAMINATION OF FETAL AND PLACENTAL TISSUES Trueperella (Arcanobacterium) pyogenes, Bacillus spp., Brucella spp., Campylobacter spp., Histophilus somnus, Salmonella, Listeria monocytogenes, etc. Fungi Aspergillus, Phycomycetes Protozoa Neospora caninum (see comments), Toxoplasma gondii Viruses IBR, BVD AGENTS REQUIRING SPECIAL TESTS (BY REQUEST) Bacteria Leptospira (see comments below), Ureaplasma, Mycoplasma (culture) Tritrichomonas fetus infection is best diagnosed by placing preputial wash Protozoa or fetal fluids/stomach contents directly into TF pouch for culture Bacteria Eyeball (aqueous) Nitrate/nitrite COMMENTS • • If leptospirosis is suspected, extra effort should be made to deliver freshly aborted, chilled fetuses directly to the lab. PCR and FA tests can be conducted on kidney. Serology on dam sera is very helpful. Diagnosis of Neospora caninum abortion is based on histopathologic examination of brain, heart, skeletal muscle, liver, lung, and placenta for characteristic lesions. Presence of the organism can be confirmed by immunohistochemistry. Absence of serum antibody in the cow would rule out neosporosis. BOVINE CENTRAL NERVOUS SYSTEM DISORDERS Specimens to submit: Tissues from euthanized or dead animals including: Blood sample EDTA tube for lead analysis or cholinesterase inhibition Eyeball (aqueous) Cations (calcium); nitrite Brain (including brain stem) 1/2 brain divided longitudinally, fresh/chilled 1/2 brain, formalin-fixed Optional, nervous coccidiosis. Several partial loops with contents, Colon fresh/chilled. 1 cm pieces of several loops, formalin-fixed Liver Optional, lead toxicosis. Fresh/chilled. Kidney Optional, lead toxicosis. Fresh/chilled. Spinal cord Optional, locomotor involvement Entire carcass or vertebral column, fresh/chilled Dissected cord, fresh/chilled Cross-sections (1/2 cm) of cord from 4-5 levels, formalin-fixed Rumen contents Fresh/chilled SAMPLING TECHNIQUES 1. 2. 3. 4. Entire head can be submitted. Chill before shipment if possible. Do NOT freeze fresh brain or head. Fresh half of brain should be packed carefully to avoid crushing. Fixed half of brain should be incised, at least once, transversely (not longitudinally) into the lateral ventricle to aid fixation if the brain is large. AGENTS DETECTED BY ROUTINE EXAM Histophilus somnus, Listeria monocytogenes, Arcanobacterium pyogenes, etc. Polioencephalomalacia AGENTS REQUIRING SPECIAL TESTS ( BY REQUEST) Magnesium (serum, entire eyeball, fresh/chilled), calcium (serum, Deficiencies fresh/chilled) Parasites Coccidia (flotation; feces, fresh/chilled) - NO lesions in brain Lead (whole blood in EDTA, liver, stomach contents, fresh/chilled), Toxicoses organophosphate (whole blood in EDTA, brain, rumen, fresh/chilled) Sodium (whole blood in EDTA, brain, rumen, fresh/chilled) Rabies (FA), pseudorabies virus (FA, VI); bovine herpesvirus (brain, Viruses fresh/chilled) Bacteria Non-infectious COMMENTS • • Cerebellum and brain stem are affected by most infectious causes of CNS disease and should always be included in submitted samples. Many toxic, nutritional, and metabolic causes of CNS disease do not induce lesions in the brain and must be diagnosed by analysis of other tissues. For most toxicoses, submission of rumen contents, complete feed, water and feed components, liver, kidney, and whole blood (in EDTA) as well as brain would include the tissues necessary for diagnosis. 36 BOVINE ENTERITIS – CALVES < 2 MONTHS OF AGE Specimens to submit: Antemortem fecal samples are of value if collected on the first day of diarrhea. Alternatively, tissues should be removed from a euthanized calf. Abomasum Fresh/chilled and formalin-fixed Cecal contents 10 ml fluid contents, fresh/chilled Ileum Two or three 10-15 cm segments, fresh/chilled Three 1 cm pieces, formalin-fixed Jejunum Two or three 10-15 cm segments, fresh/chilled Three 1 cm pieces, formalin-fixed Liver Fresh/chilled and formalin-fixed Mesenteric lymph node Fresh/chilled and formalin-fixed Spiral colon Several partial loops, fresh/chilled Several 1 cm pieces, formalin-fixed Spleen Fresh/chilled and formalin-fixed Ear notch Formalin-fixed Because autolysis occurs very quickly in bovine intestines, samples removed at necropsy in the field are usually better than a whole dead calf submitted to the lab SAMPLING TECHNIQUES 1. 2. 3. 4. Samples must be taken as soon after death as possible (within minutes). Intestines do not need to be tied off at the ends. Flush intestinal segments for histopathologic examination with formalin and drop in fixative. Or, gently open ends of 1 cm segments with scissors or forceps to expose mucosa as immersed. Do not split open. Pool all formalin-fixed tissues from each calf in one bag; individual calves can be pooled or kept separate as desired. Package fresh intestines separately from other tissues and each calf in a separate bag. Chill fresh tissues before mailing. Do NOT freeze. AGENTS DETECTED BY ROUTINE EXAM E. coli, Salmonella spp., Clostridium spp., Enterococcus durans Cryptosporidia, Coccidia Rotavirus, Bovine coronavirus AGENTS REQUIRING SPECIAL TESTS (BY REQUEST) IHC on fixed ileum, colon, mesenteric lymph node, spleen, skin, and any BVD virus gross lesions; VI on chilled mesenteric lymph node, spleen, kidney, thymus, and lung Bacteria Parasites Viruses COMMENTS • • • In cases of necrotic enteritis, submit both necrotic and adjacent non-necrotic segments fresh and fixed. In-house quick tests (acid-fast stained impression smears) may be of value for detection of cryptosporidia. The preferred site for impression smears/mucosal scrapings for cryptosporidia is ileum. As such, it is helpful if fresh ileum is submitted in a separate container. Colon is the preferred tissue in which to identify lesions of coronavirus enteritis and for laboratory confirmation with BCV IHC. Colon should be submitted with all calf diarrhea cases. 37 BOVINE ENTERITIS - CALVES > 2 MONTHS OF AGE, FEEDLOT CATTLE, ADULTS Specimens to submit: Fecal samples may be of value if collected on the first day of diarrhea. From euthanized or dead animals, tissues should include: Abomasum Fresh/chilled and formalin-fixed Any other gross lesions Fresh/chilled and formalin-fixed Colon Several partial loops, fresh/chilled Three 1 cm pieces, formalin-fixed Colon contents 10 ml fluid contents, fresh/chilled Ileum Two or three 10-15 cm segments, fresh/chilled Three 1 cm pieces, formalin-fixed Jejunum Two or three 10-15 cm segments, fresh/chilled Three 1 cm pieces, formalin-fixed Liver Fresh/chilled and formalin-fixed Mesenteric lymph node Fresh/chilled and formalin-fixed Rumen Fresh/chilled and formalin-fixed Rumen contents Fresh/chilled for pH Spleen Fresh/chilled and formalin-fixed Samples removed in the field are better than a whole dead animal submitted to the lab. SAMPLING TECHNIQUES 1. 2. 3. 4. Samples must be taken as soon after death as possible (within minutes). Intestines do not need to be tied off at the ends. Flush intestinal segments for histopathologic examination with formalin and drop in fixative. Or, gently open ends of 1 cm segments with scissors or forceps to expose mucosa as immersed. Do not split open. Pool all formalin-fixed tissues from each calf in one bag; individual calves can be pooled or kept separate as desired. Package fresh intestines separately from other tissues and each calf represented in a separate bag. Chill fresh tissues before mailing. Do NOT freeze. AGENTS DETECTED BY ROUTINE EXAM Bacteria Salmonella spp., Clostridium perfringens Parasites Coccidia Viruses BVD virus (see comments below) AGENTS REQUIRING SPECIAL TESTS (BY REQUEST) Culture of feces, mesenteric lymph nodes, and intestine; histopath and Bacteria acid fast-stains on intestines and mesenteric lymph nodes Parasites Coccidia and GI nematodes; (feces, fresh/chilled for fecal flotation) Bovine coronavirus (feces for ELISA, fixed ileum and colon for histopath Viruses and IHC) COMMENTS • • • BVD mucosal disease diagnosis: Fixed ileum, spleen, mesenteric lymph nodes, skin, heart, lung, and ANY GROSS LESIONS for immunohistochemistry. Fresh/chilled spleen, lung, thymus mesenteric lymph node, and kidney for virus isolation. Coccidiosis is a common cause of diarrhea in this age group. It is necessary to submit feces and/or colon to diagnose coccidiosis. 38 BOVINE PNEUMONIA Specimens to submit: From euthanized or dead animals, tissues should include: Bronchial lymph node Fresh/chilled only Entire lung (one side), or generous portion of lesion and adjacent Lung unaffected lung, fresh/chilled. Sample 3-4 areas of lung, including both cranial and caudal lobes Four or more thin slices (1 cm) through affected and adjacent unaffected lung, formalin-fixed Nasal swabs, Deep nasoUse a long, Dacron-tipped swab that reaches deep into the nasal cavity pharyngeal swabs, Swabs to be used for virus FA or PCR should penetrate the mucous layer to retrieve epithelial cells (rub hard, roll swabs on slides to deposit nasal Tracheal wash/lavage mucosal epithelial cells for FA test). Submit separate swabs for bacterial culture and virus isolation in saline or transport media. Do not freeze. Swabs and / or lavage material can be submitted for PCR respiratory panel (bacteria and viruses) as antemortem samples. See video on sampling techniques. Acute and convalescent serum from 5-10 affected and 5-10 normal Serum samples calves. Hold acute samples and submit with convalescent. Optional, if lesions are observed. Affected portion (10 cm) with larynx, Trachea fresh/chilled. Several rings at edge of lesion, formalin-fixed. SAMPLING TECHNIQUES 1. 2. 3. Fresh tissues should be chilled before shipping. Do NOT freeze. Samples for virus detection need to be taken from ACUTE animals at the onset of respiratory signs. Swabs must be kept moist and cold before and during shipment. AGENTS DETECTED BY ROUTINE EXAMINATION Arcanobacterium pyogenes, Histophilus somnus, Mannheimia hemolytica, Pasteurella multocida, Mycoplasma bovis IBR, BRSV, BVD, BRCV, PI-3 (PCR on lung, swabs, and/or lavages; also Viruses FA, VI, IHC; lung, trachea, bronchial lymph node, fresh/chilled and formalin-fixed) AGENTS REQUIRING SPECIAL TESTS (BY REQUEST) Mycoplasma Mycoplasma dispar (Culture on fresh/chilled lung; histopath) Bacteria COMMENTS • • • • Acute lesions are most likely to hold active causative agents are usually at the diseased/normal interface. Chronic lesions in dependent tips or lobes may no longer hold primary pathogens. Nasal swabs may pick up resident bacterial flora but may be of value in acute cases. Nasal swabs may also be of value to identify viruses if sampled in the early stages (exhibiting serous nasal discharge). Tracheal washes submitted on ice may be used for both virus and bacteria identification. 39 PORCINE ABORTION Specimens to submit: Entire fetuses with placenta, minimally contaminated, fresh/chilled are preferred specimens. Do NOT freeze. Send 4-6 representative fresh fetuses and all mummified fetuses. Alternatively, remove the following tissues from 3 fetuses per litter: Thoracic fluid 0.25 - 1 ml per aborted pig, may pool within litter for PRRS virus, PCV2 Brain 1/2 brain, fresh/chilled and formalin-fixed Heart 1/2 of organ fresh/chilled, 1/2 cm slice formalin-fixed Kidney Fresh/chilled, formalin-fixed (1/2 cm slices) Liver Fresh/chilled (1/3 of organ), formalin-fixed(1/2 cm slice) Lung Fresh/chilled (1 entire lung), plus formalin-fixed (1/2 cm slice) Stomach contents 1-3 ml in sterile syringe or tube, fresh/chilled Placenta Fresh/chilled and several pieces formalin-fixed Umbilicus Formalin-fixed, several 1/2 cm slices Sow serum Optional, see notes on abortion serology. 1-3 ml from affected sows SAMPLING TECHNIQUES 1. 2. 3. Do NOT freeze tissue. Submit placenta whenever possible Thorough investigation of abortion should include serology. Submit dam's sera. Retain 1/2 of sample frozen. AGENTS DETECTED BY ROUTINE EXAMINATION Arcanobacterium pyogenes, Bacillus spp., Brucella spp., E. coli, Salmonella spp., Erysipelothrix, Streptococcus spp., etc Parvovirus, PCV2, PRRSV, PRV, (see comments) Bacteria Viruses AGENTS REQUIRING SPECIAL TESTS (BY REQUEST) Leptospira PRRS virus Toxicosis If leptospirosis is suspected, extra effort should be made to deliver freshly aborted, chilled fetuses directly to the lab for PCR or FA tests which are on kidney or IHC. Serology on sow sera is probably the most reliable method for diagnosing leptospirosis. PRRSV virus is not present in all aborted fetuses. Virus isolation is not routinely conducted but PCR on pooled tissues or thoracic fluids is routine. Histopath may occasionally demonstrate lesions suggestive of PRRS virus in umbilical cords, lungs, heart or other tissues. Preferred samples are lung or serum from weakborn littermates or from pigs that develop pneumonia shortly after birth. PCR on serum from sick sows is often valuable. A serologic survey of the sow herd may be useful, but may be difficult to interpret in PRRS-endemic or vaccinated herds. Carbon monoxide (heart blood in EDTA; clotted heart blood or thoracic fluid as second choice). COMMENTS • • Parvovirus and PCV2 usually do not cause abortion but may be present in mummified fetuses. Mummified fetuses may harbor parvovirus, PCV2 or PRRSV. Lungs and hearts from mummified fetuses are useful for detection of these viruses by PCR. Fetal serology may aid in the diagnosis of porcine parvovirus, PCV2, Leptospira and PRRSV. 40 PORCINE CENTRAL NERVOUS SYSTEM DISORDERS Specimens to submit: One or more acutely affected live pigs. Alternatively, tissues from field necropsy should include: Brain (including brain stem) Swab of brain stem and base of cerebellum (for bacterial culture) 1/2 brain divided longitudinally, fresh/chilled 1/2 brain, formalin-fixed Intestine Optional, edema disease. One 10-15 cm slice of ileum and jejunum, fresh/chilled Several 1/2 cm slices of ileum, formalin-fixed Spinal cord Optional, locomotor problems. Entire carcass or vertebral column, fresh/chilled Dissected cord, fresh/chilled Cross-sections (1/2 cm slices) of cord from 4-5 levels, formalin-fixed Spleen Fresh/chilled and formalin-fixed Tonsil Fresh/chilled and formalin-fixed SAMPLING TECHNIQUES 1. 2. 3. 4. 5. Entire head can be submitted. Chill before shipment if possible. Do NOT freeze fresh brain or head. Fresh half of brain should be packed carefully to avoid crushing. Fixed half of brain should be incised transversely (not longitudinally) into the ventricle to aid in fixation if brain is large. CSF can be collected prior to removing the skull. When a bacterial meningitis is suspected, CSF is an excellent sample as there is less opportunity for contamination compared to most methods of opening the skull. AGENTS DETECTED BY ROUTINE EXAMINATION Streptococcus suis, Haemophilus parasuis, Arcanobacterium pyogenes, E. coli (intestine, edema disease) Viruses Pseudorabies virus, PRRS virus, PCV2 Non-infectious Water deprivation/sodium toxicity AGENTS REQUIRING SPECIAL TESTS (BY REQUEST) Toxicosis Selenium (liver and spinal cord - lumbar intumescence, fresh/chilled) Organophosphate (whole blood in EDTA, brain, stomach contents, fresh/chilled) Rabies (FA on brain); other viruses (e.g. HEV, porcine Teschovirus, Viruses paramyxovirus, herpesviruses detected by PCR or VI on fresh/chilled brain and spinal cord) Bacteria COMMENTS • • • Cerebellum and brain stem are affected by most infectious causes of CNS disease and should always be included in submitted samples. Many toxic causes of CNS disease do not induce lesions in the brain and must be diagnosed by analysis of other tissues. For most toxicoses, submission of stomach contents, liver, kidney, feed, water, and whole blood (in EDTA), as well as brain, would include the tissues necessary for diagnosis. Spinal cord is essential for diagnosis of causes of posterior paresis or paralysis. 41 PORCINE ENTERITIS – NURSING PIGS Specimens to submit: The best specimens are acutely-ill (<24 hours) live untreated pig(s). Alternatively, necropsy of euthanized pig(s) with intestines collected in formalin within 10 minutes of death. Colon/cecum contents 2-10 ml fresh/chilled Colon and cecum Entire organ, fresh/chilled Several 1 cm pieces, formalin-fixed Ileum 10-15 cm segments, fresh/chilled Three 1 cm pieces, formalin-fixed Jejunum 10-15 cm segments, fresh/chilled Three 1 cm pieces, formalin-fixed Lesions 10-15 cm segments, fresh/chilled Several 1 cm pieces, formalin-fixed Samples removed at necropsy in the field are better than a whole dead pig submitted to the lab. SAMPLING TECHNIQUES 1. 2. 3. 4. 5. Samples must be taken as soon after death as possible (within minutes). Intestines do not need to be tied off at the ends. Flush intestinal segments for histopathologic examination with formalin and drop in fixative. Or, gently open ends of 1/2" segments with a scissors or forceps to expose mucosa as immersed. Pool all formalin-fixed tissues from each pig in one bag; individual pigs can be pooled or kept separate as desired. Package fresh intestines separately from other tissues and each pig in a separate bag. Chill fresh tissues before mailing. Do NOT freeze. Do not send whole, dead pigs (intestines autolyze quickly). AGENTS DETECTED BY ROUTINE EXAMINATION Bacteria Parasites Viruses Clostridium difficile, Clostridium perfringens, E. coli, Enterococcus durans, Salmonella spp. Isospora (coccidia), Cryptosporidia Rotavirus, PED (Porcine Epidemic Diarrhea) virus, TGE virus, PRRSV COMMENTS • • • • • • • Accurate diagnosis of diarrhea in suckling piglets usually requires submission of tissues. Feces from acutely affected pigs are useful for detection of epidemic agents such as TGEV or PEDV by PCR. Results of tests on feces only (both positive and negative) may not be completely definitive and must be evaluated with consideration of clinical signs. Samples (10-20 ml) should be taken on the first day of diarrhea. Accurate diagnosis of endemic agents requires both the detection of the offending agent(s) as well as the presence of compatible histologic lesions. Brachyspira hyodysenteriae can occasionally be isolated from feces (swabs are even less reliable). Wet mounts of intestinal impression smears or fecal flotation may be of value for quick in-house detection of Isospora In cases where mesocolonic edema is prominent, Clostridium difficile is a differential and the entire colon or colon contents should be submitted for a C. difficile toxin ELISA. In cases of necrotic enteritis, submit both necrotic and adjacent non-necrotic segments, fresh and fixed. 42 PORCINE ENTERITIS - WEANED PIGS Specimens to submit: The best specimen is an acutely-ill (< 24 hours) live untreated pig(s). Alternatively, tissues may be removed from euthanized pigs. Colon and cecum Several 10 cm sections, fresh/chilled Several 1 cm pieces, formalin-fixed Feces/colon content 2-10 ml fluid contents, fresh/chilled Ileum 10-15 cm segment, fresh/chilled Three 1 cm pieces, formalin-fixed Jejunum 10-15 cm segment, fresh/chilled Three 1 cm pieces, formalin-fixed Lesions 10-15 cm segment, fresh/chilled Several 1 cm pieces, formalin-fixed Samples removed at necropsy in the field are often better than a whole, dead pig submitted to the lab. SAMPLING TECHNIQUES 1. 2. 3. 4. 5. Samples must be taken as soon after death as possible (within minutes). Intestines do not need to be tied off at the ends. Flush intestinal segments for histopathologic examination with formalin and drop in fixative. Or, gently open ends of 1/2" segments with a scissors or forceps to expose mucosa as immersed. Pool all formalin-fixed tissues from each pig in one bag; individual pigs can be pooled or kept separate as desired. Package fresh intestines separately from other tissues and each pig in a separate bag. Chill fresh tissues before mailing. Do NOT freeze. Do not send whole, dead pigs (intestines autolyze quickly). AGENTS DETECTED BY ROUTINE EXAMINATION Parasites E. coli, Salmonella spp., Clostridium perfringens, Enterococcus durans, Brachyspira spp. Lawsonia intracellularis Coccidia, roundworms, whipworms Viruses Rotavirus, TGE virus, PED virus, PRRSV, PCV2 Bacteria COMMENTS • • • • • • Feces can be used to detect TGEV and PEDV by PCR and is diagnostic when expected negative. Detection of endemic agents from feces does not provide a definitive diagnosis. Detection of Lawsonia or rotaviruses detected by PCR or Salmonella or hemolytic E. coli detected by culture or parasite ova/oocysts detected by fecal flotation should be interpreted in context of clinical signs. Histopathology on tissues for compatible pathologic lesions is required for definitive diagnosis. Fecal samples (10-20 ml) should be taken on the first day of diarrhea. Call the laboratory to discuss the value of feces for diagnosis or monitoring for specific pathogens of interest. Colitis associated with Brachyspira hyodysemeteriae and Brachyspira pilosicoli should be confirmed by culture and histopathology for a definitive diagnosis. Porcine Proliferative Enteritis (PPE) associated with Lawsonia intracellularis can be confirmed by IHC or PCR. Ill-defined conditions such as dietary hypersensitivity or nonspecific colitis may be implied but cannot be confirmed by routine diagnostic investigations. In cases of necrotic enteritis submit both necrotic and adjacent non-necrotic segments, fresh and fixed. 43 PORCINE MULTISYSTEMIC DISEASE INVESTIGATIONS Specimens to submit: The best specimen is a live untreated pig(s). Alternatively, tissues should include: Brain Turbinate Colon and Cecum Heart Intestine Kidney Liver Lung Joint swabs/synovium Lymphoid tissues Spinal cord Whole blood/serum 1/2 fresh and 1/2 formalin-fixed Turbinate swab (chilled) and turbinate in formalin Entire organ, fresh chilled Several 1 cm pieces, formalin fixed Fresh/chilled and formalin-fixed/swabs of fibrin if present Two 10-15 cm slices of ileum and two jejunum, fresh/chilled Several (6-10) 1/2 cm slices ileum and jejunum, formalin fixed Fresh/chilled and formalin-fixed Fresh/chilled and formalin-fixed Entire lung (one side) or generous portion of lung containing the lesions and adjacent unaffected lung, fresh/chilled 4-6 thin slices (1 cm) through affected and adjacent unaffected lung, formalin-fixed Swabs chilled; synovium fresh/chilled and in formalin Spleen, tonsil, and lymph nodes Fresh/chilled and formalin-fixed Entire carcass or vertebral column, fresh/chilled, or Dissected cord, fresh/chilled Cross-sections (1/2 cm slices) of cord from 4-5 levels, formalin-fixed Chilled; useful for clin-path, PCR, serology, chemistry SAMPLING TECHNIQUES 1. 2. Fresh tissues should be chilled before shipping. Do NOT freeze. Pool all formalin-fixed tissues from each pig in one bag; individual pigs can be pooled or kept separate as desired. Package fresh intestines separately from other tissues and keep each pig in a separate bag. AGENTS DETECTED BY ROUTINE EXAMINATION Bacteria Parasites Viruses Mycoplasma Non-infectious Pasteurella multocida, Streptococcus suis, Actinobacillus pleuropneumoniae, Actinobacillus suis, Arcanobacterium pyogenes, Bordetella bronchiseptica, Haemophilus parasuis Erysipelothrix, Salmonella spp., E. coli, Clostridium perfingens type A and C, Enterococcus durans, Lawsonia intracellularis, Brachyspira spp. Coccidia, Cryptosporidia, round worms, whip worms PCV2, PRRS virus, SIV, cytomegalovirus/inclusion body rhinitis (only if turbinates are submitted), PRV, PEDV, rotavirus, TGE virus, Teschovirus, and more. Mycoplasma hyopneumoniae/hyorhinis/hyosynoviae by PCR Water deprivation, toxicities, deficiencies COMMENTS • PRRS virus is often best isolated from lung lavage fluids. The lung can be lavaged (with cell culture growth media or Lactated Ringers Solution) and the fluid submitted or the lavage can be done at the lab if at least one half of the lung is submitted without holes or slices in it. 44 PORCINE PNEUMONIA / RHINITIS Specimens to submit: Live acutely affected pig(s). Alternatively, tissues should include: Brain 1/2 fresh, and 1/2 formalin-fixed Entire lung (one side) or generous portion of lesion and adjacent Lung unaffected lung, fresh/chilled 4 to 6 thin slices (1 cm) through affected and adjacent unaffected lung, formalin-fixed. At least 3-4 cross sections through anteroventral lung are recommended Swab of deep airway, chilled (Dacron-tipped, slightly moistened, for Nasal swab bacterial and viral cultures) Snout or turbinate Turbinate scroll from one side removed at junction with midline septum, formalin-fixed Tonsil 1/2 fresh, and 1/2 formalin-fixed SAMPLING TECHNIQUES 1. 2. 3. 4. 5. Fresh tissue should be chilled before shipping. Do NOT freeze. Do not submit fresh samples in glycerin (glycerin has not proven to be of value and prevents freezing of sections for FA tests). Samples for virus detection need to be taken at the onset of respiratory signs. Nasal swab preservation: swabs must be kept moist and cool before and during shipment. Fixed turbinate must be submitted to confirm the presence of porcine cytomegalovirus (inclusion body rhinitis) AGENTS DETECTED BY ROUTINE EXAMINATION Pasteurella multocida, Streptococcus suis, Salmonella choleraesuis, Actinobacillus pleuropneumoniae, Actinobacillus suis, Arcanobacterium pyogenes, Bordetella bronchiseptica, Haemophilus parasuis PCV2, PRCV, PRRS virus, SIV, cytomegalovirus/inclusion body rhinitis Viruses (if turbinates are submitted for histopath) Mycoplasma hyopneumoniae Mycoplasma AGENTS REQUIRING SPECIAL TESTS (BY REQUEST) Viruses Isolation, sequencing Bacteria COMMENTS • • • • • Cultures for Mycoplasma hyopneumoniae can be done on fresh/chilled lung but are not routine because of the difficulty of recovering these fragile organisms in the presence of heavy contamination or concurrent bacterial or other mycoplasmal infection. Porcine respiratory coronavirus is more readily detected from nasal swabs than lung tissues. High serum antibody titers to TGE virus in herds with no evidence of TGE diarrhea also may suggest the presence of PRCV. A differential ELISA serology test is available through the VDL. PCR is used on nasal swabs, oral fluids or lung tissues for detection of swine influenza virus with subtyping to determine hemagglutinin (H) and neuraminidase (N) subtypes routine. Sequencing has higher success rate from specimens with lower cycle threshold values. PRRSV is often best isolated from lung lavage samples or serum. The lung can be lavaged (with cell culture growth media or Lactated Ringers Solution) and the fluid submitted. Lung lavages can be done at the lab if at least one half of the lung is submitted without holes of slices. PRRSV sequencing has higher success rate from specimens with lower cycle threshold values. 45 OVINE ABORTION Specimens to submit: Entire fetus and placenta are the preferred specimens. Fetal tissues should include: Brain 1/2 of organ, formalin-fixed Ewe serum Optional, see notes on abortion serology. 3-5 ml ewe's serum Heart 1/2 cm slice, formalin-fixed Kidney 1 entire kidney, fresh/chilled Liver 1/8-1/4 of organ, fresh/chilled 1/2 cm slice, formalin-fixed Lung 1/8-1/4 of organ, fresh/chilled 1/2 cm slice, formalin-fixed Placenta 3 cotyledons, fresh/chilled 2 cotyledons, formalin-fixed Spleen 1/2 of organ, fresh/chilled Stomach contents 1-3 ml syringe or tube, fresh/chilled Thoracic fluid Clear, uncontaminated, fresh/chilled Thymus Fresh/chilled; 1/2 cm slice formalin-fixed Vaginal swabs Optional, select recently aborted ewes SAMPLING TECHNIQUES 1. 2. 3. Do NOT freeze tissues. Submit placenta whenever possible. Submit ewe’s sera, retain 1/2 of sample frozen. AGENTS DETECTED BY ROUTINE EXAMINATION Bacteria Parasites Viruses Trueperella (Arcanobacterium) pyogenes, Bacillus, Campylobacter, Chlamydia, Listeria monocytogenes Toxoplasma gondii (see comments), Neospora Border disease virus COMMENTS • • • • Diagnosis of toxoplasmal abortion can be accomplished through detection of characteristic lesions in placenta and brain and/or detection of antibody in fetal thoracic fluid. Detection of antibody in ewe serum is not evidence for abortion, only infection at some time; antibody titers persist for months. Absence of antibody in the ewe would rule out toxoplasmosis. Diagnosis of Chlamydial abortion is most readily accomplished through an ELISA test conducted on fresh placenta. The ELISA can also be conducted on swabs of fetal fluids or liver or vaginal swabs from affected ewes (within 5 days after abortion). Histopathology is also useful. Cache Valley virus infection can only be identified by serological studies conducted at a reference laboratory. Fetal fluids or precolostral serum from live-born affected lambs can be examined. Analysis of serological results from paired ewes (affected/unaffected) also may be helpful. Chlamydia and Toxoplasma gondii are 2 of the 3 most common infectious causes of ovine abortion. Without placenta, brain, and/or fetal thoracic fluid, we cannot properly address the primary differentials. 46 ABORTION SEROLOGY - GENERAL COMMENTS Fetal serology from fetal thoracic fluids has not proven to be of much value in abortion diagnosis. In sheep, the diagnosis of toxoplasma abortion can be accomplished by detection of antibody in fetal fluids. Fetuses are rarely capable of producing antibody until the last trimester. Fetal thoracic fluids are useful for detection of viruses and should be included in submissions. Serological analysis of maternal serum can aid in the diagnosis of abortion under limited circumstances with some diseases. The lack of antibody can be considered evidence ruling out a given disease. It is more difficult to establish guidelines that point toward a diagnosis. Additional points to consider would include the following: 1. Paired serum samples (at the time of abortion and 2-3 weeks later) may provide more information (i.e., a 4 fold increase in titer) than a single sample. However, as a general rule, the dam has seroconverted at the time of abortion and titers are often maximal or declining. 2. As an alternative to paired samples, comparing mean antibody titers and percent seropositivity between affected and unaffected groups of animals may be helpful. Samples should include serum from approximately 20 aborting animals and at least an equal number of matched (as closely as possible by age and stage of gestation) unaffected animals. 3. With some diseases, many herds are endemically infected and a majority of the animals will have titers. Examples: parvovirus in swine, toxoplasma in sheep, BVD in cattle. Presence of antibody against these organisms in aborting animals is not sufficient evidence of cause. However, high titers may indicate recent infection. The absence of antibody titer could rule out several agents as causal. 4. Response to vaccination also can complicate interpretation of titers. SPECIFIC DISEASES BVD virus Most cattle herds are infected with BVD virus. Virus can circulate within a herd without observable problems. In such herds, many animals will have (SN) titers (2 to >4,096). Vaccination titers vary considerably with the type (killed vs live) vaccine used. Individual animal tests usually are of little value. Paired samples on a portion of the herd may provide information on virus activity within the herd. Antibody titers to both type I and type II BVD viruses should be determined and evaluated in light of the herd vaccination history. Antibody titers in precolostral neonates and unvaccinated calves over 6 months of age are almost always significant. IBR virus Vaccination (SN) antibody titers in cattle usually range from <2-32, occasionally 32-128 after boosters. Recent infection may stimulate antibody titers of 64-256. Paired samples on a number of animals to determine virus activity within the herd may be of more benefit than single samples. Aborted fetuses are usually autolyzed in utero, so fetal serology is not generally useful. Leptospirosis In cattle, infection with serovars other than hardjo usually will stimulate (MAT) antibody titers of 1:800 or greater that will stay high for a few months. Cows aborting due to leptospirosis may be ill, though illness may not be obvious, especially with serovar hardjo. Vaccination antibody titers usually will peak at 1:800 for a short time, but higher titers may be seen occasionally in recently vaccinated animals. Serovar hardjo infection does not stimulate a strong antibody response. Serology is not reliable in diagnosing infection with this serovar. An antibody titer of 1:400 would suggest recent infection with this serovar. In swine, similar comments would apply, but aborting sows and gilts usually will not be ill. Even higher antibody titers (1:32,000) may be seen occasionally in infected swine. In this species, serovar bratislava is the host-adapted strain to which minimal antibody response is expected and antibody titers resulting from infection with serovar bratislava rarely exceed 1:400. Parvovirus (PPV) Nearly all swine herds are infected with PPV. Natural infection usually stimulates (HI) titers, greater than 256 (occasionally over 10,000) that persist for months. Vaccination titers rarely exceed 512 and begin to drop by 2 months. A titer >512 or greater suggests exposure to natural infection at some time. PRRS virus Results of the commercial ELISA currently used are reported as S/P ratios, not as actual antibody titers. An S/P ratio of 0.40 or greater is considered positive with strong positives reported in the 1.5-4.0 range. Recent or active infection may result in value up to 2.5. Differential tests for distinguishing field infection from vaccine are currently NOT available. Toxoplasmosis Not all sheep herds are infected with Toxoplasma gondii. Antibody titers remain for years after infection, thus the presence of antibody is not evidence for current problems. A serum antibody titer merely indicates the animal was infected at some time. 48 SEROLOGY LIST OF SEROLOGY ASSAYS The VDL website (www.vdpam.iastate.edu) contains a complete list of serology tests currently offered, testing schedule and fee for each. TESTING SCHEDULE Our goal is to provide accurate results and timely service. However, many diagnostic assays are not performed daily and/or testing schedules may change. If your case involves testing animals for export, testing show animals, testing animals to be sold, and/or testing a large number of samples, please contact the VDL (515-294-1950) prior to submission to verify the availability and turnaround time of specific assays. A phone call will often shorten turnaround time by allowing us time to prepare for testing prior to receipt of samples. SUBMISSION GUIDELINES Please submit samples according to the following guidelines in order to expedite testing and shorten turn-around time. 1. Provide 0.5 ml of serum per assay requested. Submit serum only. Never submit blood. Even if you use serum separation tubes, serum must be poured off into plastic snap cap tubes. A charge of $.25 per sample will be assessed for processing blood samples. 2. Submit serum in plastic snap-cap tubes (Falcon 2054 or equivalent). Laboratory equipment is designed to fit these tubes - glass tubes are too large. Avoid leakage - be sure to ‘double snap’ the caps on tubes! 3. Number the tubes in consecutive order 1 through 'X' matching the tube number with the tube number on the serology submission form. Use permanent marker to identify tubes. Grease pencil marks are easily rubbed off and labels fall off during the heating process necessary for some assays. 4. Place the tubes in consecutive numerical order in cardboard boxes designed to hold snap-cap tubes (e.g. brucellosis boxes). Do NOT submit in bags. 5. Keep samples from each case or client separate. Save a portion of each serum sample in your freezer. 6. Keep a copy of the paperwork for your files. 7. Optimize the interpretation of test results by collecting acute and convalescent samples. Paired sera (acute and convalescent) should be submitted together. Alternatively, comparing serum antibody levels in affected vs unaffected animals can assist in interpretation. 8. Serology samples submitted to comply with EIA, Brucellosis, and PRV Disease Programs require special submission forms. To obtain forms for EIA and Brucellosis 49 please contact USDA APHIS at 515-284-4140. For other forms, please see the Iowa Department of Agriculture website at: http://www.iowaagriculture.gov/departmentForms.asp. 9. Because of the number of samples we receive, serum samples can only be saved for 2 weeks after testing. We will gladly return samples to you at your expense upon request. SEROLOGY FOR EXPORT CASES Please use the following guidelines for submission of sera for export cases: 1. To minimize testing delays, contact the VDL at least one week before sending sera for testing. Provide the date sera will be delivered to the VDL, the number of samples, the specific assays to be run, and the date results are needed. 2. Indicate on the Serum Submission Form that the testing is for export purposes (check the box) and indicate the importing country. 3. On the Serum Submission Form, clearly indicate the specific tests needed, including the agent, test type, and dilution required. Include a copy of the importing country's requirements, if available, to avoid confusion. Example: Vesicular stomatitis virus (VS) SVN test at 1:8 dilution. 4. If export requirements specify a minimum change in antibody titer, the sera from different bleed dates must be tested as paired sera on the same test date. This information must be communicated to the VDL prior to sending in sera from the initial bleed dates. 5. Never assume test results will be done by a certain date. Follow up your written test requests with a phone call to verify that the correct tests are being performed and that results will be available in the required time frame. REPORTING SEROLOGY RESULTS Serology results will be reported as requested on the Serum Submission Form. The fastest methods of reporting results are by web, email, and/or FAX. If you are not already a web client you may sign up on our web site: http://www.vdpam.iastate.edu/signlims/ 50 INTERPRETATION OF SELECTED SEROLOGIC TESTS ASSAY Actinobacillus pleuropneumoniae CF Anaplasma marginale CF Bovine respiratory syncytial virus VN COMMENTS The CF is a highly specific test for detecting ant-APP antibodies. Samples with antibody titers ≥1:4 are suspect; ≥1:8 are considered positive. Antibodies develop 7 to 10 days after infection, peak at 1:4 to 1:256, and last for several months. Maternal antibody may be detected until 8 to 9 weeks of age. Positive results indicate exposure. Sample quality is important to avoid nonspecific reactions (AC). Vaccine antibody titers are usually ≤1:128. To aid in interpretation, compare titers in clinically affected vs unaffected animals, or acute vs convalescent. BVD virus VN BVD type 1 and type 2 are run in separate assays, but comments apply to both Antibody response is highly variable, in part because strains vary antigenically. Field strains generally produce high neutralizing antibody titers (≥1:2,048). Titers from inactivated-virus vaccines are often ≤1:256 and against MLV vaccines ≤1:2,048. Exposure to vaccine vs field virus cannot be reliably differentiated on the basis of antibody titiers. Compare titers from clinically affected vs unaffected animals to aid in interpretation. Titers ≥1024 in unvaccinated calves >6 months of age suggest exposure to field virus. Histophilus somnus CF 1:64 to 1:128 suggestive of vaccinated animals. 1:64 to 1:256 suggestive of animals vaccinated more than once. 1:256 to 1:512 suggestive of early active or chronic infection. 1:1040 to 1:4096 suggestive of recent active infection. For interpretation, use paired samples or compare affected vs unaffected. Inactivated vaccine antibody titers are usually <1:16 and MLV vaccine antibody titers are usually < 1:64. Serology is of limited diagnostic use in bovine abortions caused by IBR, especially in well-vaccinated herds. Cows generally abort 23 – 53 days post exposure, i.e., cows reach maximum IBR antibody titers prior to aborting. IBR virus (BHV-1) VN Leptospira interrogans MAT Mycoplasma hyopneumoniae ELISA Parainfluenza virus type 3 VN Diagnostically significant antibody titers are usually ≥1:800. Titers have usually developed by the time abortion occurs, so consider comparing samples from clinically affected vs unaffected animals to aid in interpretation. Interpretation requires vaccination history. Vaccine antibody titers may be 1:400 to 1:800. In dogs, if icterohaemorrhagiae and canicola both react, it is probably vaccination. Dogs infected with grippotyphosa may have titers of 1:200 to 1:800. In cattle with reproductive failure associated with hardjo, hardjo antibody titers may be 1:100 to 1:200. The Serology Section of VDL has implemented a USDA-licensed ELISA: Mycoplasma hyopneumoniae antibody test manufactured by IDEXX Laboratories, Inc. Serum samples with S/P ratios <0.3 are considered negative; 0.3 to 0.4 are classified as suspect; and >0.4 are considered positive. Cross-reaction is possible with M. hyorhinis, or M. fluocculare. M. hyopneumoniae resides on the surface of the respiratory tract and may colonize without stimulating an immune response. When the organism does begin to proliferate and induce disease, the immune response is slow to develop. A ubiquitous virus. Positive results indicate exposure. Compare titers in clinically affected vs unaffected animals, or acute vs convalescent. 51 ASSAY Porcine circovirus IFA Porcine parvovirus HI PRRS virus ELISA PRV gI ELISA PRV latex agglutination assay PRV screen ELISA Swine influenza HI TGE virus VN TGE virus/PRC virus differential ELISA COMMENTS The VDL assay utilizes PCV Type 2 as the antigen in the IFA. For screening, samples are run at dilutions of 1:20 and 1:40. The sample is considered positive if reactive at either dilution. Vaccination titers usually ≤1:256. Maternal antibody may persist for months. Although considered ubiquitous, dams in commercial herds frequently have not been exposed to field virus. S/P values ≥0.40 are positive; <0.40 are negative. The manufacturer estimates sensitivity of 100% (90 to 100); specificity of 99.5% (98.3 to 99.9). The commercial ELISA detects antibody against either North American or European strains of PRRS virus. Antibodies are detectable within 14 days of infection or vaccination with modified-live vaccine. The gI ELISA differentiates field virus infection from vaccination. Serum samples with S/N values ≤0.60 are positive for antibodies against field virus; samples >0.60 to 0.70 are suspect, and samples with S/N >0.70 are negative. The PRV latex agglutination assay does not differentiate between field infection and vaccination. A latex agglutination index of <4.5 is negative for PRV antibodies; >8.0 is positive; and intermediate values are suspect. Serum samples with S/P values <0.4 are negative; samples ≥0.40 are positive. The PRV screen ELISA does not differentiate between field infection and vaccination. Interpretation of HI results is the same for SIV H1 and SIV H3. Antibody titers ≥1:40 are positive, although clinically relevant titers are usually ≥1:80. Titers rise to ≥1:320 by 14 days post infection and persist for approximately 4 weeks before declining. Antibody ≥1:40 interfers with inactivated vaccine. Maternal antibody in pigs from unvaccinated sows will last up to 8 weeks; in pigs from vaccinated sows up to 20 weeks of age. Neutralizing antibody titer of ≥1:4 is considered positive, although recent infection usually results in titers ≥1:32. Vaccination titers are usually ≤1:16. Antibodies against TGE virus and PRC virus are indistinguishable by VN. Antibodies against TGE virus can be differentiated from antibodies against PRC virus using ELISA. The test is not accurate for recent infection (<28 days). 52 TOXICOLOGY/NUTRITION LIST OF TOXICOLOGY ASSAYS The VDL website (www.vdpam.iastate.edu) contains a complete list of chemistry tests currently offered, fee for each test, and prefered samples to submit. Our goal is to provide accurate results and timely service. However, many diagnostic assays are not performed daily and/or testing schedules may change. If your case involves testing animals for export, testing show animals, testing animals to be sold, and/or testing a large number of samples, please contact the VDL (515-294-1950) prior to submission to verify the availability and turnaround time of specific assays. A phone call will often shorten turnaround time by allowing us time to prepare for testing prior to receipt of samples. SUBMISSION GUIDELINES The purpose of the diagnostic toxicology section is to provide consultation, suggestions, and interpretation regarding suspected toxicoses. This consultation includes information about specific samples or specimens useful in confirming diagnoses suspected by the attending clinician. To best accomplish this, a thorough interchange of information between clinician and diagnostician is important. A written or telephone history from the attending veterinarian is useful if the analyses are to be suggested by the toxicologist. A complete account of history (including management and feed type), clinical signs, and lesions submitted with specimens for laboratory evaluation is very important. Currently, the practice of diagnostic toxicology requires that specific analyses or chemical categories be selected. There is no practical or affordable way to check for all possible poisons. The more information provided, the more efficient and useful is the selection process. The VDL offers services for nutritional monitoring through analysis of feeds, serum and liver for minerals and selected vitamins. Water quality analysis is also available. The VDL does not perform proximate analysis (TDN, protein, etc.) on feeds or forages. Selecting specimens for toxicology and chemical evaluation requires three main criteria: 1. The correct specimens must be provided. 2. Adequate amounts must be available. 3. Specimens must be properly preserved. Choice and condition of specimens is extremely important when diagnostic support is required for a suspected toxicology case. The following are guidelines to increase the usefulness of toxicologic submissions. 53 1. Specimens should be free of chemical contamination and debris. Contamination of samples with hair, vomitus, dust, dirt, etc., may contaminate the sample and produce erroneous results. 2. Freeze animal and tissue specimens for chemical analysis and package them to arrive at the laboratory while still frozen. Do NOT freeze whole blood samples, but keep them refrigerated. 3. Serum, if separated from the clot and not hemolyzed, may be frozen. In some cases, such as analysis for ammonia, this is essential. 4. Always package specimens from various organs and fluids separately. 5. Use clean glass or plastic containers that can be tightly sealed. 6. Label each specimen (container) so it can be clearly identified. 7. Never add preservative, such as formalin, unless there is a specific reason. If preservatives are added, include a sample of the preservative along with the specimen. 8. Submit samples for chemical analysis of low level organic contaminants, such as pesticide residues or PCB's, in glass containers (line lids with foil), not plastic. Solid samples for this purpose may be wrapped in aluminum foil. 9. If ammonia, urea, or cyanide toxicosis is suspected, freeze rumen contents and blood or serum immediately after collection. Keep them frozen. 10. On all toxicology cases involving a dead animal, submit both fresh and fixed tissues. MAINTAINING PACKAGE INTEGRITY In situations where integrity of a package must be maintained for legal or insurance purposes, sealing of the package and transmittal is recommended according to the Federal Bureau of Investigation Guidelines as follows: 1. 2. 3. 4. 5. 6. Place specimens securely in box. Seal box. Place copy of transmittal letter in envelope and mark ‘invoice.’ Stick envelope to outside of sealed box. Wrap sealed box in outside wrapper and seal with gummed paper. Address to laboratory, with attention to a specific person if possible. TISSUE SPECIMENS Analyses are directed toward exposure sites (skin and digestive tract), areas of metabolism and excretion (liver, kidney, urine), and accumulation in specific affected organs or storage sites (e.g., brain, fat, bone). 54 SPECIMENS TO SUBMIT FROM A LIVE ANIMAL: Milk (if appropriate) Serum (clot removed) Urine Vomitus or feces Whole blood (in anticoagulant) 100 ml 5 ml 50 ml 200 grams 10 ml SPECIMENS TO SUBMIT FROM A DEAD ANIMAL: Body fat Bone Brain Eyeball Kidney Liver Rumen or stomach contents Serum or whole blood (if available) Urine 100 grams 100 grams 1/2 frozen, 1/2 formalin-fixed (divide by midline sagittal section) Whole eye or aqueous humor 100 grams, and formalin-fixed 100 grams, and formalin-fixed 200 grams 10 ml 50 ml FEEDS AND ENVIRONMENTAL SAMPLES Sampling error or collection of a non-representative sample is often a weak link in submitting feeds or forages for analysis. General rules for sampling follow: Grain, mixed feed 1. Take multiple samples from a moving stream of grain/feed or by probe sampling of a bin. Mix these samples thoroughly and retain a representative 1 kilogram sample for analysis. 2. To prevent condensation and fungal growth during transport, do not ship high-moisture samples unless feed is first dried or frozen. Cloth or paper packaging may be better suited to maintain grain/feed sample integrity during shipping, since plastic bags promote fungal growth. Forage 2. Sample size for hay and silage should be at least 1 quart. Cut all forages to a length of 3" or less. 3. Pack samples tightly to exclude air, and seal airtight in plastic bags. Very dry samples may be submitted in paper bags. 4. Transport samples to the laboratory as quickly as possible. For silage or green chop, freezing is essential. 55 5. Take multiple samples from baled or loose hay. A Penn State forage sampler or equivalent core sampling device is recommended. 6. For square bales, sample from the end of the bale the full length of the sampler tube. 7. For round bales, sample across the bale at the center. 8. Take standing pasture and row crop samples from a minimum of 8-10 locations within a field. Remove forage from a four-foot area at grazing height. Mix all collected forage and take a representative sample (500 grams) for analysis. 9. In some cases, it might be advisable to retain larger samples of feed or forage in case feeding of test animals becomes important. For this purpose, a minimum of 5-10 pounds or more may be required depending on the animal desired for experimental feeding. Feeding trials may require a 10-14 day (or more) supply of feed. If a feeding trial or bioassay is desired, please contact the laboratory for further details. Water 1. Water samples should represent the water to be tested. Let hydrant or faucet run for a few seconds before collecting sample. 2. Rinse container with the water to be tested prior to collecting the sample to be tested. 3. Submit water samples for inorganic analyses in clean containers of either glass or plastic. Containers should be sterile if microbiologic testing is requested. 4. Submit all water samples for organic analyses in clean glass jars with clean aluminum foil placed over the mouth of the jar before attaching the lid. 5. Transport samples to the laboratory as soon as possible. RESULTS AND INTERPRETATION The interpretation of the clinical significance of chemical data should be done carefully taking into consideration other evidence presented in the case. Positive chemical findings are not always evidence of intoxication nor are negative findings always indicative that toxicosis did not occur. For example, chlorinated hydrocarbon insecticides may accumulate to high levels in fatty tissues of animals without being associated with clinical signs. On the other hand, organophosphate insecticides rapidly disappear from body tissues and their absence would not guarantee the animal had not been poisoned. 56 VIROLOGY LIST OF VIROLOGY ASSAYS The VDL website (www.vdpam.iastate.edu) contains a complete list of virology tests currently offered, testing schedule and fee for each. TESTING SCHEDULE Our goal is to provide accurate results and timely service. However, many diagnostic assays are not performed daily and/or testing schedules may change. If your case involves testing animals for export, testing show animals, testing animals to be sold, and/or testing a large number of samples, please contact the VDL (515-294-1950) prior to submission to verify the availability and turnaround time of specific assays. A phone call will often shorten turnaround time by allowing us time to prepare for testing prior to receipt of samples. SUBMISSION GUIDELINES Successful isolation and/or detection of viruses in clinical materials depends largely on proper collection and handling of specimens. Care should be taken to protect the virus in specimens from environmental damage and maintain virus infectivity by using the proper transport system. In general, specimens intended for virological testing should be collected as early as possible in the course of the disease, i.e., within the first 7 days after the onset of illness. Samples collected during the acute phase of viral infection usually contain adequate amounts of virus for detection in available assays. Samples collected later in the course of infection usually require more laboratory time and often yield poor or negative results. Since certain viral infections may predispose the host to secondary viral or bacterial infections, samples collected late in the disease process may lead to a misdiagnosis when secondary infection is involved. The appropriate samples for virus detection include bodily fluids and secretions (e.g., nasal or conjunctival secretion, genital swabs, urine, saliva, vesicle fluid, semen, milk), feces, blood samples, and skin biopsies from infected, live animals (i.e., antemortem), and relevant tissues and organs from necropsied animals (i.e., postmortem). The collection of antemortem samples from sick animals should be based on clinical manifestation. For example, nasal or nasopharyngeal swabs should be collected from animals with respiratory diseases; fecal samples from animals with enteric diseases; cerebrospinal fluid (CSF), nasal secretion and feces from animals with CNS signs; vesicle fluid and biopsies from animals with skin lesions. The same principle can be applied to the collection of postmortem samples. As a general practice, whole blood* and serum should always be collected from animals with suspected viral diseases, regardless of clinical manifestations. Secondary lymphoid tissues (e.g., tonsil, lymph nodes, spleen) are always good specimens for viral diagnosis. 57 *For collecting whole blood, citrate is preferable to EDTA as an anticoagulant because EDTA may inactivate some viruses due to its chelating activity. For best results in isolation and detection of viruses, clinical specimens should be aseptically collected, kept fresh, and transported immediately to the laboratory. If delays are unavoidable or any detrimental affects on virus in samples are anticipated during transport, samples should be refrigerated at 40ºF (4ºC) for no more than 2 days. For longer storage periods, freeze samples at minus (-) 70ºC, but NEVER at minus (–) 20ºC. Self-defrosting freezers in conventional refrigerators are not appropriate for storage. NEVER freeze whole blood samples. Ideally, frozen samples should be submitted on dry ice, but commercial refrigerant packs can be used if necessary. Unbleached swabs (e.g., Dacron swabs are available from Baxter) are strongly recommended for collecting nasal and fecal swabs. Standard cotton swabs contain residual bleach that can inactivate viruses. Swabs MUST be prevented from drying. For that reason, swabs may need to be placed in a viral transport system. Ideally, swabs should be placed in a broth medium or balanced salt solution supplemented with 0.5% gelatin, serum, or bovine serum albumin, to protect the viability of viruses in transit plus antibiotics to prevent bacterial and fungal growth. Minimally, physiological saline or Lingo solution could be used on an emergency basis. Specimen collection and transport systems (e.g., Viral Culturette, Becton Dickinson) are available. It is important to choose not only the most appropriate specimen, but also to collect an adequate amount of specimen for virological testing. Submit a minimum of 3-4 ml serum and 3-5 grams or 5 ml wet volumne of fecal material. Insufficient amounts of sample are a potential cause of inconclusive diagnosis or false-negative result. When it is necessary to ship a specimen, use a leak-proof container (e.g., tubes, plastic bags) enclosed in a second watertight container containing absorbent material. Ideally, the specimen container should be placed in a Styrofoam box with commercial refrigerant packs or dry ice. Avoid using wet ice because it will melt and leak from the package. Dry ice is preferable if transport requires more than 3 days. All specimens should be packed in a manner to avoid leakage or breakage, and to withstand the trauma of mailing. Shipping agencies may choose not to deliver leaking packages because of new Department of Transport regulations on shipment of potentially biohazardous materials. In addition, a special permit may be required for interstate transportation of certain veterinary viruses within the United States. Practitioners are referred to published federal guidelines and regulations for details pertaining to packaging, labeling, and interstate shipping of infectious agents (Title 42 CFR Part 72; Title 49 CRF Part 173.386-388). All specimens should be properly labeled and accompanied by the proper VDL submission form. Please provide all information requested on the form: animal identification, age or body weight, gender, date of onset of disease, major clinical signs, days of gestation if samples originate from abortions, herd size, number of animals affected or dead, date of collection, 58 animal source and location, vaccination history, and differential diagnosis. This latter information is essential for the selection of the most sensitive test system in the laboratory when samples are submitted by mail. CONVENTIONAL VIROLOGICAL ASSAYS Virus isolation (VI) Virus isolation consists of two steps - the attempted recovery of virus and identification of the isolate. Isolation is attempted using in vitro cell culture. Identification of the isolate is done using immunofluorescence microscopy, electron microscopy, or molecular techniques. Although isolation of virus followed by identification is considered to be the definitive diagnosis, VI is often laborious, expensive, and time consuming. Results are not generally available for 1-2 weeks after submission. Therefore, VI should primarily be attempted under specific circumstances: 1. When other detection methods fail or when trying to isolate virus(es) from previously unrecognized diseases. 2. If there is no other detection method of similar or greater sensitivity. 3. If the virus is required for other purposes, such as differentiation (e.g., RFLP, sequencing), characterization (e.g., typing), and/or for production of autogenous vaccines. Aseptic collection and proper handling of clinical specimens is critical for VI. Although certain viruses (e.g., parvovirus, circovirus) can withstand harsh environmental changes, this is not true for most viruses, particularly enveloped viruses. Virus isolation can be done on most clinical specimens, including biopsy and necropsy tissues, blood, secretions, and excretions. However, some clinical materials, e.g., urine, feces and semen, are difficult to work with because they are toxic to cell cultures. The quantity of sample required for VI is usually more than that needed for rapid diagnostic assays so collect and submit the adequate amount of sample. To avoid unnecessary expense, be specific in your VI requests. Refer to specimen selection guidelines in the User’s Guide or call the laboratory for advice. Fluorescent antibody examination of frozen tissue section (FA) FA is a rapid, specific laboratory diagnostic test for detecting viral antigen in tissues and cell smears. The test utilizes virus-specific antibody labeled with a fluorochrome. When viewed with a fluorescence microscope, complexes of viral antigen and labeled antibody appear as fluorescent green areas. For best results, FA requires very fresh clinical specimens. Freezing and thawing tissues can be detrimental to the test. If autolysis of tissues is anticipated, other antigen detection methods, such as immunohistochemistry (IHC) on formalin-fixed tissues, should be considered. It is important to recognize that VI is a nonspecific test that can detect a variety of viruses. In contrast, FA and IHC are very specific tests that, because they are use virus-specific reagents 59 (e.g., antibody), only detect the targeted virus. Furthermore, FA and IHC cannot be performed on certain specimens, such as serum or feces. Antigen-capturing ELISA (AgELISA) AgELISA is a rapid, sensitive laboratory diagnostic test for detecting virus or viral antigen(s) in a variety of clinical materials, e.g., tissues, serum, secretions, and excretions. The assay is a variant of the ELISA format in which virus-specific antibody is used to coat the surface of the plates instead of antigen. Therefore, virus or antigen in clinical specimen is captured by antibody. The use of virus-specific monoclonal antibody in AgELISAs provides high specificity. The presence of antibody-antigen complexes is subsequently visualized by a colorimetric reaction. In general, color development is proportional to the amount of viral antigen present in the specimen. One advantage of the AgELISA format is that it detects both infectious and inactivated viruses; however, expense is a consideration. EXPORT CASES Submitters should clearly indicate on the submission form that the testing is for export purposes. Clearly indicate the specific test(s) needed, including the agent, test type, the number of passages needed if virus isolation is requested, and any special requirements imposed by the importing country. SUBMISSIONS FOR RABIES EXAMINATION Please use the VDL Rabies Examination Submission Form found on the web at: http://vetmed.iastate.edu/diagnostic-lab/forms when submitting samples for rabies virus testing. Provide a complete history and indicate clearly if human exposure is involved. The absence of a complete history may delay the rabies examination until the laboratory can compile and verify the necessary information. Information to be provided includes: 1. 2. 3. 4. Name of submitting veterinarian or physician. The species of animal submitted and any pertinent description. The name of the person making the request or the name of the person exposed. If a human exposure, the date of exposure and route of exposure. RABIES SPECIMENS The entire animal, chilled (not frozen), should be delivered by private carrier. Alternatively, submit the intact head, properly sealed to prevent leakage, and identified as a rabies suspect. 60 To safely prepare a rabies head: 1. Remove the head by disarticulation of the occipito-atlantal joint. Gloves, face, and eye protection are recommended for this procedure. 2. Refrigerate and package in a leak-proof container with refrigerant packs. 3. Deliver by private or commercial carrier with the package identified as ‘Rabies Suspect.’ 4. Rabies suspects should be euthanized prior to delivery to the VDL, only in exceptional cases and with prior notice should a live animal be submitted. Note: According to the Centers for Disease Control and Prevention (CDC) guidelines, the cerebrum, cerebellum, and hippocampus are REQUIRED for the appropriate diagnostic evaluation of rabies virus infection. RABIES TEST PROCEDURES 1. A microscopic direct fluorescent antibody (DFA) test is performed routinely Monday through Friday on all rabies submissions. Results are available the same day if received before 11 a.m. Samples received after noon may be tested on the next business day. 2. Emergency and weekend rabies test service is provided by the University Hygienic Laboratory (UHL) (319-335-4500) in Iowa City. The UHL is a state-codified lab for rabies testing and provides 7-days-a-week, 24-hours-a-day service for rabies testing at no charge. 3. After-hours submissions to the ISU Vet Diagnostic Lab can now be placed directly in a refrigerator located inside the VDL submission door foyer. Please consult our website www.vdpam.iastate.edu for further information. RABIES RESULTS AND REPORTING 1. All rabies results are reported by telephone as soon as they become available. 2. Test results are reported as ‘inconclusive’ when, as per CDC guidelines, any of the three parts of the brain required for the test are missing and the FA result is negative. 3. Cases are reported as ‘unsuitable’ when autolysis is evident and the FA test result is negative. 4. In all cases, a written report will be provided to the submitter. Additional tests, if requested, will not be performed on a rabies suspect case until the rabies examination is completed. This will usually cause a one-day delay. For this reason, it is important that the carcass and tissues be handled correctly if the differential diagnosis includes agents in addition to rabies virus. 61 ABBREVIATIONS USED IN THE VDL USER’S GUIDE AgELISA AGID ALA BAPA BRCV BRSV BVD cELISA CF EDTA ELISA FA FA FFN HI IBT IFA IHC IPT LAT Mab MAT NADC NVSL PCFIA PCR PCV2 PED PI PPV PRCV PRRS virus PRV RAP RFLP RIV RSAT SIV SPT STT TGEV VI VME VMRI VN Antigen-Capture Elisa (Antigen Detection Assay) Agar gel immunodiffusion Automated latex agglutination Buffered acidified plate antigen test Bovine Respiratory coronavirus Bovine respiratory syncytial virus Bovine viral diarrhea Competitive (blocking) ELISA Complement fixation Ethylenediaminetetraacetate Enzyme-linked immunosorbent assay Fluorescent antibody Fluorescent antibody tissue section Fluorescent focus neutralization Hemagglutination inhibition Immunoblot assay Indirect fluorescent antibody Immunohistochemistry Immunoperoxidase test Latex agglutination Monoclonal antibody Microscopic agglutination test National Animal Disease Center (USDA:ARS) National Veterinary Services Laboratories (USDA:APHIS) Particle concentration fluorescence immunoassay Polymerase chain reaction Porcine circovirus type 2 Porcine epidemic diarrhea virus Persistent infection Porcine parvovirus Porcine respiratory coronavirus Porcine reproductive and respiratory syndrome virus Pseudorabies (Aujeszky’s disease) virus Rapid automated presumptive test Restriction fragment length polymorphism Rivanol precipitation – plate agglutination test Rapid slide agglutination test Swine influenza virus Standard plate agglutination test Standard tube agglutination test Transmissible gastroenteritis virus Virus isolation Veterinary Medicine Extension, Iowa State University Veterinary Medical Research Institute, Iowa State University Serum-virus neutralization 62 INDEX OF ENTRIES Abortion, bacterial ...................................................................... 20 Abortion, mycotic ....................................................................... 20 Actinobacillus pleuropneumoniae ........................................... 49, 56 Actinobacillus suis ........................................................................ 49 Actinomyces bovis ......................................................................... 20 Actinomycosis ............................................................................. 20 Ammonia ...................................................................................... 59 Anaerobic infections ................................................................... 20 Anaplasma marginale .................................................................... 56 Anthrax ........................................................................................ 20 Antibiotics..................................................................................... 63 Antinuclear antibody titer (ANA) ............................................ 20 Antrophic rhinitis ....................................................................... 20 Arcanobacterium pyogenes................ 20, 38, 39, 42, 43, 44, 49, 51 Arthritis .................................................................................. 20, 21 Aspergillus ............................................................................ 20, 38 Atrophic rhinitis.......................................................................... 20 Bacillus ....................................................................... 20, 38, 43, 51 Bacillus anthracis .......................................................................... 20 Bacteroides................................................................................... 20 Black leg ....................................................................................... 21 Border disease virus ................................................................... 51 Bordetella bronchiseptica ......................................................... 20, 49 Botulism ....................................................................................... 21 Bovine coronavirus ..................................................................... 40 Bovine herpesvirus ..................................................................... 39 Bovine respiratory disease......................................................... 21 Bovine respiratory syncytial virus............................................ 56 Brachyspira ............................................................................ 18, 24 Brachyspira hyodysenteriae ..................................................... 24, 46 Brachyspira pilosicoli .................................................................... 47 BRCV ............................................................................................ 42 BRSV ............................................................................................. 42 Brucella....................................................................... 20, 21, 38, 43 Brucella ovis .................................................................................. 21 Brucellosis .............................................................. 10, 11, 13, 21, 54 BVD virus ............................................... 32, 33, 36, 40, 41, 52, 56 Calcium ........................................................................................ 39 Campylobacter ......................................................... 20, 21, 38, 51 Campylobacteriosis .................................................................... 21 Carbon monoxide ....................................................................... 43 Chlamydia .......................................................................21, 21, 51 Chlamydia psittaci ......................................................................... 21 Chlamydial infections ................................................................ 21 Chlorinated hydrocarbon insecticides ...................................... 61 Cholinesterase ............................................................................. 39 Clostridium ................................................... 21, 22, 40, 41, 46, 47 Clostridium difficile ....................................................................... 46 Clostridium perfringens .................................................... 22, 41, 47 Coccidia .......................................................... 39, 40, 41, 46, 47, 48 Colibacillosis ................................................................................ 21 Coliform bacteria ......................................................................... 24 Coronavirus .................................................................... 40, 41, 50 Corynebacterial pneumonia of foals ......................................... 21 Corynebacterium ............................................................... 21, 23, 24 Corynebacterium renale ................................................................. 24 Cryptococcus neoformans .......................................................... 23 Cryptosporidia............................................................................. 40 Cyanide......................................................................................... 59 Cystitis .......................................................................................... 21 Cytomegalovirus ......................................................................... 49 Dermatomycosis .......................................................................... 22 Dermatophilosis .......................................................................... 22 Dermatophilus congolensis ............................................................ 22 E. coli ...................................................... 21, 22, 40, 43, 44, 46, 47 Edema disease....................................................................... 22, 44 Enteric disease ............................................................................. 62 Enterococcus .............................................................22, 40, 46, 47 Enterococcus durans ......................................................... 40, 46, 47 Enterotoxemia .............................................................................. 22 Erysipelas ..................................................................................... 22 Erysipelothrix rhusiopathiae ................................................... 21, 22 Eubacterium ................................................................................... 20 Exudative epidermitis................................................................. 22 Fusobacterium ............................................................................. 20 Gangrene disease......................................................................... 21 Greasy pig disease....................................................................... 22 Haemophilus ...............................................................21, 22, 44, 49 Haemophilus parasuis ....................................................... 22, 48, 49 Hemagglutinating encephalomyelitis virus ............................ 45 Histophilus somnus ....................................... 21, 23, 38, 39, 42, 56 IBR virus .............................................................. 33, 38, 42, 53, 56 IgG level, single radial immunodiffusion ................................ 20 IHC ............................................... 31, 32, 35, 36, 38, 40, 41, 42, 64 in situ hybridization .................................................................... 25 Insecticides ................................................................................... 61 Keratoconjunctivitis, bovine ...................................................... 22 L. interrogans serovar bratislava................................................. 53 L. interrogans serovar hardjo ...................................................... 53 Lawsonia intracellularis ................................................................. 23 Lead................................................................................... 18, 39, 62 Leptospira..................................................... 20, 22, 38, 43, 44, 56 Leptospira interrogans ................................................................... 56 Leptospirosis .............................................................22, 38, 43, 53 Listeria monocytogenes .................................... 20, 22, 38, 39, 51 Listeriosis...................................................................................... 22 Magnesium .................................................................................. 39 Malessezia pachydermatis......................................................... 23 Mannheimia hemolytica .......................................................... 21, 42 Mastitis ................................................................................... 18, 23 Meningitis .................................................................................... 23 Microsporum ............................................................................... 22 Mineral .......................................................................................... 58 Moraxella bovis ............................................................................. 22 Mycobacterium paratuberculosis................................................... 22 Mycoplasma .................................... 21, 21, 23, 38, 42, 49, 50, 56 Mycoplasma bovis ......................................................................... 42 Mycoplasma dispar........................................................................ 42 Mycoplasma hyopneumoniae.............................................49, 50, 56 Nematodes .......................................................................41, 46, 48 Neospora caninum ................................................................... 33, 38 Nocardiosis .................................................................................. 23 Oral fluids ..................................................................................... 27 Organophosphate ........................................................... 39, 44, 61 Otitis externa ............................................................................... 23 Parainfluenza virus type 3 ................................................... 42, 57 Paramyxovirus ...................................................................... 13, 45 Parvovirus .................................................... 43, 44, 52, 53, 57, 64 Pasteurella multocida ................................................. 20, 21, 42, 49 PCR ................................................................. 25, 26, 27, 43, 47, 50 PCV2 ............................................................................................. 44 PED ................................................................................... 46, 47, 67 Phycomycetes .............................................................................. 38 Pleuropneumonia ....................................................................... 23 Porcine circovirus .................................. 29, 30, 43, 44, 47, 49, 57 Porcine Epidemic Diarrhea ..............................................See PED Porcine parvovirus ......................................................... 44, 53, 57 Porcine proliferative enteritis .................................................... 23 Porcine respiratory coronavirus ......................................... 50, 57 Proteus .................................................................................... 21, 23 PRRS virus .............................................. 33, 34, 43, 44, 49, 53, 57 Pseudorabies virus ....................................... 10, 13, 39, 44, 54, 57 Pseudotuberculosis ......................................................................... 23 Pyelonephritis, bovine ................................................................ 24 Rabies ................................................................... 10, 39, 45, 65, 66 Rheumatoid Factor test............................................................... 20 Rhodococcus equi ........................................................................... 21 Rotavirus ...................................................................40, 46, 47, 48 Salmonella ............................................. 24, 40, 41, 43, 46, 47, 49 Salmonella choleraesuis .................................................................. 49 Salmonellosis ............................................................................... 24 Selenium ....................................................................................... 44 Staphylococcus aureus ................................................................... 22 Staphylococcus hyicus ................................................................ 22 Strangles ....................................................................................... 24 Streptococcus .......................................... 20, 22, 23, 24, 43, 44, 49 Streptococcus equi ....................................................................... 24 Streptococcus suis .......................................................20, 23, 44, 49 Sulfadimethoxine ........................................................................ 18 Sulfathiazole................................................................................. 18 Swine dysentery .................................................................... 13, 24 Swine influenza virus .................................................... 49, 50, 57 Teschovirus ........................................................................... 45, 48 TGE virus............................................................ 46, 47, 48, 50, 57 Toxoplasma gondii................................................... 38, 51, 52, 53 Trichomonas fetus .......................................................................... 38 Trichomoniasis ............................................................................ 24 Trichophyton ............................................................................... 22 Trueperella (Arcanobacterium) pyogenes ............................... 38, 51 Urea ............................................................................................... 59 Ureaplasma ........................................................................... 24, 38 Vesicular stomatitis virus ........................................................... 55 Water deprivation ....................................................................... 44 Water quality ......................................................................... 24, 58 Whipworms .......................................................................... 46, 48