Download S - College of Veterinary Medicine

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