Washington Animal Disease Diagnostic Lab

  

Molecular diagnostic assays detect nucleic acid (DNA or RNA) of infectious disease agents within test specimens. Test specimens may be swabs, fluids, blood, or tissues. A test simply designated as “PCR” is a polymerase-chain-reaction test to detect DNA and is composed of 3 basic parts:

1. Extraction of the DNA from the sample,
2. Addition of sample DNA, one set of DNA nucleotide primers and other reagents to a PCR-cycler machine for amplification of target DNA
3. Detection of target DNA by gel-electrophoresis

RT-PCR is a reverse-transcriptase PCR test to detect RNA and is composed of the same 3 basic parts as PCR and an additional step using reverse-transcriptase enzyme to synthesize complementary DNA from the target RNA. The complementary DNA is then run in the PCR test.

Nested PCR is a modification that uses 2 sets of nucleotide primers and 2 complete cycles of amplification; the second cycle of amplification further amplifies a target fragment of DNA originating within an already amplified larger target fragment of DNA. Nested PCR results in higher sensitivity than simple PCR or RT-PCR and is used for diseases that have very little target nucleic acid in tissue samples.

Real Time PCR (also called quantitative PCR) is the same as PCR except that it simultaneously amplifies and quantitates the amount of DNA after each PCR cycle. It is based on the detection and quantitation of fluorescence of a reporter dye attached to DNA probe which is specific for the amplified DNA of interest (target) The test is very specific and about 10,000 more sensitive than conventional PCR in detecting DNA.

General guidelines for sample submission

Preferred specimens for each test are listed in the table below. Under special circumstances, we may test other specimens; however, please contact the lab before submitting these. Submit all samples as quickly as possible and keep them chilled during transport to the lab for best results (refer to “WADDL Sample Submission”). Formalin can degrade DNA or RNA over time, so paraffin-embedding should be done as quickly as possible for best results from fixed tissues.

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BVD-PI (Persistent Infection) Ear Notch Testing Program
Number of Cattle Tested	PCR Pool Size	PCR Charge per Head	Ag-ELISA charge per Head
<12 samples	No Pooling	NA	$5.00
12-35 samples	12	$3.95	$1.75 *
36-99 samples	up to 36	$2.95	$1.75 *
100 or more samples	up to 36	$1.95	$1.75 *

* BVD Ag-ELISA testing on individual samples within a PCR positive pool
   Non-Washington samples will be assesed a 50% surcharge. (See General Policies #2, above)

Also see: Bovine Viral Diarrhea Virus Persistent Infection (BVD-PI) Ear Notch Testing Program for Cattle Herds

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Test Specifics

Fee Schedule

Test	Fees	Specimen	Turnaround (days)		Comments
			Ave.	Max.
Anaplasma marginale	fee schedule	EDTA blood, fresh tissue (lung, spleen) fixed tissue block	3	8
Avian influenza	fee schedule	Tracheal or cloacal swabs (up to 5 pooled samples) in 2 ml BHI. Fresh lung, spleen, or intestine (no pooling for fresh tissue). Whole bird.	2	7	NAHLN Assay Contact Lab
Avian paramyxovirus (Newcastle's Disease)	fee schedule	Tracheal or cloacal swabs (up to 5 pooled samples) in 2 ml BHI. Fresh lung, spleen, brain, or intestine (no pooling for fresh tissue). Whole bird.	2	7	NAHLN Assay Contact Lab
Bluetongue virus	fee schedule	EDTA blood, fresh tissue (lung, spleen) fixed tissue block	3	7
BVD (blood)	fee schedule	EDTA blood	3	6	Pooled testing of EDTA samples available; contact lab
BVD (ear notch)		Ear notch skin	3	6	See BVD-PI (Persistent Infection) Ear Notch Testing Program
BVD (milk or tissue)	fee schedule	milk, fresh tissue (lung, spleen) fixed tissue block	3	7
Classical swine fever (CSF, Hog Cholera)	fee schedule		2	7	NAHLN Assay Contact Lab
Epizootic hemorrhagic disease virus (EHD)	fee schedule	EDTA blood, fresh tissue (lung, spleen) fixed tissue block	3	7
Canine influenza virus	fee schedule	Tracheal wash, BAL, fresh tissue, nasal swab, oropharyngeal swab, fixed tissue block	2	7	Lung or bronchial lymph nodes are best tissues
Clostridium perfringens	fee schedule	Feces, intestinal tract tissue, isolate	3	7	Genotyping is done from bacterial isolates. Detection of types A, B, C, D, E, Beta 2, and enterotoxin gene
E. coli virulence	fee schedule	Feces, intestinal tract tissue, isolate	3	6	Assay is performed from bacterial isolates. Routine assay detects F5(K99), F41, eaeA (intimin), stx1, and stx2 genes.
Equine Herpesvirus-1 (EHV-1) neuropathogenic and wild type	fee schedule	EDTA blood, nasal swab, lung tissue	1	7	Shipping:  Ice pack
Flavobacterium psychrophilum	fee schedule	Tissue, isolate on slant or plate	2	7	Assay is performed from bacterial isolates.
Francisella Tularensis (Tularemia)	fee schedule	EDTA blood Culture isolate	2	7	Zoonotic Disease Surveillance Program Contact Lab
Fungal identification by universal primers and sequencing	fee schedule	Fixed tissue block (affected organ), fresh tissue, isolate on SAB slant	7	20
Helicobacter spp.	fee schedule		3	7
Malignant Catarrhal Fever virus (MCF)	fee schedule	EDTA blood, Fresh or fixed tissues. Lymphoid tissues such as spleen, lymph nodes, lung, or kidney are best. Liver and intestine may be tested, but sometimes contain PCR inhibitors. Brain may be tested, but false negatives may occur due to very low viral replication in the brain.	2	7	Species being tested and clinical history is essential for the lab to perform the proper assay. Real time PCR specific for ovine herpesvirus-2 is used for clinical cases only. Nested PCR is used for non-clinical carrier species or for other MCF viruses.
Mycobacterium avium	fee schedule	Fresh or fixed tissues, culture	3	7
Mycobacterium avium/ M. genavense duplex	fee schedule	Fresh or fixed tissues, culture	3	7	For birds or other species where one of these Mycobacterium species is suspected.
M. avium ssp. paratuberculosis (Johnes)	fee schedule	Culture slant	3	7	Assay is performed from bacterial isolate
M. avium ssp. paratuberculosis (Johnes)	fee schedule	Feces or fresh intestine	4	8	Contact laboratory for information on pooled fecal testing.
Mycobacterium spp. identification by universal primers and sequencing	fee schedule		7	20
Mycoplasma bovis	fee schedule	Culture broth (preferred) or plate	3	7	Culture may be done in WADDL Bacteriology lab. Speciation of bacterial isolate by PCR.
Myxobolus cerebralis (Whirling Disease)	fee schedule		3	7
Neospora caninum	fee schedule	Fixed tissue block (brain or affected organ)	4	7
Renibacterium salmoninarum (BKD)	fee schedule	Tissue pool (processed at WADDL)	4	7	WADDL Aquaculture Section preprocessed samples for PCR. Samples which test negative may be further tested by nested PCR as required by OIE or Blue Book standards for pre-shipment, at extra charge.
Rhodococcus equi	fee schedule	Tracheal wash	1	5
Toxoplasma gondii	fee schedule	Fixed tissue block (brain or affected organ)	4	7
Tritrichomonas foetus	fee schedule	Prepucial wash or vaginal scraping using In-Pouch transport/culture system	3	7	Shipping:  Must be sent to WADDL by FEDEX or other 48 hour shipping method.  Keep sample at room temperature or 37 degrees C before shipping and ship without ice
West Nile virus	fee schedule	Whole bird. Brainstem from horse. Brain, tissue pool from others. Mosquito pool (25 or less in 2.0 ml microfuge tube)	3	8
Yersinia pestis(Plague)	fee schedule	Culture isolate	2	7	Zoonotic Disease Surveillance Program Contact Lab
Yersinia ruckeri	fee schedule	Slant of plate with isolate. Tissue.	3	7	PCR done on isolated colonies.
Direct PCR / Sequencing	fee schedule	Fresh or fixed tissue. Isolate (fungi, bacteria, mycobacteria) on slant	7	20
Cloning / Sequencing	fee schedule		15	40

Fee Schedule

NAHLN - National Animal Health Laboratory Network

Contact Lab for large volume testing