National Veterinary Services Laboratories (NVSL) Update - Dr. Paul Hauer, USDA/APHIS/NVSL, from the 2012 Annual Conference of the National Institute for Animal Agriculture, March 26 - 29, Denver, CO, USA. More presentations at: http://www.trufflemedia.com/agmedia/conference/2012-decreasing-resources-increasing-regulation-advance-animal-agriculture

1. Safeguarding Animal Health
National Veterinary Services
Laboratories Update

2. Safeguarding Animal Health
Director
Elizabeth Lautner
Diagnostic Virology Laboratory
Director
Beverly Schmitt
Equine + Ovine Viruses
Eileen Ostlund
NAHLN
Barbara Martin
Program & Administrative
Services
Carl Nagle
Laboratory Resources Unit
Lorie Walsh
Avian Viruses
Vacant
Bovine, Porcine + Aquaculture
Viruses
Sabrina Swenson
Diagnostic Bacteriology Laboratory
Director
Matthew Erdman
Mycobacteria + Brucella
Suelee Robbe-Austerman
Serology
David Kinker
Bacterial Identification
Vacant
Foreign Animal Disease
Diagnostic Laboratory
Director
Vacant
Diagnostic Services
Fernando Torres-Velez
Reagents +
Vaccine Services
Wei Jia
Proficiency +
Validation Services
Mike McIntosh
Pathobiology Laboratory
Director
Arthur Davis
Pathology, Parasitology +
Entomology Section
S. Mark Hall
Chemistry +
Analytical Services
Walter Hyde
Associate Director
Paul Hauer
Chief of Staff
Beth Harris
Administrative Unit
Michael Sibert
Quality Manager
Tina Buffington
Parasitology Sub-section
Jack Schlater
Technicians Sub-section
Bruce Thomsen
Reagents Sub-section
Randy Capsel
NVSL Structure

3. Safeguarding Animal Health
NVSL Mission
• To safeguard U.S. animal health and contribute to
public health by ensuring that timely and accurate
laboratory support* is provided by a nationwide
animal health diagnostic system.
 Reference and confirmatory laboratory for USDA
FY 2011 (Oct 2010 – Sept 2011): 66,800 accessions and 533,400
tests approved.
* Includes both domestic and foreign animal diseases

4. Safeguarding Animal Health
NVSL Activities
• Conduct diagnostic testing
• Supply reference reagents to other laboratories
 Exchange diagnostic reagents with other laboratories to standardize and harmonize testing
• Provide training in diagnostic techniques
 State, university, private and foreign government laboratory personnel
 APHIS or other agency personnel
• Conduct proficiency testing of other laboratories
• Consult on agents/techniques
• Conduct developmental projects to improve diagnostic techniques for diseases
of significance
• Participate in World Organization for Animal Health (OIE) Collaborating Centre
for the Diagnosis of Animal Diseases and Vaccine Evaluation in the Americas –
CVB, ISU
• Serve as OIE reference laboratory
 high pathogenicity avian influenza, anthrax, pseudorabies, bluetongue, contagious equine metritis,
equine encephalomyelitis, equine infectious anemia, leptospirosis, Newcastle disease, swine
influenza, vesicular stomatitis and West Nile encephalitis
• Serve as FAO reference center for vesicular diseases, classical swine fever,
African swine fever
4

5. Safeguarding Animal Health
Diagnostic Virology Laboratory
• Dr. Bev Schmitt,
Director
• Dr. Sabrina
Swenson,
BPA Section Head
• Dr. Eileen Ostlund,
EO Section Head
• [Vacant],
Avian Section Head
5

6. Safeguarding Animal Health
Schmallenberg Virus Update
• First identified in Europe in August, 2011
• Affects sheep, goats, cattle, possibly
other ruminants.
• Impact on wild ruminants unknown at this
time.
• Fever, anorexia, diarrhea, and decreased
milk production seen in adult cattle
• Clinical signs non-specific; fetal
malformations in sheep, goats or cattle
6

7. Safeguarding Animal Health
Schmallenberg Virus - Agent
• Closely resembles viruses in the genus
Orthobunyavirus.
• Other closely related viruses are Shamonda,
Aino and Akabane viruses.
• Not considered a human disease risk by CDC
at this time.
• Vaccine development underway; not
available at this time.
7

8. Safeguarding Animal Health
Schmallenberg Virus – Vector Range
• Most likely transmitted by biting midges, thus
transmission occurs during seasons when
insects are most active. Fetus affected when
dam infected during early pregnancy.
• Belgian study – identified positive pools of
Culicoides obsoletus and C. dewulfi
• C. dewulfi not recorded in North America
• C. obsoletus occurs in most of the U.S. and
southern Canada
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9. Safeguarding Animal Health
Schmallenberg Virus – U.S. Response
• Has not been found in the U.S. at this time
• APHIS has issued an import alert which requires
specific collection conditions for semen, embryos
and other genetic material from the EU.
• Trade restrictions are in addition to those already
in place for the EU because of BSE.
• USDA developed general information sheets to
share with stakeholders and general public.
• www.aphis.usda.gov/publications/animal_health/
2012/schmallenberg_virus.pdf
9

10. Safeguarding Animal Health
Schmallenberg Virus – Laboratory
Capacity
• NVSL has obtained the virus, protocols and
reagents for PCR testing from Germany.
• Have ability to conduct diagnostic tests (PCR,
VI and VN) for Schmallenberg at NVSL
(Ames and Plum Island).
• NVSL-Ames can accept samples to test for
Schmallenberg; work with AVIC and State
Veterinarian, call NVSL for information on
sampling procedures
10

11. Safeguarding Animal Health
Schmallenberg Virus – Sampling
Criteria for Submission to NVSL
• Submit cases in ruminants where more than one
dam produces fetus or neonate displaying signs
of arthrogryposis hydranencephaly syndrome:
stillbirths, mummies, muscle atrophy, joint
malformations, etc.
• Differential diagnostic testing for other causes of
reproductive problems should be handled by
local diagnostic laboratory.
 Ames will only test for Schmallenberg virus and Cache
Valley Fever virus
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12. Safeguarding Animal Health
Schmallenberg Virus – Submitting
Samples
• Initially requesting samples associated with
reproductive signs
 Fetal tissues: brain, heart blood, serum
 Dam: serum ONLY
• Submit to NVSL-Ames on our 10-4 submission
form; indicate “Schmallenberg virus” as exam
requested
• Contact the VS AVIC and State Animal Health
Official when submitting
• If case is a FAD suspect, handle as FAD
investigation as per VS Memo 580.4
12

13. Safeguarding Animal Health
2011 Q Fever: Washington & Montana
4/2011 99 goats tested QF CFT 73/99-74% positive
99 goats tested QF ELISA 77/99-78% positive
Tested at NVSL submitted by owner
Noted by owner 14 does aborted from Dec 2010
6/2011 Epi-Aid request to CDC for assistance
Request from a state begin process
Rapid and focused action on trace outs
567 goats tested QF ELISA 62/567-11% positive
Tested at NVSL submitted by CDC
Farms in WA/MT/OR purchased goats from index

14. Safeguarding Animal Health
Swine Influenza Update
14
• Investigation of suspected pig lineage influenza in
humans; H3N2v
• Collaboration with NAHLN labs to identify and
obtain isolates H3N2 isolates as part of SIV
surveillance program
• Full genome sequencing from repository of SIV
isolates using new Ion Torrent method

15. Safeguarding Animal Health
Avian Influenza Update
Wild Birds: 420 specimens received for confirmation and identification from individual
state wildlife service surveillance programs, research, etc.
• No HPAI H5N1 detected
• LPAI H5N1 (North American lineage) detected in 4 states (ID, OH, MN and WA)
• LPAI H5N2 – Predominate subtype (52 isolations)
• LPAI H7N3 – Predominate subtype (27 isolations)
• Other subtypes detected
 H1(4), H2 (3), H3 (36), H4 (38), H5 (62), H6 (29), H7 (49), H8 (1), H9 (1), H10 (5),
H11 (35), H12 (4), H13 (1), H14 (3)
 The H14 isolation represents the first isolation of H14 in North America. Previous
to this H14 avian influenza was isolated one time and that is from the Caspian
Sea in 1982.
Commercial Poultry: Two cases reported to OIE in FY 2011

16. Safeguarding Animal Health
Live-bird Market Surveillance – FY11
State
NJ
PA
MA
MD
NY
RI
CT
FL
OH
WA
NH
NE
Specimens
3580
26
125
8
19
49
28
18
200
22
4
318
Submissions
544
22
29
7
11
11
7
4
46
5
1
74
761 (8.8% pos) 4,397 (2.6% pos)Total - 12

17. Safeguarding Animal Health
Diagnostic Bacteriology Laboratory
17
• Dr. Matt Erdman, DBL
Director
• Dr. Suelee Robbe-
Austerman, MB Section
Head
• Dr. Dave Kinker,
Serology Section Head
• [Vacant]: BI Section Head

18. Safeguarding Animal Health
Diagnostic Bacteriology Laboratory
• Implemented ovine tissue matching and sex determination
microsatellite assays.
• Harmonized NVSL TB genotyping (VNTR) to match CDC
genotyping protocols for human isolates.
• Initiated a whole genome sequencing project on Mycobacterium
bovis and Brucella spp to improve epidemiological traces.
• Provided Proficiency tests for CEM
• Proficiency tests for Group D Salmonella in poultry, Salmonella serotyping,
and general bacteriology will be distributed in April and May 2012
• Leptospira MAT proficiency test and training course planned for summer
2012
• Implemented new testing scheme and PCR for Campylobacter spp
identification
• Implemented luminex xMAP Salmonella Assay for molecular serotyping of
Salmonella isolates
18

19. Safeguarding Animal Health
• Collaborated with Ohio State University to screen Salmonella
isolates for antimicrobial resistance
• Successfully completed initial large scale production of TB
tuberculin bulks.
• Continuing Caudal Fold Tuberculin distribution in multiple fill
volumes to accommodate multiple herd size testing (10 ml, 5 ml,
and 1 ml)
• Brucellosis Reagents - Large volume production of Card, RAP,
and BAPA antigen. Smaller volume production of Tube, Plate,
and Rivanol antigens.
• Successful production of Dourine CF antigen, Glander’s CF
antigen, Anaplasma CF antigen, and various control serum.
Diagnostic Bacteriology Laboratory

20. Safeguarding Animal Health
• TB Cervid Stat-Pak Project: 1654 serum samples from Elk,
White-Tailed Deer, and Reindeer were tested on the Cervid
Stat-Pak. The goal of this project is to develop sensitivity
and specificity values so that this test could potentially be
used as a screening test or official test instead of the skin
test for bovine TB. This project is ongoing and will continue
in FY2012.
• Samples provided by NVSL Serum Bank :
~ 425 Cervid samples
~ 250 Bovine samples
3734 Cervid samples and 2891 Cattle samples in bank
20
Diagnostic Bacteriology Laboratory

21. Safeguarding Animal Health
Pathobiology Laboratory
• Dr. Art Davis,
Director
• Dr. Walter Hyde,
CAS Section Head
• Dr. Mark Hall,
PPE Section Head
21

22. Safeguarding Animal Health
Pathobiology Laboratory
22
• The Pathobiology group receives
approximately 10,000 samples annually for
histopathological examination in support of
the USDA Bovine Tuberculosis eradication
program. Of these samples approximately
85-90% are from slaughter surveillance
stream.
• Also a participant in the evaluation of
Permethrin for use in dip vat solutions
against cattle fever ticks.
• Conducting ongoing evaluation of three
IHC TSE testing platforms (Ventana,
BioCare, Leica) for use in VS Programs.

23. Safeguarding Animal Health
CWD Rectal Biopsy Study
• Test sensitivity for the rectal biopsy samples was approximately
70% compared to the gold standard of CWD testing on brain and
or lymph nodes samples
• Test sensitivity was influenced by animal genotype and stage of
disease
 RB test sensitivity is highest in prion protein codon 96 GG
deer, with lower RB test sensitivity in 96GS and 96SS deer
 Deer later in the course of the disease (as defined by
increasing amounts of prion protein in the brain) have the
greatest likelihood of a positive RB test
 Conversely, deer early in the course of the disease that lack
prion in the brain or have low amounts of prion, more
frequently have false negative RB results

24. Safeguarding Animal Health
Foreign Animal Disease Diagnostic Laboratory
• [Vacant], Director
• Dr. Fernando
Torres-Velez, DSS
Section Head
• Dr. Mike McIntosh,
PVS Section Head
• Dr. Wei Jia,
RVS Section Head
24

25. Safeguarding Animal Health
FADDL - International Collaborations
Dx Assistance & Capacity Building
• Ecuador
 Characterization of O/ECU FMD strain
• Dominican Republic
 Low pathogenic CSF
 PTV-1
• Haiti
 PTV-1
• Mexico
 Reference lab Capacity building (CSF)
• Mongolia
 OIE twinning program (FMD)
International Transboundary
Animal Disease Course
FY 10 FY 11
FADD
Trained 19 23
• Harmonization
 US/Mexico CSF Dx
harmonization
 Proficiency panels
(Canada)
9

26. Safeguarding Animal Health
FADDL: Emerging Disease Investigations
• October 2011
 Vesicular-like to ulcerative disease
among Ringed seals in the
northwest Alaska Arctic region
 Animals from Canada and Russia
also reported with same symptoms
 FADDL provided support through
conventional and state of the art
diagnostic techniques to rule out
vesicular diseases
 The etiologic agent still unknown as
of January 2012

27. Safeguarding Animal Health
FADDL: CSF Surveillance in the Americas
• 2011 – CSF outbreak in
Guatemala near MX border
FADDL monitoring the “re-
emergence” of Classical Swine
Fever in Guatemala for
possible low virulence strain
variations
We provided molecular
characterization and phylogeny
analysis to complement the field
clinical observations.
Mexico
Guatemala
Belize

28. Safeguarding Animal Health
Diagnostic Development
Projects
Assay
Development
• FMD 3ABC ELISA
• FMD 3D ELISA (DIVA)
• FMD LPBE
• CSF Competitive ELISA
• Isothermal LAMP assay*
 Pan-mycoplasma
 Capripox
 Lumpy skin disease
• Panviral microarrays
• Multiplex platforms
*not DHS funded
Assay
Validation
• FMD Penside test
(SVANODIP® FMDV-Ag)
• Detection of FMDV in
bulk tank milk
• Sample preservation &
transport in FTA paper
(CSF & FMD)
• Ropes for collection of
oral fluid (Dx CSF &
FMD)
Reagent
Development &
Production
• Vesicular Ag ELISA
reagent production
• Monoclonal production
 ASF
• FMD antisera bank
28

29. Safeguarding Animal Health
Assay Validation:
• Evaluation of FMD Penside Dx Assays-Lateral Flow Device
Investigate performance characteristics of a penside FMD assay
pending licensing in the USA
• Bulk Tank Milk PCR for Foot-and-Mouth Disease
 FMD PCR in Milk: Inter-laboratory Comparison study
(currently underway)
 FMD PCR in Milk: Negative Cohort study (to begin in
Summer 2012)

30. NAHLN- A State and Federal Partnership to Safeguard Animal Health

31. Safeguarding Animal Health
NAHLN Strategic Planning
February 2011 -
NAHLN structure
options were
discussed during
Coordinating
Council Meeting
July 2011 –
Structure options
provided to
USAHA/AAVLD
Joint NAHLN
Committee for
input
August 2011 – Edits
discussed by
Coordinating Council
and proposed model
document finalized.
• 4 models based on:
state, region, function,
or geographic area
August 2011 –
AAVLD
Executive
Committee
solicited input
from stakeholder
groups including
laboratory
personnel,
industry, SAHOs,
and VS.
• 138 individuals
responded
September 2011
– Coordinating
Council
developed
concept paper
shared at
USAHA/AAVLD
• “A Vision for
National Animal
Health
Laboratory
Network
Structure-
Current
Thinking”
February 2012–
All comments on
concept paper
received and
shared with
NAHLN
Coordinating
Council
May 2012–
Concept paper to
be finalized and
laboratory related
policies reviewed
for finalization
• This is an
important step
to codification of
NAHLN in the
9CFR

32. Safeguarding Animal Health
NAHLN Surveillance Update– Highlights
SIV Report
testing numbers October 2010 January 2012
• 6855 Total samples submitted under program
• 2733 accessions (approximate number of herds)
• 1058 Matrix positive accessions
• 171 accessions positive for pN1 gene
• Total of 629 isolates sequenced
 Sequences deposited in Gen Bank for reference for vaccine development researchers
• Currently in process of changing the testing algorithm to a broader focus of
swine influenzas and emerging virus strains by removing N1 PCR and adding
standardized subtyping PCRs.
 Expected to implement in the next few weeks
• Since 2008, VS has been collaborating with CDC on SIV by regularly sharing
isolates mainly for human vaccine development purposes, and also sharing
SOPs methods and molecular expertise.
32

33. Safeguarding Animal Health
Upcoming NAHLN Training
• May 2012– Planned QMS training in
collaboration with AAVLD and
International Services
• August 2011 – NAHLN IT messaging
training with 20 participants from NAHLN
labs and VS OCIO
• February and March 2012– Funding
mechanism webinar series for NAHLN
labs
• May 2012– VS Memo 580.4
implementation training for laboratory
and field staff

34. Safeguarding Animal Health
NVSL Proficiency Tests 2011
• African Swine Fever
• Anaplasmosis
• Avian Influenza (rRT-PCR
and AGID)
• Bacteriology
• Bluetongue
• Bovine Leukosis
• Bovine Spongiform
Encephalopathy
• Brucellosis
• Classical Swine Fever
• Contagious Equine Metritis
• Equine Infectious Anemia
• Equine Viral Arteritis
• Foot and Mouth Disease
• Johne’s Disease (organism,
serum, milk)
• Leptospira
• Newcastle Disease
• Pseudorabies (serology,
latex aggulation, gI ELISA,
gB ELISA)
• Rinderpest
• Salmonella Type D
• Scrapie/Chronic Wasting
Disease
• Vesicular Stomatitis
34

35. Safeguarding Animal Health
NVSL Scientific Publications
• (NVSL Authors: Ostlund E and Emery M) 2011. Notes from the field: Q
fever outbreak associated with goat farms--Washington and Montana, Morb
Mortal Wkly Rep. 2011 Oct 14;60(40):1393.
• Scoles GA, Hutcheson HJ, Schlater JL, Hennager SG, Pelzel AM,
Knowles DP. 2011. Equine piroplasmosis associated with Amblyomma
cajennense Ticks, Texas, USA. Emerg Infect Dis. Oct;17(10):1903-5.
• Miller M, Terrell S, Lyashchenko K, Greenwald R, Harris B, Thomsen BV,
Fontenot D, Stetter M, Neiffer D, and Fleming G. 2011. Mycobacterium
kansasii Infection in a Bontebok (Damaliscus pygaragus dorcas) Herd:
Diagnostic Challenges in Differentiating from the Mycobacterium
tuberculosis Complex. J Zoo Wildl Med 42(3):468-472.
• Donahue BC, Petrowski HM, Melkonian K, Ward GB, Mayr GA, Metwally
S. 2012. Analysis of clinical samples for early detection of classical swine
fever during infection with low, moderate, and highly virulent strains in
relation to the onset of clinical signs. J Virol Methods. Jan;179(1):108-15.
Epub 2011 Oct 20.
35

36. Safeguarding Animal Health
NVSL Scientific Publications
• Nolting J, Fries, AC, Courtney C, Hines N, Pedersen J, Killian ML. Recovery of H14 influenza A
virus isolates from sea ducks in the Western Hemisphere [Internet]. Version 5. Knol. 2011 Dec 13.
Available from: http://knol.google.com/k/jacqueline-nolting/recovery-of-h14-influenza-a-
virus/1gl1uoak9qgic/1
• Killian ML, Zhang Y, Panigrahy B, Trampel D, Yoon KJ. 2011. Identification and Characterization
of H2N3 Avian Influenza Virus from Backyard Poultry and Comparison with Novel H2N3 Swine
Influenza Virus. Avian Dis. 55(4):611-619.
• Carleton RE, Mertins JW, Yabsley MJ. 2012. Parasites and Pathogens of Eastern Bluebirds
(Sialia sialis): A Field Survey of a Population Nesting Within a Grass-Dominated Agricultural
Habitat in Georgia, U.S.A., with a Review of Previous Records. Comp Parasit. 79(1):30-43.
• Short MA, Clark CA, Harvey JW, Wenzlow N, Hawkins IK, Allred DA, Knowles DP, Corn JL,
Grause JL, Hennager SG, Kitchen DL, Traub-Dargatz JL. 2012. Outbreak of equine
piroplasmosis in Florida. JAVMA 240(5):588-595.
• Deng,MY, Millien M, Jacques-Simon R, Flanagan JK, Bracht AJ, Carrillo C,Barrette RW,
Fabian A, Mohamed F, Moran K, Rowland J,Swenson SL , Jenkins-Moore M, Koster L,
Thomsen BV, Mayr G, Pyburn D, Morales P, Shaw J, Burrage T, White W, McIntosh MT, and
Metwally S. 2012. Diagnosis of porcine teschovirus encephalomyelitis in the Republic of Haiti. J
Vet Diagn Invest, Vol. 24, Number 4, in press.
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37. Safeguarding Animal Health
Websites
• National Veterinary Services Laboratories
www.aphis.usda.gov/animal_health/lab_info_services
• National Animal Health Laboratory Network
www.aphis.usda.gov/animal_health/nahln