National Veterinary Services Laboratories Update...• Implemented luminex xMAP Salmonella Assay for molecular serotyping of Salmonella isolates 18 Safeguarding Animal Health • Collaborated

Safeguarding Animal Health

National Veterinary Services Laboratories Update


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


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


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


Diagnostic Virology Laboratory

• Dr. Bev Schmitt, Director

• Dr. Sabrina Swenson,

BPA Section Head

• Dr. Eileen Ostlund, EO Section Head

• [Vacant], Avian Section Head

5


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

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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


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

8


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


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


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

11


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

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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


Swine Influenza Update

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• 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


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


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



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• Dr. Matt Erdman, DBL

Director

• Dr. Suelee Robbe-

Austerman, MB Section

Head

• Dr. Dave Kinker, Serology Section Head

• [Vacant]: BI Section Head

http://images.google.com/imgres?imgurl=http://static.dailystrength.org/groupfiles/0/5/8/0/10000850/g_1406341031.jpg&imgrefurl=http://www.dailystrength.org/groups/cdc/media/444739&usg=__1EoEQk35xYMIq_rEsjhdsmMZZ6Y=&h=448&w=449&sz=39&hl=en&start=9&um=1&tbnid=1-OAAqa5TDq8YM:&tbnh=127&tbnw=127&prev=/images?q=bacterial+culture+salmonella&hl=en&rls=com.microsoft:*&um=1


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

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• 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.



• 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

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• Dr. Art Davis, Director

• Dr. Walter Hyde, CAS Section Head

• Dr. Mark Hall,

PPE Section Head

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• 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.


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


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

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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


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


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

Beliz

e


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

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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)

NAHLN- A State and Federal Partnership to Safeguard Animal Health

http://inside.aphis.usda.gov/vs/


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


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.

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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


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

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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.

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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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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

Documents

National Veterinary Services Laboratories Update...• Implemented luminex xMAP Salmonella Assay for molecular serotyping of Salmonella isolates 18 Safeguarding Animal Health • Collaborated