AOAC CASP Meeting - MYM 2020
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Ca nabis Ana ytical Science Program
AOAC INTERNATIONAL
CANNABIS ANALYTICAL SCIENCE PROGRAM (CASP) MEETING
WEDNESDAY, MARCH 11, 2020 8:00AM - 2:00PM
at the
Gaithersburg Marriott Washingtonian Center 9751 Washingtonian Blvd. Gaithersburg, Maryland, 20878 SALON C/D/E
Pioneer Members
PerkinElmer R-Biopharm AG SōRSE Technology TEQ Analytical Titan Analytical
Association of Food and Drug Officials (AFDO) Applied Food Sciences BioRad MilliporeSigma
Partner Members
CV Sciences
Eurofins Scientific
Affiliate Members
Alkemist Labs BIOTECON Diagnostics Canopy Growth Corporation CEM Corporation
Charm Sciences Crystal Diagnostics Hygiena Institute of Food
Technologists (IFT)
WiFi: Marriott Conference Password: aoac2020 Sign-In Here: http://bit.ly/casp-mym
AOAC INTERNATIONAL Cannabis Analytical Science Program (CASP)
Wednesday, March 11, 2020 | 8:00AM – 2:00PM ET Gaithersburg Marriott Washingtonian Center – Salon C/D/E
MEETING AGENDA
I. WELCOME, INTRODUCTIONS AND ANNOUNCEMENTS (8:00AM – 8:15AM) David Schmidt, AOAC INTERNATIONAL
II. PROGRAM REVIEW (8:15AM – 8:30AM) Scott Coates, AOAC INTERNATIONAL
III. CONSENSUS BUILDING AT AOAC INTERNATIONAL (8:30AM – 8:45AM) Deborah McKenzie, AOAC INTERNATIONAL
IV. UPDATE ON THE USDA DOMESTIC HEMP PROGRAM (8:45AM – 9:30AM) Kerry Smith , Director, Laboratory Approval and Testing Division, Agricultural Marketing Service, USDA
V. REPORT FROM THE MICROBIAL CONTAMINANTS WORKING GROUP (9:45AM – 10:30AM) Julia Bramante, Colorado Department of Public Health & Environment
VI. REPORT FROM THE CHEMICAL CONTAMINANTS WORKING GROUP (10:30AM – 11:15AM) Susan Audino, Audino and Associates and Julie Kowalski, Consultant
VII. REPORT FROM THE CANNABOIDS IN CONSUMABLES WORKING GROUP (11:15AM – 12:00PM) Holly Johnson, American Herbal Products Association
- Lunch 12:00pm – 1:00pm -
VIII. INTRODUCTION TO TRAINING & EDUCATION WORKING GROUP (1:00PM – 1:30PM) Susan Audino, Audino and Associates and Toby Astill, PerkinElmer
IX. DISCUSSION ON NEXT STEPS FOR CASP (1:30PM – 1:45PM) Scott Coates, AOAC INTERNATIONAL
X. SUMMARY/WRAP UP (1:45PM – 2:00PM) Scott Coates & Palmer Orlandi, AOAC INTERNATIONAL
02-10-2020 Version 3 – Subject to Change Without Notice
Morning Break at 9:30AM
March 11, 2020 AOAC CASP Meeting – Presenter Bios
AOAC INTERNATIONAL CASP MEETING – SPEAKER BIOS
Susan Audino, Ph.D
Audino & Associates
Dr. Susan Audino is a chemist/chemometrician and independent consultant to chemical and biological laboratories. On behalf of Accreditation Bodies, she assesses laboratories to and is an instructor for multiple ISO/IEC standards such as ISO 17025. She is recognized as a leader in the cannabis industry, focusing on consumer safety and sound science in the development of official and consensus analytical test methods. Susan serves as a scientific advisor to several scientific organizations, regulatory bodies, and sits on expert review panels. Dr. Audino has chaired the AOAC cannabis advisory panel and currently chairs the chemical contaminants working group, and is a board member for the Center for Research on Environmental Medicine. She has provided seminars, workshops, webinars, and facilitated symposia domestically and internationally.
March 11, 2020 AOAC CASP Meeting – Presenter Bios
Julia Bramante, Ph.D.
Colorado Department of Public Health and Environment Lead Scientist
Julia began her career in the cannabis industry in 2014 at Gobi Labs, one of the first cannabis testing facilities to open in Colorado. She then transitioned to the Colorado Department of Public Health and Environment’s Marijuana Reference Laboratory where she currently serves as Lead Scientist. Julia is also the Chair of the Cannabis Chemistry Subdivision of the American Chemical Society and the Co-Chair of the AOAC CASP Microbial Contaminants Working Group.
March 11, 2020 AOAC CASP Meeting – Presenter Bios
Scott Coates, M.S.
AOAC INTERNATIONAL Senior Director of the AOAC Research Institute
Scott was appointed as the Senior Director of the AOAC Research Institute on July 1, 2018. He is responsible for daily management of and business development for the AOAC Research Institute. Scott also serves as the Program Lead for the Cannabis Analytical Science Program. Scott served as the Chief Science Office from 2009 until June 2018. In this capacity, he served as the technical lead for many AOAC projects. Scott led the writing and development of Appendix F in the Official Methods of Analysis of AOAC INTERNATIONAL that describes validation requirements and the development of Standard Method Performance Requirements . Before joining AOAC, he worked for 10 years as the Operations Manager for an in-vitro diagnostic manufacturer making medical test kits such as Strep tests and specialized bacterial culture media. Scott holds a B.S. in Microbiology (1978) and a M.S. in Biotechnology Management (1994) from the University of Maryland. Contact Scott at: scoates@aoac.org, (301) 924-7077 x137
March 11, 2020 AOAC CASP Meeting – Presenter Bios
Holly Johnson, Ph.D.
American Herbal Products Association
Chief Science Officer
Holly E. Johnson Ph.D., is the Chief Science Officer for the American Herbal Products Association (AHPA). She previously served for three years as Laboratory Director for Alkemist Labs, an ISO 17025 accredited natural products testing lab specializing in botanical dietary supplements. Dr. Johnson took her Ph.D. in Pharmacognosy at the College of Pharmacy, University of Illinois – Chicago (UIC), under renowned Pharmacognosist and researcher Dr. Norman Farnsworth. Holly was awarded a National Institutes for Health (NIH) Fellowship and trained at the UIC/NIH Center for Botanical Dietary Supplements. She was a Postdoctoral Research Fellow at the Institute for EthnoMedicine studying the etiology of neurodegenerative disease, and also worked for Waters Corporation conducting technical training and regulatory consulting for pharmaceutical and supplements companies. She is currently a Research Associate with the National Tropical Botanical Garden and serves on AOAC working groups, stakeholders’ panels, and expert review panels for Foods and Dietary Supplements. She is a member of the United States Pharmacopeia’s (USP) Medical Cannabis Expert Panel, the Editorial Board of the Journal of AOAC International, and she serves on the Advisory Boards of the American Botanical Council and the American Herbal Pharmacopeia. Holly has over 20 years’ experience working with natural products & botanicals and spent many happy years conducting research on medicinal plants and giving courses at the University of Hawaii.
March 11, 2020 AOAC CASP Meeting – Presenter Bios
Julie Kowalski, Ph.D.
jkSS llc Consultant
Julie Kowalski is a technical consultant primarily serving the cannabis and hemp testing market. She earned her graduate degree in Analytical Chemistry from Pennsylvania State University. Her professional experience includes troubleshooting, method development and validation for GC, GC-MS, LC, and LC-MS/MS in addition to pesticide residue analysis and chromatography method development. She has previously served as the President of the North American Chemical Residue Workshop, served on AOAC Expert Review Panels, the Cannabis Scientific Task Force for Washington State and is currently chairing the AOAC CASP Chemical Contaminants Working Group.
March 11, 2020 AOAC CASP Meeting – Presenter Bios
Kerry Smith, Ph.D.
United States Department of Agriculture (USDA) Director, Laboratory Approval and Testing Division
Kerry Smith, Ph.D., is the Director of the Laboratory Approval and Testing Division (LATD). LATD provides laboratory testing and approval services to facilitate domestic and international marketing of food and agricultural commodities. Kerry was critical in the reorganization and standardization of the Agricultural Marketing Services’ (Agency) laboratory services. She has led significant process and service enhancements, making LATD a leader in residue, mycotoxin, and adulteration testing. In addition, LATD serves as the scientific arm of the Agency, providing subject matter expertise in many areas, including bioengineered foods and hemp production. Kerry has worked for the United States Department of Agriculture (USDA) for 15 years.
Scott Coates Senior Director
AOAC Research Institute AOAC INTERNATIONAL
March 11, 2020
Pioneer
Association of Food and Drug Officials
Titan Analytical
Applied Food Sciences
PerkinElmer BioRad
TEQ Analytical
R- Biopharm AG
SōRSE Technology
MilliporeSigma
Partner
Eurofins Scientific
CV Sciences
Affiliate
Alkemist Labs Alkemist Labs
BIOTECON Diagnostics
CEM Corporation
Charm Sciences
Hygenia
Institute of Food Technologists (IFT)
Canopy Growth Corporation
Crystal Diagnostics
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Current CASP Working Groups and Projects
Microbiology in Cannabis Initial focus on Aspergillus . SMPR completed. Call for methods issued. Chemical Contaminants in Cannabis Residual Solvents in Cannabis. SMPR completed. Call for methods issued. • Heavy metals. SMPR completed. Pending review and adoption. Cannabinoids in Consumables Initial focus on cannabinoids in hemp plant materials. SMPR completed. Call for methods issued. Recommendation on reporting total THC (THC + THCA). Completed, in SMPR. • Recommendation on dry weight. SMPR completed. Pending review and adoption.
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Priorities March 2020 Chemical Contaminants Working Group
Pesticides Residual Solvents Heavy metals Mycotoxins
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Priorities March 2020 Cannabis in Consumables Working Group
Hemp plant materials Dry weight Extracts Foods/ beverages Personal care products Veterinary products
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Priorities March 2020 Microbiology in Cannabis Working Group
Aspergillus Listeria Salmonella E.coli ( STEC)
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New Working Groups
Training and Education Training Working Group • Dr. Toby Astill [Perkin Elmer] recruited to be Chair.
Proficiency Testing Working Group • To be organized in April.
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CASP Team Scott Coates, MSBTM CASP Program Lead Senior Director AOAC Research Institute scoates@aoac.org 301-924-7077 x137 Jonathan Goodwin, , SHRM-SCP, SPHR Deputy Executive Director & Chief HR Officer AOAC INTERNATIONAL jgoodwin@aoac.org 301-924- 7077 x104 Palmer A. Orlandi, Jr., Ph.D. Deputy Executive Director and Chief Science Officer AOAC INTERNATIONAL
Christopher Dent Manager, Standards Development & Official Methods of Analysis® AOAC INTERNATIONAL cdent@aoac.org 301-924- 7077 x119. Alicia Meiklejohn Governance and Business Development
AOAC INTERNATIONAL ameiklejohn@aoac.org 301-924-7077 x101
porlandi@aoac.org 301-924- 7077 x163.
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AOAC Standards Development
Approval of AOAC Standards & Consensus Documents for CASP draft SMPRs
Deborah McKenzie Sr. Director, Standards & Official Methods SM AOAC INTERNATIONAL
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ANALYTICAL AOAC Products, Services, and Analytical Excellence
Standards & Methods Development
A Complete & Harmonized Quality System Through
Official Methods of Analysis SM (OMA) & Performance Tested Methods SM (PTM)
Laboratory Proficiency Testing & Quality Systems
Analytical Excellence
Publications, Training, Educational Outreach & Horizon ‐ scanning
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Examples: AOAC Consensus Products
Basic Principles
• Transparency • Openness • Balance of Interests • Due Process • Consensus • Appeals
• Performance Requirements • Guidelines
• Sampling Standards • Methods of Analysis • Best Practices • Operational Documents
AOAC Consensus & Products
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To Date – Opportunities to Participate Balance of Perspectives & Due Process
TARGETED COMMUNICATION
INVITATIONS TO SMES
EMAIL BLASTS & WEBSITE NOTIFICATIONS
PARTICIPATION IN MEETINGS – AOAC AND EXTERNAL MEETING
ASSOCIATION NEWS ARTICLES
ONLINE & WRITTEN ‐ PUBLIC COMMENT FORMATS
BRIEFINGS & PUBLIC HEARINGS
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Global Perspectives Included
Contract Research Laboratories Independent Contractors US Rule Makers Academia Commodity Producers Product Manufacturers Instrument & Technology Providers State Regulators and Laboratories Reference Material Organizations Proficiency Testing Programs Trade Organizations Scientific Associations Rapid Method Developers Accreditation Organizations
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CASP Standards Development Activity
3 working groups launched new standards work: September ‐ October 2019
February 2020
Community Consensus via electronic consensus and approval March – April 2020
Comments on draft standard method performance requirements Comment period for all 3 documents began on February 2020 through March 2020. Online Open Comment Session held on February 17, 2020
‐ Heavy Metals ‐ Dry moisture ‐ Salmonella
Deliberate and reach consensus on a final versions of the documents WG chairs will present summaries of WG draft standards for deliberation and input March 11, 2020
Working groups developed draft documents: Working groups met to begin their work and continued drafting documents via web conference October 2019 – January 2020
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Approving the Draft Standards
Program Lead and Standards Manager will oversee the approval process
STEP 2 : Program Lead will conclude deliberation and Standards Manager will record and verify with WG chair(s) any revisions, if needed
STEP 1: WG chair(s) introduced to present the draft standard along with how comments were reconciled followed by discussion on the draft standard
STEP 3 : After final revisions are complete, the WG chair will make a motion for stakeholder acceptance of the standard and recommend approval. A second to the motion may be entertained, but is not necessary STEP 6: Standards Manager will walk attendees through the general consensus process. All attendees will be able to participate in the demonstration of consensus
STEP 4: Program lead will acknowledge the motion (and second, if offered) and offer time for discussion/questions on the motion.
STEP 5: After any due discussion, Program Lead will call for a vote on the motion.
NOTE: 2/3 vote in favor of a motion will pass a motion. 2/3 of those voting will demonstrate consensus in passing a motion Negative votes need to be recorded
If the motion is approved, a consensus standard is formed.
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Documentation and Communication
AOAC carefully documents the actions of CASP and the Working groups
AOAC will prepare summaries of the meetings Communicate summaries to the stakeholders Publish status and summaries in the Referee section of AOAC’s Inside Laboratory Management Publish documents in Journal of AOAC INTERNATIONAL, Official Methods of Analysis of AOAC INTERNATIONAL
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Questions?
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Julia Bramante Chair, CASP Microbial Contaminants Working Group SMPR Presentation
March 11, 2020 Gaithersburg Marriott Washingtonian Center 9751 Washingtonian Blvd., Gaithersburg, MD, 20878
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CASP Microbial Contaminants: Working Group Members (as of January 2020)
Organization
Organization
Organization
Organization Think20Labs LabsMart Inc,
Name
Name
Name
Name
Melissa Aldwin
Ambler Anterola
BreezeTrees, LLC
Stephen Bridgett
Goldman Graham Griffin Hansen Haskell Helbert Hildreth Hoffman Hordyk Hudalla Hughes Jayanthi Johnson Hayakawa Hom
PhytaTech
Raymundo
Lerma
Phytatech
Edward
Sawicki
Southern IllinoisUniversityCarbondale
OrganaKannalytics
Cynthia
LeVesque Lorenzen
CaligreenLaboratory
Devin
Sears
Toby Susan Brian
Astill
Perkinelmer
Todd Scott
G2Analytical
Kyle
YoungLivingEssentialOils
CARLOS Rachel Sheryl Casey Sidney SungOui Neil
SEPULVEDA
AGROLABMEXICO SoRSETechnology
Audino
S.A.Audino&Associates
BotanacorLaboratories DesignGroupCollaborative
Eva
Lynch
RockRiverLaboratory
Shegog
Beck
Microbiologics
Bradford
Lena
Madden Maqsood McKernan
Limerick InstituteofTechnology
Shepherd Silverman Simmons
NATA
Cornelius
Berka
BIOTECONDiagnostics
Darin
HawaiiDOHState LaboratoriesDivision
Madeeha
ProVerde Laboratories MedicinalGenomics
PacificStarLabs LLC
Pat
Bird
PMBBiotek SORA Labs
Yvonne
MedicinalGenomics
Kevin
Hygiena
Tammy Rafael
Blakemore Bombonato Bramante Brauninger Brodnick Campbell Boyar
Jana
SelfEmployed
Ronald Megan
Miller Murn Nelson
TherapeuticHealthChoices
Sudberg
AlkemistLabs
Curaleaf
Shannon Sherman
SteepHill
Microbiologics
Suh
ATCC
Kyle Julia
MedicinalGenomics
NJDeptofHealth,PublicHealth&Environmental LaboratMaria
AOAC
Christy
Swoboda Thomas
RomerLabs, Inc.
CDPHE
Nathan
Industrial Laboratories ProVerde Laboratories
Dustin
Newman Niehaus
Instituteof FoodSafety&Defense
Katherine
NJDepartmentofHealth VivariantLaboratories SartoriusCorporation
Roger Robert
A2LA (accreditationbody)
Chris
Gary
CrystalDiagnostics
Anand Tricia Gordon
Thota
TitanAnalytical
Daniel Srinivas
MCRLaboratories
Melissa Shawn
Nutter O'Leary
TitanAnalytical
Vail
Shari Mike
KaychaLabs
BiotechPharmacal Inc.
NJDOH
Vrdoljak
StateofCADeptofPublicHealth
Clark
BioRadLaboratories
Ron
BioMerieux
Shaun
Opie
E4Bioscience Think20Labs
Christopher
Waggener
VirginiaDCLS
Bob
Clifford
Shimadzu
James
Jursich
CenteraBioscience
Ben Jess
Orsburn Paoletti Parish Parisi Pfaller Phillips
Matthew
Ward
ColoradoDeptofPublicHealthandEnvironment
Pearl
D'Cruz
PEARLConsultingLLC
Ben
Katchman
PathogenDx
TEQAnalytical Labs
Jane
Weitzel
IndependentConsultant
Danielle
Deschene
SCLaboratories
Jason Kati Julie
Kircos
Neogen
Zachary
LevelOne Labs
Daniel Jeffrey
Wene
New JerseyDepartmentofHealthPHEL
Lori
Dodson
MarylandMedicalCannabisCommission
Kiss
ATCC
Salvatore
AlBalqaAppliedUniversity
Wigton
WesternAlternative
Wilfredo
Dominguez
3M
Kowalski
TraceAnalytics
Mike
Universityof Iowa
AnnaWilliams Williams
A2LA NIST
Janie Mike Ross
Dubois
ContractAnalytical Services
Kelsey Nikhil Robert
Kropp Kumar
CDPHE
Melissa
NIST
Walter
Wilson Wong
Esposito Franklin
MCRLabs
Canalysis Laboratories
Juan
Rodriguez Rodriguez Salfinger
GreenHillsAnalytics Lab
Seth
TEQAnalytical Laboratories/Industrial Laboratories
MCRLabs. LLC
LaBudde
Alena
Rm3Labs
Victoria Joshua Wendi
Wu
ABCTesting&MateriaMedicaLabs
Ted
Gatesy
MichiganDept.ofAgriculture
Jasen
Lavoie
U.S.CannabisPharma NeogenCorporation
Yvonne
AFDO
Wurzer Young
SCLaboratories MileHighLabs HardyDiagnostics
Quynh ‐ Nhi
Le
Nandakumara Sarma
USPharmacopeialConvention
Jessa
Youngblood
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Microbial Contaminants Working Group: Work Since Last CASP Meeting
• Six teleconferences (October 2019 – February 2020) • One SMPR drafted ( Salmonella ), one other started (STEC) • Public comment period (February – March, 2020) • SMPR made ready for review and approval
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SMPR Development
Standard Method Performance Requirements ® (SMPRs) for Detection of Salmonella in Cannabis and Cannabis Products
Applicability: Candidate methods used to detect Salmonella species and their serovars in cannabis (plants/flowers) and/or cannabis products (concentrates, infused edibles, and infused non ‐ edibles). Candidate methods may be validated for specific matrices, categories, or broader claims.
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Salmonella species • Straight rods, 0.7 – 1.5 x 2-5 μ m. • Facultative anaerobic, gram negative bacteria. • D-glucose and other carbohydrates are catabolized with the production of acid and usually gas. • Occur in humans, warm and cold blooded animals, food, and the environment. • Pathogenic for humans and many animal species. • Shown to survive well under dessication. • Causative agent of typhoid fever, enteric fevers, gastroenteritis, and septicemia.
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Definitions • POD, Probability of Detection.— The portion of positive analytical outcomes for a qualitative method for a given matrix at a given analyte level or concentration • dPOD CP – This difference in POD values between presumptive and confirmed results • LPOD – The POD value obtained from combining all valid collaborator data sets for a given matrix at a given analyte level or concentration • LCL, Lower confidence limit.— Calculated to determine 95% confidence interval of various POD values • UCL, Upper confidence limit.— Calculated to determine 95% confidence interval of various POD values • CFU, Colony forming unit.— Number of viable microorganisms, presented per a specific quantity. (ex. CFU/mL) • MPN, Most probable number.— Method used to estimate contamination levels
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Method Performance Requirements
Table 1. Validation Acceptance Criteria (Plants/Flowers, Concentrates, Infused Edibles, Infused Non ‐ Edibles)
Target Test Concentration a
Parameter
Parameter Requirements
Minimum Acceptable Results
Single Laboratory Validation with artificial contamination
Fractional positive results, 25 ‐ 75% (5 ‐ 15 positive test replicates) dPOD CP 95% CI: LCL < 0 < UCL b
Replicates per matrix: 20 Inoculation procedure: AOAC Appendix J
Low level to produce fractional positive results Ex. 0.2 ‐ 2 CFU/Test Portion
Fractional Concentration (low level)
Replicates: 5 Inoculation procedure: AOAC Appendix J
High level to produce consistently positive results Ex. 2 ‐ 10 CFU/Test Portion
High Concentration
POD of 1.00 c
Non ‐ Inoculated (Zero) concentration
Replicates: 5
0 CFU/Test Portion
POD of 0.00 c
Single Laboratory Validation with natural contamination
Fractional positive results, 25 ‐ 75% (5 ‐ 15 positive test replicates) for minimum 1 lot dPOD CP 95% CI: LCL < 0 < UCL b
Acceptable minimum detection level (low level)
2 separate lots of 20 replicates
N/A
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Method Performance Requirements
Table 1. Validation Acceptance Criteria (Plants/Flowers, Concentrates, Infused Edibles, Infused Non ‐ Edibles)
Target Test Concentration a
Parameter
Parameter Requirements
Minimum Acceptable Results
Multi Laboratory Validation
0.15 ≥ LPOD ≥ 0.85 dPOD CP 95% CI: LCL < 0 < UCL b
Replicates: 12
1 ‐ 10 CFU/Test Portion
LPOD
Replicates: 12
10 ‐ 50 CFU/ Test Portion
LPOD ≥ 0.95
Replicates: 12
0 CFU/Test Portion
LPOD ≤ 0.05
LPOD (0)
a Determined through MPN Procedures (see Table 4)
b The range between the lower and upper confidence interval should encompass 0, if not, the results must be investigated, and an explanation provided.
c If acceptance criteria is not observed, results must be investigated, and an explanation provided.
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Method Performance Requirements Table 7. Inclusivity/Exclusivity Performance Requirements
Final Test Concentration (CFU/mL)
Minimum Acceptable Results
Parameter
Parameter Requirements
Inclusivity Single ‐ laboratory validation (SLV) study: A minimum of 100 strains is required to be
10 ‐ 100 x limit of detection of the candidate method
100% positive results a
cultured by the candidate method enrichment procedure (including those detailed in Table 8).
Exclusivity SLV study: At least 30 non ‐ target organisms, cultured under optimal conditions for growth b
Overnight growth undiluted
100% negative results a
a. 100% correct analyses are expected. All unexpected results are to be retested following internationally recognized guidelines (ISO 16140, AOAC OMA Appendix J, The Compendium of Analytical Methods of Health Canada). Some unexpected results may be acceptable if the unexpected results are investigated, and acceptable explanations can be determined and communicated to method users b. In instances where an exclusivity culture produces a positive result by the candidate method, the culture may be reanalyzed after culture following the candidate method enrichment procedure. Both results (optimal growth conditions and candidate method enrichment) must be reported.
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Method Performance Requirements • Use of live (viable) cultures (liquid stressed/non-stressed, lyophilized) is required. • To screen samples for the presence or
• Final confirmation can be achieved via matrix assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectroscopy, sequencing, or other suitable confirmatory procedures (e.g., biochemical analysis). • One matrix must contain microflora at 10x the level of the target microorganism. • A minimum three level MPN analysis to determine concentration of target microorganism. Use of matrix study replicates is encouraged.
absence of the target analyte, two methods that employ different technologies (e.g., agar plate, PCR, ELISA) must be used.
• To ensure the viability of the inoculating organism (both confirming presumptive
results or determining false negative results) a secondary enrichment followed by plating of the sample to a minimum of two types of agar plates, one of which is recommended to be chromogenic agar, is required (Table 6). Bulk inoculation of test material is required.
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Method Performance Requirements
Table 3. Acceptable Matrix Claims
Table 2. Category Test Portion Requirements
Minimum Test Portion Size a
Criteria
Category
Matrix Claim
Plants & Flowers
10 g
Number of Matrices Minimum Number of Categories
15 (minimum 3 matrices/category) ≥ 10 (minimum 2 matrices/category)
Concentrates
5 g
Broad Range
4 categories
Infused Edibles
25 g
Variety
4 categories 2 categories 1 category
Infused Non ‐ Edibles 10 g a Minimum test portion size required for validation. Alternatively, larger test portions may be validated.
Select
≥ 5 ≥ 5 ≥ 1
Specific Category Specific Matrix (s)
1 category RE: AOAC Technical Bulletin: TB02MAY2016: Acceptable Validation Claims for Proprietary/Commercial Microbiology Methods for Foods and Environmental Surfaces.
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Method Performance Requirements
Table 5. Condition of Inoculating Culture and Stabilization of Matrix Table 6: Recommended Secondary Selective Broths and Agar
Stabilization Conditions 4°C, 48 ‐ 72 h 4°C, 48 ‐ 72 h
Matrix
Inoculating Cells
Media Name
Media Type
Perishable product Heat processed perishable product
Liquid non ‐ stressed culture
Liquid heat stressed
Rappaport ‐ Vassiliadis (RV) (alternately Rappaport ‐ Vassiliadis R10)
Broth
Liquid non ‐ stressed culture (If frozen food is processed, cells must be heat stressed)
‐ 20°C, 2 weeks
Broth Broth
Tetrathionate (TT)
Frozen Product
Selenite cysteine (SC)
Ambient Temperature (20 ‐ 25 o C), 2 weeks Ambient Temperature (20 ‐ 25 o C), 2 weeks
Agar Agar Agar Agar Agar
Xylose lysine desoxycholate (XLD)
Dried culture
Hektoen enteric (HE) Bismuth sulfite (BS) Chromogenic Salmonella
Shelf stable dry product
Liquid non ‐ stressed culture (If shelf stable product is processed, cells must be heat stressed)
Shelf stable liquid product (heat processed)
MacConkey
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Comments Submitted
Comment Received
Response to Comment
Section 6 : Reference Materials Update 1st sentence to remove fungal spores
Fungal spores reference maintained in SMPR to account for potential use of fungal spores in exclusivity and/or artificial contamination.
Table 1, MLV Section, Column 2: Add Replicates in front of 12
MLV table updated to include “Replicates:” in front of 12 to provide clarity.
Table 6: Update title to read “Recommended Secondary Selective Broths and Agar Table 7, Inclusivity: Update strain requirements, Delete at least 10 strains per required Salmonella spp. Table 8: Update column 3 title to strains (not serovars) and add footnote indicating that it’s a requirement only if method claims
Table 6 title updated per comment recommendation.
Table 7, Inclusivity updated to read: A minimum of 100 strains is required to be cultured by the candidate method enrichment procedure (including those detailed in Table 8). Table 8 title updated to read: Minimum Number of Strains Included* Table 8 footnote updated to read: *Required number of strains per subspecies, per method claims
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Motion: Move to accept the Standard Method Performance Requirements ® (SMPR ® ) for Detection of Salmonella in Cannabis and Cannabis Products as presented.
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Discussion?
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Pre-decisional draft, do not distribute
AOAC Standard Method Performance requirements 2019.XX, Version 7; March 11, 2020. Method Name: Detection of Salmonella species in Cannabis and Cannabis Products Purpose: AOAC SMPRs describe the minimum recommended performance characteristics and suggested inclusivity/exclusivity organisms to be used during the evaluation of a method. The evaluation may be an on-site verification, a single-laboratory validation, or a multi-site collaborative study. SMPRs are written and adopted by AOAC Stakeholder Panels composed of representatives from the industry, regulatory organizations, contract laboratories, test kit manufacturers, and academic institutions. AOAC SMPRs are used by AOAC Expert Review Panels in their evaluation of validation study data for methods being considered for Performance Tested Methods or AOAC Official Methods of Analysis , and can be used as acceptance criteria for verification at user laboratories.
Approved Body: Approval Date: Final version date: 1. Intended Use :
AOAC Cannabis Analytical Science Program
Consensus-based Reference method.
2. Applicability:
Alternative methods used to detect Salmonella species and their serovars in
cannabis and cannabis products.
3. Analytical Technique : Any analytical technique that can meet the requirements. 4. Definitions :
Candidate Method . — The method submitted for validation [Appendix J: AOAC INTERNATIONAL Methods Committee Guidelines for Validation of Microbiological Methods for Food and Environmental Surfaces , Official Methods of Analysis of AOAC INTERNATIONAL , (2019) 21 st Ed., AOAC INTERNATIONAL, Rockville, MD, USA] Candidate Method Presumptive Result . —Preliminary result for a test portion produced by following a candidate method’s instructions for use. Candidate Method Confirmed Result . —Final result obtained for a test portion after cultural confirmation of a candidate method. Cannabis .—genus of flowering plants within the Cannabinaceae family that commonly contain 9- tetrahydrocannabinol (THC), cannabidiol (CBD), and other cannabinoids and terpenes. Cannabis includes, but is not limited to, high-THC and high-CBD cultivars. Cannabis Concentrates . —Extracts (primarily composed of cannabinoids and/or terpenes) manufactured through the extraction and concentration of compounds derived from the cannabis plant or flower. Final products can be many forms including oils, wax, or hash (Category II). Cannabis Infused Edibles .—Food and drinks containing extracts of cannabis and/or cannabis materials (Category III).
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Draft Salmonella SMPR v6
Pre-decisional draft, do not distribute
Cannabis Infused Non-Edibles .—Products containing extracts of cannabis and/or cannabis materials intended to be applied to the human body or any part thereof. Final products can be many forms including creams, ointments, cosmetics and therapeutic pads (Category IV). Cannabis Plant and Flower . —General terms for the structural and flowering unadulterated parts of the cannabis plant (Category I). Cannabis Products .—Products (edible and non-edible) extracted or infused with compounds derived from the cannabis plant including but not limited to CBD and THC. Probability of detection (POD) .—The portion of positive analytical outcomes for a qualitative method for a given matrix at a given analyte level or concentration. The difference in POD values between presumptive and confirmed results is termed dPOD CP . Exclusivity .—Study involving pure nontarget strains, which are potentially cross-reactive, that shall be not detected or enumerated by the candidate method. See Table 10 for a list of recommended nontarget strains. [Appendix J: AOAC INTERNATIONAL Methods Committee Guidelines for Validation of Microbiological Methods for Food and Environmental Surfaces , Official Methods of Analysis of AOAC INTERNATIONAL , (2019) 21 st Ed., AOAC INTERNATIONAL, Rockville, MD, USA] Fractional positive .—Validation criterion that is satisfied when an unknown sample yields both positive and negative responses within a set of replicate analyses. The proportion of positive responses should fall within 25 and 75% and should ideally approximate 50% of the total number of replicates in the set. A set of replicate analyses are those replicates analyzed by one method. Only one set of replicates per matrix is required to satisfy this criterion. Inclusivity .—Study involving pure target strains that shall be detected or enumerated by the candidate method. See Tables 8 and 9 for a list of recommended target strains. [Appendix J: AOAC INTERNATIONAL Methods Committee Guidelines for Validation of Microbiological Methods for Food and Environmental Surfaces , Official Methods of Analysis of AOAC INTERNATIONAL , (2019) 21 st Ed., AOAC INTERNATIONAL, Rockville, MD, USA] Laboratory probability of detection (LPOD) .—The POD value obtained from combining all valid collaborator data sets for a method for a given matrix at a given analyte level or concentration. [Appendix H: Probability of Detection (POD) as a Statistical Model for the Validation of Qualitative Methods , Official Methods of Analysis of AOAC INTERNATIONAL , (2019) 21 st Ed., AOAC INTERNATIONAL, Rockville, MD, USA]
LCL .—Lower confidence limit.
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Salmonella Straight rods, 0.7 – 1.5 x 2- 5 μm. Gram negative. Usually motile by peritrichous flagella. Facultative anaerobic. Chemoorganotrophic, having both a respiratory and fermentative metabolism. D- glucose and other carbohydrates are catabolized with the production of acid and usually gas. Oxidase negative, catalase positive, indole and Voges-Proskauer negative, methyl red and Simmons citrate positive. Lysine and ornithine decarboxylase positive, there is a variable arginine dihydrolase reaction. H 2 S is produced, urea is not hydrolyzed, and growth on KCN and utilization of malonate are variable. Reduce nitrates. Carbohydrates usually fermented include L-arabinose, maltose, D- mannitol, D-mannose, L-rhamnose, D-sorbitol, trehalose, and D-xylose. Occur in humans, warm and cold blooded animals, food, and the environment. Pathogenic for humans and many animal species. Causative agent of typhoid fever, enteric fevers, gastroenteritis, and septicemia. 1 Test portion . —The test portion is the sample size used in most validation studies. For cannabis flower/plant and cannabis infused non-edible products a 10 g test portion is used. For cannabis concentrates, a 5 g test portion is used. For cannabis infused edibles, a 25 g test portion is used. A larger test portion can be used in validation studies when appropriate. See Table 2 for minimum test portion requirements. 5. System suitability tests and/or analytical quality control: Positive and negative controls shall be embedded in assays as appropriate. Inhibition controls should be used for method verification for each new matrix. Manufacturer must provide written justification if controls are not appropriate to an assay. 6. Reference Material(s): The use of live cultures and/or fungal spores (liquid stressed/non-stressed, lyophilized) is required for inclusivity and exclusivity testing and for inoculation of test matrices during the matrix studies. Extracted DNA is not suitable for use in validating methods against this SMPR but may be used to develop supplemental information. 7. Validation Guidance: Appendix F: Guidelines for Standard Method Performance Requirements; 19 th Edition of the AOAC INTERNATIONAL Official Methods of Analysis (2012). Available at: http://www.eoma.aoac.org/app_f.pdf Appendix J: AOAC INTERNATIONAL Methods Committee Guidelines for Validation of Microbiological Methods for Food and Environmental Surfaces [ Official Methods of Analysis of AOAC INTERNATIONAL (2019) 21 st Ed., AOAC INTERNATIONAL, Rockville, MD, USA]; or ISO 16140-2:2016. UCL . —Upper confidence limit.
United States Pharmacopeia. Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests (61), USP 40. United States Pharmacopeia.
1 Bergey's Manual of Determinative Bacteriology Ninth edition edited by John G. Holt.
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United States Pharmacopeia. Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms (62), USP 40. United States Pharmacopeia. Feng, P., Weagant, S.D., Grant, M.A., Burkhardt, W. (2017)
Bacteriological Analytical Manual: Chapter 4 Enumeration of Escherichia coli and the Coliform Bacteria
https://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm064948.htm Andrews, W. H., Wang, H., Jacobson, A., Hammack, T. (2018) Bacteriological Analytical Manual Chapter 5: Salmonella https://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm070149.htm
At the time of the publication, no national reference method exists for the confirmation of Salmonella spp. from cannabis products. Until a suitable reference method is established the following is recommended for method developers:
To screen samples for the presence or absence of the target analyte, two methods that employ different technologies (agar plate, PCR, ELISA) must be used.
To ensure the viability of the inoculating organism (both confirming presumptive results or determining false negative results) a secondary enrichment followed by plating of the sample to a minimum of two types of agar plates, one of which is recommended to be chromogenic agar, is required (Table 6). Final confirmation can be achieved via matrix assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectroscopy, sequencing, or other suitable confirmatory procedures (e.g., biochemical analysis).
When performing the validation, bulk inoculation of test material is required. In certain instances (e.g., therapeutic patches) individual item inoculation may be required.
For the Single Laboratory Validation with artificial contamination, matrix naturally contaminated with non-target organisms (when available) shall be used. For at least one matrix evaluated during the single laboratory validation, competing non-target microflora must be at least 10x the level of the target microorganism. If the concentration of competing microflora does not exceed 10x the target organism for any matrix, artificial contamination of one matrix with non-target organism(s) is required. A minimum three level most probable number (MPN) study should be performed to determine the concentration of the target organism used in the validation. If possible, the use of test portions included in the matrix study should be included as a level in the MPN study. See AOAC Appendix J guidelines for details on performing the MPN study.
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8. Maximum Time-To-Determination: None 9. Method Performance Requirements
See Table 1 for acceptance criteria for validation See Table 2 for category test portion requirement See Table 3 for matrix claims acceptance criteria See Table 4 for descriptions of MPN analysis See Table 5 condition of inoculating culture and stabilization of matrix for inoculation See Table 6 for selective broth and agar recommendations See Table 7 for inclusivity and exclusivity performance requirements
See Tables 8 & 9 for inclusivity organisms See Table 10 for exclusivity organisms
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Table 1. Validation Acceptance Criteria (Plants/Flowers, Concentrates, Infused Edibles, Infused Non- Edibles)
Parameter Requirements
Target Test Concentration a
Parameter
Minimum Acceptable Results
Single Laboratory Validation with artificial contamination
Low level to produce fractional positive results Ex. 0.2-2 CFU/Test Portion High level to produce consistently positive results Ex. 2-10 CFU/Test Portion 0 CFU/Test Portion
Fractional positive results, 25-75% (5-15 positive test replicates)
Replicates per matrix: 20 Inoculation procedure: AOAC Appendix J Replicates: 5 Inoculation procedure: AOAC Appendix J
Fractional Concentration (low level)
dPOD CP 95% CI: LCL < 0 < UCL b
High Concentration
POD of 1.00 c
Non-Inoculated (Zero) concentration
Replicates: 5
POD of 0.00 c
Single Laboratory Validation with natural contamination
Fractional positive results, 25-75% (5-15 positive test replicates) for minimum 1 lot
Acceptable minimum detection level (low level)
2 separate lots of 20 replicates
N/A
dPOD CP 95% CI: LCL < 0 < UCL b
Multi Laboratory Validation
0.15 ≥ LPOD ≥ 0.85 dPOD CP 95% CI: LCL < 0 < UCL b
Replicates: 12
1-10 CFU/Test Portion 10-50 CFU/ Test Portion 0 CFU/Test Portion
LPOD
Replicates: 12 Replicates: 12
LPOD ≥ 0.95 LPOD ≤ 0.05
LPOD (0)
a Determined through MPN Procedures (see Table 4) b The range between the lower and upper confidence interval should encompass 0, if not, the results must be investigated, and an explanation provided. c If acceptance criteria is not observed, results must be investigated, and an explanation provided
Table 2. Category Test Portion Requirements
Category
Minimum Test Portion Size a
Plants & Flowers Concentrates Infused Edibles
10 g
5 g
25 g
Infused Non-Edibles 10 g a Minimum test portion size required for validation. Alternatively, larger test portions may be validated.
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Table 3. Acceptable Matrix Claims
Matrix Claim
Criteria Number of Matrices 15 (minimum 3 matrices/category) ≥ 10 (minimum 2 matrices/category)
Minimum Number of Categories
Broad Range of Cannabis & Cannabis Products Variety of Cannabis & Cannabis Products Select Cannabis Products
4 categories
4 categories 2 categories 1 category
≥ 5 ≥ 5 ≥ 1
Specific Category Specific Matrix (s)
1 category RE: AOAC Technical Bulletin: TB02MAY2016: Acceptable Validation Claims for Proprietary/Commercial Microbiology Methods for Foods and Environmental Surfaces.
Table 4. Minimum Most Probable (MPN) Number Recommendation
Medium Test Portions
Inoculation Level
Large Test Portions 20 x 10 g* 5 x 10 g*
Small Test Portions
Category
Plants & Flowers Concentrates Concentrates
Low High Low High Low High Low High
3 x 5 g 3 x 5 g
3 x 1 g 3 x 1 g 3 x 1 g 3 x 1 g 3 x 5 g 3 x 5 g 3 x 1 g 3 x 1 g
20 x 5 g 5 x 5 g*
3 x 2.5 g 3 x 2.5 g 3 x 10 g 3 x 10 g
20 x 25 g* 5 x 25 g* 20 x 10 g* 5 x 10 g*
Infused Edibles
3 x 5 g 3 x 5 g
Infused Non- Edibles
*Test portions from matrix study
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Table 5. Condition of Inoculating Culture and Stabilization of Matrix
Stabilization Conditions 4°C, 48-72 h 4°C, 48-72 h
Matrix
Inoculating Cells
Liquid non-stressed culture
Perishable product Heat processed perishable product
Liquid heat stressed
Liquid non-stressed culture (If frozen food is processed, cells must be heat stressed)
-20°C, 2 weeks
Frozen Product
Ambient Temperature (20-25 o C), 2 weeks Ambient Temperature (20-25 o C), 2 weeks
Dried culture
Shelf stable dry product
Liquid non-stressed culture (If shelf stable product is processed, cells must be heat stressed)
Shelf stable liquid product (heat processed)
Table 6: Recommended Secondary Selective Broths and Agar
Media Type
Media Name
Rappaport-Vassiliadis (RV) (alternately Rappaport-Vassiliadis R10)
Broth Broth Broth Agar Agar Agar Agar Agar
Tetrathionate (TT) Selenite cysteine (SC)
Xylose lysine desoxycholate (XLD)
Hektoen enteric (HE) Bismuth sulfite (BS) Chromogenic Salmonella
MacConkey
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Table 7. Inclusivity/Exclusivity Performance Requirements
Final Test Concentration (CFU/mL)
Minimum Acceptable Results
Parameter
Parameter Requirements
Inclusivity Single-laboratory validation (SLV) study: A minimum of 100 strains is required to be cultured by the candidate method enrichment procedure (including those detailed in Table 8). Exclusivity SLV study: At least 30 non-target organisms, cultured under optimal conditions for growth b
10-100 x limit of detection of the candidate method
100% positive results a
Overnight growth undiluted
100% negative results a
a 100% correct analyses are expected. All unexpected results are to be retested following internationally recognized guidelines (ISO 16140, AOAC OMA Appendix J, The Compendium of Analytical Methods of Health Canada). Some unexpected results may be acceptable if the unexpected results are investigated, and acceptable explanations can be determined and communicated to method users b In instances where an exclusivity culture produces a positive result by the candidate method, the culture may be reanalyzed after culture following the candidate method enrichment procedure. Both results (optimal growth conditions and candidate method enrichment) must be reported.
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Tables 8 & 9: Inclusivity Panel The following are lists of required and suggested subspecies and serovars that method developers can use to validate their methods. It is recommended that method developers reference CDC’s revised Atlas on Salmonella (https://www.cdc.gov/salmonella/pdf/salmonella-atlas-508c.pdf) to incorporate as many serovars listed therein as possible. A minimum of 100 serovars are required for AOAC adoption. Additionally, the requirements in Table 8 must be met.
Table 8: Required Salmonella subspecies for Inclusivity
Minimum Number of Strains Included*
SALMONELLA
2 3 3 3 3 3
1 2 3 4 5 6 7
Salmonella bongori
Salmonella enterica subsp. arizonae Salmonella enterica subsp. diarizonae Salmonella enterica subsp. houtenae Salmonella enterica subsp. indica Salmonella enterica subsp. salamae Salmonella enterica subsp. enterica
1 strain per serovar
*Required number of strains per subspecies, per method claims
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Table 9: Suggested Salmonella serovars for Inclusivity
Center for Disease Control and Prevention Top 20**
Food and Drug Adminstration Ranking Top 40
Antigenic Properties SEROTYPE
Year OUTBREAK
SALMONELLA (serovar included)
O
H
1 2 3 4 5 6 7 8 9
66 66 47 50 53 55 56 57 58 59 60 40 51 62 63 65 35 47 48 61
z 41 :-
Salmonella bongori, Serotype Brookfield
Salmonella bongori
Salmonella enterica subsp. Salamae Salmonella enterica subsp. Salamae Salmonella enterica subsp. salamae Salmonella enterica subsp. salamae
Salmonella enterica subsp. salamae serovar Artis
Salmonella enterica subsp. salamae
Salmonella enterica subsp. salamae serovar Basel
10 Salmonella enterica subsp. salamae 11 Salmonella enterica subsp. salamae 12 Salmonella enterica subsp. Arizonae # 13 Salmonella enterica subsp. Arizonae 14 Salmonella enterica subsp. Arizonae 15 Salmonella enterica subsp. Arizonae 16 Salmonella enterica subsp. Arizonae 17 Salmonella enterica subsp. diarizonae 18 Salmonella enterica subsp. diarizonae 19 Salmonella enterica subsp. Diarizonae #
41
29
20 Salmonella enterica subsp. diarizonae serovar Eilbek 21 Salmonella enterica subsp. houtenae serovar Halmstad
3,{10}{15}{15,34}
g,s,t:-
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