PRIPPP
WORK INSTRUCTIONS
TEMPLATE FOR AVIAN INFLUENZA DIAGNOSIS
1
Work Instruction for avian influenza diagnosis in the Pacific
SPC LRD - AHP / PRI PPP
Vers ion 2 6 Nov 09
2
Work Instruction for avian influenza diagnosis in the Pacific
SPC LRD - AHP / PRI PPP
Vers ion 2 6 Nov 09
Table of Contents
Page
Abbreviations
1
Introduction
2
Pre-diagnostic procedures
WI 1.0
Minimum protective clothing for avian influenza surveillance team
4
WI 2.0
Basic hygiene practices for avian influenza surveillance team
5
WI 3.1
Wearing of personal protective equipment (using cover alls)
6
WI 3.2
Wearing of personal protective equipment (using back-tying gown)
7
WI 4.1
Removing of personal protective equipment (using cover alls)
8
WI 4.2
Removing of personal protective equipment (using back-tying gown)
9
WI 5.0
Proper attire inside the avian influenza laboratory
10
WI 6.0
Post-mortem examination for avian influenza diagnosis
11
WI 7.1
Collection of tissue specimen for avian influenza diagnosis (for virus
isolation)
12
WI 7.2
Collection of tissue specimen for avian influenza diagnosis (for polymerase
chain reaction)
13
WI 8.0
Collection of blood specimen for avian influenza diagnosis
15
WI 9.1
Proper handling of serum specimen for avian influenza diagnosis (using
blood collection tubes)
17
WI 9.2
Proper handling of serum specimen for avian influenza diagnosis (using
syringes)
19
WI 10.1
Collection of cloacal swabs for avian influenza diagnosis (for virus
isolation)
21
WI 10.2
Collection of cloacal swabs for avian influenza diagnosis (for polymerase
chain reaction)
23
WI 11.0
Collection of tracheal swabs for avian influenza diagnosis
24
WI 12.0
Use and maintenance of biosafety cabinet
26
WI 13.0
Handling spills inside the laboratory
27
WI 14.0
Packaging of whole animal (poultry) using double bag method
28
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Work Instruction for avian influenza diagnosis in the Pacific
SPC LRD - AHP / PRI PPP
Vers ion 2 6 Nov 09
Page
WI 15.0
Shipping specimen to overseas laboratory
29
WI 16.0
Packaging of specimen for transport to overseas laboratory
30
WI 17.0
Proper documentation for shipping specimen overseas
31
Reagent preparation procedures
WI 18.0
Preparation of Alsever’s solution (anticoagulant)
32
WI 19.0
Preparation of virus transport medium (VTM) for avian influenza testing
33
WI 20.0
Preparation of 0.5% gelatin in minimum essential medium (MEM)
35
WI 21.0
Preparation of agar gel for avian influenza agar gel immunodiffusion
(AGID) testing
36
WI 22.0
Phosphate buffered saline (PBS) preparation
38
Laboratory diagnostic procedures
WI 23.0
Screening test for avian influenza using Anigen® AI rapid test kit
39
WI 24.0
Using IDEXX® avian influenza A ELISA antibody test kit
41
WI 25.0
Agar gel immunodiffusion (AGID) / Agar gel precipitation test (AGPT)
procedure for detection of serum antibody against avian influenza
43
WI 26.0
Preparation of chicken RBC for hemagglutination hemagglutinationinhibition (HA-HI) test
45
WI 27.0
Adsorption of test serum with standardized chicken RBS for HA-HI test
46
WI 28.0
Treatment of test serum with receptor destroying enzyme (RDE)
47
WI 29.0
Opening a glass ampoule of avian influenza A viral antigen
48
WI 30.0
Hemagglutination test (titration of viral antigen)
49
WI 31.0
Back titration of viral antigen dilution for hemagglutination-inhibition (HI)
test
51
WI 32.0
Hemagglutination-inhibition (HI) test for avian influenza
53
WI 33.0
Immunofluorescence (IF) antibody detection test for avian influenza
56
Laboratory forms
Form 1.a
Avian influenza sample identification worksheet
59
Form 1.b
Sample identification worksheet
60
Form 1.c
Sample identification worksheet
61
Form 2.a
HA-HI worksheet
62
Form 2.b
HA-HI worksheet
63
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Work Instruction for avian influenza diagnosis in the Pacific
SPC LRD - AHP / PRI PPP
Vers ion 2 6 Nov 09
Page
Form 3
ELISA worksheet
64
Form 4.a
AGID worksheet
65
Form 4.b
AGID worksheet
66
Form 5
IFA worksheet
67
Form 6
Rapid test kit worksheet
68
Form 7
Laboratory supplies inventory worksheet
69
Form 8
Laboratory equipments inventory worksheet
70
Annex 1
References
72
Annex 2
Terminology
73
Annex 3
Hand washing poster
74
Annex 4
Animal health post-mortem kit checklist
75
Annexes
iv
Work Instruction for avian influenza diagnosis in the Pacific
SPC LRD - AHP / PRI PPP
Vers ion 2 6 Nov 09
Abbreviations
AGID
agar gel immunodiffusion
AGPT
ELISA
agar gel precipitation test
Animal Health and Production team of the Secretariat of the Pacific
Community, Land Resources Division
enzyme link immunosorbent assay
HA
hemagglutination
HA-HI test
hemagglutination hemagglutination-inhibition test
HI
hemagglutination-inhibition
IF
immunofluorescence
IFA
immunofluorescence assay
LRD
Land Resources Division
PCR
polymerase chain reaction
PPE
personal protective equipment
RBC
red blood cells
RDE
receptor destroying enzyme
RTK
rapid test kit
SPC
Secretariat of the Pacific Community
SPF
specific pathogen free
VTM
virus transport medium
WI
work instruction
AH&P
1
Work Instruction for avian influenza diagnosis in the Pacific
SPC LRD - AHP / PRI PPP
Vers ion 2 6 Nov 09
Introduction
The Pacific Regional Influenza Preparedness Project (PRIPPP) has been designed to build the
capacity of Pacific Island Countries and Territories (PICTs) to deal with the potential threat of avian
influenza, pandemic influenza and other emerging diseases. PRIPPP is a four year project with
duration from 1st July 2006 to 30 June 2010. One of the key findings of the PRIPP project is that
animal health (AH) laboratory capacity in the region are in need of much support to develop
proficiency in handling and detecting infectious animal diseases.
These work instruction templates for diagnosing avian influenza were developed by the Animal
Health and Production team of the Secretariat of the Pacific Community, Land Resources Division.
These templates are in line with specific objectives of the Pacific Regional Influenza Pandemic
Preparedness Project (PRIPPP), which include preparing countries for responding to emerging
infectious diseases such as avian influenza. This document serves as baseline information and a
format for Pacific Island countries and territories (PICTs) in developing their own laboratory standard
operating procedures. It is designed for use in selecting diagnostic procedures that are appropriate
for the available in-country resources.
How to use this document. Revisions are to be made by laboratory staff and/or Pacific Animal
Health Laboratory Network (PAHLNet) country focal people. Adaptation of this document will require
changing the agency logo, name and addresses. Pages have spaces provided for signatures of
initiator (the person who will revise the work instructions), reviewer (the person who will review the
work instructions), and approver (head of the agency or division). The revision number and dates are
also to be changed accordingly.
All procedures are compiled from diagnostic manuals developed by the World Health Organization
(WHO), the Food and Agriculture Organization (FAO) and the World Organisation for Animal Health
(OIE) and other references. Protocols found in this document can be changed according to
recommendations of reference and referral laboratories identified by the country. Templates for
laboratory forms are also included for monitoring individual performances and recording
results. While the procedures outlined are specific for avian influenza, country versions can include
work instructions for diagnosis of other animal diseases.
Standard operating procedures, guidelines, manual of procedures and work instructions are
important documents that guide employees and staff in performing actual duties in the workplace. For
the purpose of this document, each term is defined and differentiated in the Terminology section in
Annex 1.
It is assumed that individuals using this document are knowledgeable in basic laboratory skills, have
undergone basic laboratory training, and have carefully read other references and standard operating
procedures.
2
Work Instruction for avian influenza diagnosis in the Pacific
SPC LRD - AHP / PRI PPP
Vers ion 2 6 Nov 09
Work instructions
3
Work Instruction for avian influenza diagnosis in the Pacific
SPC LRD - AHP / PRI PPP
Vers ion 2 6 Nov 09
Insert name of laboratory here
Insert agency logo
here
AVIAN INFLUENZA DIAGNOSIS
Document Type:
Insert name and
address of ministry
here
Title:
Work Instructions
Page
1 of 1
Minimum protective clothing for avian influenza
surveillance team
Document No.:
WI 1.0
Revision 1
Insert date
PURPOSE

Minimize direct skin contact with animals and inhalation of particles during specimen
collection.

Eliminate risk of spreading any form of animal disease from one site (e.g. farm, town
or area) to another.
PROCEDURE
1. It is strongly recommended for members of any avian influenza surveillance team to
have been previously immunized with human seasonal influenza vaccine (containing
H1, H3 and B virus).
2. Each member of the avian influenza surveillance team, particularly those who will be
assigned to restraining animals and collecting specimens, should wear the following
minimum protective clothing:
□ Scrub shirt or coveralls
□ Scrub pants, or any long pants or coveralls
□ Pair of examination gloves
□ Face mask
□ Rubber boots
Prepared by:
Reviewed with:
Authorized for use by:
Date prepared:
Name, laboratory staff
Date reviewed:
Name, laboratory manager
Name, Chief veterinarian/Director for animal health
Insert date here
Insert date here
4
Work Instruction for avian influenza diagnosis in the Pacific
SPC LRD - AHP / PRI PPP
Vers ion 2 6 Nov 09
Insert name of laboratory here
Insert agency logo
here
AVIAN INFLUENZA DIAGNOSIS
Document Type:
Insert name and
address of Ministry
here
Title:
Work Instructions
Page
1 of 1
Basic hygienic practices for avian influenza surveillance
team
Document No.:
WI 2.0
Revision 1
Insert date
PURPOSE

Prevent ingestion of potentially infectious materials after specimen collection.

Minimize the risk of spreading any form of animal disease from one location to
another.
PROCEDURE
1. The blood collector and the assistant must remove and properly dispose of their
examination gloves and/or facial masks before transferring from one area to another.
If necessary, change gloves before transferring to the next household within the
area.
2. After removing the examination gloves, properly wash hands up to the elbows with
soap and water using the proper hand washing procedure (see hand washing poster
in Annex 3)
3. Disinfect hands with 70% alcohol.
4. The surveillance team should disinfect all footwear before leaving the area. This can
be done by spraying disinfectant on all surfaces of the footwear or stepping in a
portable footbath.
5. Place disposable needles, syringes and cotton applicator sticks in a biohazard
container or an empty plastic bottle. Tightly seal the container before disposing it.
6. All disposable materials (syringes, needles, cotton, applicator sticks, examination
gloves, facial masks) used by the surveillance team should be placed in a biohazard
bag. This biohazard bag must be processed in an incinerator or autoclaved and
buried on a secured dumping site.
Prepared by:
Reviewed with:
Authorized for use by:
Date prepared:
Name, laboratory staff
Date reviewed:
Name, laboratory manager
Name, Chief veterinarian/Director for animal health
Insert date here
Insert date here
5
Work Instruction for avian influenza diagnosis in the Pacific
SPC LRD - AHP / PRI PPP
Vers ion 2 6 Nov 09
Insert name of laboratory here
Insert agency logo
here
AVIAN INFLUENZA DIAGNOSIS
Document Type:
Insert name and
address of Ministry
here
Title:
Work Instructions
Page
1 of 1
Wearing personal protective equipment — PPE (using
coveralls)
Document No.:
WI 3.1
Revision 1
Insert date
PURPOSE

Protect laboratory staff or field personnel from possible exposure to contaminants
when handling potentially infected specimens.
MATERIALS
□ Coveralls
□ Shoe cover
□ Face mask
□ Goggles
□ Head cap
□ Disposable gloves (2 pairs)
□ Rubber or cotton gloves
PROCEDURE
It is recommended that PPE materials be worn using the following order:
1. Gloves — inner latex pair
2. Coveralls. Extend the wrist cuffs of the coveralls over the wrists of the latex gloves.
3. Rubber boots.
4. Mask.
5. Goggles or face shields.
6. Hood of coveralls, or hair cover. Ensure that hair strands are well-covered.
7. Plastic apron.
8. Gloves — second latex pair. Extend wrist cuff of gloves over the wrist cuff of
coveralls.
9. Gloves — cotton or rubber if using any.
Prepared by:
Reviewed with:
Authorized for use by:
Date prepared:
Name, laboratory staff
Date reviewed:
Name, laboratory manager
Name, Chief veterinarian/Director for animal health
Insert date here
Insert date here
6
Work Instruction for avian influenza diagnosis in the Pacific
SPC LRD - AHP / PRI PPP
Vers ion 2 6 Nov 09
Insert name of laboratory here
Insert agency logo
here
AVIAN INFLUENZA DIAGNOSIS
Document Type:
Insert name and
address of Ministry
here
Title:
Work Instructions
Page
1 of 1
Wearing of personnel protective equipment (using backtying lab gown)
Document No.:
WI 3.2
Revision 1
Insert date
PURPOSE

Protect laboratory staff or field personnel from possible exposure to contaminants
when handling potentially infected specimens.
MATERIALS
□ Duct tape (optional)
□ Back-tying lab gown (long sleeved)
□ Shoe cover
□ Face mask
□ Goggles
□ Head cap
□ Disposable examination gloves
□ Rubber or cotton gloves
PROCEDURE
1. Ensure that individuals are wearing comfortable inner working garments such as
scrub suits and appropriate laboratory footwear (WI 5.0).
2. Gloves — inner latex pair.
3. Back-tying gown. Extend the wrist cuffs of the gown over the wrists cuffs of the latex
gloves.
4. Cover laboratory footwear with shoe cover. Seal off the leg area using tape if
necessary.
5. Face mask.
6. Goggles or face shields.
7. Head cap.
8. Plastic apron if necessary.
9. Gloves – second latex pair. Extend wrist cuffs of the gloves over the wrist cuffs of the
gown. Seal off the wrist using tape if necessary.
10. Gloves — cotton or rubber if using any.
Prepared by:
Reviewed with:
Authorized for use by:
Date prepared:
Name, laboratory staff
Date reviewed:
Name, laboratory manager
Name, Chief veterinarian/Director for animal health
Insert date here
Insert date here
7
Work Instruction for avian influenza diagnosis in the Pacific
SPC LRD - AHP / PRI PPP
Vers ion 2 6 Nov 09
Insert name of laboratory here
Insert agency logo
here
AVIAN INFLUENZA DIAGNOSIS
Document Type:
Insert name and
address of Ministry
here
Title:
Work Instructions
Page
1 of 1
Removing of personnel protective equipment — PPE
(using coveralls)
Document No.:
WI 4.1
Revision 1
Insert date
PURPOSE

Properly remove and disinfect possibly infected clothing to minimize the risk of
contact with viruses present in the materials.

Prevent spread of infection due to infected clothing.
MATERIALS
□ Biohazard disposal bag
PROCEDURE
Remove PPE in the following order and dispose immediately in biohazard bags or
containers:
1. Rubber or cotton gloves (if using any)
2. Scrub and disinfect boots
3. Remove rubber boots
4. Gloves — second latex pair
5. Coveralls
6. Goggles or face shield
7. Mask
8. Gloves — inner pair
9. Perform hand hygiene (see Annex 3)
10. Shower
Prepared by:
Reviewed with:
Authorized for use by:
Date prepared:
Name, laboratory staff
Date reviewed:
Name, laboratory manager
Name, Chief veterinarian/Director for animal health
Insert date here
Insert date here
8
Work Instruction for avian influenza diagnosis in the Pacific
SPC LRD - AHP / PRI PPP
Vers ion 2 6 Nov 09
Insert name of laboratory here
Insert agency logo
here
AVIAN INFLUENZA DIAGNOSIS
Document Type:
Insert name and
address of Ministry
here
Title:
Work Instructions
Page
1 of 1
Removing of personnel protective equipment — PPE
(using back-tying gown)
Document No.:
WI 4.2
Revision 1
Insert date
PURPOSE

Properly remove and disinfect possibly infected clothing to minimize the risk of
contact with viruses present in the materials.

Prevent the spread of infection due to infected clothing.
MATERIALS
□ Biohazard disposal bag
PROCEDURE
Remove PPE in the following order and dispose immediately in biohazard bags or
containers:
1. Remove the cotton or rubber gloves.
2. Remove the second gloves.
3. Remove goggles, head cap and mask.
4. Remove cover-all, rolling down inside-out.
5. Remove shoe cover.
6. Remove first gloves.
7. Take a shower and change clothing (if possible) prior to boarding any vehicle to
leave the laboratory.
Prepared by:
Reviewed with:
Authorized for use by:
Date prepared:
Name, laboratory staff
Date reviewed:
Name, laboratory manager
Name, Chief veterinarian/Director for animal health
Insert date here
Insert date here
9
Work Instruction for avian influenza diagnosis in the Pacific
SPC LRD - AHP / PRI PPP
Vers ion 2 6 Nov 09
Insert name of laboratory here
Insert agency logo
here
AVIAN INFLUENZA DIAGNOSIS
Document Type:
Insert name and
address of Ministry
here
Title:
Work Instructions
Page
1 of 1
Proper attire inside the avian influenza laboratory
Document No.:
WI 5.0
Revision 1
Insert date
PURPOSE
 Prevent introduction of infectious substances into the laboratory.
 Prevent spread of infectious substances from the laboratory to the outside
environment.
PROCEDURE
1. Laboratory staff, personnel and visitors are required to change their street clothes
and footwear before entering the primary door to the laboratory.
2. Laboratory staff should wear and change laboratory gowns allocated for each room
of the main laboratory.
3. All laboratory gowns and suits must be placed inside an autoclavable biohazard
plastic bag when transporting to another location for autoclaving. If this is not
applicable, laboratory gowns and suits should be disinfected by soaking in detergent
or disinfectant before taking outside the laboratory.
4. Laboratory gowns and other protective suits should not be worn outside the
laboratory.
Prepared by:
Reviewed with:
Authorized for use by:
Date prepared:
Name, laboratory staff
Date reviewed:
Name, laboratory manager
Name, Chief veterinarian/Director for animal health
Insert date here
Insert date here
10
Work Instruction for avian influenza diagnosis in the Pacific
SPC LRD - AHP / PRI PPP
Vers ion 2 6 Nov 09
Insert name of laboratory here
Insert agency logo
here
AVIAN INFLUENZA DIAGNOSIS
Document Type:
Insert name and
address of Ministry
here
Title:
Work instruction
Page
1 of 1
Post-mortem examination for avian influenza diagnosis
Document No.:
WI 6.0
Revision 1
Insert date
PURPOSE

Gross identification of lesions for avian influenza diagnosis in freshly dead birds (less
than 24 hours).
MATERIALS
□ Class 2 biosafety cabinet (BSC)
□ Sprayer with disinfectant
□ Biohazard disposal bag
□ Incinerator or autoclave (dirty)
□ Basin or pail with water
□ Post-mortem kit (complete list in Annex 4)
PROCEDURE
For live animals, perform neck dislocation directly before performing the post-mortem
procedure.
1. Disinfect Class II BSC.
2. Allow BSC blower to run for five minutes.
3. Wear complete personal protective equipment (WI 3.1 or WI 3.2).
4. Remove bird carcass from biosafety bag.
5. Wet the ventral surface of the bird with clean running water or by dipping it in a
bucket of water.
6. Place carcass in a non-absorbent pan or tray.
7. Place inside a Class II BSC.
8. Pluck feathers as necessary to further minimize risk of cross-contamination.
9. Open the abdomen using necropsy scissors, taking care to avoid incising any
internal organs.
10. Cut along the ribs until the keel and breast can be lifted up towards the birds head.
11. Expose the thoracic cavity.
12. Observe and note any abnormality in the internal organs and collect tissue
specimens for avian influenza diagnosis (WI 7.1 and WI 7.2).
13. After examination and sample collection, spray disinfectant over the carcass.
14. Place carcass in an autoclavable biohazard plastic bag using double-bag method
(WI 14.0).
15. Remove from the laboratory room for autoclaving or incineration.
16. Disinfect all necropsy instruments, pans and BSC.
Prepared by:
Reviewed with:
Authorized for use by:
Date prepared:
Name, laboratory staff
Date reviewed:
Name, laboratory manager
Name, Chief veterinarian/Director for animal health
Insert date here
Insert date here
11
Work Instruction for avian influenza diagnosis in the Pacific
SPC LRD - AHP / PRI PPP
Vers ion 2 6 Nov 09
Insert name of laboratory here
Insert agency logo
here
AVIAN INFLUENZA DIAGNOSIS
Document Type:
Insert name and
address of Ministry
here
Title:
Work instruction
Page
1 of 1
Collection of tissue specimen for avian influenza
diagnosis (for virus isolation)
Document No.:
WI 7.1
Revision 1
Insert date
PURPOSE
 Collect tissue specimens for avian influenza diagnosis.
MATERIALS
□ Post-mortem kit (Annex 4)
□ Class 2 biosafety cabinet (BSC)
□ Sterile scissors
□ Sterile specimen containers
□ Virus transport medium (WI 19.0)
□ 100% ethanol or non-toxic ribonucleic acid (RNA) preservative
PROCEDURE
Birds suspected of being infected with non-toxic RNA preservative should be killed by
cervical dislocation (neck wringing) only.
1. Aseptically open carcass (follow steps 1–12 of WI 6.0).
2. Using sterile scissors, collect at least 1 cm3 of tissue from trachea, lungs, spleen, air
sacs, kidney, brain, liver and heart and any obviously abnormal tissues.
3. Place specimens in containers using the following steps:
a) Without virus transport medium (VTM): Place tissue specimens in sterile
containers (without transport medium) either pooled or in separate containers.
b) With VTM: Using sterile scissors (not those used during the post-mortem
examination), cut tissues into finely minced pieces and put into transport medium
as 10–20% (weight/volume) suspension.
c) Place specimens from the digestive tract separately in individual containers with
or without VTM following instructions a) and b), respectively.
4. Label all specimens.
5. Disinfect instruments and BSC after use.
6. Record all collected specimens on the Sample Identification Worksheet (Form 1.a or
Form 1.b).
7. Specimens should be processed in the laboratory. Allow at least 1–2 hours of
incubation at room temperature (22–25oC) for specimens in VTM.
8. Specimens may be stored at 4oC for up to 4 days and at -70oC for prolonged
storage.
9. Ideally, specimens should be transported to an overseas laboratory using a -70oC (or
below) cold chain procedure.
10. In the absence of cold chain procedures, properly package specimen with ice packs
(WI 16.0).
Prepared by:
Reviewed with:
Authorized for use by:
Date prepared:
Name, laboratory staff
Date reviewed:
Name, laboratory manager
Name, Chief veterinarian/Director for animal health
Insert date here
Insert date here
12
Work Instruction for avian influenza diagnosis in the Pacific
SPC LRD - AHP / PRI PPP
Vers ion 2 6 Nov 09
Insert name of laboratory here
Insert agency logo
here
AVIAN INFLUENZA DIAGNOSIS
Document Type:
Insert name and
address of Ministry
here
Title:
Work instruction
Page
1 of 2
Collection of tissue specimen for avian influenza
diagnosis (for polymerase chain reaction)
Document No.:
WI 7.2
Revision 1
Insert date
PURPOSE
 Collect tissue specimen for avian influenza diagnosis through polymerase chain
reaction (PCR).
MATERIALS
□ Post-mortem kit (Annex 4)
□ Class 2 biosafety cabinet (BSC)
□ Sterile scissors
□ Sterile specimen containers
□ Virus transport medium (WI 19.0)
□ 0.9% normal saline or RNAlater® RNA stabilization reagent
PROCEDURE
Birds suspected of infection with highly pathogenic avian influenza (HPAI) should be
killed by cervical dislocation (neck wringing) only.
1. Aseptically open carcass (follow steps 1–12 of WI 6.0).
2. Using sterile scissors, collect at least 1 cm3 of tissue from trachea, lungs, spleen, air
sacs, kidney, brain, liver and heart and any obviously abnormal tissues.
3. Cut tissues into slices of around less than 0.5 cm thick.
4. In the absence of a cold chain procedure or -70oC freezer, preserve specimens in
containers with either of the following preservatives:
a) 0.9% normal saline.
b) RNAlater® RNA stabilization reagent. Place approximately 10 µl of reagent
per 1 mg of tissue and 500 µl reagent per 0.5 cm3 tissue.
5. Label all specimens.
6. Disinfect instruments and BSC after use.
7. Record all collected specimens on the Sample Identification Worksheet (Form 1.a or
Form 1.b).
8. Best results are obtained if specimens are processed in the laboratory immediately
after collection.
9. Specimens may be stored or transported to overseas laboratories at room
o
temperature (22–25ooC) for certain periods depending on the preservative used:
 0.9% normal saline – 24 hours
 RNAlater® RNA stabilization reagent – 7 days
13
Work Instruction for avian influenza diagnosis in the Pacific
SPC LRD - AHP / PRI PPP
Vers ion 2 6 Nov 09
Insert name of laboratory here
Insert agency logo
here
AVIAN INFLUENZA DIAGNOSIS
Document Type:
Insert name and
address of Ministry
here
Title:
Work instruction
Page
2 of 2
Collection of tissue specimen for avian influenza
diagnosis (for polymerase chain reaction)
Document No.:
WI 7.2
Revision 1
Insert date
10. For prolonged periods:
 0.9% normal saline – store in 4oC (refrigeration) for 3–4 days or -70oC for longer
storage.
 RNAlater® RNA stabilization reagent – store in 4oC for 4 weeks; -20oC to
-80oC for longer storage.
Prepared by:
Reviewed with:
Authorized for use by:
Date prepared:
Name, laboratory staff
Date reviewed:
Name, laboratory manager
Name, Chief veterinarian/Director for animal health
Insert date here
Insert date here
14
Work Instruction for avian influenza diagnosis in the Pacific
SPC LRD - AHP / PRI PPP
Vers ion 2 6 Nov 09
Insert name of laboratory here
Insert agency logo
here
AVIAN INFLUENZA DIAGNOSIS
Document Type:
Insert name and
address of Ministry
here
Title:
Work Instructions
Page
1 of 2
Collection of blood specimen for avian influenza
diagnosis
Document No.:
WI 8.0
Revision 1
Insert date
PURPOSE
 Minimize animal stress during blood collection.
 Efficiently collect avian blood specimen from the wing vein.
 Obtain good quality serum for avian influenza laboratory testing.
MATERIALS
□ Disposable syringes (3 ml)
□ Sterile cotton applicators (with plastic handle)
□ Needle, G25 (for chicken under age 4 weeks); G23 (for older chickens)
□ Needle, 1.5 inch
□ 70% alcohol
□ Cotton
□ Avian Influenza Sample Identification Worksheet (Form 1.a)
□ Masking tape
□ Clip board
□ Scissors
□ Disinfectant
□ Sprayer (for disinfectant)
□ Marking pens or any means of labeling
PROCEDURE
Live birds
1. Restrain animal with the help of an assistant. The assistant holds the bird in a
sideways position, immobilizing the feet, neck area and one wing.
2. Holding a clean disposable syringe on one hand, the blood collector uses his/her
other hand to pull the other wing towards his body.
3. Gently pluck away small feathers on the underside of the wing at the collection site.
4. Disinfect the area using a cotton swab and 70% alcohol solution
5. Insert the needle under the white tendon of the pronator muscle, found just below
the area where the wing vein splits into two blood vessels.
6. An amount of blood can be observed to enter the tip of the syringe if the needle is
properly inserted inside the wing vein. Collect 2–5 ml of blood by gentle suction.
7. Withdraw the needle and apply gentle pressure to the vein for a few seconds to stop
further bleeding.
8. Unscrew uncapped needle from the syringe directly inside the sharp disposal
container. Take the necessary precautions to prevent needle prick injury.
9. Separate serum from the blood clot by transferring the collected blood into a tube
(WI 9.1) or by letting the blood stand in the syringe (WI 9.2).
15
Work Instruction for avian influenza diagnosis in the Pacific
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AVIAN INFLUENZA DIAGNOSIS
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Collection of blood specimen for avian influenza
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10. In the Avian Influenza Sample Identification Worksheet (Form 1.a), record blood
sample and cloacal swab label in the appropriate boxes. A serum sample without a
corresponding cloacal swab and surveillance form cannot be processed for
laboratory testing.
Dead birds
Immediately after the bird is dead, perform cardiac bleed to collect blood using the
following steps:
1.
Select a needle size in proportion to the size of the bird (e.g. duck, 1.5 inch
needle).
2.
Aim needle just below the keel (breast bone).
3.
Withdraw blood with minimal negative pressure.
4.
Unscrew uncapped needle from the syringe directly inside the sharp disposal
container. Take the necessary precautions to prevent needle prick injury.
5.
Separate serum from the blood clot by transferring the collected blood into a tube
(WI 9.1) or by letting the blood stand in the syringe (WI 9.2)
6.
In the Avian Influenza Sample Identification Worksheet (Form 1.a), record blood
sample and cloacal swab label in the appropriate boxes. A serum sample without
a corresponding cloacal swab and surveillance form cannot be processed for
laboratory testing.
7.
Treat blood sample as described in work instructions for handling serum specimens
(WI 9.1 or WI 9.2).
Prepared by:
Reviewed with:
Authorized for use by:
Date prepared:
Name, laboratory staff
Date reviewed:
Name, laboratory manager
Name, Chief veterinarian/Director for animal health
Insert date here
Insert date here
16
Work Instruction for avian influenza diagnosis in the Pacific
SPC LRD - AHP / PRI PPP
Vers ion 2 6 Nov 09
Insert name of laboratory here
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here
AVIAN INFLUENZA DIAGNOSIS
Document Type:
Insert name and
address of Ministry
here
Title:
Work Instructions
Page
1 of 2
Proper handling of serum specimen for avian influenza
diagnosis (using blood collection tubes)
Document No.:
WI 9.1
Revision 1
Insert date
PURPOSE
 Collect good quality serum from blood specimens.
 Effectively handle and store serum samples.
MATERIALS
□ 5–10 ml tubes
□ Biohazard bag
□ Permanent marker
□ Basin with detergent or disinfectant
□ Tube stand
□ Resealable plastic bag
□ Working table
□ Disposable transfer pipettes
□ 70% alcohol or any disinfectant
□ Sterilized 1–2 ml screw-cap or snaplock microtubes
PROCEDURE
Collected blood specimens should be brought to the laboratory immediately for processing.
Ideally, serum separation is done inside the laboratory.
1. Immediately after collection, transfer collected blood into 5 ml or 10 ml tubes.
2. Label the tubes and place them in the upright position in a stable container.
3. Alternatively, stabilize tubes in an upside down position (rubber cork side down).
This will conveniently hold clot in the rubber cover and will allow easy separation of
serum and clot.
4. Immediately transport blood specimens to the laboratory.
5. Let blood sample stand in room temperature (22–25oC) for 1–2 hours or until the
blood clots (maximum of 12 hours). Putting the blood sample in a cold
environment will hinder the separation of serum. Exposure to direct heat or
hot location will cause hemolysis.
6. Centrifuge the blood tubes at about 2,500 rpm for 10–15 minutes.
7. Prepare the working table by cleaning and disinfecting the surface with 70% alcohol
or any kind of disinfectant.
8. Spread a clean piece of disposable kitchen towel or any absorbent paper on top of
the working table. This will absorb any spills from the separation process.
9. Prepare a plastic bag (preferably of biohazard quality) and a basin with disinfectant.
10. Place enough sterilized microtubes on a clean surface or in a container.
11. Wear minimum personal protective equipment (WI 1.0) before starting to separate
the serum.
12. Aspirate the serum from the blood clot using a disposable transfer pipette. Be
careful not to disturb the blood clot or hemolysed blood. At least 0.5 ml of
serum is necessary for laboratory testing.
17
Work Instruction for avian influenza diagnosis in the Pacific
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Vers ion 2 6 Nov 09
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Work Instructions
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Proper handling of serum specimen for avian influenza
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Document No.:
WI 9.1
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13. Dispense serum to a clean microtube.
14. Securely cover the microtubes. Press cover until a clicking sound is heard.
15. Label the microtubes.
16. Place serum tubes in a sealable plastic bag.
17. Properly label the plastic bag with the farm owner’s name, location, number of serum
samples and the date of collection.
18. If serum samples will be transported immediately to the laboratory, place them in a
cooler with an ice pack. For longer storage, keep in freezer (at -20oC).
19. Dispose of blood clots and all used materials in a biohazard bag for autoclaving or
incineration and burial at a secure dumping site.
20. Place glass tubes in basin with detergent or disinfectant.
21. Soak for at least 30 minutes; wash and autoclave.
Prepared by:
Reviewed with:
Authorized for use by:
Date prepared:
Name, laboratory staff
Date reviewed:
Name, laboratory manager
Name, Chief veterinarian/Director for animal health
Insert date here
Insert date here
18
Work Instruction for avian influenza diagnosis in the Pacific
SPC LRD - AHP / PRI PPP
Vers ion 2 6 Nov 09
Insert name of laboratory here
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here
AVIAN INFLUENZA DIAGNOSIS
Document Type:
Insert name and
address of Ministry
here
Title:
Work Instructions
Page
1of 2
Proper handling of serum specimen for avian influenza
diagnosis (using syringes)
Document No.:
WI 9.2
Revision 1
Insert date
PURPOSE

Collect good quality serum from blood specimens in syringes.

Effectively handle and store serum samples.
MATERIALS
□ 5–10 ml tubes
□ Permanent marker
□ Tube stand
□ Working table
□ 70% alcohol or any disinfectant
□
□
□
□
□
Biohazard bag
Basin with detergent or disinfectant
Resealable plastic bag
Disposable transfer pipettes
Sharps (needles, blades, syringes)
disposal container
□ Parafilm® sealing tape
□ Sterilized 1–2 ml screw-cap or snaplock microtubes
PROCEDURE
Collected blood samples should be transported to the laboratory immediately for
processing. Ideally, separation of serum is done inside the laboratory.
1. Immediately after collection, unscrew uncapped needle from the syringe directly
inside the sharp disposal bin. Take the necessary precautions to prevent needle
prick injury.
2. Cover syringe opening with sealing tape. Alternatively, attach a new, well-capped
needle to the syringe.
3. Leave around 15 mm of air space between the blood and the tip of the syringe, to
give space for the separation of the serum.
4. Immediately label the syringes and place in a stable container with the well-covered
opening pointing upwards. If a new needle is re-attached, take the necessary
precautions to prevent needle prick injury.
5. Immediately bring blood specimen to the laboratory.
6. Let blood sample stand at room temperature for 1–2 hours or until the blood clots
(maximum of 12 hours). Putting the blood sample in a cold environment will
hinder the separation of the serum. Exposure to direct heat or a hot location
will cause hemolysis.
7. Prepare the working table by cleaning and disinfecting the surface with 70% alcohol
or any kind of disinfectant.
8. Spread a clean piece of disposable kitchen towel or any absorbent paper on top of
the working table. This will absorb any spills from the separation process.
9. Prepare a plastic bag (preferably of biohazard quality) and a sharps disposal
container or an empty plastic bottle for the disposal of needles and syringes.
10. Place enough sterilized microtubes on a clean surface or in a container.
19
Work Instruction for avian influenza diagnosis in the Pacific
SPC LRD - AHP / PRI PPP
Vers ion 2 6 Nov 09
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AVIAN INFLUENZA DIAGNOSIS
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Work Instructions
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Proper handling of serum specimen for avian influenza
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Document No.:
WI 9.2
Revision 1
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11. Wear minimum protective clothing before starting to separate the serum from the
collecting syringes.
12. Remove the covering tape or capped needle from the syringe and dispose in a sharp
container bin or empty plastic bottle.
13. Let the tip of syringe touch the rim of the microtubes.
14. Gently press the plunger to allow the serum to flow out of the syringe careful not to
disturb the blood clot or hemolysed blood. At least 0.5 ml of serum is needed for the
laboratory test.
15. Securely cover the microtubes. Press the cover until a clicking sound is heard.
16. Label the microtubes.
17. Place serum in a sealable plastic bag.
18. Properly label the plastic bag with the farm owner’s name, location, number of serum
samples and the date of collection.
19. If serum samples will be used immediately for laboratory testing, place them at 4oC
(refrigeration temperature) or in a cooler with an ice pack. For longer storage, keep
in freezer.
20. Dispose of all used materials in a biohazard bag for autoclaving or incineration and
burial at a secured dumping site.
Prepared by:
Reviewed with:
Authorized for use by:
Date prepared:
Name, laboratory staff
Date reviewed:
Name, laboratory manager
Name, Chief veterinarian/Director for animal health
Insert date here
Insert date here
20
Work Instruction for avian influenza diagnosis in the Pacific
SPC LRD - AHP / PRI PPP
Vers ion 2 6 Nov 09
Insert name of laboratory here
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here
AVIAN INFLUENZA DIAGNOSIS
Document Type:
Insert name and
address of Ministry
here
Title:
Work Instructions
Page
1 of 2
Collection of cloacal swabs for avian influenza diagnosis
(for virus isolation)
Document No.:
WI 10.1
Revision 1
Insert date
PURPOSE

Minimize animal stress during swab collection.

Collect cloacal swab from healthy and sick birds for virus isolation.

Effectively handle and store cloacal swab samples.
MATERIALS
□ Virus transport media – VTM (WI 19.0)
□ Sterile cotton applicators (rayon, dacron or any plastic-handled swab)
□ Masking tape
□ Clip board
□ Marking pens or any means of labeling
□ Cooler with ice packs
PROCEDURE
1. Store VTM in cooler with ice packs at all times.
2. Insert sterile cotton applicator stick in the vent of the bird.
3. Gently rotate the stick, swabbing against the mucosal wall.
4. Make it deep enough to get at least 1 g of fecal sample.
5. Dip the collected cloacal swab in VTM. Cloacal swab specimen can be pooled for up
to five samples per 3 ml (of VTM), provided samples are from the same species and
the same area.
6. Cut the plastic handle of the applicator stick to a length long enough to allow the
screw cap to close properly.
7. Leave the cotton with the cut handle of the applicator stick inside the VTM tube.
8. If several samples are to be pooled in one tube, dip the collected cloacal swab in the
VTM for at least 30 seconds.
9. Press cotton on the sides of the tube and properly dispose in a biohazard bag or in
an empty plastic bottle. Do not leave any cotton applicators inside the tube when
pooling samples.
10. Label the tubes.
11. Place in sealable plastic bag with farm owner’s name, location and date of collection.
12. Fill up the applicable boxes in the Avian Influenza Sample Identification Worksheet
(Form 1.a or Form 1.b). Make sure it is aligned with the corresponding blood sample
label.
21
Work Instruction for avian influenza diagnosis in the Pacific
SPC LRD - AHP / PRI PPP
Vers ion 2 6 Nov 09
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AVIAN INFLUENZA DIAGNOSIS
Document Type:
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Title:
Work Instructions
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Collection of cloacal swabs for avian influenza diagnosis
(for virus isolation)
Document No.:
WI 10.1
Insert date
Revision 1
13. Immediately return transport media with cloacal swab inside the cooler.
14. Bring the transport media to the laboratory with the corresponding blood/serum
sample and completed Sample Identification Worksheet.
15. If samples are not to be processed immediately for diagnosis, samples may be
stored accordingly:
 at 4oC for up to 4 days.
 for prolonged storage keep at -70oC.
Prepared by:
Reviewed with:
Authorized for use by:
Date prepared:
Name, laboratory staff
Date reviewed:
Name, laboratory manager
Name, Chief veterinarian/Director for animal health
Insert date here
Insert date here
22
Work Instruction for avian influenza diagnosis in the Pacific
SPC LRD - AHP / PRI PPP
Vers ion 2 6 Nov 09
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here
AVIAN INFLUENZA DIAGNOSIS
Document Type:
Insert name and
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here
Title:
Work Instructions
Page
1 of 2
Collection of cloacal swabs for avian influenza diagnosis
(for polymerase chain reaction)
Document No.:
WI 10.2
Revision 1
Insert date
PURPOSE

Minimize animal stress during swab collection.

Collect cloacal swab from healthy and sick birds for polymerase chain reaction
testing.

Effectively handle and store cloacal swab samples.
MATERIALS
□ Commercially available non-toxic ribonucleic acid (RNA) preservative
(RNAlater®) or guanidine buffer (Invitrogen®)
□ 2 ml snaplock or screw-cap microtube
□ Sterile cotton applicators (rayon, dacron or any plastic handled swab)
□ Masking tape
□ Clip board
□ Marking pens or any means of labeling
□ Cooler with ice packs
PROCEDURE
1. Place around 1.2 ml of RNA preservative or guanidine buffer in a microtube.
2. Insert sterile cotton applicator stick in the vent of the bird.
3. Gently rotate the stick, swabbing against the mucosal wall.
4. Make it deep enough to get at least 1 g of fecal sample.
5. Dip and agitate the collected cloacal swab in the preservative/buffer for 30 seconds.
6. Squeeze out the liquid by pressing the cotton swab at the sides of the tube.
7. Place one sample per tube.
8. Label the tubes.
9. Place in a sealable plastic bag with farm owner’s name, location and date of
collection.
10. Swabs can be transported to overseas laboratory or stored at room temperature for
2 weeks.
11. Fill in the applicable boxes in the Avian Influenza Sample Identification Worksheet
(Form 1.a or Form 1.b). Make sure it is assigned with the corresponding blood
sample label.
Prepared by:
Reviewed with:
Authorized for use by:
Date prepared:
Name, laboratory staff
Date reviewed:
Name, laboratory manager
Name, Chief veterinarian/Director for animal health
Insert date here
Insert date here
23
Work Instruction for avian influenza diagnosis in the Pacific
SPC LRD - AHP / PRI PPP
Vers ion 2 6 Nov 09
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AVIAN INFLUENZA DIAGNOSIS
Document Type:
Insert name and
address of Ministry
here
Title:
Work Instructions
Page
1 of 2
Collection of tracheal swab for avian influenza diagnosis
(for virus isolation)
Document No.:
WI 11.0
Revision 1
Insert date
PURPOSE

Minimize animal stress during swab collection.

Collect tracheal swab from live and dead birds for virus isolation.

Effectively handle and store tracheal swab samples.
MATERIALS
□ Transport media
□ Sterile cotton applicators (rayon, dacron or any plastic handled swab)
□ Avian Influenza Sample Identification Worksheet
□ Masking tape
□ Clip board
□ Marking pens or any means of labeling
□ Cooler with ice packs
PROCEDURE
Live birds
1. The procedure requires close contact with possibly infected birds and requires
proper protective clothing.
2.
The assistant holds the bird against his chest with the wings folded.
3.
The individual taking the specimen pries open the beak with his/her free hand.
4.
Holding the swab in the same fashion as holding a pencil, insert the swab into the
trachea.
5.
Gently swab the wall.
6.
Withdraw the swab with a gentle rotation and place in a virus transport medium
(VTM).
7.
Dip the collected swab in VTM. Cloacal swab specimen can be pooled for up to five
samples per 3 ml of VTM provided the specimens are from the same species and
the same area.
8.
Cut the plastic handle of the applicator stick to a length long enough to allow the
screw cap to close properly.
9.
Leave the cotton with the cut handle of the applicator stick inside the VTM tube.
10. If several tracheal swabs are to be pooled in one tube, dip the collected swabs in
the VTM for at least 30 seconds.
11. Press cotton on the sides of the tube and properly dispose in a biohazard bag or in
an empty plastic bottle. Do not leave any cotton applicators inside the tube when
pooling specimens.
12. Label the tubes.
24
Work Instruction for avian influenza diagnosis in the Pacific
SPC LRD - AHP / PRI PPP
Vers ion 2 6 Nov 09
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AVIAN INFLUENZA DIAGNOSIS
Document Type:
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Title:
Work Instructions
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Collection of tracheal swab for avian influenza diagnosis
Document No.:
WI 11.0
Revision 1
Insert date
13. Place in sealable plastic bag with farm owner’s name, location and date of
collection.
14. Fill in the applicable boxes in the Avian Influenza Sample Identification Worksheet
(Form 1.a or Form 1.b). Make sure it is assigned a corresponding blood/serum
label.
15. Immediately return the transport media with swab inside the cooler. If swabs are
not to be processed immediately for diagnosis, VTM may be stored accordingly:


at 4oC (refrigeration temperature) for up to 4 days.
for prolonged storage keep at -70oC (ultra low temperature freezer).
16. Bring transport media to the laboratory with the corresponding blood/serum
specimen and completed Sample Identification Worksheet.
Dead birds
1. During post-mortem examination (WI 6.0), remove lungs and trachea from carcass.
2. Hold the trachea in a gloved hand.
3. Insert the swab into its maximal length inside the trachea.
4. Vigorously swab the wall and place in a VTM.
5. Follow step # 7 above onwards.
Prepared by:
Reviewed with:
Authorized for use by:
Date prepared:
Name, laboratory staff
Date reviewed:
Name, laboratory manager
Name, Chief veterinarian/Director for animal health
Insert date here
Insert date here
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Work Instruction for avian influenza diagnosis in the Pacific
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Vers ion 2 6 Nov 09
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AVIAN INFLUENZA DIAGNOSIS
Document Type:
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Title:
Work Instructions
Page
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Use and maintenance of biosafety cabinet
Document No.:
WI 12.0
Revision 1
Insert date
PURPOSE
 Use a biosafety cabinet (BSC) for safe handling of virus specimens.
 Maximize longevity and function of a BSC.
PROCEDURE
1. BSC certification must be performed
□ Before initial BSC use
□ After moving the BSC
□ After high efficiency particulate air (HEPA) filter replacement
□ At least annually
□ After internal BSC repairs
2. When cleaning the BSC, use a disinfectant that is appropriate for the infectious
agent being handled in the laboratory.
Good general disinfectants include
iodophores, bleach and quaternary ammonium compounds.
3. Containers and equipment should be surface decontaminated and removed from the
BSC when work is completed.
4. Schedule uninterrupted work time. Place a sign on the door that says work in the
BSC is being conducted.
5. Place the necessary materials in the BSC before beginning work.
6. Move arms in and out of the BSC slowly, perpendicular to the BSC’s face opening.
7. Other activity — such as opening and closing doors, or individuals walking past the
BSC — can cause air curtain disruption.
8. All materials should be placed as far back in the BSC as practical (e.g. 4–6 inches
back from front grille).
9. Work should flow from the clean to the contaminated area across the work surface.
10. A final surface decontamination of all BSC work surfaces should be performed at the
end of the day. This should include the BSC's sides and back, and the interior of the
glass.
Prepared by:
Reviewed with:
Name, laboratory staff
Name, laboratory manager
Date prepared:
Date reviewed:
Insert date here
Insert date here
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Work Instruction for avian influenza diagnosis in the Pacific
SPC LRD - AHP / PRI PPP
Vers ion 2 6 Nov 09
Authorized for use by:
Name, Chief veterinarian/Director for animal health
Insert name of laboratory here
Insert agency logo
here
AVIAN INFLUENZA DIAGNOSIS
Document Type:
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address of Ministry
here
Title:
Work Instructions
Page
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Handling spills inside the laboratory
Document No.:
WI 13.0
Revision 1
Insert date
PURPOSE

Minimize hazards and exposure of laboratory staff and immediate environment to
any laboratory spills.
MATERIAL
□ Disposable absorbent material
□ Disinfectant
□ Biohazard disposal bag
PROCEDURE
1. Carefully cover the spill with absorbent material (spill pads, etc.).
2. Gently pour 1:10 solution of 5.25% sodium hypochlorite or other appropriate
disinfectant over the absorbent material.
3. Let sit for at least 20–30 minutes depending on the recommended contact time for
the disinfectant.
4. For large spills in the biosafety cabinet involving liquids going down the front or back
grille(s):
o Ensure petcock is closed
o Pour disinfectant onto work surface and through grille(s)
o Absorb disinfectant on work deck and place in biohazard waste
o Empty drain pan into collection vessel containing disinfectant
5. Record activity in laboratory log book.
Prepared by:
Reviewed with:
Authorized for use by:
Date prepared:
Name, laboratory staff
Date reviewed:
Name, laboratory manager
Name, Chief veterinarian/Director for animal health
Insert date here
Insert date here
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Work Instruction for avian influenza diagnosis in the Pacific
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Vers ion 2 6 Nov 09
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AVIAN INFLUENZA DIAGNOSIS
Document Type:
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Title:
Work Instruction
Page
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Packaging of whole animal (poultry) using double bag
method
Document No.:
WI 14.0
Revision 1
Insert date
PURPOSE

Minimize the spread of infectious disease during the transport of possibly infected
dead birds to the local laboratory.
MATERIALS
□
□
□
□
Personal protective equipment (PPE)
Plastic bag (ideally of biohazard quality)
Sealing tape
Cooler with ice packs
PROCEDURE
1. Wear appropriate PPE.
2. Invert a plastic bag around your gloved hand.
3. Surround the animal with the bag so that you do not directly touch the animal.
4. Seal the bag tightly with sealing tape or by tying a knot.
5. Insert sealed bag inside another plastic bag.
6. Seal the second bag tightly with sealing tape or by tying a knot.
7. Place in a cooler with an ice pack.
8. Record in the sample collection Sample Identification Worksheet (Form 1.a or 1.b).
9. Immediately bring to the laboratory.
10. Store in refrigeration temperature (22–25oC) and examine as soon as possible.
Prepared by:
Reviewed with:
Authorized for use by:
Date prepared:
Name, laboratory staff
Date reviewed:
Name, laboratory manager
Name, Chief veterinarian/Director for animal health
Insert date here
Insert date here
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Work Instruction for avian influenza diagnosis in the Pacific
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Vers ion 2 6 Nov 09
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AVIAN INFLUENZA DIAGNOSIS
Document Type:
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here
Title:
Work Instruction
Page
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Shipping specimen to overseas laboratories
Document No.:
WI 15.0
Revision 1
Insert date
PURPOSE
 Transport suitable animal (biological) specimen classified as UN 3373 to overseas
laboratory for disease diagnosis.
MATERIALS
□ Packaging materials
□ Shipping documents
PROCEDURE
1. Inform receiving laboratory of the shipment. Provide an advanced list of specimens
and other necessary information.
2. Inform the courier service provider about the shipment.
3. Prepare all necessary documentations (shipping protocol template).
4. Package the specimen (WI 32.0) about an hour before the scheduled pick up by
courier. Attach all necessary documents in the packaging of the specimen (WI 16.0).
5. Forward the packaged specimen with necessary documentations to courier service
provider.
6. Update receiving laboratory of the status of shipping, final list of specimen and
expected arrival date.
7. Record activity in laboratory log book.
8. Follow up results from receiving laboratory.
9. Record results in laboratory log book.
Prepared by:
Reviewed with:
Authorized for use by:
Date prepared:
Name, laboratory staff
Date reviewed:
Name, laboratory manager
Name, Chief veterinarian/Director for animal health
Insert date here
Insert date here
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Work Instruction for avian influenza diagnosis in the Pacific
SPC LRD - AHP / PRI PPP
Vers ion 2 6 Nov 09
Insert name of laboratory here
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AVIAN INFLUENZA DIAGNOSIS
Document Type:
Insert name and
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here
Title:
Work Instruction
Page
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Packaging of specimen for transport to overseas
laboratory
Document No.:
WI 16.0
Revision 1
Insert date
PURPOSE

Properly package suitable serum, swab and tissue (biological) specimen classified
as UN 3373 for transport to overseas animal health laboratories.
MATERIALS
□ Personal protective equipment (PPE)
□ Bio boxes including:
o Primary receptacle
o Secondary receptacle
o Outer packaging
o Ice packs (if necessary)
□ Absorbent and cushioning material
□ Shipping documents (shipping protocol template)
PROCEDURE
1. Wear appropriate PPE before handling the specimen.
2. Ensure that all primary receptacles containing the specimen are well labeled
including specimen identifications.
3. Secure cap of receptacle using sealing tape or Parafilm® tape.
4. Wrap primary receptacle in absorbent material enough to absorb entire contents in
case of leaks.
5. Place primary receptacle in secondary receptacle.
6. Ensure that total volume of specimen does not exceed the capacity of the
containers.
7. Attach itemized list of contents in the secondary receptacle (shipping protocol
template).
8. Cushion the secondary receptacle and place in outer packaging.
9. Securely arrange ice packs (if necessary) around the secondary receptacle.
10. Ensure that the total content of the outer packaging does not exceed 4 L or 4 kg.
11. Place required markings and labels appropriately (shipping protocol template).
12. Attach two sets of documentation including list of contents, import permits and other
documents as required (shipping protocol template).
Prepared by:
Reviewed with:
Authorized for use by:
Date prepared:
Name, laboratory staff
Date reviewed:
Name, laboratory manager
Name, Chief veterinarian/Director for animal health
Insert date here
Insert date here
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Work Instruction for avian influenza diagnosis in the Pacific
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Vers ion 2 6 Nov 09
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AVIAN INFLUENZA DIAGNOSIS
Work Instruction
Document Type:
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Title:
Page
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Proper documentation for shipping specimen overseas
Document No.:
WI 17.0
Insert date
Revision 1
PURPOSE
 Provide complete documentation for the efficient transport of animal (biological)
specimen to overseas laboratory for disease diagnosis.
MATERIALS
□ Packaging list
□ Import and export permit
□ Import and export declaration (if required)
PROCEDURE
1. Read the Pathogen Safety Data Sheet (PSDS) for avian
(www.inspection.gc.ca/english/sci/bio/anima/disemala/avflue.shtml)
influenza
2. Before packaging the specimen (an hour before expected transport), ensure that all
documents are ready.
3. Ensure that the primary receptacle is labeled appropriately.
4. The document required in the secondary receptacle is as follows:
□ Specimen list (shipping protocol template)
5. Ensure that two sets of the following documents are attached in the outer packaging:
□ Specimen list (shipping protocol template)
□ Import permit (to be provided by receiving laboratory)
□ Airway bill (to be provided by freight service provider)
6. Provide the following documents to the courier service provider:
□ Completed sender’s information sheet (to be provided by courier)
□ Specimen list
□ Import permit
Prepared by:
Reviewed with:
Authorized for use by:
Date prepared:
Name, laboratory staff
Date reviewed:
Name, laboratory manager
Name, Chief veterinarian/Director for animal health
Insert date here
Insert date here
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Work Instruction for avian influenza diagnosis in the Pacific
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AVIAN INFLUENZA DIAGNOSIS
Work Instruction
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Title:
Page
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Preparation of Alsever’s solution (anticoagulant)
WI 18.0
Document No.:
Insert date
Revision 1
REAGENTS
Reagent
Citric acid (C6H5O7)
Glucose (C4H12O6)
Sodium chloride (NaCl)
Sodium citrate (Na3C6H5O7)
Distilled water
Sodium hydroxide (NaOH)
Hydrogen chloride (HCl)
MATERIALS
□ Analytical balance
□ Weighing boats
□ Measuring spoons
□ Erlenmeyer flasks
□ Marking pen or any means
labeling
Quantity
0.55 g
20.50 g
4.20 g
8.0 g
1L
Specifications
1N
1N
□
□
□
□
Graduated cylinder
pH meter
Autoclave
Dispensing bottles
of
PROCEDURE
1. Measure out a volume of distilled water that is a little less than the desired final
volume.
2. Dissolve all reagents in the measured distilled water.
3. Measure pH. Desired pH is 6.1 + 0.1.
4. Adjust pH if needed. If pH is below 6.0 add 1N of NaOH; if above 6.2 adjust with 1N
HCl.
5. Add distilled water to make up 1 L solution.
6. Dispense in small volumes.
7. Autoclave for 15 minutes at 121oC.
8. Allow to cool.
9. Label bottles including date and lab staff initials.
10. Record activity in laboratory log book.
Prepared by:
Reviewed with:
Authorized for use by:
Date prepared:
Name, laboratory staff
Date reviewed:
Name, laboratory manager
Name, Chief veterinarian/Director for animal health
Insert date here
Insert date here
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Work Instruction for avian influenza diagnosis in the Pacific
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Preparation of virus transport medium (VTM) for avian
influenza testing
Document No.:
WI 19.0
Revision 1
Insert date
REAGENTS
Reagent
Penicillin powder for injection
Streptomycin powder for injection
Fungizone
Sodium bicarbonate (NaHCO3)
0.5% gelatin-MEM (WI 20.0)
Sterile distilled water
Quantity
1 M IU
1g
10 ml
7 ml (max)
500 ml
5.5 ml
Specifications
250 µl/ml
MATERIALS
□ Sterile screw-cap tubes (5 ml capacity)
□ Sterile screw-cap tubes (2 ml capacity)
□ Tube stand (for 5-ml tubes)
□ Glass pipette (at least 10 ml capacity)
□ Rubber bulb aspirator or electronic multistep pipettor
□ Syringe or micropipettor (1,000 µl capacity) with sterile tips
PROCEDURE
1. Prepare aseptically in a biosafety cabinet.
2. Using a sterile syringe or micropipettor, dissolve 1 million IU of penicillin injectable
powder in 2 ml sterile distilled water.
3. Using a sterile syringe or micropipettor, dissolve 1 g of streptomycin injectable
powder in 3.5 ml sterile distilled water to make up 4 ml.
4. Aliquot streptomycin solution in 1.0 ml volumes using 2 ml tubes.
5. Store unused streptomycin solutions in freezer.
6. Mix 1 M IU of penicillin solution, 1 ml of streptomycin solution and 10 ml of
Fungizone 250 µl/ml in 0.5% gelatin-MEM (WI 20.0) to make up 500 ml solution.
7. Measure pH. Desired pH is 7.2–7.4.
8. If pH is below the desired amount, adjust by slowly adding drops of 7% NaHCO3.
9. Dispense in 3.0 ml volumes using 5 ml tubes.
10. Store in clean freezer (at -20oC). Alternatively, VTM can be stored at room
temperature (22–25oC) or refrigeration temperature (4oC) for 1–2 days only.
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11. Test for contamination by placing 1–2 tubes of aliquots at room temperature (22–
25oC) or at 37oC for 24 hours. A yellowish change in color and turbidity indicates
contamination.
*Note: When frozen, transport medium is yellow in color. When thawed out, medium should
have the original pink color, discard if yellow and turbid.
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Work Instruction for avian influenza diagnosis in the Pacific
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Title:
Page
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Preparation of 0.5% gelatin in minimum essential
medium (MEM)
Document No.:
WI 20.0
Revision 1
Insert date
REAGENTS
Reagent
Minimum essential medium powder (MEM)
Nutrient gelatin
Distilled water
Quantity
9.39 g
0.5 g
1L
Specifications
MATERIALS
□ Autoclave
□ Erlenmeyer flask (50–2,000 ml capacity)
□ Hot plate with magnetic stirrer
□ Microwave (optional)
□ Analytical balance
□ Weighing boats
□ Measuring spoons
□ Reagent bottle (500 ml capacity)
□ Refrigerator
PROCEDURE
1.
2.
3.
4.
Measure out a volume of distilled water that is less than the desired final volume.
Dissolve 9.39 g of MEM powder in distilled water by stirring.
Place some of this solution in a smaller beaker/flask and stir 0.5 g of nutrient gelatin.
Heat in microwave oven or on a hot plate for 20 seconds or until solution turns clear
yellow. Avoid boiling.
5. Add distilled water to make a final volume of 1 L.
6. Dispense in 500 ml volumes.
7. Autoclave for 15 minutes at 121oC.
8. Label bottle, including date and lab staff initials.
9. Cool and store at 4oC.
10. Record activity in laboratory log book.
Prepared by:
Reviewed with:
Authorized for use by:
Date prepared:
Name, laboratory staff
Date reviewed:
Name, laboratory manager
Name, Chief veterinarian/Director for animal health
Insert date here
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Work Instruction for avian influenza diagnosis in the Pacific
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Preparation of agar gel for avian influenza agar gel
immunodiffusion (AGID) testing
Document No.:
WI 21.0
Revision 1
Insert date
REAGENTS
Reagent
Quantity
Specifications
Agarose
9g
Type II medium grade (Sigma Chemical Co.
Cat # A-6877 or equivalent)
Sodium chloride (NaCl)
80 g
Phosphate buffered saline
1L
0.01 M, pH 7.2–7.4
(PBS)
MATERIALS
□ Analytical balance
□ Weighing boats
□ Laboratory spatula or laboratory spoons
□ Autoclave (clean)
□ Hot plate / stirrer
□ Top loading balance (capable of measuring 0.1 g differences)
□ Glass pipette (for loading agar gel to glass slides)
□ Glass slides or Petri dish (100 x 15 mm or 60 x 15 mm)
□ Erlenmeyer flasks 1.5–2 L
□ Graduated cylinders
□ Dispensing bottles (for storing excess agar gel)
□ Gel punch
PROCEDURE
Gel plates
1. Mix the three reagents together in an Erlenmeyer flask.
2. Dissolve the mixture by heating on a hot plate using a magnetic spin bar.
3. Heat until mixture becomes homogenous, swirling once in a while to prevent boiling.
4. Autoclave at 121oC for 15 minutes.
5. Allow the agar to cool to room temperature (approximately 25oC) for 10–15 minutes
before dispensing into plates.
6. Dispense 15–17 ml of melted agar to 100 x 15 mm Petri plates or 5–6 ml to a 60 x
15 mm plate using a 25 ml pipette. The agar’s thickness should be approximately 2–
3 mm.
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Work Instruction for avian influenza diagnosis in the Pacific
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Preparation of agar gel for avian influenza AGID testing
Document No.:
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7. Allow plates to cool in a relatively dust-free environment with lids off to permit the
escape of water vapor. The lids should be let off for at least 15 minutes but not
longer than 30 minutes, as electrolytic concentration of the agar may change
due to evaporation and adversely affect formation of precipitin lines.
8. Using a gel punch template, cut the agar after it has hardened. Up to seven template
patterns can be cut in a 100 x 15 mm plate and two patterns for a 60 x 15 mm plate.
9. Remove the agar plugs.
10. Record activity in laboratory log book.
Gel slides
1. Follow steps 1–4 in preparing gel plates.
2. Place a 75 mm x 25 mm glass slide in a clean flat surface and a relatively dust-free
environment.
3. Aspirate at least 5 ml of liquid agar using a glass pipette.
4. Dispense around 5 ml of liquid agar, covering the entire surface of the glass slide.
5. Allow the agar to solidify.
6. Create two sets of wells per agar slide.
7. Record the activity in laboratory log book.
Additional notes:
 Agar plates and slides should be used within 24 hours after they are poured.
 Agar can be dispensed into small quantities (daily working volumes) before
autoclaving. Store in airtight containers at 4oC for several weeks. Gels are melted in
a water bath and dispensed into plates as needed.
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Phosphate buffered saline (PBS) preparation
Document No.:
WI 22.0
Revision 1
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REAGENTS
Reagent
Sodium chloride (NaCl)
Potassium chloride (KCl)
Disodium hydrogen phosphate (Na2HPO4)*
Potassium dihydrogen phosphate (KH2PO4)
Distilled water, to make
Quantity
8.0 g
0.2 g
1.15 g
0.2 g
1L
Specifications
anhydrous
*same as sodium phosphate, dibasic (anhydrous) or disodium hydrogen orthophosphate (anhydrous)
MATERIALS
□ Analytical balance
□ Weighing boats
□ Measuring spoons
□ Erlenmeyer flask
□ Autoclave
□ Reagent bottles
□ Magnetic stirrer or stirring rod
□ pH meter
PROCEDURE
1. Measure out an amount of distilled water that is less than the required volume.
2. Add reagents in the above order, dissolving each thoroughly before adding the next.
3. Add distilled water to make the final volume.
4. Check the pH and adjust accordingly to pH 7.2–7.4 by using HCl (to lower the pH) or
saturated NaCl (to increase pH).
5. Autoclave at 121oC for 20 minutes.
6. Allow to cool to room temperature.
7. Label bottle with date and lab staff initials.
8. Store at 4oC.
9. Record activity in laboratory log book.
Prepared by:
Reviewed with:
Authorized for use by:
Date prepared:
Name, laboratory staff
Date reviewed:
Name, laboratory manager
Name, Chief veterinarian/Director for animal health
Insert date here
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Work Instruction for avian influenza diagnosis in the Pacific
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Page
®
Screening test for avian influenza using Anigen avian
inlfuenza rapid test kit
Document No.:
WI 23.0
Revision 1
Insert date
PURPOSE

Initial screening test for avian influenza.
MATERIALS
□
□
□
□
Anigen avian influenza A rapid test kit
70% alcohol or any disinfectant
Biohazard disposal bag
Disposable kitchen towel or absorbent paper
PROCEDURE
1.
Collect trachea or cloacal swab (WI 10.0 and WI 11.0) specimen using sterile
applicator stick provided in the kit.
2.
Dip applicator stick (with swab) to the assay diluent provided in the kit, stirring for
about 60 seconds.
3.
Prepare the laboratory working table. If to be performed in the field, select a flat
surface in an area with a minimal amount of people.
4.
Prepare the working table by cleaning and disinfecting the surface with 70% alcohol
or any kind of disinfectant.
5.
Spread a clean piece of disposable kitchen towel or any absorbent paper on top of
the working table. This will absorb any spills from the separation process.
6.
Prepare a plastic bag (preferably of biohazard quality) for disposal of used materials.
7.
On top of the absorbent paper, place the specimen (in assay diluent) and needed
rapid test kit (RTK) materials.
8.
Label RTK test plates. Place back on top of absorbent mat.
9.
Aspirate enough specimens, using the disposable plastic dropper included in the kit.
10. Place five drops of specimen at the sample hole of the RTK test plate.
11. Allow the test plate to stand for 20–30 minutes.
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®
Screening test for avian influenza using Anigen avian
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WI 23.0
Document No.:
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12. Read and interpret results as follows:
C - line
T - line
Result
Action
Yes
No
Negative
Report
Yes
Yes
Positive
Report & confirm
No
No
Invalid
Repeat test
No
Yes
Invalid
Repeat test
11. Record results in Rapid Test Kit Worksheet (Form 6).
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AVIAN INFLUENZA DIAGNOSIS
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®
Using IDEXX ELISA avian influenza virus antibody test
kit Flockchek MultiS-Screen Ab
Document No.:
WI 24.0
Revision 1
Insert date
PURPOSE

Measure the relative level of avian influenza type A antibody in serum samples from
a chicken, turkey, duck, ostrich, goose, horse or pig.
MATERIALS
□
□
□
□
□
□
□
□
□
□
IDEXX ELISA avian influenza virus antibody test kit
ELISA Worksheet
ELISA reader, 650 nm filter
ELISA washer (optional)
Single-channel micropipettor with corresponding tips (1–20 µl capacity)
Multi-channel micropipettor with corresponding tips (20–300 µl capacity)
Reagent reservoir
Paper towel
96 well microtiter plates
Distilled water
PROCEDURE
1. Allow reagents and ELISA plate(s) to come to room temperature.
2. Dilute wash concentrate 1:10 with Milli-Q water before use (e.g. 30 ml of concentrate
plus 270 ml water is sufficient for one plate).
3. Arrange test sera on racks and record specimen identification positions on the
ELISA Worksheet (Form 3).
4. Label a blank microtiter plate as plate 1.
5. Prepare 1:10 dilution of test serum and sample diluents in using plate 1 by mixing 15
µl of test serum with 135 µl of sample diluents.
6. Change tips between each serum specimen.
7. When all test sera are diluted in the plates, mix by gently tapping on the sides of the
plate.
8. In the IDEXX ELISA test plate, dispense 100 µl of undiluted negative control into
wells A1 and B1.
9. Dispense 100 µl of undiluted positive control into wells of C1 and D1.
10. Dispense 100 µl of serum samples from plate 1 (diluted 1:10) into corresponding
wells of the IDEXX ELISA test plate from E1 to H12.
11. Incubate for 60 minutes at room temperature (18–25oC).
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Using IDEXX ELISA Avian Influenza virus antibody test
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12. At the end of the incubation period, aspirate the contents of the plate in a pan with
disposable absorbent paper.
13. Tap the test plates in a dry disposable absorbent paper to remove remaining
droplets.
14. Wash each well with 300 µl of diluted wash solution five times manually or using an
automated plate washer.
15. Dispense 100 µl of anti-AI horseradish peroxidase conjugate into all wells.
16. Incubate for 30 minutes at room temperature (18–25oC).
17. Repeat wash cycle (steps 12–14).
18. Dispense 100 µl of tetramethylbenzidine (TMB) substrate solution into all wells.
19. Incubate for 15 minutes at room temperature (18–25oC)..
20. Add 100 µl of stop solution into all wells.
21. Immediately measure absorbance at 650 nm.
22. Print and record results in ELISA Worksheet (Form 3).
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Date prepared:
Name, laboratory staff
Date reviewed:
Name, laboratory manager
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AVIAN INFLUENZA DIAGNOSIS
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1 of 2
Title: Agar gel immunodiffusion test (AGID)/Agar gel
precipitation test (AGPT) procedure for serum antibody
detection
Document No.:
Revision 1
WI 25.0
Insert date
Document Type:
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PURPOSE
 Detect circulating antibodies sensitive to type A influenza.
 Identify isolates of type A influenza viruses.
MATERIALS
□ Avian influenza AGID antigen
□ Immunodiffusion template cutter
□ AI AGID positive control antiserum
□ Closed chamber (for incubation)
□ Agar plates
□ Damp towel
□ Sample Identification Worksheet (Form 1.a or 1.b)
□ Microscope illuminator or other appropriate light source for viewing results
PROCEDURE
1. Record the sample identification, reagent lot numbers, test date, and identification of
personnel performing and reading the test.
2. Using a gel punch template, cut the agar after it has hardened. Up to seven template
patterns can be cut in a 100 x 15 mm plate and two patterns for a 60 x 15 mm plate
and 75 x 15 mm glass slides.
3. Remove the agar plugs.
4. Place 25 µl of test serum in alternate peripheral wells
5. Place 25 µl reference serum/positive control serum in the remaining three peripheral
wells. This arrangement provides a positive control line on each side of the test
serum thereby facilitating accurate determination of lines of identity. Three samples
can be tested in each pattern.
6. Place 25 µl of antigen (Ag) in the center well. Serum or antigen should not run on top
of the agar.
7. Cover each plate after filling all wells.
8. Incubate the plate at room temperature (approximately 25oC) in a closed chamber to
prevent evaporation.
9. Humidity should be provided by placing a damp paper towel in the incubation
chamber. Temperature changes during migration may lead to artifacts.
10. Read the results after 24 hours over a beam of light against a dark background. If
precipitin lines are absent, continue incubation up to 48 hours.
11. Record your results in the Agar Gel Immunodiffusion test (AGID)/ Agar Gel
Precipitation test (AGPT) Worksheet (Form 4.a or Form 4.b).
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Title: Agar gel immunodiffusion test (AGID)/ Agar gel
precipitation test (AGPT) procedure for detection of
serum antibody against avian influenza
Document No.:
Revision 1
WI 25.0
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INTERPRETATION OF RESULTS
Negative reaction. The control lines continue into the test sample well without bending or
with slight bend away from the antigen well and toward the positive control serum
well.
Positive reaction. The control lines join with, and form a continuous line, with the line
between the test serum and antigen. The location of the line will depend on the
concentration of antibodies in the test serum
Weak positive reaction. It may not produce a complete line between the antigen and test
serum but may only cause a tip or end of the control line to bend inward toward the
test well.
Non-specific lines. Lines occasionally observed between the antigen and test serum
well. The control lines will pass through the non-specific line and will continue on
into the test serum well. It does not form a continuous line with positive control
lines.
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Preparation of chicken red blood cells for HA-HI test
WI 26.0
Document No.:
Revision 1
Insert date
PURPOSE

Prepare red blood cells (RBC) for the adsorption of test sera for non-specific
agglutinins.

Prepare 0.5% chicken RBC for HA-HI test.
MATERIALS AND REAGENTS
□
Phosphate buffered saline —
PBS (WI 19.0)
□
Alsever’s solution (WI 15.0)
□
Centrifuge
□
Microhematocrit tube
□
□
Hematocrit centrifuge
50 ml screw-cap tubes
□ Working table
PROCEDURE
1. Collect blood from any chickens that are antibody-free to avian orthomyxoviruses or
paramyxoviruses
2. Mix collected blood with Alsever’s solution. The minimum ratio of blood to
Alsever’s solution is 1:4.
3. Re-suspend blood in Alsever’s solution by gentle agitation.
4. Decant required volume into a clean 50 ml centrifuge tube. Wash only the required
volume of cells as they will have a maximum usable life of 1 week once in
phosphate buffered saline (PBS).
5. Fill the tube with PBS.
6. Centrifuge at 1,500 g (approximately 1,200 rpm) for 5 minutes.
7. Decant supernatant.
8. Refill tube with PBS.
9. Repeat steps 4 and 5 a minimum of three times. At the conclusion of the final wash
discard the supernatant.
10. Make cells up to a final volume of 5 ml with PBS.
11. Re-suspend by gentle agitation.
12. Collect wash cells in a microhematocrit centrifuge tube.
13. Centrifuge wash cells in a hematocrit centrifuge for 5 minutes.
14. Measure packed cell volume (PCV).
15. Adjust the concentration of RBC to 10%.
Formula: Final volume = (PCV / 10 ) x 5
16. Store washed and standardized RBC at 4oC.
17. Prepare 0.5% chicken RBC. For one plate, add 250 µl of the 10% chicken RBC in
4.75 ml PBS.
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Date prepared:
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Date reviewed:
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Adsorption of test serum with standardized chicken
RBC for HA-HI test
Document No.:
WI 27.0
Insert date
Revision 1
PURPOSE


Remove non-specific agglutinins from serum samples.
Make a 1:4 dilution of test serum for HI test.
MATERIALS AND REAGENTS
□ Specific pathogen free (SPF) chicken red blood cells (RBC)
□ 2 ml microcentrifuge tubes
□ 4oC incubator
□ Single-channel micropipette with corresponding tips (100–400 µl, 10–30 µl)
□ Bench top centrifuge
PROCEDURE
1. In a 1.5-ml microtube, mix 100 µl of serum and 300 µl of PBS.
2. Add 25 µl of packed, washed chicken RBC.
3. Shake samples to re-suspend the cells.
4. Incubate samples at +4oC or on ice for 30–60 minutes
5. Intermittently shake samples every 10 minutes to re-suspend the cells.
6. Centrifuge the samples at 1,000 g for 10 minutes
7. Decant the supernatant. The serum is tested as a 1:4 dilution of the original serum.
* Note: The RBC used to adsorb sera must be the same RBC that will be used in the HA-HI
test.
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Date reviewed:
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Treatment of test serum with receptor destroying
enzymes (RDE)
Document No.:
WI 28.0
Revision 1
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PURPOSE

Inactivation of non-specific agglutinins which can contribute to false negative results.
MATERIALS AND REAGENTS
□ Lyophilized receptor destroying enzyme (RDE)
□ Physiological saline
□ Single-channel micropipette with corresponding tips (100–500 µl)
□ 37oC and 57oC incubator
PROCEDURE
1. Reconstitute the RDE with physiological saline (0.85% NaCl) according to label
instructions.
2. Aliquot and store at -20oC (freezer temperature) to -70oC (ultra low freezer).
3. Add 1 volume of serum to 3 volumes of RDE (0.9 ml RDE + 0.3 ml serum).
4. Incubate overnight in a 37oC water bath.
5. Inactivate in a 56oC water bath for 30 minutes. The serum is retested for
hemagglutination-inhibition (HI) as 1:5 dilution of the original.
6. Record your activity in laboratory logbook.
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Date reviewed:
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Opening a glass ampoule of avian influenza antigen
Document No.:
WI 29.0
Insert date
Revision 1
PURPOSE
 Avoid harm to the technician and contamination to the reference antigen during
opening of glass ampoule.
MATERIALS
□ Glass cutter
□ Masking tape
□ Cold block
□ Sterile distilled water
□ Disposable absorbent towels
□ Screw cap tube
□ Parafin film
□ Sharps disposal unit
□ Micropipette with tips 1,000 µl capacity or syringe 3 ml capacity
PROCEDURE
1. Disinfect biosafety cabinet (BSC).
2. Prepare the materials needed for opening the glass ampoule inside the BSC.
3. Wear personal protective equipment.
4. Remove label of AI antigen glass ampoule. Set aside.
5. Put masking tape around the middle area of the ampoule.
6. Tap ampoule gently to collect material at the bottom end.
7. Place glass ampoule on top of ice block covered with absorbent towel.
8. Score or etch the glass at the top of the masking tape to mark the area of the
glass that will be cut.
9. Cut the glass. Take care that no glass particles fall into the ampoule and no
material is lost from the ampoule.
10. Using a micropipette or a syringe, dilute the antigen powder with 1 ml of sterile
distilled water.
11. Mix until all antigen powders are diluted.
12. Aspirate diluted antigen and transfer to a sterile screw-cap tube.
13. Refrigerate immediately.
14. Record activity in the laboratory log book.
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Name, laboratory manager
Date prepared:
Date reviewed:
Insert date here
Insert date here
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Work Instruction for avian influenza diagnosis in the Pacific
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AVIAN INFLUENZA DIAGNOSIS
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Hemagglutination test (titration of viral antigen)
Document No.:
WI 30.0
Revision 1
Insert date
PURPOSE
 Determine hemagglutinating titer of purchased avian influenza viral antigen.
MATERIALS
□ Single-channel micropipettes (25–50 µl volumes) with tips
□ Multi-channel micropipettes (25–50 µl volumes) with tips
□ “U” or “V” 96-well microtiter plates
□ Plate shaker
□ 4oC storage
□ Plate covers
PROCEDURE
Reminder: The red blood cells (RBC) used to adsorb sera must be the same RBC
that will be used in the hemagglutination hemagglutination-inhibition
(HA-HI) test.
1. Put 25 µl of phosphate buffered saline (PBS) to all wells in a row of a microtiter plate.
(Make duplicates by doing the steps in two rows.)
2. Add 25 µl of virus (antigen) into the first well of each row.
3. Make two-fold serial dilutions of the virus from column 1 to 11 and discard 25 µl from
the last well. Column 12 is the RBC control.
4. Add an additional 25 µl of PBS at all wells.
5. Add 50 µl of 0.5% standardized chicken RBC to all wells.
6. Cover wells and shake gently for 10–15 seconds.
7. Incubate at 4oC (refrigeration temperature) for 30–45 minutes.
8. Examine the plate for hemagglutination.
9. Record results in HA-HI Worksheet (Form 2.a or 2.b).
49
Work Instruction for avian influenza diagnosis in the Pacific
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AVIAN INFLUENZA DIAGNOSIS
Work Instruction
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Hemagglutination test (titration of viral antigen)
WI 30.0
Document No.:
Revision 1
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INTERPRETATION OF RESULTS
 End point is the highest virus dilution at which there is complete agglutination. At
this dilution, the virus is said to contain 1 hemagglutinating unit (HAU) per 25 µl.
Sera are tested against 4 HAU of virus.
 Working dilution of the virus is calculated by dividing the endpoint dilution by 4. If
the endpoint of the titration is at a dilution of 512, the working dilution of this antigen
preparation is 1:128.
1
2
3
4
5
6
7
8
9
10
11
12
RBC
Control
PBS
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
Ag
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
PBS
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
0.5%
RBC
50 µl
50 µl
50 µl
50 µl
50 µl
50 µl
50 µl
50 µl
50 µl
50 µl
50 µl
50 µl
Ag
Dilution
2
4
25 µl
Incubate for 45 minutes at 4oC
End point is taken as the highest dilution to produce 100% RBC agglutination
8
16
32
64
128
256
512
1024
2048
Figure WI 26.0: Guide to antigen titration
Prepared by:
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Date prepared:
Name, laboratory staff
Date reviewed:
Name, laboratory manager
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Insert date here
Insert date here
50
Work Instruction for avian influenza diagnosis in the Pacific
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AVIAN INFLUENZA DIAGNOSIS
Document Type:
Insert name and
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here
Title:
Work Instruction
Page
Back
titration
of
viral
antigen
hemagglutination-inhibition (HI) test
Document No.:
WI 31.0
1 of 2
dilution
Revision 1
for
Insert date
PURPOSE
 Confirm hemagglutinating units (HAU) in standardized antigen dilution.
MATERIALS
□ Single-channel micropipettes (25–50 µl volumes)
□ Multi-channel micropipettes (25–50 µl volumes)
□ “U” or “V” 96-well microtiter plates
□ Plate shaker
□ 4oC storage
□ Plate covers
PROCEDURE
1. Put 25 µl of phosphate buffered saline (PBS) from column 1 to 6 of a microtiter plate.
(Make duplicates or triplicates.)
2. Add 25 µl of virus (antigen) into the five wells.
3. Make two serial dilutions of the virus from column 2 to 5.
4. Discard 25 µl from the last well. Column 6 is the red blood cells (RBC) control.
5. Add an additional 25 µl of PBS from columns 2 to 6.
6. Add 50 µl of 0.5% standardized chicken RBC to all wells.
7. Cover the wells and shake gently for 10–15 seconds.
8. Incubate at 4oC (refrigeration temperature) for 45 minutes.
9. Examine the plate for hemagglutination.
10. Record the results in the HA-HI Worksheet (Form 2.a or 2.b).
51
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AVIAN INFLUENZA DIAGNOSIS
Work Instruction
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2 of 2
Page
Back titration of viral antigen dilution for HI test
WI 31.0
Document No.:
Revision 1
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INTERPRETATION OF RESULTS

The antigen titer is acceptable if results show a hemagglutinating unit (HAU) of
between 2 and 8.
1
2
3
4
5
6
7
8
9
10
11
RBC
Control
PBS
Ag
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
50 µl
50 µl
50 µl
50 µl
50 µl
PBS
0.5%
RBC
Ag
Dilution
50 µl
2
4
8
16
25 µl
discard
Incubate for 45 minutes at 4oC
32
Figure WI 27.0: Guide to back titration for hemagglutination test
Prepared by:
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Date prepared:
Name, laboratory staff
Date reviewed:
Name, laboratory manager
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52
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12
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AVIAN INFLUENZA DIAGNOSIS
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Hemagglutination-Inhibition (HI) tests for avian influenza
Document No.:
WI 32.0
Revision 1
Insert date
PURPOSE
 Determine hemagglutinating antibody titer of test serum (specimen) from suspected
flocks.
MATERIALS
□
□
□
□
□
□
□
□
□
□
□
AI virus antigen dilution
Positive control avian influenza antigen
Sterile phosphate buffered saline (PBS) solution
Single-channel micropipettes (25 to 50 µl volumes)
Multi-channel micropipettes (25-50 µl volumes)
Micropipette tips
Reagent reservoir
“U” or “V” 96-well microtiter plates
Plate shaker
4oC storage
Plate covers
PROCEDURE
Note: The red blood cells (RBC) used to adsorb sera must be the same RBC that will be
used in the hemagglutination hemagglutination-inhibition (HA-HI) test.
1. Prepare worksheets by listing sera and controls on each plate.
2. Perform back titration of all viruses (antigens) to be used.
3. Put 25 µl of PBS from columns 1 to 12 of a microtiter plate.
4. Add 25 µl of 1:4 dilution of test serum to columns 1 to 2. Make two-fold dilution of
each serum from the second well and discard 25 µl from the 11th well. The last well
will serve as the RBC control.
5. Add 25 µl of the working dilution of the virus (antigen) from second to the highest
dilution of each serum. The first well will serve as the serum control.
6. Cover plate and gently shake for 10–15 seconds and incubate at room temperature
(22–25oC) for 1 hour or at 37oC for 30 minutes.
7. Add 50 µl of 0.5% standardized chicken RBC to all wells.
8. Cover plate.
9. Shake gently for 10–15 seconds.
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Hemagglutination-Inhibition (HI) tests for avian influenza
Document No.:
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10. Incubate at 4oC (refrigeration temperature) for 45 minutes.
11. Examine the plate for the presence or absence of hemagglutination.
12. Record the results on the HA-HI Worksheet (Form 2.a or Form 2.b).
INTERPRETATION OF RESULTS

Negative result. Serum control wells showing no non-specific agglutination and the
test wells showing no inhibition of the serum are considered negative for antibody.
Serum samples showing inhibitions at dilutions of 1:8 or less is negative for antibody.

Positive result. Sera showing inhibition at dilutions of 1:16 or greater against 4
HAU of antigen are considered positive for antibody and may be re-tested by HI or
treated with receptor destroying enzyme (RDE) before retesting.

Non-specific positive reaction. Agglutination of serum controls may result to nonspecific positive reactions. Sera should be adsorbed with chicken red blood cells and
re-tested for HI. This type of reaction rarely happens with chicken samples while
sera from other species may cause agglutination of chicken red blood cells.
QUALITY CONTROL

Back titration. The antigen titer to be used for the test should be between 2 and 8
hemagglutinating units.

Negative control. The titer of the negative control serum should be 8 or less.

Positive control. The titer of the positive control serum should be 32 or more.
54
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Hemagglutination-Inhibition (HI) tests for avian influenza
WI 32.0
Document No.:
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Revision 1
1
2
3
4
5
6
7
8
9
10
11
12
RBC
Control
PBS
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
Test
Serum
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
25 µl
50 µl
50 µl
50 µl
1:512
1:1024
1:2048
Antigen
Incubate for 30 minutes at 37oC or 1 hour at room temperature
0.5%
RBC
50 µl
50 µl
50 µl
50 µl
50 µl
50 µl
50 µl
50 µl
50 µl
Incubate for 45 minutes at 4oC
Ag
Dilution
1:4
1:2
1:8
1:16
1:32
1:64
1:128
1:256
* End point is taken as the highest dilution to produce 100% RBC inhibition
Figure WI 28.0: Guide to hemagglutination test for avian influenza
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Date prepared:
Name, laboratory staff
Date reviewed:
Name, laboratory manager
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Immunoflourescence (IF) antibody detection test for
avian influenza
Document No.:
WI 33.0
Revision 1
Insert date
PURPOSE

Detect the presence of antibody against influenza group A.
MATERIALS AND REAGENTS
□ Cover slip (22 x 50 cm or equivalent)
□
Marking pencil
□ Pipette and disposable tips (25–300 µl)
□
Hydrophobic marker
□ Slide rack
□
Test tube for preparing dilution
□ Staining chamber
□
Washing container
o
□ Fluorescent microscope
□ Incubator (37 C)
□ Phosphate buffered saline (PBS)
□ Timer
□ Mounting medium (PBS with 10% glycerol)
□ Anti-species IgG fluorescence conjugate
□ Acetone fixed slides containing AI-infected cells (commercially available)
PROCEDURE
1. Take out the appropriate number of influenza A infected slides from freezer (-20 to
-70oC) and warm at room temperature (22–25oC) prior to use.
2. Add 50 µl of test serum to the well of the slide.
3. Place a positive control in the last well of the slide.
4. Incubate the slide for 30 minutes at 37oC in a humidified staining chamber.
5. Wash slide two times, 5 minutes each wash, in PBS buffer.
6. Wash slide with distilled water. If available, a rotary shaker set at a low speed (30–
40 rpm) should be used.
7. Air dry the slide.
8. Fill the wells with appropriately diluted anti-species fluorescein isothiocyanate (FITC)
conjugate (i.e. anti-chicken IgG) with or without counterstain.
9. Wash by repeating steps 7–8.
10. Air dry slide.
11. Add a drop of mounting medium.
12. Place cover slip on top.
13. Examine under fluorescence microscope.
14. Interpret and record results on the Immunofluorescence Assay Worksheet (Form 5).
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Immunoflourescence (IF) antibody detection test for
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INTERPRETATION OF RESULTS


Negative result. Under an FA microscope, images appear dark or have a dull green
fluorescence.
Positive result. Images under the microscope appears with bright
green fluorescence of the nucleus or cytoplasm depending on the stage of cell
infection. Positive fluorescence should be present in at least 30% of the cells.
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Laboratory Forms
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AVIAN INFLUENZA
SAMPLE IDENTIFICATION WORKSHEET
Form 1.a
Date of collection:
Date of acceptance:
Laboratory #:
Name of farm:
Name of owner:
Location:
Type of farm:
GPS:
Backyard
Semi-commercial
Commercial
Farm animal population (species, # of heads):
Disease history
Species
affected
Onset of
signs
Clinical signs
%
mortality
%
morbidity
Remarks
Sample collection
Avian type
Sample ID
Serum
Swab
Age
Sex
Remarks
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Form 1.b
SAMPLE IDENTIFICATION WORKSHEET
Laboratory #:
Name of farm:
Name of owner:
Location:
Type of farm:
Date of collection:
Date of acceptance:
Backyard
Semi-commercial
Commercial
Farm animal population (species - # of heads):
Disease history:
Treatment and prevention:
Name of medicine
Date of 1st
administration
Quantity
Date of last
administration
Sample Collection
Species
Type of
sample
Age
Sex
Remarks
Requested examinations:
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Form 1.c
SAMPLE IDENTIFICATION WORKSHEET
Date of collection:
Date :
Laboratory technician:
Lab No.
Sample Id
Species
Age
Sex
Owner
Location
Remarks
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HA-HI WORKSHEET
Form 2.a
Plate No. ___________________________
Operator _________________________________
Ag type / Lot # _______________________
Date set up __________
Date prepared (Ag) ___________________
Pos serum HI titer ________
Ag HA titer __________________________
Remarks: _________________________________
1
2
3
4
5
6
7
8
Date read __________
9
Valid test Yes No
10
11
12
A
B
C
D
E
F
G
H
Plate No. ___________________________
Operator _________________________________
Ag type / lot # _______________________
Date set up __________
Date prepared (Ag) ___________________
Pos serum HI titer ________
Ag HA titer __________________________
Remarks: _________________________________
1
2
3
4
5
6
7
8
9
Date read __________
Valid test Yes No
10
11
12
A
B
C
D
E
F
G
H
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HA-HI WORKSHEET
Form 2.b
Plate No. ___________________________
Operator _________________________________
Ag type / Lot # _______________________
Date set up __________
Date prepared (Ag) ___________________
Pos serum HI titer ________
Ag HA titer __________________________
Remarks: _________________________________
1
2
3
4
5
6
7
8
9
Date read __________
Valid test Yes No
10
11
12
A
B
C
D
E
F
G
H
Sample #
Titer
Comments
Sample #
Titer
Comments
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ELISA WORKSHEET
Form 3
Technician ___________________________________
Kit / Manufacturer _______________________________________
Plate No. ____________________________________
Lot / Serial No. _________________________________________
Date ________________________________________
Expiry date:
1
2
3
4
5
6
7
___________________________________________
8
9
10
11
12
A
B
C
D
E
F
G
H
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AGID WORKSHEET
Form 4.a
1
1
1
6
2
6
2
6
2
5
3
5
3
5
3
4
4
4
Plate #
Plate #
Plate #
Date prepared
Date prepared
Date prepared
Date read
Date read
Date read
Ag lot #
Ag lot #
Ag lot #
+ Control lot #
+ Control lot #
+ Control lot #
Lab technician:
Lab technician:
Lab technician:
1
1
1
6
2
6
2
6
2
5
3
5
3
5
3
4
4
4
Plate #
Plate #
Plate #
Date prepared
Date prepared
Date prepared
Date read
Date read
Date read
Ag lot #
Ag lot #
Ag lot #
+ Control lot #
+ Control lot #
+ Control lot #
Lab technician:
Lab technician:
Lab technician:
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AGID WORKSHEET
Form 4.b
1
1
1
6
2
6
2
6
2
5
3
5
3
5
3
4
Sample ID
4
Results
Sample ID
4
Results
Sample ID
1
1
1
2
2
2
3
3
3
4
4
4
5
5
5
6
6
6
1
1
Results
1
6
2
6
2
6
2
5
3
5
3
5
3
4
Sample ID
4
Results
Sample ID
4
Results
Sample ID
1
1
1
2
2
2
3
3
3
4
4
4
5
5
5
6
6
6
Results
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Form 5
Immunofluorescence Assay (IFA) WORKSHEET
Date:
Date:
Lot #:
Lot #:
Technician:
Technician:
Sample ID & Results
Sample ID & Results
1
6
1
6
2
7
2
7
3
8
3
8
4
9
4
9
5
10
5
10
Date:
Date:
Lot #:
Lot #:
Technician:
Technician:
Sample ID & Results
Sample ID & Results
1
6
1
6
2
7
2
7
3
8
3
8
4
9
4
9
5
10
5
10
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Form 6
RAPID TEST KIT WORKSHEET
Kit / Plate #:
Kit / Plate #:
Date:
Lab
Technician:
Date:
Lab
Technician:
Sample ID:
Sample ID:
Result :
Result :
Kit / Plate #:
Kit / Plate #:
Date:
Lab
Technician:
Date:
Lab
Technician:
Sample ID:
Sample ID:
Result :
Result :
Kit / Plate #:
Kit / Plate #:
Date:
Lab
Technician:
Date:
Lab
Technician:
Sample ID:
Sample ID:
Result :
Result :
Kit / Plate #:
Kit / Plate #:
Date:
Lab
Technician:
Date:
Lab
Technician:
Sample ID:
Sample ID:
Result :
Result :
68
Work Instruction for avian influenza diagnosis in the Pacific
SPC LRD - AHP / PRI PPP
Vers ion 2 6 Nov 09
Insert agency logo
here
Insert name and address of agency here
Form 7
LABORATORY SUPPLIES INVENTORY WORKSHEET
Name of Laboratory / Station
Address
Supplies Inventory (consumables)
Supplies
Description (model, brand, etc)
Prepared by: _______________________Position: ______________________
Quantity
Date of last
purchase
Date: ____________
69
Work Instruction for avian influenza diagnosis in the Pacific
SPC LRD - AHP / PRI PPP
Vers ion 2 6 Nov 09
Insert agency logo
here
Insert name and address of agency here
Form 8
LABORATORY EQUIPMENTS INVENTORY WORKSHEET
Name of Laboratory / Station
Address
Equipment
Equipment
Description (model, brand,
etc.)
Condition
Date of last
calibration
Prepared by: ______________________________________________ Date: _____________________
Position: __________________________________________________
70
Work Instruction for avian influenza diagnosis in the Pacific
SPC LRD - AHP / PRI PPP
Vers ion 2 6 Nov 09
Annexes
71
Work Instruction for avian influenza diagnosis in the Pacific
SPC LRD - AHP / PRI PPP
Vers ion 2 6 Nov 09
Annex 1. Selected references for further information
1. Avian influenza. OIE manual of standards for diagnostic tests and vaccines. May 2005. World
Organisation for Animal Health. http://www.oie.int/fr/normes/mmanual/A_00037.htm
2. Collecting, preserving and shipping specimens for avian influenza A (H5N1) virus infection,
Guide to field operations. World Health Organization. October 2006.
http://www.who.int/csr/resources/publications/surveillance/WHO_CDS_EPR_ARO_2006_1/
en/index.htL
3. Dangerous goods regulations manual (50th ed.) International Air Transport Association. 2009.
4. FAQs. Praxiom Research Group Limited.
http://www.praxiom.com/faq2.htm#What's%20the%20difference%20between%20a%20pro
cedure%20and%20a%20work%20instruction.
5. Farlex. The Free Dictionary. http://www.thefreedictionary.com
6. Guidance for the transport of Infectious Substances 2009–2010. World Health Organization.
January 2009.
http://www.who.int/csr/resources/publications/biosafety/WHO_HSE_EPR_2008_10.pdf
7. Manual of avian influenza diagnostic techniques. Bureau of Animal Industry Department of
Agriculture of the Philippines. August 2007.
8. PPE information manual. Pacific Regional Influenza Pandemic Preparedness Project
Secretariat of the Pacific Community. 2009.
https://www.spc.int/prippp/index.php?option=com_docman&task=cat_view&gid=52&dir=D
ESC&order=name&Itemid=102&limit=5&limitstart=5.
9. Preparing for highly pathogenic avian influenza. FAO Animal Production and Health. Food
and Agriculture Organization of the United Nations. Rome, 2006.
http://www.offlu.net/OFFLU%20Site/HPAI_manual.pdf
10. Reference manual on avian influenza for field personnel. Bureau of Animal Industry
Department of Agriculture of the Philippines. 2007.
11. Sampling methods. OIE manual of diagnostic tests and vaccines for terrestrial animals. World
Organisation for Animal Health. May 2005.
www.oie.int/eng/normes/mmanual/A_00011.htm.
12. Shipping protocol template. Pacific Regional Influenza Pandemic Preparedness Project
Secretariat of the Pacific Community. June 2009.
13. WHO guidance on regulations for the transport of infectious substances 2009–2010. World
Health Organization. 2008.
http://www.who.int/csr/resources/publications/biosafety/WHO_HSE_EPR_2008_10/en/ind
ex.htL
14. WHO manual on animal influenza diagnosis and surveillance. World Health Organization.
2002. http://www.who.int/vaccine_research/diseases/influenza/WHO_manual_on_animaldiagnosis_and_surveillance_2002_5.pdf
15. Effect of preservative on recoverable RT-PCR amplicon length from Influenza A virus in bird
feces. David L Evers, Richard D. Selmons, Jeffery K. Taubenberger. 2007.
www.pubmedcnetral.nih.gov/articlerender/fcgi?artid=2504707 .
16. Preservation of tissue RNA in normal saline. Vladimir Vincek, Mehdi Nassiri, jennean
Knowles, Mehrdad Nadji, Azorides R. Morales. Oct 2002.
www.nature.com/labinvest/journal/v83/nl/full/3780599a.htL .
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Work Instruction for avian influenza diagnosis in the Pacific
SPC LRD - AHP / PRI PPP
Vers ion 2 6 Nov 09
Annex 2. Terminology
competency
erythrocytes
the quality of being able to perform a certain task
red blood cells or red blood corpuscles. These
components of the blood are responsible for carrying
oxygen to tissues and removing carbon dioxide from
tissues.
guidelines
group of standard operating procedures used to carry out
a given operation in a given situation
hemagglutination inhibition
test
synonymous to HI or HAI test; an assay for the presence
of specific antiviral antibodies in a test serum. The serum,
usually a twofold dilution series, is mixed with a standard
number, usually 4 to 8 HA units, of virus and incubated
prior to the addition of a standard suspension of
erythrocytes (red blood cells). The highest dilution of
serum that inhibits hemagglutination is the HI titer of the
serum.
hemagglutination test
synonymous to HA test; hemagglutinating viruses directly
agglutinate (clumping) erythrocytes (red blood cells) by
binding to specific receptor sites on the surface of the
erythrocyte and this characteristic can be used in
detection, identification and quantitation of the virus
hemagglutination viruses
viruses capable of agglutinating red blood cells of a variety
of animals, e.g. adenoviruses, parvoviruses, togaviruses,
some coronaviruses, picornaviruses, orthomyxoviruses
and paramyxoviruses. Useful in classifying viruses and
assaying amounts of virus and antibody.
manual
A reference text that can include guidelines, operating
procedures and work instructions
standard operating
procedures
A set of specific steps to be followed in carrying out a
given procedure or activity that also provides relevant
background information for the procedure or activity. As a
minimum a standard operating procedure document
should answer the question of why, how, and when a
procedure or activity is to be performed and outline any
safety risks.
work instructions
Step-by-step instructions that describe how a specific task
is to be performed. Work instructions are a simpler
document than a standard operating procedure document.
.
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Work Instruction for avian influenza diagnosis in the Pacific
SPC LRD - AHP / PRI PPP
Vers ion 2 6 Nov 09
Annex 3. Hand washing poster
http://www.spc.int/prippp/index.php?option=com_docman&task=cat_view&gid=46&Itemid=102
74
Work Instruction for avian influenza diagnosis in the Pacific
SPC LRD - AHP / PRI PPP
Vers ion 2 6 Nov 09
Annex 4. Animal health post-mortem kit checklist
The following equipments are necessary for post-mortem examination and sample collection
in animals:
Item
1
2
3
Apron
Blood collection needle
holder / sleeve
Blood collection needles
4
Blood collection tubes
5
6
7
Blood collection tubes
Blood collection tubes
Bone cutters
Culture tubes with sterile
swabs
Culture tubes with sterile
swabs
9
10
11
12
13
14
15
16
Digital camera
Fixative - 10% buffered
formalin
Fixative - 70% alcohol
Forceps, pointed
Forceps, rat toothed
Gloves, latex examination
17
18
GPS, handheld
Ice packs/bricks
19
Knife scabbard
20
21
22
23
24
25
Knife, boning
Knife, skinning
Masks, surgical
Microscope slides
Needles, sterile disposable
Pipettes, disposable
26
Plastic bags
27
Plastic bags, biohazard
Sample vial, glass, screw
cap
Sample vial, plastic, screw
cap
Scabbard chain
Scalpel blades
Scalpel handle
28
29
30
31
32
Description
reusable waterproof full length
apron
reusable plastic sleeve to use with
Vacutainer™ assembly
to suit Vacutainer tubes
Vacutainer™ type, plastic, purple
top (anticoagulant)
Vacutainer™ type, plastic, red top
(clot activator)
serum tubes (black tops) plain
heavy duty secateurs
containing viral transport medium
(VTM)
containing Stewart's solution form
bacteria
Unit or size
Quantity
per kit
1
10
100
5 ml
50
5 ml
10 ml
pair
50
50
1
10
10
1
pre-diluted, ready to use
ready to use
pointed tissue forceps
serrated forceps
variable sizes
sturdy, waterproof, standard
batteries
solid brick type
to fit the 2 knives - about 250 mm
deep
curved skinning knife
23 g x 1" and 21 g x 1"
plastic, 3 L transfer pipettes
sturdy press sealed or zip-lock
type medium (250 mm) and large
(400 mm)
large biohazard waste disposal
bags
clear glass, leakproof screw-cap
bottles
plastic, screw-cap vials,
CryoTube™ type
for knife scabbard
size 15, 22
small, to fit blades above, size 3, 4
100 ml bottle
100 ml bottle
150 mm
150 mm
box 100
1
1
1
1
1
each
1
4
each
150 mm (6") to
200 mm (8")
200 mm (8")
box 100
box 100
box 100
1
1
1
1
1
1 each
50
10 each
5
20 ml
5
2–3 ml
50
1
1 each
2 each
box 100
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Work Instruction for avian influenza diagnosis in the Pacific
SPC LRD - AHP / PRI PPP
Vers ion 2 6 Nov 09
33
34
Item
Scissors, dissecting,
curved
Scissors, straight
35
36
Scrubbing brush - boots
Scrubbing brush - nails
37
Sharpening steel
38
Sharpening stone
39
Sharps container
40
41
42
43
Specimen collection jar
Spray bottle
Swabs, sample collection
Syringes
44
Tape
45
Tool box, plastic box
46
Burdizzo
Specimen Transport
Packaging - IATA
compliant for Category B
ambient
Description
stainless steel
scrubbing brush with handle
(dishwashing style)
nail scrubbing brush
Unit or Size
Quantity
per kit
150 mm
100 mm
2
1
each
each
300 mm at
least
100 mm x 20
mm
1
1
durable plastic, 0.5–1 L
50 ml leakproof, screw-cap plastic,
with label
1
1
1
500 ml
packet 100
box 100
12
1
1
1 each
roll
1
50 L
1
each
1
each
5
each
1
1 roll
1
49
Specimen Transport
Packaging - IATA
compliant for Category B
chill pack
Absorbent packing
material
cotton tipped, plastic handle, 15cm
sterile disposable 2 ml and 5 ml
Duct tape, 100 mm wide, heavy
duty
sturdy heavy duty plastic container
with lid and handle to hold these
items
castration clamp (for cervical
dislocation of poultry), bloodless
type, 36 cm
bio-bottle or similar system, 850
ml capacity, including bottle, lid,
absorbent pad, labels, secondary
carton etc
outer carton , thick walled
thermally efficient polybin, BioPouch® 5NZ, 2 x gel packs,
absorbent pad and bubble wrap
bag
cotton wool, paper towel,
newspaper
50
Bucket, plastic
10 L with handle
1
51
Esky medium
hard, small and medium
1
52
53
54
55
56
57
Face Shield
Marker, permanent
Paper
Pen
Soap
Sodium hypochlorite 0.5%
47
48
clear plastic, reusable, to below
chin
surveillance worksheet/documents
disinfectant if possible
household bleach
1 bar
1 L bottle
2
2
1
2
1
1
76
Work Instruction for avian influenza diagnosis in the Pacific
SPC LRD - AHP / PRI PPP
Vers ion 2 6 Nov 09
77
Work Instruction for avian influenza diagnosis in the Pacific
SPC LRD - AHP / PRI PPP
Vers ion 2 6 Nov 09