changchun tu - world organisation for animal · pdf fileemail: [email protected] 1. ......
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International standards
for rabies diagnosis——
more than the book
Changchun Tu(Ph.D & DVM)
Email: [email protected]
http://cvrirabies.bmi.ac.cn
1. Diagnostic Laboratory on Rabies and Wildlife Associated Zoonoses (DLR), Chinese Ministry of Agriculture.
2. Changchun Vet Res Institute (CVRI), Chinese Academy of Agri Sci
3. OIE Reference Laboratory for Rabies
May 17-19, 2016, Bangkok, Thailand
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In summery
1. Sample collection
2. Shipment
3. Laboratory tests
(1) Detection of viral antigen
a. FAT
b. dRIT
c. antigen ELISA
d. Immunochromatographic test strip (ICT)
(2) Detection of viral replication
a. Cell culture test (RTCIT)
b. Mouse inoculation test (MIT)
(3) Molecular techniques
a. RT-PCR
b. real-time PCR
c. genotyping (phylogeny)
d. microarray
4. Serology tests
(1) virus neutralisation: FAVN and RFFIT
(2) antibody ELISA
(3) ICT
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Collection of brain samples
1
987
654
32
Opening of the skull
By courtesy of APHA,UK.
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Straw method:
• WHO、OIE reccommendated
• Suitable for field sampling
• Simple
• low risk to produce aerosol
A 5 mm drinking straw is inserted into the occipital foramen in the direction of an eye. Samples can be collected from the rachidian bulb, the base of the cerebellum, hippocampus, cortex, and medulla oblongata.
Occipital foramen route
Collection of brain samples
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Expose the occipital foramen
Insert plastic straw
Occipital foramen route for brain sampling
Collection of brain samples
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Collection of brain samples
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Collection of brain samples
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• Golden standard of OIE and WHO
• Using fluorescein isothiocyanate (FITC)-conjugated mAbs.
• Suitable for brain tissue; cell monolayers
• Simple with high sensitivity and specificity
• Reliable and 98-100% accurate
• Most widely used in the world
• Requisite for rabies laboratory
• National standard of China (GB/T18639-2002)
• But needs fluorescent microscope, trained personnel
Rabies fluorescent antibody test (FAT)
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pick up a tiny part of specimen (0.02 g) with a tip, place on a wooden spatula
1
Imprint on a slide
2
Rabies fluorescent antibody test (FAT)
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Cold aceton fixation for 20 min
3
Observe under FM
6
Incubation at 37℃ for 30 min
5
Place a drop of FITC-conjugate
4
Rabies fluorescent antibody test (FAT)
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dog
Negative staining
cow sheep pig neuron
Rabies fluorescent antibody test (FAT)
Positive staining
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A. Bioveta (Czech Republic)B. Milipore (United Kingdom)C.Fujirebio (USA)D. SIFIN (Germany)E. Bio-rad (France)
E. Robardet et al. Journal of Virological Methods. 191 (2013) 88–94.
Rabies fluorescent antibody test (FAT)
Bioveta Milipore Fujirebio SIFIN Bio-rad
Potency comparison of five comercial FICT-conjugates
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Fujiribio(USA) Milipore (UK) SIFIN (Germany)
RABV ++++ ++++ ++++
LBV + ++ ++++
MOKV + ++++ ++++
DUVV + ++ +
EBLV-1 + +++ +++
EBLV-2 ++ +++ +
ABLV +++ ++++ ++
RABV
Fujiribio Milipore SIFIN
4 ++++: very bright green fluorescence3 +++ bright green fluorescence2 ++ dull green fluorescence1 + dim but detectable green fluorescence
Scores:
Rabies fluorescent antibody test (FAT)
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(Lembo, et al. Evaluation of a Direct, Rapid Immunohistochemical Test for Rabies Diagnosis. Emerging Infectious Diseases, 2006)
Direct rabies immunohistochemistry test (dRIT)
dRIT FAT
Using Biotin-conjugated mAbs.
As the same specific and sensitive as FAT
Used as an alternative to FAT
Use only light, not fluorescent, microscope
Suitable for field work
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Direct rabies immunohistochemistry test (dRIT)
~100% coincident with FAT
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Biotin-conjugated mAbs against N protein of RABV
Positive Negative
Direct rabies immunohistochemistry test (dRIT)
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FAT dRIT
Direct rabies immunohistochemistry test (dRIT)
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donkey
Positive control Negative control
camel bovine
dog sheep
fox
Test on brain tissues of different animals
Direct rabies immunohistochemistry test (dRIT)
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Comparision of dRIT with FAT
animal totalno. of positive no. of negative
FAT dRIT FAT dRIT
Dog 82 32 31 50 51
Sheep 6 6 5 -- 1
Cow 7 7 7 -- --
Camel 4 4 4 -- --
Donkey 1 1 1 -- --
Foxe 2 2 2 -- --
mouse 3 3 3 -- --
合计 105 55 53 50 52
Coincidence: 99.05%
Direct rabies immunohistochemistry test (dRIT)
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• OIE and WHO standard
• National standard of China: GB/T18639-2002
• high sensitivity
• used for virus detection and isolation
• disadvantage: time consuming, high-containment facility
• N2A and BHK-21 cell lines
Rabies Tissue Culture Isolation Test (RTCIT)
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• OIE and WHO standard
• Use of 3-4 weeks old young mice (12-14
g) or 2 day-old suckling mice
• high sensitivity
• used for virus detection and isolation,
virulence determination, vaccine potency
test (NIH)
• Advantage: easily and practicably applied
when RTCIT not available
• Disadvantage: long time (up to 28 days), high-containment facility, animal welfare issue, high cost etc
• Use is reduced (3R principles)
Mouse inoculation test (MIT)
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Target region
RT-nPCR of 7 LVs
Molecular methods
RT-nPCR
• OIE and WHO reccommended
• high sensitivity and specificity
• detection of virus gene,
• Genotyping: phylogenetic analysis
• Advantage: screening of large scale
samples; can be automated
• Disadvantage: high levels of false
positive or false negative; very stringent
quality control (partition of room).
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Tagman RT-PCR
• OIE and WHO reccommended
• Rapid, sensitive, specific, reproducible
• Advantage: automatic; screening of large
scale samples, quantification of the virus
• Disadvantage: false positive or false
negative; very stringent quality control
(partition of room).
Molecular methods
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Room partition for PCR: Four-room principle
• Room 1: Reagent storage and pre-mixture prep
• Room 2: Prep of viral nucleic acid and PCR reaction
• Room 3: PCR
• Room 4:Gel running and documenting
Molecular methods
Room 1Room 2
Room 3
Room 4
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Day post
decompositionFAT* RT-nPCR* Tagman RT-PCR* MIT*
1 +/+ +/+ +/+ -/-
2 +/+ +/+ +/+ -/-
3 +/+ +/+ +/+ -/-
4 +/+ +/+ +/+ -/-
5 +/+ +/+ +/+ -/-
6 +/+ +/+ +/+ -/-
7 +/- +/+ +/+ -/-
8 -/- +/+ +/+ -/-
9 -/- +/+ +/+ -/-
10 -/- +/+ +/+ -/-
11 -/- +/+ +/+ -/-
12 -/- +/+ +/+ -/-
13 -/- +/+ +/+ -/-
14 -/- +/+ +/+ -/-
15 -/- +/+ +/+ -/-
16 -/- +/+ +/+ -/-
17 -/- +/+ +/+ -/-
注:“+”positive;“-”negative *TJ105 strain/CQQJ-09 strain
Comparison of 4 tests in detection of two decomposed samples
Test of decomposed brain tissue
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Rabies virus neutralisation (VN) includes FAVN and RFFIT
Serology tests
• Golden standard of OIE and WHO
• Prescribed test for international trade or travel of live
animals
• Quantification of antibody level (0.5 IU/ml)
• Most widely used in the world
• Requisite for professional rabies laboratory
• Disadvantage:time-and-labor-consuming, high-containment facility, high cost.
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OIE recommended
Suitable to detect the herd immunity
Advantage: rapid, no live virus, large scale
screening
Commercialized
Disadvantage: not well correlates with VN
Antibody ELISA
Serology tests
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Company Kit
BIO-RAD (France)
Recognized and prescribed by OIEBio-Rad Platelia TM Rabies II kit
BioPro (Czech Republic) BioPro Rabies ELISA Ab kit
Alpha Diagnostic International(ADI) Rabies virus antibody (IgG) ELISA Kit
TSZ (USA) Rabies virus antibody (IgG) ELISA Kit
R&D (USA) Rabies virus antibody (IgG) ELISA Kit
IBL (German) Rabies virus antibody (IgG) ELISA Kit
Antibody ELISA
Serology tests
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1. Sample collection and processing
2. FAT or dRIT
3. RT-nPCR (real time RT-PCR)
4. Sequencing and genotyping
5. MIT
6. RTCIT
7. FAVN or RFFIT
8. Sample storage
Minimum requirement for national and regional laboratory
Basic laboratory capacity
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Minimum requirement for local and
provincial laboratory
1. Sample collection and processing
2. FAT or dRIT
3. RT-nPCR (real time RT-PCR)
4. Sample strorage
5. Antibody ELISA
Basic laboratory capacity
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BSC class II in BSL-2
Laboratory biosafety regulation in China (RABV)
Name Biosafety level
Risk category II
Culture of street virus BSL-3
Animal challenge with street virus ABSL-3
VN with fixed virus BSL-2
Original infectious sample (without
artificial culture)
BSL-2
Killed sample BSL-1
Non-infectious operation BSL-1
Laboratory biosafety
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Web:www.cvrirabies.bm.ac.cn
Tel:+86 431 80887698
Acknowledgement
2014.08