Changchun Tu - World Organisation for Animal · PDF fileEmail: [email protected] 1. ......

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

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

Collection of brain samples

1

987

654

32

Opening of the skull

By courtesy of APHA,UK.

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


Expose the occipital foramen

Insert plastic straw

Occipital foramen route for brain sampling


• 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)

pick up a tiny part of specimen (0.02 g) with a tip, place on a wooden spatula

1

Imprint on a slide

2


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


dog

Negative staining

cow sheep pig neuron


Positive staining

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.


Bioveta Milipore Fujirebio SIFIN Bio-rad

Potency comparison of five comercial FICT-conjugates

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:


(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


~100% coincident with FAT

Biotin-conjugated mAbs against N protein of RABV

Positive Negative


FAT dRIT


donkey

Positive control Negative control

camel bovine

dog sheep

fox

Test on brain tissues of different animals


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%


• 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)

• 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)

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).

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

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

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

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.

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

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

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

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

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

Web：www.cvrirabies.bm.ac.cn

Tel：+86 431 80887698

Acknowledgement

2014.08

Changchun Tu - World Organisation for Animal · PDF fileEmail: [email protected] 1. ......

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