Poultry disease diagosis

Practical approaches for diagnosis of poultry
viral diseases
1. Clinical diagnosis
2. Rapid field diagnostic tests
3. Serological diagnosis (HI& ELISA)
4. Molecular diagnosis (PCR technologies)
5. Isolation and characterization (the gold standard methods limited to
reference laboratories)

1. Clinical diagnosis
Clinical diagnosis was made on the basis of:
1. Case history.
2. Clinical signs.
3. External examination of live and dead, birds.
4. Gross pathological lesions of the dead and sick bird

Respiratory infections
Agent Respirator
y infection
GIT NEU. Renal
HPAI √ √ √ √
LPAI √ √ √ √
NDV √ √ √ √
IBV √ ?
√
ILTV √
aMPV √
M.g √
M.s √
E.coli √ √ √ √

Are clinical symptoms enough for confirmative
diagnosis of infection?
Symptoms are not confirmative, for example:
1. HPAI can share Velogenic NDV in high mortality number (90-100%),
nervous and/or digestive signs, Haemorrhages of proventricular glands and
haemorrhages of Peyer's patches.
2. LPAI can share IB in respiratory signs (caseous plugs) and kidney affection
or other respiratory pathogens that affect egg production and quality.
3. Don’t forget presence of mixed (AI with IB or NDV ) and subclinical
infection (IBDV- CAV-AE-REO).
4. So you need quick accurate tool to confirm and differentiate your suspected
diagnosis.

How to confirm your clinical diagnosis?
Range of Detection of AIV in a Flock (Unvaccinated)VirusLevel
Days Post-Infection
0 7 14 21 28
Rapid field tests
B. Antibody detection
rRT-PCR
Virus Isolation
HI (IgG)
ELISA (IgG)
A. Direct Virus detection

1. Limitation of rapid antigen tests
1. Most useful for sick and dead birds (acute)
2. Time of application: at beginning of clinical signs (high titter of the
virus) in case of AI up to 5 days after beginning of clinical signs.
3. AI detection limit: 103.5-104.5 EID50/ML.
4. Sensitivity depend on the titter of virus shedding, so it confirm
positive result not negative result.
5. The tests should only be interpreted on a flock basis and not as an
individual bird test (up to10 test/house).
6. Preliminary tests: so need further tests---------(Molecular diagnosis-
isolation).

3. Limitation of Serological diagnosis
(B. Antibody detection)
Ex. HI& ELISA.
1. Vaccination programs.
2. Disease monitoring
• Baseline establishment
• Experience in interpretation
• Delayed Action.
• Multiple reagents and in some cases live antigens
• ELISA Reader and High skillful technical
Diagnosis can only be established when one combines VI
scores with clinical symptoms and isolation of the pathogen
and/or confirmation testing with PCR.

4. Molecular Diagnosis
(A. Virus detection)
PCR technologies
• Detecting the nucleic acids of the pathogens.
• High sensitivity.
• High specificity.
• Samples can be the environmental samples and biological materials.

PCR application
 Screen the most concerned pathogens.
 Identify the pathogen from the common clinical sign (ex. Differential diagnosis for
respiratory clinical signs).
 Monitoring the pathogen before the vaccination (subclinical infection with
IBDV, AIV H9 and CIAV, Mycoplasma).
 Detect the pathogen from weak birds prior the ELISA antibody result turns to be
positive.
 Enhancing biosecurity.

The PCR Lab that you know
• Sensitive
• Great for diagnostics
• Confirmation test
• Time
• Performed by
• well-trained personnel
• On expensive devices
• With complicated procedures

ARTAT with GeneReach Provide a new solution
for complete PCR system (pocket Combo)

Traditionnel PCR lab to mobile PCR lab
Does not
require high
biosafety
level

POCKIT combo (Mobile PCR Lab in suitcase)
Specification
1) 1 POCKIT™ Nucleic
Acid Analyzer
2) 1 taco™ mini
Automatic Nucleic Acid
Extraction System
3) 1 cubee™ Mini-
Centrifuge
4) 2 Pipettes included
(p1000 & p50)
4
3
1
2

QUICKER TEST RESULTS AND QUICKER RESPONSE:
Pen-side Test: iiPCR POCKIT Portable system

GeneReach – Compliance of Quality
System
 Human medical device manufacturing process
Certification: OIE (2008, 2013)
ISO 9001:2008
ISO 13485:2003
GMP Factory

Certificates of ISO and GMP
ISO 9001:2008 ISO 13485:2003 GMP

GeneReach Biotechnology Corporation
Worldwide Coverage
America 30%

Safe your time by POCKIT Combo system
From sample to result only 90 minutes
DNA/RNA Amplification &
Detection
tacomini Automatic DNA/RNA Extraction
Instrument & Reagents
1.5 hoursSamples Results
25 minutes
60 minutes

Technical principles
• GeneReach depend on 2 techniques
1. For RNA/DNA extraction:
Principle of Magnetic Particle Extraction (e.g. Taco™mini System)
2. For nucleic acid amplification and qualitative detection
Insulated Isothermal PCR (iiPCR) (POCKIT™ system)

1. Principle of Magnetic Particle Extraction
(taco™mini System)
taco™/ taco™mini System is
based on the magnetic
separation technology, with
the special design of the
disposable taco™ Plate &
Comb which is provided with
the reagent kit. Samples and
reagents including the
magnetic particles are
dispensed into the wells
according to the
corresponding kit instructions.
Magnetic beads are surface-modified
Magnetic beads are capable for binding nucleic acid
Lysis by lysis buffer in blue well Washing from well 2-5 by washing buffer (A 2
times &B 2 times) Release DNA/RNA in Eluting buffer (green well)

taco™ Nucleic Acid Extraction Reagents
taco™ Preloaded DNA/RNA Extraction Kit
Preloaded 48-Well Plate for DNA/RNA extraction
48 rxns
(6 plates)
Preloaded Kit
Get DNA/RNA in 25 minutes
Open the seal and
load the samples
Load the mixing comb and
extraction plate for extraction
mixing comb

Efficiency of RNA extraction with taco DNA/RNA extraction kit was equivalent to that
of RNeasy mini kit (Qiagen) with the most common types of avian samples for the
diagnosis and the surveillance.
Swabs Tissue homogenate
Correlation of results (Ct values) between different RNA extraction kits for various types of avian samples

2. Principle of Insulated Isothermal PCR (iiPCR)
(POCKIT™ system)
• POCKIT uses Taqman probe chemistry (similar to RT-PCR)
• POCKIT uses Thermal convection in capillary reaction tube to drive NA amplification.

Principle of Insulated Isothermal PCR (iiPCR)

Consistent uniform temperature profile can be
generated by POCKITTM (iiPCR)
Temperature Program for Heater
1.50 ℃ 10 minutes for RT reaction
2.95 ℃ 30 minutes for PCR

Optic
collection
apparatus
Digital Signal
Processing
Chip
LED
excitation
FAM (520 nm)
JOE (550 nm)
Fluorescent PCR
TaqMan reagent
Automatic Data Interpretation Process
One-touch operation without programming
Band-pass filter
Optical Fiber

Traditional PCR/ RT PCR iiPCR
Time-consuming (usually > 1 h) Time-saving (< 1 h)
Instrument is heavy Light (Portable)
Complicated set-up (Different programs for
each disease)
Single default program
Well trained personnel No specific training is required
High Investment Affordable system: make molecular detection in
the out reach of all customers
Specific lab design, ventilation system &
workflow
Open system , small bench is sufficient
Quantitative for RT PCR Qualitative System: +ve/-ve, but master mix
uses same technology of RT-PCR & can be used
on any RT-PCR Universal System.
Traditional PCR / RT PCR v.s. Insulated Isothermal PCR (iiPCR)

48 tests/package
Analytical sensitivity up
to 10 copies/reaction
Storage at 4°C
iiPCR Specific Reagents for poultry
(Ready to use)
1) Avian Influenza H5 Subtype
2) Avian Influenza H7 Subtype
3) Avian Reovirus
4) Chicken Infectious Anemia Virus (CIAV)
5) Influenza A Virus
6) Salmonella spp. (After enrichment)
7) Infectious Bronchitis Virus (IBV)
8) Infectious Laryngotracheitis Virus (ILT)
9) Infectious Bursal Disease Virus (IBDV)
10) Mycoplasma gallisepticum
11) Mycoplasma synoviae
12) Newcastle Disease Virus (class I-class II- LaSota Strain)
13) Under developing: Marek's disease virus and Avian influenza H9 subtype

iiPCR suggestive Application
1. Diagnosis of acute cases of poultry diseases.
2. Differential diagnosis of multiple respiratory diseases in the same time (eg.
IBV& NDV& AIV A/H5/H9 & ILT and Mycoplasma).
3. Monitoring of subclinical infection eg. IBDV, AIV H9, Reo virus, CIAV, Marek’s,
and Mycoplasma (especially before administration of vaccines- causes of
vaccination failure
4. Pathogens affect Hatchability: (eg. Mycoplasma and Salmonella
5. Baby chicks: Vertical transmitted diseases (eg. Mycoplasma, Salmonella, Reo
virus and AE).).
6. Tracking the source of infection from biological and environmental samples (eg.
Salmonella and AIV).
7. Screening layer/ breeder flocks 3 weeks before transfer to production houses
(Salmonella, mycoplasma, AE, AIV, EDS, Reo)

1) Sampling (swabs or tissues):
a) swab sample pool:
up to 5 swab samples/5ml PBS or Storage buffer.
b) Tissue sample:
40 mg into a clean 1.5 ml micro centrifuge tube with grinding in 0.5 ml PBS or storage buffer
grinding using disposable grinder.
2) Centrifugation with cubee™ Mini-Centrifuge for 1 minute
3) Take 200 µl supernatant for extraction in 48-well taco™ Preloaded DNA/RNA
Extraction plate Kit (48 rxns)
How to use POCKIT™combo
in poultry diseases Diagnosis?

4. Open the door of the instrument, insert Mixing Comb and Preloaded
48-Well Extraction Plate with sample (extraction will be finished in
25 minutes)

Pilot study of Pen-side PCR for Avian
influenza surveillance at live bird market in
Northern Vietnam
FAO Vietnam and DAH Vietnam

Stop Influenza spreading
Quicker PCRresults in onehour

2017 New outbreak of avian influenza in China

Present danger to Vietnam
H7N9?

Test
Test 1: Test results with H7N9 virus diluted by 10-fold
Virus
dilution
-3 + + + 25.4 25.3 25.4
-4 + + + 28.7 28.6 28.5
-5 + + + 31.8 32.1 32.2
-6 + + + 35.3 35.3 36.2
-7 + + + 40.0 37.0 39.9
-8 + - - neg neg neg
Pockit M qPCR at Lab (Ct value)
Sensitivity and specificity of POCKIT Influenza A reagent for
Flu A detection
Positive Negative
Pos 23 0 23
Neg 1 18 19
24 18 42
Specificity (%)=
Sensitivity & specificity of Pockit M compared with qPCR M
100.0
94.7
Pockit M
qPCR M*
Total
Total
Sensitivity (%) =

Sensitivity and specificity of POCKIT H7 for H7N9 detection
Test 2: Results of Pockit H7 with samples of Ct value between 27-37
qPCR*
Pos Neg Tested
27 1 0 1 100
28 2 0 2 100
29 2 0 2 100
30 1 0 1 100
31 3 0 3 100
32 2 0 2 100
33 2 0 2 100
34 3 0 3 100
35 3 1 4 75
36 1 0 1 100
37 0 1 1 0
Negative 0 8 8 100
Total 20 10 30
Pockit H7
% AgreementNumber of samples
Ct value
Test
Test 1: Test results with H7N9 virus diluted by 10-fold
Virus
dilution
-3 + + + 25.0 24.8 24.8
-4 + + + 28.2 28.1 28.1
-5 + + + 31.5 32.1 31.7
-6 + + + 35.3 34.8 36.1
-7 + - - 38.6 38.8 37.4
-8 - - - neg neg neg
Pockit H7 qPCR at Lab (Ct value)
Positive Negative
Pos 20 1 21
Neg 0 9 9
20 10 30Total
Sensitivity (%) = 95.2
Specificity (%)= 100.0
Sensitivity & specificity of Pockit H7 compared with qPCR M
Pockit M
Total
qPCR M*

Pen-side / Point of care PCR
• Needs
– Quick results for quick response / control measures / treatment
– Screening test in the field
• Challenge of the current system
– Takes time for transportation of samples from field to lab
• Requirements
– Easy to use: does not require high skill to operate
– Portable equipment (battery operated)
– Easy reagent storage (does not require freezers)
– Cost: not expensive
– Equivalent level of sensitivity with qPCR in the lab = no compromise

Pipe line of taco
8 samples extraction 24 samples extraction

Pipe line of POCKIT

POCKIT Central
Features
• Sample in, result out fully automatic nucleic acid detection.
• Extraction based on magnetic particle transfer technology.
• iiPCR technology with 2 optical channels (520 nm, 550 nm for
nucleic acid detection.
• Disposable preloaded reagents and consumables.
• Sample ID input.
• Built-in data storage with USB port available.
• Built-in UV light for sterilization.
• External printer optional.
• Run time: within 2 hours from extraction to
amplification/detection.
• Operator skill required: minimal.

Specifications of POCKIT Central and reagent
cassette
Specifications of POCKIT Central
Throughput 1 -8 samples
Reaction Time within 2 hours
Fluorescent
Wavelength
520 nm &550 nm
Dimensions (W xH xD) 310 x 400 x 480 mm
Weight Approx. 25 kg

THANK YOU
Dr. Moustafa Mohamed Elshazly
Master of poultry diseases
Assistant Manager of Diagnostic & Laboratory Supplies/ Poultry Sector
E-mail: moustafa@artat.com.sa

Poultry disease diagosis

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