Active and Passive Avian Influenza
Surveillance/Monitoring Programs with the Most Adequate Screening Tool
and the Right Sample Size
Avian influenza (AI) is a viral disease caused by type A Orthomyxovirus.
There are three influenza types—A, B and C—but only
influenza A viruses are known to infect birds. Diagnosis is by
serology, isolation and characterization of the virus. This is because
infections in birds can give rise to a wide variety of clinical signs
that may vary according to the host, strain of virus, the host’s
immune status, presence of any secondary exacerbating organisms and
environmental conditions, but may only be displayed as a reduction in
feed consumption.
The virus has been recovered from domestic and wild avian
species throughout the world, and impacts international trade
in poultry and poultry products. Shorebirds and ducks have been
associated with carrier status of different types of AI viruses due to
five major migratory pathways:
- The East Atlantic flyway
- The Black Sea/Mediterranean flyway
- The West Asia/West African flyway
- The Central Asia/India flyway
- The East Asian-Australasian flyway
Figure 1.
Two forms of AI exist:
-
Highly pathogenic—characterized by acute systemic disease
with rapid onset high mortality. This form of the disease should be
ruled out from the very velogenic form of Newcastle disease, as OIE
reportable diseases.
-
Low pathogenic—characterized by high morbidity and low
mortality. From the clinical perspective, the low pathogenic
conditions can be misdiagnosed with other respiratory diseases or
syndromes, even with rolling-vaccine reactions.
Low pathogenic avian influenza (LPAI) has spread
in many poultry regions, such as Mexico (1994) and Holland (2003), due
to the lack of on going active or passive surveillance/monitoring
programs.
On a continuous and random basis, a statistically valid number of
serum samples should be tested to monitor for the presence of
antibodies against avian influenza, using serological tests that can
detect antibodies against the nucleo-protein (NP) or the
matrix-protein (M) from any avian influenza virus. ELISA and agar gel
immunodiffussion (AGID) tests are suggested by the OIE as screening
tools for nonvaccinated or presumed-negative populations. Laboratories
can use ELISA as a screening tool and AGID as a confirmatory tool for
serological programs.
Surveillance and monitoring programs—A
monitoring program utilizes sample size and frequency of sampling,
among other factors. One of the main concerns in the field is the
amount of samples to be collected. The determination of the minimum
amount of samples is vital for the validity of the results.
The AI status should be determined by an ongoing surveillance and
monitoring program (carried out in conformity with the Appendix
3.8.9 published by the OIE) based on serology results, DNA
detection (real-time PCR) and virus isolation. The program may need to
be adapted to target parts of the country that are at higher risk due
to historical or geographical factors, population data, proximity to
recent outbreaks, presence of lakes or areas where migratory birds can
potentially mingle with commercial animals.1
Freedom of infection in a country or zone can be demonstrated by
serological surveillance at a minimum interval of six months. The
program should provide results based on a 95 percent confidence level
for detecting infection, assuming less than 5 percent prevalence.
A fifty-nine (59) sample size of serum samples is the most accurate
and statistically valid number per population, but if your
company/area/region is assuming lower risk of exposure, the sample
size could be taken based on 10% prevalence (29 samples per
population). All these numbers are valid for poultry farms with 500,
1,000, 2,500, 5,000, 10,000 or more birds in the same farm.
Birds should share the same biosecurity, environment and management
program to be considerd a single poultry population.
Table 1.
Sampling
Concepts
A sample is any part of a population, whereas sampling is the process
of collecting samples from a population. Learn more
about sampling and sample size.
Screening strategy with serological tests—As
all influenza A viruses have similar nucleocapsid and matrix antigens,
ELISA and AGID tests are used to detect antibodies to these antigens.
Not all birds develop demonstrable precipitating antibodies.
Hemagglutination inhibition tests (HI) have also been employed in
routine diagnostic serology, but it is possible that this technique
may miss some particular infections because the hemagglutinin is
subtype specific. ELISA tests have been accepted as a screening tool
by the OIE.1
Figure 2.
The IDEXX FlockChek*
Avian Influenza Test Kit is an enzyme-linked immunosorbent assay
(ELISA) for the detection of antibodies against type A influenza
virus, approved and licensed by the USDA. The test is an indirect
format, utilizing avian influenza virus antigen immobilized in
microtiter wells. The serum is diluted in specific sample diluent,
which is formulated to minimize the amount of nonspecific binding to
the antigen-coated wells. Following incubation with serum, unbound
materials are washed away and antigen-antibody complexes are detected
with an anti-chicken/anti-turkey immunoglobulin-enzyme conjugate.
Detection of a serologic response in the test serum is demonstrated by
conversion of chromogen by the enzyme. Results are analyzed by
calculation of an adjusted sample absorbance divided by the adjusted
positive control absorbance (S/P ratio) reported by xChek* software. IDEXX has
validated the kit with the USDA (U.S.) and the animal health
institute-Deventer (Holland). The test kit is sold in more than 60
countries and licensed in more than 14 countries, including Germany,
Brazil, Holland, Australia and Poland.
The
IDEXX FlockChek* Avian Influenza Test Kit is USDA-approved and would
be considered an official test under Code of Federal Regulations,
title 9, subchapter G - Poultry Improvement, Part 145- National
Poultry Improvement Plan, under 9 CFR 145.14(d). The ELISA test is
named as an official blood test for AI. Kits for testing in the U.S.
must be licensed by the USDA. Under 9 CFR 147.9(b), a federally
licensed ELISA may be used as a screening test for AI.
The IDEXX FlockChek* Avian Influenza Antibody Test Kit
detects exposure to the most common and prevalent avian influenza
viruses, including H5N1.
The IDEXX FlockChek Avian Influenza Test Kit detects Type A influenza
virus exposure in chickens. The test has been demonstrated to detect
antibody reactivity against 20 different subtypes of avian influenza,
including 14 hemagglutinin glycoproteins, and the H5N1 subtype.
Figure 3.
Figure 4.
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IDEXX Customer
Experiences
These six examples have been shared by IDEXX customers around the
world and have been summarized to share with our Animal Health
Updates subscribers.
| 1. |
Holland
99.3% specificity for the IDEXX AI ELISA (27,908 field sera and
108 SPF sera)7, and detectability 7–10 days
after virus or vaccine inoculation
An avian influenza monitoring program was established in Holland7
(HPAI after the spread of LPAI H7N7) for the screening of
previous low pathogenic avian influenza (LPAI H7N7) with
FlockChek* Avian Influenza Antibody Test Kit.
Methods and Materials
Year 2000: 20,000 samples, all negative
March 9–15, 2003: 28,018 blood samples from 1,193 poultry
farms (10 samples per house)
- Arranged per county; turkey, duck and
free-range farms; all serum samples were run with ELISA except
for the duck serum samples
- No indication of previous spread of LP H7N7,
but five farms infected, three with H7N3, one with H1, and
one-unknown
Sensitivity: 225 post-inoculation (H5, H6, H7 of H9) sera of
SPF birds, 108 field sera of LPAI-infected farms and 11
monospecific sera with Ig against 11 H-types were positive with
the IDEXX AI ELISA
Forty-five, 16-week-old SPF hens per group (accommodated in
isolators) were inoculated with the AI serotype H5 (inactivated
vaccine), H6 (apathogenic strain) or H9 (apathogenic strain).
Seven days before the inoculation and 7, 14, 21 and 27 days
after the inoculation, blood samples were taken and tested with
the IDEXX AI ELISA (two batches, as above).
Results are shown in Tables 2 and 3.8
Table 2.
After inoculation with the apathogenic live H5, H6, H7 and H9
strains, positive reactions were already detected seven days
after infection (cutoff value of 0.5) with the IDEXX AI ELISA
(A). At 7, 14 and 21 days after inoculation, the average
percentages of positive sera were 65%, 77% and 87%,
respectively. The two positive sera on day 7 were probably
"singleton reactors" or "false-positives."
The serological response after inoculation with the
inactivated H5 antigen was slower than with the live antigens.
At 7, 14, 21 and 27 days after the injection, 14%, 40%, 100%,
respectively, and 100% of the reactions were positive.
Table 3.
After inoculation with the apathogenic live H5, H6, H7 and H9
strains, positive reactions were already detected seven days
after infection (cutoff value of 0.5) with the IDEXX AI ELISA.
The average percentages of positive sera 7, 14 and 21 days after
inoculation were 63%, 78% and 86%, respectively.
The serological response after inoculation with the
inactivated H5 antigen was slower than with the live antigens.
At 7, 14, 21 and 27 days after the injection, 11%, 40%, 87 and
100%, respectively, of the reactions were positive.
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| 2. |
Germany.
Testing chicken and turkey samples
A target surveillance program was established between August
1998 and March of 1999 in a German poultry area where some
respiratory and egg drop clinical signs were correlated with
infectious bronchitis virus, but the presence of avian influenza
was included in the rule-out list. A total of 723 serum samples
were received from 30 turkey farms (522 serum samples) and 12
commercial layer farms (201 serum samples). Eight of the thirty
turkey farms were positive on the FlockChek* Avian Influenza
Antibody Test Kit and confirmed positive by AGID. All twelve
commercial layer farms were negative by both the IDEXX AI ELISA
and AGID.4
Table 4.
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| 3. |
Mexico
Good correlation with hemagglutination inhibition (HI) test
The IDEXX AI ELISA was evaluated in Mexico to demonstrate the
correlation with the traditional HI test used locally. Based on
the H5N2 avian influenza virus prevalence, the range of
correlation for the ELISA vs. HI was between 96.3 and 100%. The
IDEXX AI ELISA demonstrated a specificity of 99.5%, and the
overall correlation between the two tests was 98.3%, with a
kappa value of 0.97.2
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| 4. |
Pakistan, United States
and Mexico
Obtaining quantitative results after inactivated vaccine use
The IDEXX AI ELISA has been used in different countries for the
monitoring of flocks that have been vaccinated with inactivated
avian influenza vaccines against: H9N2, H6N2 and H5N2.2,3,5
Figure 5.
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| 5. |
Canada.
Different avian influenza viruses tested
The National Centre for Foreign Animal Disease in Winnipeg,
Manitoba, is the Canadian Food Inspection Agency’s reference
laboratory for avian influenza.6 They have tested the
following HI-characterized antisera on the IDEXX AI ELISA:
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| 6. |
China
Good correlation with H5N1 detection vs. hemagglutination
inhibition test
A comparative study was conducted on the sensitivity of the
IDEXX FlockChek* Avian Influenza Antibody Test Kit vs.
hemagglutination inhibition (HI) tests for detecting H5N1
antibodies.
Objective: To compare the sensitivity of the
IDEXX AI ELISA and HI in detecting H5N1 antibodies
Method and Materials:
- 10 positive sera from SPF chickens vaccinated with H5N1
inactivated vaccine
- Two negative sera from SPF chickens
- The antigen used for the HI test was sourced from H5N1.
All the samples were double serial-diluted from 1:1 to 1:256
and tested to analyze the detectability by the IDEXX AI ELISA
and/or HI tests.
For interpretation the IDEXX AI ELISA results with S/P>0.5 are
positive. The HI interpretation followed the China standard
procedure: HI≥5 is positive, HI= 4 is suspect and HI ≤3 is
negative.
Results: The IDEXX AI ELISA results are
shown on Table 5, the HI results are shown on Table 6. Table 7
shows the original HI titer for each positive sera. Table 8
shows the raw S/P data from the IDEXX AI ELISA.
These results showed that the IDEXX AI ELISA had positive
results at a higher dilution than HI using homologous virus
antigen.
Summary: The IDEXX AI ELISA was able to
detect the H5N1 antibodies as well as the HI test.
Table 5.
Table 6.
Table 7.
Table 8.
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Summary: The FlockChek* Avian Influenza Antibody
Test Kit:
- Should be your number ONE screening tool for any avian influenza
surveillance/monitoring program to prevent the potential spread of
the virus after the detection of an "index case and/or infection."
- Detects any of the most prevalent avian influenza viruses found
worldwide, including H5N1.
- Has been used successfully by different countries and customers
around the world with reliable results.
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