General avian serology

Vol. 1. Results interpretation of serological tests

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1 VOL.1 RESULTS INTERPRETATION OF SEROLOGICAL TESTS
General avian serology

Vol. 1. Results interpretation of serological tests

Introduction to practical avian serology - from the basics to the interpretation

Vol. 2.
of test results

Vol. 3. Principles of serological diagnosis in poultry

Vol. 4. Introduction and practical observations on serovaccinology in poultry

Vol. 5. Practical cases of serovaccinology in poultry

2 VOL.1 RESULTS INTERPRETATION OF SEROLOGICAL TESTS

Prof. Piotr Szeleszczuk
Founder of the Polish school of serological monitoring in poultry farming.
A valued expert in the field of avian vaccinology, for 10 years he has been
implementing his own project, the Warsaw Academy of Serological Monitoring.
Prof. Dr. Piotr Szeleszczuk is a veterinarian, specialist in poultry diseases, former
head of the Department of Pathology and Veterinary Diagnostics and the Department
of Avian Diseases of the Faculty of Veterinary Medicine of the Warsaw University of
Life Sciences.

Contents
Serology – definition.................................................................................................................................................................. 4

Serological tests ......................................................................................................................................................................... 5

ELISA tests .................................................................................................................................................................................... 6

Statistics Box 01 ........................................................................................................................................................................ 13

Statistics Box 02 ....................................................................................................................................................................... 14

How to read ELISA test results obtained with xChekPlus ............................................................................................ 19

How to read hemagglutination inhibition test results .................................................................................................. 35

3 VOL.1 RESULTS INTERPRETATION OF SEROLOGICAL TESTS

Serology -
definition
Serology is dealing with the interactions between
antigens and antibodies that occur in vitro. Serology
includes different methods to demonstrate the pre­
sence of antigens and antibodies in serum or other
biological material, thus, by identifying the pathogen
or antibodies, it enables the diagnosis of infectious
diseases (Gołąb et al., 2017).

Serological methods are commonly used in veteri­

nary medicine, they are also one of the important
tools for managing the health of poultry flocks
(Butcher, 2018; Szeleszczuk, 1995).

Serological tests have been used in the diagnosis of

infectious diseases of poultry for many decades.
According to the review of the literature, tube
hema­gglutination, introduced nearly 110 years ago
by Jones, was the first serological method used
to diagnose Salmo­nella Pullorum-infected indivi­
duals, and at the same time the first serological test
practically used in the diagnosis of poultry diseases
(Jones, 1913). Over time, other serological tests
were introduced to identify antibodies and antigens
in serum, swabs or albumin (e.g. seroneutralization
test, precipitation test, hema­gglutination inhibition
test). A major breakthrough in the use of serological
methods in poultry practice took place in the second
half of the 1980s, when the first commercial IDEXX
ELISA tests appeared on the US market (7).

4 VOL.1 RESULTS INTERPRETATION OF SEROLOGICAL TESTS

Serological tests
In avian pathology, serological tests are used to diagnose infections (serodiagnostics) and to assess post-vaccination
response (serovaccinology). These tests are also very often used by poultry pathologists to determine the prevalence
of diseases in a population (seroepidemiology).

As mentioned, several methods can be used to detect antibodies and antigens in avian serology (Fig. 1.), but in practice
ELISA and the hemagglutination inhibition (HI) test are most often used, which allow not only to answer what antibodies
are present in the serum, but also how many antibodies are contained in it (ELISA) or what is their hemagglutination
activity. Currently, the plate agglutination test and the agar gel precipitation test (qualitative tests) are less frequently
used, although they can still be very useful in some diseases. This applies in particular to the plate agglutination test
used in the diagnosis of mycoplasmas or the agar gel plate test (AGPT) test used to diagnose Marek’s disease
(Szeleszczuk, 1995).

The scientific basis for the introduction of ELISA for the diagnosis of poultry diseases was developed by a team of
researchers from the US University of Maryland (Snyder et al., 1983; Snyder et al., 1983; Snyder et al., 1984; Snyder
et al., 1986).

The use of the ELISA test for serological diagnosis of poultry diseases enabled the emergence of a new strategy in
veterinary care over large-scale poultry farming by introducing serological monitoring to the broad practice (Snyder
et al. 1985; Szeleszczuk, 1997).

Functional

Agglutination
- Plate Agglutination Test (RPA)

Hemagglutination
- Hemagglutination inhibition (HI) test

Serological tests Precipitation

- Agar Gel Precipitation (AGP) Test

Neutralization
- Seroneutralization test (SN)

Quantitative
Chemical and physical methods ELISA

Fig. 1. Tests used in practical poultry serology

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Immunoenzymatic
test (ELISA)
In the serological diagnosis of poultry diseases, the
enzyme immunoadsorption test is commonly used,
most ofter referred as ELISA (Enzyme-Linked Immu­
nosorbent Assay). The principle of the enzyme immu­
noassay was developed in 1971 inde­pen­dently by
teams of scientists from the University of Stockholm
(Engvall and Perlmann, 1971) and the research labo­
ratory of the Dutch company NV Organon in Oss (van
Weemen and Schuurs, 1971; Lequin, 2005). In a very
short time, this test was applied to both scientific and
practical research in veterinary medicine and food
hygiene (Mallinson et al., 1988) and on viruses found in
poultry like Newcastle disease virus, Gumboro disease
virus, Reoviruses (Thayer et al., 1987). Currently, ELISA
is a commonly used test in the diagnosis of many viral
diseases in poultry and some bacterial infections (8).
Since its introduction in 1995, this method has become
a useful tool in the system of integrated veterinary care
for intensive poultry production (Szeleszczuk, 1996).

ELISA tests are characterized by high sensitivity and

repeatability of results. Therefore, they make it possi­ble
to detect even low concentrations of antibodies in
the tested serum, undetectable by other serological
tests. They are also characterized by a high degree of
specificity, i.e. a low number of false positive results.
In addition, it is a safe, quick and easy method that
does not require expen­sive equipment; it is also techni­
cally simple, easy to automate and affordable.

The wide application of this test in practice was mainly

determined by the possibility of full automation of its
conduct and the speed of performing determinations;
it is possible to test 92 sera with different antigens in
approx. 2 hours. The use of microtiter plates and com­

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mercially available multi-channel pipettes, washers and
spectrophotometers adapted to this format enable quick
and precise testing even in small laboratories (Kępska et
al., 2018). On the other hand, with the use of specialized
laboratory robots in large diagnostic facilities, the tests
can be fully automated. All these advantages mean that
ELISA has an advantage over such classic techniques
as the precipitation test or the neutralization test. Since
the development of the enzyme immunoassay, many
variants have been developed, which have been used
both in clinical diagnostics and in basic research (14).
In poultry serology, IDEXX kits use to detect antibodies
indirect ELISA (Fig. 2) and blocking format test (Fig. 4),
and for antigen detection use the sandwich test format.

Basic characteristics of the ELISA test (Gut-Winiarska

et al., 2001):
a) antigen or antibody are bound in the solid phase by
passive adsorption to the plastic well surface of the
microtiter plate;
b) while one of the reagents is bound in the solid
phase, further reagents are added and co-incubated,
and their excess is removed by washing;
c) one of the reagents (serum against chicken anti­
bodies - indirect ELISA or specific antigen serum-
blocking and indirect sandwich format) used in the
test is covalently bound to the enzyme. The addition
of a substrate suitable for the enzyme results in
a colored, soluble product that can be quantified by
optical density (OD) measurement. In the indirect
test, antibody concentration (titer) increases with
increasing OD value (Fig. 3.), while in the direct
competition test - blocking ELISA, the relationship is
inver­se, the concentration of anti­bodies increases
with the decrease in the value of optical density
(Fig. 5.).

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 Antibodies

Antigens

Add sample

Wash + conjugate

Wash + substrate

Clear Stop Blue

Read optical density

Fig. 2. Scheme of a non-competitive indirect ELISA*

*The conjugate is enzyme-labeled anti-chicken antibodies

OD

Positive result

Borderline titer

Negative result

Titer

Fig. 3. Relationship between antibody concentration (titer) and optical density (OD) in an indirect test

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 Antibodies

Antigens

Add sample

Wash + conjugate

Wash + substrate

Blue Stop Light blue or clear

Read optical density

Fig. 4. Scheme of a direct competition test - blocking ELISA

** The conjugate is enzyme-labeled anti-platelet antigen antibodies

OD

Negative result

Borderline titer

Positive result

Titer

Fig. 5. Relationship between antibody concentration (titer) and optical density (OD) in the blocking ELISA

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 xChekPlus* Name of Validated S/P or S/N
Pathogen Test description
test acronym the test species ratio cut-off

ELISA test for the detection of antibody

Avian encepha­ IDEXX AE
AE* to avian encephalomyelitis Chicken S/P > 0,2
lomyelitis virus Ab Test
virus (AE) in chicken serum.

ELISA test for the detection

Avian IDEXX AI
AI* of antibody to Avian Influenza Chicken S/P > 0,5
influenza virus Ab Test
Virus (AI) in chicken serum.

IDEXX AI ELISA test for the detection of antibody Chicken, turkey,

Avian
Aims* MultiS-Screen to avian influenza virus (AI) in chicken, duck, ostrich, S/P > 0,5
influenza virus
Ab Test turkey, duck, ostrich, and goose serum and goose

Avian ELISA test for the detection of antibody

IDEXX ALV-J
ALV-J* leukosis virus to avian leukosis virus, subgroup J Chicken S/P > 0,6
Ab Test
subgroup J in chicken serum.

ELISA test for the detection of antibody

Avian IDEXX APV Chicken
APV* to avian pneumovirus (APV) in chicken S/P > 0,2
pneumovirus Ab Test and turkey
and turkey serum.

ELISA test for the detection of antibody

Chicken IDEXX CAV
CAV** to Chicken Anemia Virus (CAV) Chicken S/N < 0,6
anemia virus Ab Test
in chicken serum

ELISA test for the detection of antibody

Infectious IDEXX IBV
IBV* to infectious bronchitis virus (IBV) Chicken S/P > 0,2
bronchitis virus Ab Test
in chicken serum.

Infectious ELISA test for the detection of antibody

IDEXX IBD
IBD* bursal to infectious bursal disease virus (IBD) Chicken S/P > 0,2
Ab Test
disease virus in chicken serum.

ELISA test with enhanced dynamic

Infectious IDEXX
range for the detection of antibody
IBD-XR* bursal IBD-XR Chicken S/P > 0,2
to infectious bursal disease (IBD)
disease virus Ab Test
in chicken serum

Tab. 1. IDEXX ELISA tests for serodiagnosis of poultry diseases (8)

*Indirect (no-competitive) ELISA
**Blocking (competitive) ELISA
***Antigen Capture ELISA (Indirect Sandwich ELISA)

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 xChekPlus* Name of Validated S/P or S/N
Pathogen Test description
test acronym the test species ratio cut-off

Chicken, turkey,
falcon, flamingo,
IDEXX ELISA test for the detection quail, Japanese
Avian
INFAvian* Influenza A of antibody to influenza A virus quail, laughing S/P > 0,5
influenza virus
Ab Test in animal serum gull, house
finch, starling,
and herring gull

ELISA test for the detection

Avian leukosis
IDEXX ALV of antibody to avian leukosis virus
LLAB* virus subgroups Chicken S/P > 0,4
Ab Test (ALV-subgroups A and B)
A and B
in chicken serum

ELISA test for the detection

Avian leukosis
IDEXX ALV of avian leukosis virus Antigen p27
LLAG*** virus all Chicken S/P > 0,2
Ag Test in chicken serum, cloacal swabs,
subgroups
or albumin samples

ELISA test for the detection of antibody

Mycoplasma IDEXX MG Chicken
MG* to Mycoplasma gallisepticum (Mg) S/P > 0,5
gallisepticum Ab Test and turkey
in chicken and turkey serum.

ELISA test for the detection

Mycoplasma IDEXX MM
MMt* of antibodies to Mycoplasma Turkey S/P > 0,5
meleagridis Ab Test
meleagridis (Mm) in turkey serum.

ELISA test for detecting of antibodies

Mycoplasma IDEXX MS Chicken
MS* to Mycoplasma synoviae in chicken and S/P > 0,5
synoviae Ab Test and turkey
turkey serum.

Mycoplasma ELISA for for the detection of antibody

synoviae/ IDEXX MG/MS to Mycoplasma gallisepticum Chicken
MS/MG* S/P > 0,5
Mycoplasma Ab Test and Mycoplasma synoviae (Mg/Ms) and turkey
gallisepticum in chicken and turkey serum

Tab. 1. IDEXX ELISA tests for serodiagnosis of poultry diseases (8)

*Indirect (no-competitive) ELISA
**Blocking (competitive) ELISA
***Antigen Capture ELISA (Indirect Sandwich ELISA)

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 xChekPlus* Name of Validated S/P or S/N
Pathogen Test description
test acronym the test species ratio cut-off

ELISA test for the detection of antibody

Newcastle IDEXX NDV
NDV* to Newcastle disease virus (NDV) Chicken S/P > 0,2
disease virus Ab Test
in chicken serum.

ELISA test for the detection of antibody

Newcastle IDEXX NDV-T Ab
NDVt* to Newcastle disease virus (NDV) Turkey S/N < 0,2
disease virus Test
in turkey serum.

Ornitho­ ELISA test for the detection of antibody

IDEXX ORT Chicken
ORT* bacterium to Ornithobacterium rhinotracheale (ORT) S/P > 0,4
Ab Test and turkey
rhinotracheale in chicken and turkey serum

ELISA test for the detection

Pasteurella IDEXX PM
PM* of antibody to Pasteurella Chicken S/P > 0,2
multocida Ab Test
multocida (Pm) in chicken serum

ELISA test for the detection

Pasteurella IDEXX PM-T
PMt* of antibody to Pasteurella Turkey S/P > 0,2
multocida Ab Test
multocida (Pm) in turkey serum

ELISA test or the detection

IDEXX REO
REO* Avian reovirus of antibody to avian reovirus (REO) Chicken S/P > 0,2
Ab Test
in chicken serum.

ELISA test for the detection

Reticuloendo­ IDEXX REV
REV* of antibody to Reticuloendotheliosis Chicken S/P > 0,2
theliosis virus Ab Test
virus (REV) in chicken serum.

ELISA test for the detection

Salmonella IDEXX SE
SE Ab X2* of antibody to Salmonella Chicken S/P > 0.2
enteritidis Ab X2 Test
Enteritidis (SE) in chicken serum.

Tab. 1. IDEXX ELISA tests for serodiagnosis of poultry diseases (8)

*Indirect (no-competitive) ELISA
**Blocking (competitive) ELISA
***Antigen Capture ELISA (Indirect Sandwich ELISA)

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 In poultry serology, due to the large number of birds
in a flock, up to tens of thousands, it is necessary to
use the methods used in statistics to determine the
number of samples to be tested.

Statistic deals with the principles and methods of

gene­ralizing the results obtained from a random
sample to the entire population (that is, the popu­lation
from which the samples were taken). This procedure
is called statistical inference. Proper random selection
of appropriate quality samples is important (Moczko
et al., 1998). The first element facilitating the analysis
of the obtained results is the division into titer groups,
statistically called a distribution series; it is the basic
statistical way of presenting the distribution of test
results. In poultry serology using ELISA, it is created
by dividing (assigning) the obtained results into (up
to) 18 categories (groups of titers) and specifying the
number of sera belonging to each of these categories.
Imaging the results in the form of a histogram allows
for a clear presentation and enables quick orientation
in the variability of titers, which facilitates the evalu­
ation of the obtained result.

For practical purposes in poultry serology the statis­

tics parameters are most often used. These inform
us about the average tirer value (AMT, GMT), i.e. the
average number of antibodies in a specific volume
of serum, and they measure the variability/dispersion
of antibodies level in the population under study
(SD, CV%).

Box 1. Statistics

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 Selected statistical parameters used in serological tests graphs

In addition to the central tendency described by the mean titer (AMT, GMT), scatter is a fundamental
sample characteristic in serology testing in poultry. A poultry flock consists of many individuals whose
reaction to contact with the antigen leads to different levels of serological response. This diversity is
a natural pheno­menon resul­ting from the genetic structure of a given individual or the current efficiency
of its immune system. Therefore, the obtained, different results of serological tests must be processed
using indicators describing the degree of their differentiation. In the case of elaborating the results of
serological tests from a given popu­lation (flock), it is assumed that they have the form of the so-called
normal distribution, otherwise known as the Gaussian distribution, illustrated by the Gaussian curve,
which distribution is the most important theoretical probability distribution in statistics (Moczko et al.,
1998). The normal distribution is also the most intuitive statistical distribution. In short, it describes
a situ­ation in which most cases are close to the average result, and the more a given result deviates from
the average, the less numerous it is. The simplest way to say is that in populations with a normal distribu­
tion, most serum titers are close to the mean value, and the further we move away from the mean result,
the fewer sera there are (Fig. 6).

The most commonly used measures of dispersion in medical research are: variance, standard deviation
and coefficient of variation (Andrasiak et al., 2018). Dispersion (scattering, scattering, dispersion) is the
variation in the observed values of a variable; it is the greater, the more these values deviate from the cen­
tral tendency, i.e., the mean titer. The desired probability function of the random variable has the shape of
a normal curve (Gaussian Function). Gaussian curve (Fig. 6.) describes a normal distribution in which the
curve is symmetrical about the axis determined by the mean value and reaches its maximum here (high
and low titer values occur less frequently, the more they differ from the sample mean).

Box 2. Statistics

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 X

Incidence

0,15% 2,35% 13,5% 34% 34% 13,5% 2,35% 0,15%

-3 -2 -1 0 +1 +2 +3

68%

95%

99,7%

Standard deviation

Fig. 6. Theoretical Gaussian curve describing the normal distribution

As you can see, about 68% of the observations are close to the mean, within one standard deviation of the mean
(a measure of distance in the language of statistics). As you move away from the mean, the Gaussian curve descends.
As much as 95% of observations are in the range of -2 to +2 standard deviations from the mean value. Extreme values
(at the ends of the Gaussian curve) are represented by a negligible percentage of observations (Fig. 8.). This means that
when the distribution of antibody levels is close to the normal distribution, we can say that there are no anomalies in the
tested sample, the obtained data are normal, there are few extreme observations, and a significant part of the observa­
tions is concentrated around the mean. The occurrence of such anomalous distributions means that the properties of the
normal distribution cannot be applied to them, and thus many statistical tests cannot be used, because their results may
be distorted by the occurrence of a non-standard distribution of results (Fig. 9.). In practice, the normal distribution of
results is most often disturbed by combining chicks from different parent flocks with different immunological status in the
hatchery. This practice, sometimes referred to as chicks mixing, makes it very difficult to determine the proper timing of
vaccinations, e.g., against Gumboro disease (Fig. 10.).

Fig. 7. Distribution of IBD virus antibody titers in 39 flocks of day-old broiler chicks

15 VOL.1 RESULTS INTERPRETATION OF SEROLOGICAL TESTS

As can be seen from the data in Fig. 7., of the 897 sera tested from day-old broiler chicks, only two sera belonged to group
zero and only one to group 11. More than 35% of the sera were in the 7th titer group (the range of titers in this group is
6,000 - 7,999) and the arithmetic mean titer was 7,127. In the range of 2,507 - 11,747 (about twice the SD value), there
were approx. 95% of the tested sera.

In the presented example, it can be observed that the more measurements we make, the more the resulting curve will
resemble a symmetrical bell. If (which is of course impossible) we analyzed a group with an infinite number of cases, we
would get a normal distribution. In practice, the sample we study always has a limited number of elements, but it is worth
remembering that the more elements we include in the study, the better the actual distribution of the examined variable
in the analyzed population will be reproduced. At this point, it should be strongly emphasized that too few samples tested
may cause the results of the serological test to be unreliable and misleading. In practice, it is recommended to collect
23-30 blood samples, regardless of herd size.

Fig. 8. Example of distribution of ELISA results in a flock of day-old broiler chicks from a very well IBD vaccinated parent flock

In Fig. 8. the effect of examining a flock of one-day-old broiler chicks coming from a very well-protected parent flock was
illustrated. Noteworthy is the very low value of the coefficient of variation (CV - 10.4%) and the difference between GMT
and AMT of only 50 and the very low value of the standard deviation (954). In such a herd, scheduling vaccination against
Gumboro disease will not be a problem.

16 VOL.1 RESULTS INTERPRETATION OF SEROLOGICAL TESTS

Fig. 9. An example of the distribution of results obtained in the ELISA test in a flock of day-old broiler chicks from a very poorly NDV
vaccinated parent flock.

Fig. 10. Example of the variability of serological test results in the case of chicks from different parents’ flocks.

To describe the data obtained in quantitative serological tests, it is necessary to use descriptive statistics to better
understand the level of specific antibodies in the population, and its distribution. The values obtained because of the use
of descriptive statistics are also the basis for further, more advanced methods of statistical analysis, such as testing
differences between groups or examining relationships between selected indicators (e.g., the level of specific antibodies
against a given pathogen and the performance of the flock).

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Two groups of parameters play a key role in descriptive statistics: central tendency parameters and dispersion para­me­
ters. All central tendency parameters estimate the most typical value of the parameter characterizing the studied group,
but each of them focuses on a different aspect of its distribution. In serology the most used parameters of central ten­
dency are the arithmetic mean and the geometric mean. The central tendency parameters determine a certain value that
is most representative for the tested sample, but it is obvious that characterizing the titers of a poultry flock only with this
parameter is definitely insufficient (Andrasiak et al., 2018). Therefore, the concept of dispersion parameters was intro­
duced, which characterize the degree of dispersion of results around the central tendency measure. The larger the value
of the dispersion parameter, the more the results are scattered around the value of central tendency. In general, this also
means that the value of central tendency is less representative of such results because they vary widely. Small values of
the dispersion parameter allow, in turn, to consider the central tendency value as a good representative of the entire sam­
ple. In the case of serological assays, dispersion parameters provide information on how diverse the test population is in
terms of antibody titers. The primary parameter of dispersion used in serology is variance. This parameter expresses the
degree of dispersion of the random variable (antibody titer) around the mean value. The greater the variance, the greater
the scatter of the variable (Moczko et al., 1998).

The variance of the denominators x1, x2, ..., xn is the arithmetic mean of the squared deviations from their arithmetic mean:

σ2 =( x1 − X ¯¯¯¯ ) 2 +( x2 − X ¯¯¯¯ ) 2 +...+( xn − X ¯¯¯¯ ) 2n

The standard deviation is the square root of the variance. The standard deviation determines approximately how much
all statistical units of a given population differ on average from the arithmetic mean of the variable under study. It is assu­
med that the value of the standard deviation for a normally distributed population should not be greater than 50% of the
mean value. The standard deviation provides us with the necessary knowledge about whether the results in a particular
group of results are like each other - whether the group of titers is similar or different, and if so, to what extent (Moczko
et al., 1998).

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How to read ELISA test results
obtained with xChekPlus* software
Laboratories performing tests using IDEXX ELISA kits provide the results in the form of a template printout, consisting
of a typical histogram (graph view with the given statistics and legend - description below the graph) and a data table
(statistical data, controls, internal controls, verification wells, comments, lot number and test expiration date) [Fig. 11].
This form of presenting test results enables easy analysis of the obtained results of the serological test. In addition,
a statistical summary may also be included. In the upper left corner of the test result, there is the data of the laboratory
performing the test and the date of printing the report (this is not the date of the test).

We start the analysis of the test result by reading the description of the tested material. It is located above the histogram
and can have a very different form, but it always contains the research direction in the form of an acronym (Tab. 1.).
A description identifying the flock is printed here. This is extremely important information for the correct interpretation of
the result. Although the recording solutions used by specific laboratories differ in form, they should allow full identification
of the ordering party and the tested material.

The system of describing/marking samples in serological monitoring tests of poultry flocks in the country is not unified,
which is often an obstacle to a full analysis of the serological test result. Very often, in practice, the correct interpretation
of the serological monitoring result is hindered by the lack of additional information relevant for this assessment, such as
the age of the birds or the vaccination program.
The recommended structure of material and stock description is given in Tab. 2. and the description scheme most often
used in practice is contained in Tab. 3.

1. 2. 3. 4. 5. 6.

Laboratory Test number accor- Owner

Referring
(usually with logo ding to laboratory Submitter Sampling date - identification
veterinarian
and address) procedures data

7. 8. 9. 10. 11. 12. 13.

Farm Age of birds on Purpose of Vaccination

Production type Breed Hatching Date
- location data sampling date the test program

Tab. 2. Recommended sample coding system for serological testing

Order Sampling Species/ Material Date of

Owner Principal Material Object
number date Breed description submission

Tab. 3. The most used description in practice of serologically tested samples taken from a poultry flock.

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 Histogram
Samples data
Statistics

Fig. 11. Report of IBD test results performed using xChekPlus* software

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In order to facilitate the tests coding, a proper recording system was proposed, the use of which enables the full use of
the xChekPlus* software to generate reports that are extremely useful in managing the flock health status (Szeleszczuk,
2012). If you are working with the xChekPlus* software, use the “Comments” record to encode information about your
vaccination program. The rules for coding the vaccination program are given in Tab. 4. An example of a printout of the
result, considering the full coding system of information about the herd and the vaccination program, is given in Fig. 12.

10/12/AP/AB01/NOWAK/EŁK/S3/K3/6-0/CB/F15/120301 -IBD

Count: 23
10
Mean: 8527
9
GMean: 7738
8
SD: 3278
7
%CV: 38,4
6
Count

Min: 2899
5
Max: 13219
4

0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Titer Groups

Fig. 12. A printout of the result of the test for antibodies to Gumboro disease virus

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 Code Assay Date Count GMean CV Age
AND IBD 27 23 7738 38,4 6–0

Case Comment
10/12/22/AP/AB01/Jan Nowak/Elk/S3/K3/6-0/CB/F15/120301/P/ ///M1HI//N1AS//I1MS//I10VW//G18HW/G24HW
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

Description of code elements:

1. 10/12/22/ Laboratory serial number

2. AP/ Submitter code

3. AB01/ Veterinarian code

4. Jan Nowak/ Name and surname of flock owner - we do not code!

5. Elk/ Farm location

S3/ Sector
6.
K3/ House

7. 6–0/ Age of birds on sampling day (weeks-days)

8. CB/ Type of birds (commercial broiler)

9. F15/ Production line

10. 120301/ Date of chicks hatching YY/MM/DD, e.g., March 1, 2012

11. P/ Purpose of the study - P - problem in the herd

12. /// Three slashes separate flock data from the vaccination program

13. M1HI// Vaccination against MD at day 1 with injectable HVT vaccine

14. N1AS// Vaccination against ND at day 1 with asymptomatic vaccine by spray

15. I1MS// Vaccination against IB at day 1 with Mass vaccine by spray

16. I10VW// Vaccination against IB at day 10 with a variant vaccine by drinking water

17. G18HW/ Gumboro disease vaccination at day 18 with an intermediate plus vaccine by drinking water

18. G24HW Vaccination against Gumboro disease at day 24 with an intermediate plus vaccine by drinking water

22 VOL.1 RESULTS INTERPRETATION OF SEROLOGICAL TESTS

 Code Disease Acronym Type of vaccine Vaccination technique

H = HVT
N Marek’s disease MD I = injection
R = Rispens

A = asymptomatic S = spray
N Newcastle disease ND
L = lentogenic W = in drinking water

M = Mass S = spray
I Infectious bronchitis IB
V = variant W = in drinking water

P = intermediate
G Gumboro disease IBD W = in drinking water
H = intermediate plus

S = spray
A Metapneumovirus infections APV
W = in drinking water

R Reoviral infections REO I = injection

S = spray
C Coccidiosis Coccidiosis
W = in drinking water

Tab. 4. Vaccination codes for broiler flocks

The most important advantage of the proposed sample coding system is the ability to quickly search for results of inte­
rest, stored in the databases of programs used to generate test results. With proper coding, it is possible to prepare va­
rious reports, e.g., collecting the results of blood tests from the age of 6 weeks in one chart., taken by a specific submitter,
vaccinated against Gumboro disease.

In statistics, 2D histograms are a graphical representation of the frequency distribution of a selected variable(s), in which
the columns (bars) are plotted above the group ranges (denominator groups 0-18), and the height of the columns is pro­
portional to the number of sera in each titer group. All sera with titers below the cut-off titer (negative) are in group 0.
In the given example, there are 23 tested sera in this group.

Quantity (on the Y axis)

The number of sera with a titer belonging to a given titer group.

Group of titers (on the X axis)

Titer group number. To facilitate the recording and analysis of serological monitoring data, the obtained titer was grouped
into titer groups according to the rule given in Table 6.

23 VOL.1 RESULTS INTERPRETATION OF SEROLOGICAL TESTS

 Result/antibody
Range of changes in the group
concentration

All tests except MS,

Titers group Ms/Mg, Mg REV, LLAB
MG, REV, LLAB

0 0-396 0-1076 0-844 Negative/none

Cut off titer

1 397-999 1097-1499 845-999

2 1000-1999 1500–1999 1500–1999
Positive/Low
3 2000-2999 2000–2999 2000–2999
4 3000-3999 3000–3999 3000–3999
5 4000-4999 4000–4999 4000–4999
6 5000-5999 5000–5999 5000–5999
Positive/Average
7 6000-7999 6000–7999 6000–7999
8 8000-9999 8000–9999 8000–9999
9 10000-11999 10000–11999 10000–11999
10 12000-13999 12000–13999 12000–13999
11 14000-15999 14000–15999 14000–15999 Positive/High
12 16000–17999 16000–17999 16000–17999
13 18000–19999 18000–19999 18000–19999
14 20000–21999 20000–21999 20000–21999
15 22000–23999 22000–23999 22000–23999
16 24000–27999 24000–27999 24000–27999 Positive/Very high
17 28000–31999 28000–31999 28000–31999
18 32000 32000 32000

Tab. 6. Titer groups in the IDEXX indirect ELISA.

Next to the histogram, there are basic statistical data, most often used when analyzing the test result.

Quantity

The number of sera tested in the performed test.

The recommended number of sera is 23 for the serological diagnosis of viral diseases, and 60 for the diagnosis of bacte­
rial infections. Testing less than 18 sera per flock does not give reliable information about the flock.

24 VOL.1 RESULTS INTERPRETATION OF SEROLOGICAL TESTS

Geometric mean titer – GMT

The geometric mean of the titers is the nth root of their product. The geometric mean in statistics is most often used to
calculate the average severity of changes. In serology, the geometric mean of titers is most often used, as it better reflects
the distribution of titers in the studied population of birds.
The geometric mean of the denominator x1, x2, x3, ..., xn is expressed by the formula:

x1 – serum titer 1
x2 – serum titer 2
x3 – serum titer 3
xn – serum titer n
n – quantity of tested sera

Arithmetic mean

Arithmetic mean titer (AMT), sometimes referred to as mean titer, by default - mean titer.
The arithmetic mean of the titers is the sum of these titers divided by their number. The arithmetic mean belongs to the
classic average measures and expresses the average level of the observed feature.
The arithmetic mean of x1 , x2 , x3 , ..., xn is given by:

x1 – serum titer 1
x2 – serum titer 2
x3 – serum titer 3
xn – serum titer n

25 VOL.1 RESULTS INTERPRETATION OF SEROLOGICAL TESTS

SD

Standard deviation – standard deviation is a classic measure of variability, next to the arithmetic mean, the most frequen­
tly used concept used in the language of statistics. Standard deviation (SD), defined as the square root of the quotient
of the sum of squares of deviations of the variable (X) from the arithmetic mean of the set (x¯). Intuitively, the standard
deviation tells you how widely the values of a quantity are spread around its mean, the greater the standard deviation, the
greater the variance in the studied population. It is a measure of the distance of individual results from the average. The
further a given result is from the mean in units of standard deviation, the more unusual it is, i.e., it is inconsistent with the
normal distribution.

n
i=1

% CV

Coefficient of variation - is a classic and extremely useful measure of titer distribution variability in the analysis of serolo­
gical test results. The coefficient of variation is a relative measure, i.e., it depends on the size of the arithmetic mean, as it
expresses the variability of a feature in relation to the value of this mean. The coefficient of variation is usually expressed
as a percentage.

The coefficient of variation is defined by the formula:

The coefficient of variation characterizes the variability of the result. If the CV is less than 40%, it is assumed that the
dispersion of the results, i.e., the differentiation of changes between individual individuals, is satisfactory, which may
mean that all birds in the flock reacted similarly to the vaccine administration. A high coefficient of variation means that
the variation of titers between individuals in the flock is too large, which is not a favorable phenomenon. The valuation of
the coefficient of variation value is particularly important when determining the timing of vaccination against Gumboro
disease. Reliable determination of the optimal date of vaccination is possible only when the coefficient reaches the value
40%. In samples taken from birds vaccinated with live vaccines, the value of this ratio should not exceed 60%.

26 VOL.1 RESULTS INTERPRETATION OF SEROLOGICAL TESTS

Min

It is short for “minimal” - antibody titer. The assessment of the minimum titer value is important, for example, when evalu­
ating the results of day-old chicks, as it allows to assess whether all tested individuals were sufficiently protected against
early infection.

Max

Itis short for “maximum” antibody titer. The assessment of the maximum titer value is important, for example, when evalu­
ating the results of tests in vaccinated birds suspected of infection with field virus. Very high maximal titers indicate that
such a high serological response cannot be expected from the applied vaccination program, therefore they may confirm
clinical symptoms characteristic of a given disease.

Operator

Details of the person performing the test.

Data

The date the test was performed is shown here.

27 VOL.1 RESULTS INTERPRETATION OF SEROLOGICAL TESTS

Tab. 7. IBV result data table of 23 sera collected from a flock of 40-day-old broilers.

Next to the histogram, the result of the ELISA test is presented as a table data (Tab. 7). The TEST MATERIAL field contains
information about the test, which enables its identification, the reason for testing and the code of the technician perfor­
ming the test.
The first column contains data describing the results of testing control sera - positive and negative in duplicate, followed
by the numbers of the tested sera, and in the second column their location in a specific well on the microtiter plate.

Optical density

It is worth emphasizing that the only variable determined empirically in the ELISA test is the optical density value in the
third column of the data table. Optical density (OD), sometimes called extinction or absorbance, although not quite cor­
rectly, is a physical quantity described by the tenth logarithm of the quotient of the intensity of a monochromatic beam
entering the absorbing medium and the intensity of the beam passing through this medium (according to the Lambert-
Beer law). In simpler terms, optical density for transparent materials is measured by the ratio of the intensity of light
that has passed through a particular layer of a substance to the light incident on it. Theoretically, it is assumed that the

28 VOL.1 RESULTS INTERPRETATION OF SEROLOGICAL TESTS

concentration of a substance is one of the most impor­
tant factors affecting the amount of light absorbed.
The second is the length of the path that light travels
through the sample, which is conditioned by the width of
the measuring cuvette, and the last one is the extinction
co­efficient of this substance. According to the Lambert­
-Beer law, the intensity of the light transmitted (transmit­
tance) through the test solution decreases exponentially
if each of the three factors is increased. It is therefore
assumed that the level of color intensity, depending on
the concentration of the enzyme reaction product, is
proportional to the concentration of antibodies in the
sample. As mentioned earlier, during the next stage of
the ELISA test, after adding the appropriate substrate,
the enzyme contained in the conjugate catalyzes the
reaction, the product of which can be quantified by the
spectro­photometric method that measures the color
intensity. Parallel to the tested samples, analogous reac­
tion steps are performed for calibration sera with known
concentra­tion of the tested antibody (positive and nega­
tive controls), thanks to which it is possible to determine
the concentration of antibodies in the tested samples.

S/P ratio

Since the rate of the enzyme reaction is dependent on the conditions under which the assay is performed, especially on
the temperature in the laboratory, the relative amounts of antibodies in the test samples are calculated by reference to the
positive control, corrected for the OD values of the negative control sera. In a laboratory where the test is performed at
a higher temperature, the OD values are higher than those found in a laboratory performing the test at a lower tempera­
ture. A comparison of OD values may therefore be unreliable. Regardless of the test conditions, the result expressed as
the value of the S/P ratio in indirect tests is comparable, because the proportions are maintained. For this purpose, the
OD value of the test serum, i.e., the sample taken for testing, is divided (S, sample) by the OD value of the positive control
serum of the kit (P, positive). The value of the coefficient is calculated from the formula:

OD Tested Serum - OD Mean value Negative Controls

OD Mean value Positive Controls - OD Mean value Negative Controls

29 VOL.1 RESULTS INTERPRETATION OF SEROLOGICAL TESTS

S/N ratio

In the blocking ELISA format, the S/N ratio is calculated. Regardless of the test conditions, the result expressed as the
value of the S/N ratio in the blocking format tests is comparable, because the proportions are maintained. The value of
the S/N ratio is calculated from the formula:

OD Tested Serum - OD Mean value Positive Controls

OD Mean value Negative Controls - OD Mean value Positive Controls

Cut-off titer

The so-called the cut-off titer is the value of the titer of S/P or S/N ratio at which the result is considered positive. This
value is set by the kit manufacturer. It is usually determined empirically by testing a large number of positive and negative
samples. Values are selected that maximize the sensitivity and specificity of the test. Depending on the direction of the
examination and the type of test, it ranges from 0.2 to 0.6. For example, samples with an S/P greater than 0.2 (titer greater
than 396) when tested with the IDEXX IBD Ab assay are considered positive.

Cut off titer

Negative result Positive result

396
OD value 0,0 0,2 >6,0

Shortage Short Average High Very high

Antibody concentrations

Fig. 13. Principle of borderline determination in indirect ELISA

30 VOL.1 RESULTS INTERPRETATION OF SEROLOGICAL TESTS

Titer

The titer is one of the basic parameters assessed during the interpretation of the serological result. The titer is calculated
according to the formula provided by the test manufacturer, and it varies depending on the formula used by the test manu­
facturer to calculate it. All formulas are based on the OD value and the S/P ratio calculated on this basis.
The sample algorithm has the following formula:

Titer (log10) = 1,09 (log10 S/P) + 3,36*

*Refers to the S/P ratio at a 1:500 dilution to the end point, defined as the reciprocal of the highest dilution that gives
a reading above the cut off value.

In indirect tests by IDEXX, the cutoff titer is usually 396 (dilution ­ 1: 500; S/P > 0.2) [Tab. 6.].

As shown in Figure 14, depending on the ELISA manufacturer’s titration formula, the results of the same sera from diffe­
rent manufacturers may vary significantly, which should be considered when interpreting test results. If comparisons are
necessary, use S/P (or S/N) values that are independent of the titer calculation formula.

The next column in the table of results contains information to which group of titers the test result of a given serum belongs.

The last column gives the interpretation of the result.

31 VOL.1 RESULTS INTERPRETATION OF SEROLOGICAL TESTS

 I test II test III test

25000

20000

15000
GMT

10000

5000

0
A B C A B C A B C

0-1 3-0 6-0

Age of birds

Fig. 14. The level of antibodies against IBDV in serological monitoring performed using tests from 3 manufacturers (A, B and C) in 1. day
on 3. and 6. week in a herd vaccinated against Gumboro disease (Szeleszczuk et al., 2014)

Hemagglutination inhibition test (hemagglutination inhibition assay - HI test)

In poultry practice, the hemagglutination inhibition test is also widely used for the serodiagnosis of infectious diseases
caused by hemagglutinating pathogens. It is a serological test used to detect antibodies formed after spontaneous infec­
tion, as well as post-vaccination antibodies. It uses the fact that hemagglutinin of surface viral receptors has a clumping
effect on red blood cells (Hirst phenomenon). Regarding poultry diseases, this feature has e.g., Infectious Bronchitis virus,
Newcastle disease, Egg Drop Syndrome 1976, and Avian Influenza virus. The HI test is also used in the serodiagnosis of
mycoplasma infections. Specific antibodies, which are a humoral immune response to infection or vaccination, can inhibit
agglutination by binding to hemagglutinins of pathogens, i.e., they have the ability to inhibit pathogen-induced erythrocyte
agglutination. If the test serum contains specific antibodies against haemagglutinin, the addition of the test pathogen
causes an antigen/antibody reaction. This is visible after the addition of erythrocytes. They are no longer agglutinated as
the reaction is inhibited (Fig. 15.).

The technique of performing the HI test consists in introducing the test serum into the wells of the microplate and making
its successive dilutions, and then adding the pathogen of a certain concentration (e.g., 4 hemagglutinating units) and
a suspension of red blood cells (Fig. 16.). If the tested serum contains specific antibodies directed against this patho­
gen, its hemagglutinating properties are blocked, and as a consequence the blood cells settle in a compact group at the
bottom of the well, forming a nub. If there are no antibodies specific for a specific subtype in the tested serum, then the
patho­gen, due to its ability to agglutinate blood cells, causes them to settle at the bottom of the well in the form of a cha­
racteristic mesh. The result is read visually. The antibody titer is the reciprocal of the last serum dilution that completely
inhibits hemagglutination. The strengths of the test are: the possibility of selecting a specific antigen for testing depen­
ding on the changing epizootic situation, the possibility of simultaneous detection of antibodies to different subtypes of

32 VOL.1 RESULTS INTERPRETATION OF SEROLOGICAL TESTS

pathogens and the low price of the tests. The weaknesses of the test are: subjectivity of the result due to visual reading,
the likelihood of cross-reactions and the possibility of false-negative results, and the lack of established standards for eva­
luating results for specific disease entities.

Dilution

1/1024
1/256
1/128

1/512

Neg.

Titer log2
Pos.
1/64
1/32
1/16
1/4

1/8
1/2

Titer
1 16 4

2 128 7

3 2 1

4 1024 10
Serum

5 32 5

6 8 3

7 32 5

8 256 8

1 2 3 4 5 6 7 8 9 10

Dilution log2

Positive result Negative result

Fig. 15. Rules for reading the result of the hemagglutination inhibition test

33 VOL.1 RESULTS INTERPRETATION OF SEROLOGICAL TESTS

 1. Add 0.025 ml of PBS to all wells
75
of each row; add 0.025 ml of test
50
serum (or standard) to the first well
25

µl 25 µl 25 µl 25 µl 25 µl 25 µl 25 µl 25

2. Make two-fold dilutions

75
of the serum
50

25

3. To doubling dilutions of serum

75 (0.025 ml/well) add 4 U. HA virus
50
antigen in a volume of 0.025 ml
25

4. Mix and incubate for exactly

75 20 minutes at room temperature
50

25

5. Add 0.025 ml of 1% blood cell

75 solution to all wells
50

25

6. Mix, leave until the blood cells

75 sink to the bottom of the well for
50 approx. 40 minutes at temp. room
25 (approx. 20°C) or at temp. 4°C
(when the ambient temperature is
too high) and read the result 1:4
titre 4/2 log2

Fig. 16. Hemagglutination inhibition test (HI test)

34 VOL.1 RESULTS INTERPRETATION OF SEROLOGICAL TESTS

How to read hemagglutination inhibition
test results
Laboratories performing the HI test usually present the test result in a tabular form, attaching a graphical presentation of
the results in the form of graphs (Fig. 18.). Detailed analysis of the result depends on the disease entity and type of produ­
ction as well as the age of the birds. Guidance that may be useful in analyzing hemagglutination inhibition test results is
provided in Table 8.

Serum number HI titer Log2 Titers Result

1 8 3 Neg.
2 8 3 Neg.
3 16 4 Pos.
4 8 3 Neg.
5 16 4 Pos.
6 16 4 Pos.
7 8 3 Neg.
8 16 4 Pos.
9 8 3 Neg.
10 8 3 Neg.
11 8 3 Neg.
12 8 3 Neg.
13 32 5 Pos.
14 8 3 Neg.
15 8 3 Neg.
16 8 3 Neg.
17 8 3 Neg.
18 8 3 Neg.
19 8 3 Neg.
20 8 3 Neg.
21 8 3 Neg.
22 8 3 Neg.
23 8 3 Neg.

Statistical summary:
Mean: log23.3 Min.: log23
Pos/Neg: 5/18 Interpretation of the test result:
Max.: log25 Positive sample: log2>3

Fig. 17. An example of HI test results.

35 VOL.1 RESULTS INTERPRETATION OF SEROLOGICAL TESTS

 Flock N test results in HI test

4
log2 titer

0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Serum number

Summary of flock N results in HI test

20

18

16

14
Number of samples

12

10

0
Pos. Neg.

Fig. 18. An example of HI test results.

36 VOL.1 RESULTS INTERPRETATION OF SEROLOGICAL TESTS

 Titer Log2 titers Interpretation

2 1 No immunity.
Lack of protection
Negative test result
- in case of infection
4 2 possible clinical signs

8 3

Poor immunity.
Fisrt vaccination
16 4 Partial protection
with live vaccines
- mild symptoms

32 5

64 6

128 7
Protection against
Revaccination with
mortality.
live vaccines
No clinical symptoms
256 8

512 9

1024 10
Live and inactivated vaccines
– intensive program
2048 11
Field virus
infection - disease
4096 12

8192 13

*Depending on the pathogen, titers of 4 (log22) to 8 (log23) are considered negative.

** Titers >1024 usually confirm contact with the field germ, although it is more reliable to test pairs of sera and determine the
seroconversion rate (WS >10).

Tab. 8. General indications for interpreting the results obtained in the HI test.

37 VOL.1 RESULTS INTERPRETATION OF SEROLOGICAL TESTS

Literature
1) Andrasiak I., Wróbel T.: Statistics in hematology practice. Acta Haematol. Half. 2018, 49, 121–127.
2) Butcher GD: Diagnostic and monitoring serology in commercial poultry integrations: practical applications, 2018.
https://edis.ifas.ufl.edu/
3) Engvall E., Perlmann P.: Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulins G.
Immunochem. 1971, 8, 871–874.
4) ELISA technical guide. IDEXX Laboratories, 2019.
5) Golab J., Jakóbisiak M., Lasek W., Stokłosa T.: Immunology. PWN, Warsaw, 2017.
6) Gut-Winiarska M., Szewczyk B.: Enzymatic immunosorbent assay (ELISA) as a method of detection of specific anti­
bodies in animal serum. Biotechnology, 2001, 3, 53–70.
7) IDEXX history – http://www.fundinguniverse.com/company-histories/idexx-laboratories-inc-history/
8) IDEXX poultry tests – https://www.idexx.com/en/livestock/livestock-tests/poultry-tests/
9) Jones FS: The value of the macroscopic agglutination test in detecting fowls that are harboring Bacterium pullorum.
J. Med. Res. 1913, 27, 481–495.
10) Kępska M., Futoma-Kołoch B.: ELISA enzyme immunoassay - principle of operation and reaction optimization.
Medical Laboratory, 2018, 2, 42–49.
11) Lequin RM: Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA). Clin Chem. 2005, 51,
2415 – 2418.
12) Mallinson ET, Snyder DB, Marquardt WW, Gorham SL: Immunoassays for veterinary and food analysis. In: Immuno­
assays for Veterinary and Food Analysis–1. red. BA Morris, MN Clifford, and R. Jackman, eds. Elsevier, London and
New York. 1988, 109–117.
13) Mocko I. Bręborowicz GH, Tadeusiewicz R.: Statistics in medical research. Springer PWN, Warsaw, 1998.
14) Overview ELISA. Thermo Fisher Scientific Corporate Supplies. https://www.thermofisher.com/pl/en/home/life-scien­
ce/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/overview­
-elisa.html#/legacy=www.piercenet.com
15) Snyder DB, Marquardt WW, Mallinson ET, Russek E.: Rapid serological profiling by enzyme-linked immunosorbent
assay. I. Measurement of antibody activity titer against Newcastle disease virus in a single serum dilution.
Avian Dis. 1983, 27, 161–170.
16) Snyder DB, Marquardt WW, Russek E.: Rapid serological profiling by enzyme-linked immunosorbent assay.
II. Comparison of computational methods for measuring antibody titer in a single serum dilution. Avian Dis.
1983, 27, 474–484.
17) Snyder DB, Marquardt WW, Mallinson ET, Savage PK, Allen DC: Rapid serological profiling by enzyme-linked immuno­
sorbent assay. III. Simultaneous measurements of antibody titers to infectious bronchitis, infectious bursal disease,
and Newcastle disease viruses in a single serum dilution. Avian Dis. 1984, 28, 12–24.
18) Snyder DB, Marquardt WW, Mallinson ET, Russek - Cohen E, Gorham S, Odor E, Stein JG, Bakos S: Cooperative serolo­
gic survey of Delmarva broiler flocks by enzyme linked immunosorbent assay. 20th National Meeting on
Poultry Health and Condemnations. 1985, 110–120.

38 VOL.1 RESULTS INTERPRETATION OF SEROLOGICAL TESTS

19) Snyder DB, Marquardt WW, ET Mallinson, E. Russek-Cohen, PK Savage, and DC Allen. Rapid serological profiling by
enzyme-linked immunosorbent assay. IV. Association of infectious bursal disease serology with broiler flock perfor­
mance. Avian Dis. 1986, 30, 139–148.
20) Szeleszczuk P.: Practical remarks on the interpretation of serological test results. Matt. Scientific Conference
„Veterinary Aspects of Farm and Small-scale Poultry Breeding, p. April 28, Wrocław, 1995.
21) Szeleszczuk P.: Application of serological monitoring in poultry farming. Goods warehouse. 1, 46–49, 1996.
22) Szeleszczuk P.: Application of serological monitoring in poultry practice. Vol. I. Eskulap, Gliwice, 1997. 48
23) Szeleszczuk P.: Proper coding of samples - the basis for success in interpreting the results of serological monitoring.
Monograph „Bird Diseases” Magazyn Wet. 2012, 493–496.
24) Thayer SG, Villegas P., Fletcher OJ: Comparison of two commercial enzyme–linked immunosorbent assays and co­
nventional methods for avian serology. Avian Dis.1987, 31, 120–124.
25) van Veeman BK, Schuurs AHMW: Immunoassay using antigen enzyme conjugates. FEBS Letts 1971, 15, 232–236.

39 VOL.1 RESULTS INTERPRETATION OF SEROLOGICAL TESTS

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