Avian Serological Examination Serological examinations are of major importance for monitoring vaccination response and to detect field infections in poultry flocks.

A brief history of serological monitoring in poultry:

Scientist & Year Test and /or Comment(Rettger, 1900), The discovery of PD & carrier status in hens.

Jones in 1913 Tube agglutination test was first test made and used in the first eradication programs for PD.

Runnels et. al. 1927

Rapid plate serum agglutination test is used in S.pullorum/gallinarum infection in chicken & turkeys.

Schaffer et.al. 1931 A stained whole-blood agglutination test "Pullorum test" is used in S.pullorum/gallinarum infection in chickens

Delaplane and Stuart (1943)

The discovery of cultivation of Mycoplasma spp. In ECE.

Adler in 1954. Stained rapid serum agglutination test is used in M. gallisepticum infection in chickens and turkeys.

Oudin (1946) & The first use of Agar gel precipitation test "AGPT""Tube method"

Ouchterlony (1948). The first use of Agar gel precipitation test "AGPT" "Plate method"

(Jordan and Chubb, 1962).

The first use of "AGPT" in ILT.

(Woernle, 1966). The discovery that AGPT in poultry requires much higher levels of salt in the agar than that carried out in

mammalsFaragher, 1971 The first use of "AGPT" in IBD.Burnet, 1942 The discovery of the hemagglutinating ability of NDV &

hemagglutination test.(Phillips, 1973) The discovery of the hemagglutination inhibition test.

Allan and Gough, 1974

Carrying out a standard method for HI test in

NDV, AIV, EDS-76, and MG& IBV after enzymatic

treatment.

-"alpha" → serial dilution of Ag + constant dilution of serum.

-"beta" → constant dilution of Ag + serial dilution of serum.

Alexander and Chettle, 1977

The discovery that treatment of the IBV with an enzyme caused it to become haemagglutinating opened the

door to the use of a similar HI test to detect and quantify antibodies to IB.

(Engvall and Perlmann, 1971)

ELISA was initially used to measure antibodies of various classes.

(Voller et alii, 1976).

The use of ELISA to measure antibody response to specific infections.

(Thoen et alii, 1977).

The use of ELISA to measure antibody response to TB

1980 and above The use of ELISA became familiar to all.Many other serological tests as "5" serum-neutralization, "6"immunofluorescence & "7" complement fixation played an important role in avian medicine.

We can say that serum agglutination was the main serologic test up to the 1950's, AGPT the test of the 60's, while hemagglutination-inhibition was much improved and applied to new diseases in the 70's. The 1980's and early 90's saw the dissemination of Elisa's.

When to test the birds: Broilers Layers Breeders

1st further 1st further 1st furtherDuring the 1st days of live to detect

maternal

antibodies

(Mab).

Time of slaughter

to evaluate the

vaccination program.

During the 1st days of

live to detect Mab.

Immediately at the

onset of the problem

and 3 wks later.

During the 1st days of

live to detect Mab.

10-12 wks age

Before onset of lay, testing of the birds before revaccination for ND, IB, IBD and Reo viruses.

In production each 5-6 wks to control the status of M. synoviae, M. gallisepticum, (Ms), S. gallinarum/pullorum

and S. enteritidis.

1-Rapid whole blood agglutination test = Pullorum test = Spot test: Definition: It is a serological test used for detection of both S. pullorum & S. gallinarum in apparently healthy carrier hens. Reactors are identified immediately and culled. It is not reliable in turkeys as the test results in a significant proportion of false-positive results. Chickens are tested at a minimum age of 4 months.

Equipments: - Porcelain plate stained Ag, tested blood, dropper, watch, lancet or needle, flame of Bunsen and platinum loop.

Test procedure:1-Place 1 drop (0.02 ml) of crystal-violet-stained antigen in the center of each square.2-Place an equal size drops of fresh whole blood next to a drop of antigen.3-Mix the drops of Ag & blood by a fine glass rod, which is wiped, clean between samples.4-Use a gentle agitation and avoid drying out.

Reading:The test is time dependent as follows:

-Agglutination "clumping of the Ag & blood" within a minute is +ve reaction.-Agglutination in a time more than 2 minute is -ve reaction.-Agglutination in a time ranged from 1-2 minute is doubtful reaction and must be repeated.

Decision: -Positive reactors → cull.-Negative reactors → rearing for production.-Doubtful reactors must be retested within a week. Any doubtful reactions in retesting are regarded as positive.

The tests are carried out in the farm "spot" at room temperature (20–25°C) and a dust free area.

2-Rapid plate serum agglutination test: RPSAT is closely similar to pullorum test except for:-Whole blood is replaced by serum. -Used mainly for detection of both S. pullorum & S. gallinarum in apparently healthy carrier turkeys.-The reading time factors increase by 1 minute as follows:

-Agglutination "clumping of the Ag & blood" within 2 minutes is +ve reaction.-Agglutination in a time more than 3 minute is -ve reaction.-Agglutination in a time ranged from 2-3 minute is doubtful reaction and must be repeated.

Pullorum & RPSAT are initial screening of sera followed by confirmation of positives by the tube agglutination test. 3-Tube agglutination test : Fresh serum from chickens, turkeys is used at an initial dilution of 1/25, obtained by mixing 0.04 ml of serum with 1.0 ml of antigen. Positive and negative control sera are included in each test. The antigen is prepared from unstained S. pullorum or S. gallinarum cultures diluted to a concentration of No. 1 on the McFarland scale. The mixture is incubated at 37°C for 18–24 hrs before reading. A positive reaction consists of a granular white deposit with a clear supernatant fluid; a negative reaction shows uniform turbidity. Samples positive at a dilution of 1/25 are retested at a higher range of dilutions and a titer of 1/50 is usually considered to be positive, although this figure seems to vary in the literature.

4-Stained rapid plate serum agglutination test: It is closely similar to Rapid plate serum agglutination test except for:-It is used for detection of M. gallisepticum, M.synoviae and M.meleagridis in chicken & turkeys. -Stained Ag should be used as in pullorum.-The reading time factors are similar to that in pullorum test.

5-Hemagglutination "HA" test: It is not a serological test "it detects antigen not antibody" but it is used to identify the hemagglutinating agents for avian RBCs and also used as the basis for the hemagglutination inhibition test. The hemagglutinating agents for avian RBCs are NDV, AIV, EDS virus, IBV "after enzymatic treatment with phospholipase enzyme or culture extract of Cl.perferingins containing neuramindase enzyme" & Mycoplama spp..It is used by one of two methods:-Qualitative method: used only to identify the hemagglutinating agents for avian RBCs. It has a diagnostic function and is carried out as follow:

1-Allantoic fluid (virus) from an ECE that was inoculated with suspect material is removed.2-0.1 ml of this fluid is placed on a glass plate and 0.1 ml of 5.0% suspension of washed chicken RBC's is added.3-Clumping of RBCs will occur if a hemagglutinating agent is present.

-Quantitative method: used to identify the hemagglutinating agents for avian RBCs and also to estimate the hemagglutination "HA" unit depending on which Hemagglutination inhibition "HI" test will be carried out. It is carried out as follow:

1-In a 96 v-shaped HA plate, put 0.1 ml of PBS in each well and then put 0.1 ml the allantoic fluid under test of in the 1st well and then make double fold dilution by transferring 0.1 ml of the mixture in the well to the succeeding well and so on. 2-In each well, put 0.1 ml of 5.0% suspension of washed chicken RBC's.3-Incubate the plate 37°C for half an hour and then read the test.4-Clumping of RBCs will occur if a hemagglutinating agent is present. The HA unit is estimated as the highest dilution of Ag "allantoic fluid" causing clumping of RBCs.Decision: 1-If you will carry out the HI test to viral hemagglutinating agents, you should use 4 HA units.2-If you will carry out the HI test to bacterial hemagglutinating agents, you should use 8 HA units.

No agglutination

Agglutination

6-Hemagglutination inhibition "HI" test: It is a serological test used to quantitate serum antibody to a specific avian antigen, it is easy, rapid and cheap method for the detection of Abs against ND, AI, EDS-76, IB & Mycoplasma spp.. As we mentioned before, The hemagglutinating agents for avian RBCs are NDV, AIV, EDS virus, IBV "after enzymatic treatment with phospholipase enzyme or culture extract of Cl.perferingins containing neuramindase enzyme" & Mycoplama spp.. The antibodies against these organisms will inhibit this hemagglutination.Procedure:1-Put a constant amount of PBS in each well of 96 v-shaped microtiter plate, then the test serum is placed in the first well and serially diluted.2-A constant amount of HA antigen "4 HA in viral ones and 8 HA unit in bacterial one" is added to each well in a microtiter plate.3-The plates are incubated for one hr and then the 0.1 ml of 5% washed chicken RBCs are added to each well and then further incubation for approximately 30 minutes. Reading: If specific antibodies are present in the test serum, RBCs will not agglutinate with the HA antigen, so;

-HI -ve wells will have a diffuse sheet of agglutinated RBCs covering the bottom. -HI +ve wells will have a well-circumscribed button of un-agglutinated RBCs. -The result is expressed as a log-2 antibody titer of the sample. The titer is the reciprocal to the highest dilution of serum giving +ve result.

Comparison between HA and HI tests:Tests Hemagglutination Hemagglutination Inhibition

Nature Non-serological test Serological testPurpose Identify the

hemagglutinating agents for avian RBCs viz; NDV, AIV, EDS virus, IBV "after enzymatic treatment" &

Mycoplama spp..

Quantitate serum antibody to a specific pathogen viz; NDV, AIV, EDS virus, IBV

"after enzymatic treatment" & Mycoplama spp..

Material Allantoic fluid from ECE inoculated with a suspect

material.

A known hemagglutination agent.Test serum with unknown antibody

Chicken RBCs Chicken

7-Agar Gel Precipitation test (AGPT) : The AGP is a serological test used either to detect unknown antibodies depending on their reaction against various depending on their reaction against viruses or to detect unknown viruses depending on their reaction against known antibodies. It is basically a positive/negative reaction. The test is performed in Petri plates containing noble agar and purified salt and allowed to solidify. Wells "3 mm deep, 6 mm in diameter, and 3 mm apart" are made by punching using a special rosette and the circular pieces are removed aseptically by a forceps. A central well is made surrounding wells are punched on equal distance from the central one. The known element is placed in the central well and the elements under test are placed in the peripheral wells. The AGP is a group-specific test and will detect antibodies to all virus strains regardless of the serotype. It is therefore often used for detection of IB, IBD and adenoviruses (IBH, THE & MSP). There are two protocols for carrying out this test, they are:

1-Protocol for testing antibodies: The central well is filled with a known antigen and surrounding wells with serum samples to be tested. If the tested serum contains specific precipitating antibodies against the known antigen, a white precipitation line will be visible in the agar between them after 24-48 hrs of incubation in a humid environment.

2-Protocol for testing antigens: The central well is filled with a known antibody and surrounding wells with unknown antigens to be tested. If the tested antigen is specific to the known precipitating antibodies, a white precipitation line will be visible in the agar between them after 24-48 hrs of incubation in a humid environment.

Quantitative AGPT: The AGID test can also be used to measure antibody levels by using dilutions of serum in the test wells and taking the titer as the highest dilution to produce a precipitin line. This is very useful for measuring maternal or vaccinal antibodies and for deciding on the best time for vaccination; however, this AGID quantitative determination has now been largely been replaced by the ELISA.

8-ELISA: is used to detect antibodies to different diseases. Coating the plates requires a purified, or at least semi-purified, preparation of virus, necessitating special skills and techniques. Methods for preparation of reagents and application of the assay were described by Marquardt et al. in 1980. Commercial kits are available.

The test sera are diluted according to the established protocol or kit instructions and each is dispensed into the requisite number of wells.

After incubation under the appropriate conditions, the sera are discarded from the plates, and the wells are washed thoroughly. Anti-chicken antibodies conjugated to an enzyme are dispensed into the wells and the plates are again incubated as appropriate.

The plates are emptied and rewashed before substrate containing a chromogen that gives a color change in the presence of the enzyme used is added to the plate. After a final incubation step, the substrate/chromogen reaction is stopped by addition of a suitable stopping solution and the color reactions are quantified by measuring the optical density of each well. The Sample to Positive (S/P) ratio for each test sample is calculated.

9-Immunofluorescence: Immunofluorescence is a technique whereby an antibody labeled with a fluorescent molecule (fluorescein or rhodamine or one of many other fluorescent dyes) is used to detect the presence of an antigen in or on a cell or tissue by the fluorescence emitted by the bound antibody.

a-Direct Immunofluorescence: In direct immunofluorescence, the antibody specific to the antigen is directly tagged with the fluorochrome .b-Indirect Immunofluorescence: In indirect immunofluorescence, the antibody specific for the antigen is unlabeled and a second anti-immunoglobulin antibody directed toward the first antibody is tagged with the fluorochrome (Figure 21). Indirect fluorescence is more sensitive than direct immunofluorescence since there is amplification of the signal.

10-Complement fixation test: Antigen/antibody complexes can also be measured by their ability to fix complement because an antigen/antibody complex will "consume" complement if it is present, whereas free antigens or antibodies do not. Tests for antigen/antibody complexes that rely on the consumption of complement are termed complement fixation tests and are used to quantitate antigen/antibody reactions. This test will only work with complement fixing antibodies (IgG and IgM are best).

Antigen is mixed with the test serum to be assayed for antibody and antigen/antibody complexes are allowed to form. A control tube in which no antigen is added is also prepared. If no antigen/antibody complexes are present in the tube, none of the complement will be fixed. However, if antigen/antibody complexes are present, they will fix complement and thereby reduce the amount of complement in the tube. After allowing complement fixation by any antigen/antibody complexes, a standard amount of red blood cells, which have been pre-coated with anti-erythrocyte antibodies, is added. The amount of antibody-coated red blood cells is predetermined to be just enough to completely use up all the complement initially added, if it were still there. If all the complement was still present (i.e. no antigen/antibody complexes formed between the antigen and antibody in question), all the red cells will be lysed. If antigen/antibody complexes are formed between the antigen and antibody in question, some of the complement will be consumed and, thus, when the antibody-coated red cells are added not all of them will lyse. By simply measuring the amount of red cell lysis by measuring the release of hemoglobin into the medium, one can indirectly quantitate antigen/antibody complexes in the tube. Complement fixation tests are most commonly used to assay for antibody in a test sample but they can be modified to measure antigen.