Serology is available for a range of bacterial and viral diseases, as well as for some chlamydial, mycoplasmal, rickettsial, protozoan and metazoan diseases. Such tests may be available in Regional Veterinary Laboratories, or only in Central Veterinary Laboratories at EMAI, Menangle.

A serological test is used to show the presence or absence of antibody to a specific aetiological agent or group of agents. The presence of antibody indicates exposure to the organism, which may be due to a current clinical condition or to an earlier unrelated infection.

In tests where results are expressed in titres, the best evidence of infection is the demonstration of a four-fold titre rise between samples collected early in the clinical episode and those collected 2 3 weeks later. A titre variation of less than four-fold (i.e. one dilution) is within the normal variation of a serological test and is not significant.

Since many animals in endemic areas may have antibody to a given organism, a single sample from an affected animal may not allow the serological test to be interpreted.

A single serum sample is particularly useful in eliminating a diagnostic possibility. Frequently, presence of antibody in the acute phase or absence in the convalescent phase will eliminate a diagnostic possibility.

Haemolysed or contaminated samples often give unreliable results in a complement fixation test (CFT): the serum may be anti complementary or give non-specific low titre positives, particularly in sheep and goat CFTs.

Poor quality samples will give poor quality results, necessitating retesting of the animals involved (see Avoiding haemolysis of blood samples).

Standard Containers and Equipment

Blood vacuum tubes, 10 ml

These should be silicone coated, without anticoagulant.

Sterile screw capped containers, 5 ml

For serum samples.

Collection of Specimens

Blood samples should be collected aseptically, using a 10 ml blood vacuum tube. At least 5 ml of blood should be collected. When a range of serological tests is required (particularly when viral serology and non-viral serology is to be undertaken), duplicate samples should be collected.

Contamination of the container and stopper should be avoided. Blood and faecal material should be removed prior to despatch, to reduce the risk of contamination of laboratory staff handling the specimens.

Use a separate sterile needle to avoid mechanically transmitting infectious agents from one animal to another.

Avoiding haemolysis of specimens

Haemolysis occurs as a result of poor collection techniques, contaminated equipment or poor handling of the sample once it is collected.

Common causes of haemolysis include:

1. Use of non sterile containers for collection or storage.
2. Contamination by faecal and other material due to faulty aseptic techniques.
3. Contamination of the sample by water.
4. A slow flow from the needle, due to obstruction of the needle, or failure to insert into mid-vein.
5. Forcibly expelling blood through a needle.
6. Heating of samples, usually in car boots or through back windows of car, or after prolonged exposure to direct sunlight during collection.
7. Freezing.

NB. Pig blood haemolyses quickly. Serum should always be separated from the clot within 4 hours of collection.

Labelling of specimens

Samples must be labelled serially (e.g. from 1 to 30) with a water proof pen, preferably on an adhesive label. Keep a key list which correlates sample numbers with animal identification. Do not label the stopper, which is removed during testing.

The specimen advice form submitted with the samples should list clinical details beside each sample number. This will allow the laboratory to offer an informed comment on the results and perhaps apply other relevant tests.

DO NOT label samples with tag numbers, names, etc., as this leads to confusion and errors in reading numbers in the laboratory. It also makes it very difficult for the laboratory to ensure all sample are present or to check on missing or broken samples.

DO NOT label containers with water soluble ink. It smudges when wet and may rub off if samples are chilled or frozen.

Storage and Despatch of Specimens

Samples should be allowed to clot before transporting them over any distance. Clots may not retract readily in cold weather or if they are chilled too soon after collection. Samples should be held in a warm room until the clot retracts.

Once the clot has retracted, blood samples must be held chilled to reduce contamination, haemolysis and autolysis.

If the laboratory will not get the samples within 48 hrs of collection, decant the serum into a 5 ml sterile, screw capped plastic container and submit the serum sample only. Serum can be frozen, provided there are no blood cells present in it. If virological examination of the clot is also needed (e.g Pestivirus antigen detection), submit clot separately.

Types of serological tests

Complement Fixation Test (CFT)

The test quantifies complement-fixing antibodies to a range of antigens. Test serum, complement and antigen are incubated then sensitised erythrocytes are added. If there is specific antibody present, an antigen-antibody complex is formed which binds complement and sensitised erythrocytes are not lysed. Results are given in titre form, reflecting a doubling serial dilution of serum from 1:4 upwards. The titre indicates the dilution at which 50% or more of the erythrocytes are not lysed.

Enzyme-Linked Immunosorbent Assay (ELISA)

The ELISA detects specific antibodies in serum which are allowed to bind to antigen on a solid phase. To detect the bound serum antibodies, the test uses a second antibody system (directed against antibodies of the animal species under test) to which is linked an enzyme. This enzyme catalyses a colour reaction, and the amount of colour which develops reflects the level of original serum antibody. This colour is measured quantitatively and is termed the serum absorbance or optical density.

The second antibody is usually species-specific, but may react with closely related animal species at a different concentration. Thus each ELISA is geared towards one test animal species. An absorbed ELISA (e.g. Johne's ELISA) involves the above steps, but also includes treatment of serum to absorb non-specific antibodies before testing.

The level of ELISA-reactive antibodies can be measured in a qualitative and quantitative way. The quantitative measure is relative to control sera. This may be in terms of:

1. Absorbance (Optical Density, OD) of the test serum, or,
2. ELISA ratio - compares the OD of the test serum to that of a negative control, but taking into account the performance of one or more positive control sera on the plate to give a valid test. Depending on the test, ELISA ratios of 2 or 3 are common cut-off points, or,
3. ELISA value or ELISA units - depending on the test, this can be used to reflect either
   1. the OD of the test sample relevant to a standard curve, originating from a range of positive and negative controls, or,
   2. a mathematical formula which expresses the OD of the test sample as a percentage of the positive control, with subtraction of the negative control OD from each i.e. (test OD - neg OD)/(pos OD - neg OD) X 100

Agar Gel Immunodiffusion (AGID)

Syn. Gel Diffusion Precipitin Test (GDPT)

Specific antibody is detected by placing test serums and control serum in wells in a gel around a central antigen well. Migration of antigen and antibody towards each other results in a visible line of precipitation where an antigen/antibody complex is formed. A GDPT reaction reflects the position of a specific line of reaction relevant to the serum and antigen wells, on a limited scale of trace, 1, 2 or 3. A reading of 2 indicates a line midway between the serum and antibody wells. A trace and 1 reaction are located closer to the serum well, whereas a 3 reaction is located closer to the antigen well. Trace is the weakest reading, and 3 reflects the highest level of antibody detected.

Serum Agglutination Test (SAT)

Tests for agglutinating antibody in a tube-based test, where clearing of the tube due to agglutination is used to determine an endpoint. Results are expressed as a titre, and the dilution series used (and therefore the titre) reflects the standard protocol for the test required. In some tests, the endpoint readings are converted to international units rather than titres.

Microscopic Agglutination Test (MAT)

A test used for leptospiral antibody which measures the ability of sera at varying dilutions to give an endpoint of 50% agglutination of one of a range of live leptospiral serovars.

Indirect Fluorescent Antibody Test (IFAT)

A slide-based assay using serum at varying dilutions to show specific binding to the test antigen on the slide. The fluorescent-labelled second antibody provides a measurable signal of bound antibody from the test serum.

Rapid Plate Test (RPT)

An agglutination assay used for some poultry pathogens, and performed with diluted serum on a solid base.

Latex Agglutination Test (LAT)

Antigen coated-latex beads are reacted with diluted sera, with a positive test giving a lattice of latex beads due to agglutination.

Rose Bengal Test (RBT)

A spot agglutination test performed on a solid base and used as a screening test for Br. abortus. Gives a qualitative (+/-) result only.

Summary of available serological tests for non-viral diseases