INTERPRETATION OF SEROLOGICAL RESULTS
Dr. M. Voss Lohmann Animal Health, Cuxhaven, Germany
Serological examinations are of major importance for monitoring vaccination response and to detect field infections in poultry flocks. Many serological tests are easy to perform, relatively cheap and can be performed in smaller labs. In contrast, other methods like virus isolation, histopathology and molecular biological examinations (e.g. PCR techniques) require special equipment and trained lab technicians. Serological test are used to detect antibodies against various viruses and bacteria relevant for poultry. Depending on the test system different antibody types are detected (IgG and/or IgM). With the exception of commercially available ELISA kits (Enzyme-Linked Immunosorbent Assay) furthermore antigens are mostly not standardised. Therefore, interpretation of serological results has to be done carefully and in consideration of the used test method and age of the birds. When to test the birds: Testing of chicks during the first days of live will detect maternal antibodies, transferred via the yolk from the breeder hen to the day-old chick. In order to allow complete resorption of the maternal antibodies from the yolk samples should be taken on day 3 to 4 of life. Maternal antibodies are important for the protection of the birds during the first 2 to 3 weeks of life, but will also provide information on the vaccination program and possible field infections in the breeder flocks. Measuring the level of maternal antibodies is of importance for the determination of the vaccination date for Gumboro, as maternal antibodies will interfere with the vaccine virus. This is of less importance for other diseases like Infectious Bronchitis (IB) or Newcastle Disease (ND), as vaccinations are commonly used already in day-old chicks in order to induce local protection in the respiratory system of the chicks. Testing of birds at an age of 20 to 25 days of age is usually useless as the maternal antibodies have declined and the chicks have not yet developed own antibodies. Therefore, in broilers further testing is recommended at time of slaughter, which will provide optimal time for the birds to develop antibodies against vaccine and field viruses. In breeder flocks further testing is recommended at an age of 10 to 12 weeks, as depending on the antibody status decisions for the vaccination program has to be done (e.g. vaccination against Chicken Anemia Agent (CAA). Before onset of lay breeders and layers are usually vaccinated with inactivated vaccines to booster the immunity especially for ND, IB, IBD and Reo. Therefore, testing of the birds before revaccination will allow information on the efficacy of the vaccination program in the rearing and will identify possible field infections. This is also important to adapt the vaccination program for future flocks. During production in breeder flocks sampling of the birds should be done every 5 to 6 weeks, which is important to control the status of Mycoplasma gallisepticum (Mg), Mycoplasma synoviae (Ms), S. gallinarum/pullorum and
Salmonella enteritidis (SE). In many countries also revaccination against ND is based on the actual antibody status of the flocks. Even if samples are not tested for other viruses like IB or Reo, it is recommended to freeze the samples as it might be necessary to retest the flocks in case of health and production problems during the production period. As commercial layers usually are not tested by a complete monitoring program in case of any suspected disease problems sampling of the birds is recommended immediately at the onset of the problem and 3 weeks later. By this means an optimal interpretation of the results will be possible and will enable to clearly identify the infectious cause of the problem. Which tests could be applied: There are several test methods used in poultry which are easy to perform and need no or only minimal equipment. In contrast, test systems like ELISA or serum neutralisation test (SNT) require expensive equipment and are therefore only used in bigger labs. Rapid Plate Agglutination (RPA): RPA is a very easy method to detect antibodies against Mycoplasma (Mg and Ms) and various types of Salmonella (Sg, Sp, Se, Stm). The test could be performed in any lab or even directly in the chicken house by mixing one drop of serum/blood with one drop of antigen. After shaking for 2 minutes agglutination will be visible in case of positive reactions. Agar Gel Precipitation (AGP): The AGP is a simple method to detect antibodies against various viruses but is basically a positive/negative reaction. In principal, on an agar plate one well is filled with a known antigen and surrounding wells with serum samples to be tested. In case the serum contains precipitating antibodies after 24 to 48 hours a white precipitation line between the samples and the antigen will be visible in the agar. 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 an d Fowl Adenoviruses (FAV, e.g. Inclusion Body Heptatitis). Haemagglutination Inhibition Test (HI): Several viruses have the ability to agglutinate red blood cells of the chicken. These viruses are ND, Egg Drop Syndrome (EDS), Avian Influenza (AI) and, after special enzyme treatment, also IB. The test is serotype-specific for those viruses where antigenic different virus strains occur (AI, IB). In principal on a microtiter plate serum samples are diluted twofold and are mixed with a constant amount of haemagglutinating units of the viruses. After incubation for 20 to 45 minutes red blood cells are added and, after further incubation for approximately 30 minutes, the wells are examined for haemagglutination of the red blood cells. The result is expressed as a log-2 antibody titre of the sample. The HI test is an easy, rapid and cheap method for the detection of antibodies against ND and EDS. Testing for AI and IB antibodies requires the relevant virus strains. ELISA:
Commercial ELISA tests are available for the most important virus infections and several bacteria (e.g. Salmonella spp. and Pasteurella multocida). Various companies are selling ELISA kits, with IDEXX (US) and KPL (US, recently sold to Synbiotics) being the most important. Other companies include Biochek (Netherlands), Guildhay (UK) and SVANOVA (Sweden). ELISA tests are a rapid method to test large numbers of samples. On the other hand rather expensive equipment (reader, washer, computer) is needed to perform the test. But from one single serum dilution antibodies against different agents could be tested. The ELISA is group-specific like the AGP, detecting for example antibodies to all IB serotypes. It is a useful method to monitor vaccination programs and to detect field infections. But interpretation of results need good experience and have to consider the type of test used, number of samples, vaccination program and type of birds. Serumneutralisation (SN): The SN could be used for all viruses were the growth of the virus could be made visible on the culture system. For many years the SN has been performed in embryonated eggs evaluating embryo mortality and specific lesions in the embryo. But now usually either tissue cultures or tracheal organ cultures (TOC’s) are used depending on the virus type. The SN is serotype-specific and the most precise method to detect antibodies against different serotypes of a given virus (e.g. IB, FAV), but requires expensive equipment and the use of SPF eggs for preparation of tissue or organ cultures. Immunofluoreszenz (IF): IF is a sensitive method to detect antibodies against many agents. But due to the need of good experience, reagents and equipment it is seldom used for routine serological monitoring. Interpretation of results: Interpretation of serological results depends on the test system used, age and type of birds, time after vaccination and route and type of vaccines used. Veterinarians have to achieve their own experience with results, as there is a big variation in type of antigens and methods used between different labs. Also ELISA results from different suppliers of kits are not necessarily comparable, as there might be big variations in titre calculations. Also it is important to consider the ELISA as a flock monitoring and not to test individual birds. Any interpretation of results must include the mean titre and the coefficient of variation (CV). Printing individual titres including controls for several diseases on one page could be helpful to identify either lab or vaccination mistakes. Variations in results of +/- 10% are considered as tolerable when comparing results obtained on different plates of the same supplier. Therefore, an increase of an IB titre from 8.000 to 8.500 does not necessarily confirm a field infection. In questionable cases it might be helpful to repeat samples on the same plate to exclude intra-plate variations.
Especially when testing SPF (specific pathogen free) flocks very often limitations of ELISA tests are obvious due to false positive results. But the same applies for those tests where the birds (especially breeders) should be negative for, e.g. Mycoplasma, Salmonella, Avian Leukosis). One out of ten samples positive could not confirm infection of the flock and requires retesting of the samples by a different method or new samples. Although positive and negative controls are provided with each ELISA test kit, it is strongly recommend for each lab to run also internal controls on each plate. All test methods have some advantages and disadvantages. Therefore, it is of major importance to choose the right test system for the specific question of concern. Following some examples for specific diseases are given and further examples will be given during the presentation. Mycoplasma: The method of choice to detect antibodies against both Mg and Ms is still the RPA test. Using good quality antigens (e.g. Intervet) it is a sensitive, rapid and reliable method. In contrast, most ELISA tests show to some degree false positive reactions and are much more expensive than RPA. Non-specific reactions in the RPA have been ocassionally observed after application of bacterins and some inactivated Reo vaccines. From the ELISA tests, the SVANOVA kits seems to be the most specific as it is using a blocking system, providing clear positive/negative results but without titre calculation. Infectious Bronchitis: Several different test systems could be used for the detection of IB antibodies, with AGP and ELISA being group-specific, whereas HI and SN are serotype specific. Precipitating antibodies are detectable in a flock only within 3-4 weeks after exposure to live vaccine or field virus. The AGP is therefore a cheap and easy method to screen flocks for field infections but without any information on the type of field virus. The ELISA is useful to monitor vaccination programs and also to detect field infections. After live vaccination in the IDEXX system maximal titres will reach 2.000 to 3.000 and after inactivated vaccines 6.000 to 8.000. Recently in some flocks titres of 15.000 to 20.000 were detected, clearly indicating exposure to field virus. The HI test is used in several labs to identify antibodies against different IB serotypes. But the specificity of the test is much lower compared to SN, as there is a high cross-reactivity after a second antigen contact in the birds, making the interpretation of results more difficult. Therefore, the SN test either in chicken embryo kidney cells or tracheal organ cultures is the method of choice to differentiate antibodies against various IB strains. But it is very important to keep in mind that any serological test, even the SN, can give only some indication about the type of IB field virus. The only definite conclusion could be given after isolation and identification of the field virus.
Chicken Anemia Agent (CAA): For many years detection of antibodies to CAV was limited to a few labs working with MSB-1 cells by either IF or SN techniques. After the introduction of commercial ELISA kits these became a rapid and sensitive alternative. Monitoring for CAA antibodies is important to ensure protective antibody levels at the onset of production in breeder flocks. Avian Leukosis Virus (ALV): Infections with ALV subtype J have been of major importance in many broiler breeders during the last years. In contrast to other infections, for ALV ELISA kits are available for the detection of both antigen and antibodies. The group specific antigen p27 (GSA) is present in all Avian Leukosis Viruses (both endogenous and exogenous). Detection of GSA by ELISA is the most rapid, cost-effective and simple method. But endogenous ALV sequences in broiler breeder lines may express GSA without producing infectious virus. Endogenous ALV sequences in broiler breeder lines associated with slow feathering may express GSA and infectious virus but without causing tumours. GSA testing is the most common part in ALV eradication programs, even if uninfected birds expressing GSA from benign endogenous sources are removed. Interpretation of results has to be done very carefully: neither the presence of GSA nor the actual ELISA reading is diagnostic for the infection as demonstrated by the levels of positive GSA testing in uninfected broiler lines under normal (and stress) conditions. Type of sample Cloacal swabs Albumen Meconium Serum Fast feathering lines 0-5 % (40-50%) 0-3% (10-12%) 0-3% (10-12%) 20-80% (50-100%) Slow feathering lines 0-15% (50-60%) 0-6% (0-15%) 0-6% (0-15%) 20-80% (50-100%)
Especially serum samples should not be used for the detection of GSA, as this will not confirm infection of the flock. For antibody detection ELISA kits for both subgroups A/B and J are available. As the antibody response to Retroviruses is very weak, it will take at least 6-8 weeks for a flock to develop antibodies. Furthermore chicks infected vertically or during hatch may be immune tolerant. Also up to 10% false positive samples might be present in a flock which makes isolation of the virus essential to confirm infection.