Field aspects on Avian Influenza Diagnosis &Control By Prof.Dr. Ahmed Sayed Hamouda

1. Field aspects on Avian Influenza Diagnosis &ControlByProf.Dr. Ahmed Sayed Hamouda CAAVS 7 2009

2. H5N1 avian influenza is being a considerable problem for both veterinary and public health. CAAVS 7 2009

3. When the viruses are spread over a wide area and have infected multiple avian species, culling and physical containment are not likely to be successful. CAAVS 7 2009

4. In recent years, several vaccines have been developed against highly pathogenic avian influenza virus (HPAI) in poultry. Their use has shown that it can protect chickens from: • Developing disease symptoms. • Dying from infection. • Reduce the shedding rate. CAAVS 7 2009

5. In outbreaks of HPAI control measures still by killing infected birds as it is not known whether vaccinated poultry could ‘silently’ spread the disease, increasing the risk of new outbreaks causing a serious threat to humans. CAAVS 7 2009

6. little was known about how HPAI was transmitted in chickens, or how vaccines reduced transmission vaccination does not appear to block viral transmission completely, so some slaughtering may also be necessary. CAAVS 7 2009

7. Diagnosis of AI infection CAAVS 7 2009

8. infections in animal and humans has traditionally relied on virus isolation and identification, but this delays obtaining a definitive diagnosis for 1 to 2 weeks. Recently Polymerasechain reaction PCR)rapid real-time PCR (RRT-PCR))tests have been developed and used in human and animal field diagnostic situations, respectively. CAAVS 7 2009

9. Samples Samples from live birds should include: • Tracheal swabs. • Cloacal swabs. • Serum samples. CAAVS 7 2009

10. Samples from dead birds should include: • Intestinal contents. • Faeces or cloacal swabs. • Oropharyngeal swabs. • Trachea, lungs, air sacs, intestine, spleen, kidney, brain, liver and heart may also be collected separately or in pools. CAAVS 7 2009

11. Handling of samples • The samples should be placed in isotonic phosphate buffered saline (PBS), pH 7.0-7.4, containing antibiotics. CAAVS 7 2009

12. The antibiotics for sample preservation • penicillin (2000 units/ml). • Streptomycin (2 mg/ml). • Gentamycin (50 µg/ml) • Mycostatin (1000 units/ml) should be added for tissues ,tracheal and faecal swabs, but at five-fold higher concentrations for faeces and cloacal swabs. CAAVS 7 2009

13. It is important to readjust the pH of the solution to pH 7.0-7.4 following the addition of the antibiotics. • Faeces and finely minced tissues should be prepared as 10-20% (w/v) suspensions in the antibiotic solution. • Suspensions should be processed as soon as possible after incubation for 1-2 hours at room temperature. CAAVS 7 2009

14. Storage& transport of samples • When immediate processing is impracticable, samples may be stored and or transferred at 4°C for up to 4 days. • For prolonged storage, diagnostic samples and isolates should be kept at -80°C. CAAVS 7 2009

15. Serological detection of AI • Direct RNA detection. • Agar gel immunodiffusion. • HA and HI tests. • ELISA. • RT PCR. CAAVS 7 2009

16. Direct RNA detection Rapid test • It is a field rapid test to detect the AI antigen in oropharyngeal or tracheal samples from clinically affected or dead birds which show good sensitivity. • The test can demonstrate the presence of AI within 15 minutes. • It Is an antigen-capture enzyme immunoassay system, has been used for detecting the presence of influenza A viruses in poultry. • The kit uses a monoclonal antibody against the nucleoprotein and should therefore be able to detect any influenza A virus. CAAVS 7 2009

17. The disadvantages are: • Lack sensitivity. • It has not validated for different species of birds. • Subtype identification is not achieved. • The kits are expensive. The test is interpreted as a flock and not an individual bird test. CAAVS 7 2009

18. Agar gel immunodiffusion • This test is used to detect the presence of influenza A virus by demonstrating the presence of the nucleocapsid or matrix antigens, both of which are common to all influenza A viruses. • Precipitin lines can be detected after approximately 24-48 hours, but this may be dependent on the concentrations of the antibody and the antigen. CAAVS 7 2009

19. HA&HI tests • Variations in the procedures for HA and HI tests are practiced in different laboratories. • This variation may lead to miss diagnosis and erroneous interpretation of the results. CAAVS 7 2009

20. Haemagglutination test (HA) • The titration should be read to the highest dilution giving complete HA (no streaming); this represents 1 HA unit (HAU) and can be calculated accurately from the initial range of dilutions. CAAVS 7 2009

21. Haemagglutination inhibition test HI: The haemagglutination inhibition test is serotype specific. Each H-subtype has an individual HI test. Positive HI titres (> 1:8) develop a few days later than seen in ELISA or AGID tests; and titres persist till long after the infection. The HI test is the standard test for all avian species. CAAVS 7 2009

22. Haemagglutination inhibition test (HI) • Dispense 0.025 ml of PBS into each well of a plastic V-bottomed microtitre plate. • Place 0.025 ml of serum into the first well of the plate. • Make twofold dilutions of 0.025 ml volumes of the serum across the plate. • Add 4 HAU of virus/antigen in 0.025 ml to each well and leave for a minimum of 30 minutes at room temperature (i.e. about 20°C) or 60 minutes at 4°C. •   Add 0.025 ml of 1% (v/v) chicken RBCs to each well and after gentle mixing, allow the RBCs to settle for about 40 minutes at room temperature, i.e. about 20°C, or for 60 minutes at 4°C if ambient temperatures are high, by which time control RBCs should be settled to a distinct button. • The HI titre is the highest dilution of serum causing complete inhibition of 4 HAU of antigen. The agglutination is assessed by tilting the plates. Only those wells in which the RBCs stream at the same rate as the control wells (containing 0.025 ml RBCs and CAAVS 7 2009

23. HI interpretation • HI titres may be regarded as being positive if there is inhibition at a serum dilution of 1/16 (24 or log2 4 when expressed as the reciprocal) or more against 4 HAU of antigen. Some laboratories prefer to use 8 HAU in HI tests. While this is permissible, it affects the interpretation of results so that a positive titre is 1/8 (2_3 or log2 3) or more. CAAVS 7 2009

24. The following recommendation can be applied in the test • Use of V-bottomed microwell plastic plates in which the final volume for both types of test is 0.075 ml. • Use HAU of virus/antigen • The reagents required for these tests are isotonic PBS (0.1 M), pH 7.0-7.2. • RBCs taken from a minimum of three SPF chickens and pooled in an equal volume of Alsever's solution. • Positive and negative control antigens and antisera should be run with each test, as appropriate. CAAVS 7 2009

25. Genetic Change in Influenza Viruses Influenza viruses have the propensity to change genetically, which contributes to the interspecies transmission and zoonotic potential of AI viruses Change can occur by 2 mechanisms: random mutations in the RNA genome, especially in the haem agglutinin, which occur gradually over time; and reassortment of the 8 gene segments that occurs abruptly between 2 influenza viruses that infect a single cell, resulting in progeny that are hybrid viruses. CAAVS 7 2009

26. In birds, the greatest diversity of influenza viruses (all combinations of the 16 hemagglutinin and 9 neuraminidase subtypes) is found in (ducks and geese) and other aquatic bird Infections in these species are usually subclinical. CAAVS 7 2009

27. Immune exclusion by circulating or mucosa-bound haemagglutination-inhibiting (HI) antibodies is believed to be a major contributor to protection (Suarez and Shultz-Cherry,2000). Thus, HI serum antibodies are used as a reasonably reliable test marker for measuring the protection at least in gallinaceous birds. CAAVS 7 2009

28. Transmission and Host Adaptation Influenza viruses manifest some host adaptation with frequent and easy transmission between individuals of the same species or occasionally transmission to closely related species. For example, numerous human cases of influenza occur each year, predominantly caused by human-origin Influenza virus A strains but infrequently caused by nonhuman-origin Influenza virus A strains, such as swine-origin influenza A viruses.16On even rarer occasions, AI viruses have been directly transmitted from birds to humans. Furthermore, in experimental studies, a few AI viruses have shown limited replication in the nasal cavity of humans. The difficulty for transmission and infection of AI virus to humans can be partially attributed to different binding efficiencies of hemagglutinin of influenza viruses for surface cell receptors on avian or human respiratory epithelial cells. CAAVS 7 2009

29. VACCINATION CAAVS 7 2009

30. An effective HPAI vaccine for poultry should not only protect against disease but also against virus transmission (van der Goot et al., 2005). Efficient protection seems to depend to a large extent on antibodies which neutralise the virus and are predominantly directed against the haemagglutinin (HA). However, also antibodies against neuraminidase may neutralise the virus (Swayne et al., 2000b; McNulty, 1986; Sylte et al., 2007) CAAVS 7 2009

31. Advantages of Vaccination • Vaccination reduces susceptibility to infection.A higher dose of virus is necessary to infect the vaccinated birds • Vaccinated birds shed less virus Decreased contamination of the environment Decreased risk of human infection • Used strategically vaccination compliments a stamping out strategy by slowing/stopping the spread of the virus CAAVS 7 2009

32. Traditional control measures for HPAI have centered on stamping out, which entails the large scale culling of infected flocks and contact flocks. CAAVS 7 2009

33. The expected advantages of vaccination Firstlyvaccination reduces susceptibility to infection, a higher dose of virus is necessary for establishing an infection in vaccinated birds. Secondly there is a significant reduction in the amount of virus shed by infected birds, thus less virus to contaminate the environment reducing the risk of spread to other avian species and reducing the occupational risk faced by poultry workers. CAAVS 7 2009

34. Vaccine efficacy is dependant on the vaccine antigen and field virus being of the same H type (homologous haemagglutinin). CAAVS 7 2009

35. Types of Inactivated Vaccines Inactivated homologous vaccines: These are generally autogenously vaccines prepared from the field strain. Efficacy of homologous vaccines has been proven, however the disadvantage is that no serological distinction can be made between vaccinated and field exposed birds. CAAVS 7 2009

36. Inactivated heterologous vaccines These vaccines are prepared from a virus with the same H type as the field strain but a different N type (heterologous neuramidase). The immune response to the homologous H type ensures protection, while antibodies against the neuramidase of the field virus can be used as a marker. CAAVS 7 2009

37. Vaccination Schedule for Influenza H5* a. Dosage: 0.5 ml per dose in birds older than 3 weeks of age, 0.25 mlper dose in younger birds b. Administration: subcutaneously in the lower back of the neck or intramuscularly in older birds. c. Emergency Vaccination Schedule: Primary vaccination administered to all poultry irrespective of age. Booster vaccination administered 4 – 6 weeks later. (If the primary vaccination was givento birds younger than 3 weeks of age a third vaccination is recommended at 16 – 18 weeks of age) CAAVS 7 2009

38. Vaccination of Broilers: Vaccination of broiler chickens that are slaughtered within 7 – 8 weeks is in principle discouraged , as there is not sufficient time to develop adequate immunity following a primer and booster vaccination. However in situations where live bird trade predominates and meat chickens are raised for longer periods this may be reconsidered. (In Hong Kong local meat type chickens are vaccinated at 8 and 36 days of age.) CAAVS 7 2009

39. Vaccination of Replacement Flocks Vaccination schedule is dependant on perceived risk of infection. In high risk areas (active infection) primary vaccination (0.25 ml) is recommended at day old to establish immunity as early as possible. Two booster vaccinations (0.5ml) are recommended at 4 – 6 and 16 – 18 weeks of age. In areas with high infection pressure revaccination at midlay may beindicated. CAAVS 7 2009

40. killed viral vaccines are generally more stable and do not pose the risk of reversion to virulence compared to live vaccines, but their inability to infect cells and activate cytotoxic T cells makes them much less protective. Consequently, they generally require strong adjuvants and several injections to induce the required level of immunity and are usually effective in controlling only clinical signs rather than infection. CAAVS 7 2009

41. Monitoring Efficacy of Vaccination − Assessment of vaccination should be done by HI test one month after the second vaccination. − Test 10 - 20 serum samples per flock. − Require an HI titre greater than 1:16 in more than 70% of tested samples CAAVS 7 2009

42. Monitoring for Virus Circulation in Vaccinated Flocks − Thirty to sixty clearly identified sentinels chickens left unvaccinated must be placed in each house. − Ten to twenty serum samples collected from sentinels should be tested every 30 – 45 days (ELISA or HI). − If the sentinels seroconvert the flock is considered AI positive. However, in case of HPAI infection the sentinels will most likely die within 2-3 days of infection. CAAVS 7 2009

43. Confirmation of Infection Confirmation of an AI infection in a vaccinated flock By • Mortality. • Seroconversion of sentinels. CAAVS 7 2009

44. ELISA is of limited value when multiple serotypes of virus are circulating (e.g. H9 in Asia) CAAVS 7 2009

45. CAAVS 7 2009