MXPA06011928A - Aqueous inactivated vaccine for preventing avian influenza. - Google Patents

Abstract

The present invention is related to an inactivated vaccine against Avian Influenza in an aqueous solution, which includes adjuvants for obtaining a higher local immunity response, the invention also referring to a massive vaccination method by thin drop spray of said death virus vaccine against Avian Influenza in aqueous solution for increasing antibodies of the IgA type against the Avian Influenza virus or viruses located in the respiratory tract, wherein the vaccine is administered for providing protection against field strains of Avian Influenza and reducing the viral excretion thereof, thus simultaneously reducing the spread of the disease in a flock.

Description

1NACTIVATED AQUEOUS VACCINE FOR THE PREVENTION OF AVIAN INFLUENZA. Field of the invention. The present invention relates to an inactivated vaccine to prevent the spread of poultry by the Avian Influenza virus by its application by spray in fine droplet, which is preferably applied after the administration of a live virus vaccine that is replicate in the respiratory system of the birds.

BACKGROUND OF THE INVENTION Avian Influenza is known by several names, including: Avian Plague, Avian Plague and Brunswick Disease. In 1981, during the first symposium of the International Organization of Epizootics, the name of High Pathogenicity Avian Influenza was adopted as the official name of the highly virulent form of Avian Influenza Virus (Swayne, D.E. and Halvorson, D.A. 2003). When outbreaks occur in this way, the disease is notifiable and causes trade restriction and recently the obligatory slaughter of the birds. The term Influenza, originally referred to epidemics in humans, characterized by acute catarrhal fevers, which spread rapidly and were caused by viruses of the Orthomixoviridae family. Currently, it is recognized that the Orthomixovirus cause a significant number of diseases mainly in the upper respiratory tract of humans, horses, pigs and several species of birds, as well as in some aquatic animals. Influenza virus infection in poultry produces syndromes ranging from asymptomatic infections, to respiratory diseases and severe drops in egg production, as well as mortalities in some cases of up to 100%, caused by influenza viruses. High pathogenicity The economic losses due to Avian Influenza depend on the strain of virus, species of infected bird, number of farms involved, control methods used and the speed of implementation of control or control measures. eradication. Generally, the greatest losses are generated in commercial intensive chicken farms. Losses in outbreaks of High Pathogenic Influenza include costs of disposal of dead birds and repopulation of the flock, morbidity and mortality, quarantine costs, cleaning and disinfection costs, as well as losses in the production. The Avian Influenza virus is excreted both by the respiratory tract and by the digestive tract, therefore in birds the transmission from bird to bird is by air or orally. Contaminated stools are a source of contamination between the flocks. Because of this, measures have been implemented to prevent contamination by reducing the movement of people on farms, clean the equipment used with the use of disinfectants and carry out a general disinfection scheme of the houses between flocks and flocks, However, the epidemics that are reported continuously in all regions of the world. The use of emulsified inactivated vaccines has been used to prevent clinical signs and reduce bird mortality. There is bibliographic evidence from numerous studies that demonstrate that monovalent and polyvalent vaccines with adjuvants are able to induce an antibody response and provide protection against mortality, morbidity and fall of posture, as well as to reduce virus excretion and replication. in the vaccinated bird. (Brugh, M. ef al 1979, Brugh, M. et al 1986, Karunakaran er al 1987, Stone, HD 1987, Stone, HD 1988), however vaccination does not prevent infection and therefore some of the effects of the disease 5 Another type of vaccine that has been developed is recombinant vaccines. In this type of vaccines, segments of the Influenza gene (the one that codes for hemagglutinin) are inserted into other viruses, such as, for example, the smallpox virus. The advantages of these vaccines are that the animal receiving this vaccine does not have antibodies measured by the gel or agar immunodiffusion test or by the haemagglutination inhibition test, so that the trade and mobility of the vaccinated birds are not prevented by this vaccine and that your application can be done at day of age in the incubator. However, its great disadvantages are the failure to provide consistent protection for the smallpox fraction in birds that have previously been vaccinated with this agent (Swayne, et al., 1997, Webster et al., 1996), which interferes with the protection of the smallpox vaccine, in addition that only confers protection against mortality but does not prevent infection with the influenza virus. A further disadvantage may be the fact that insofar as there is less homology between the hemagglutinin expressed in the recombinant vaccine and the haemagglutinin of the field virus, the ability to inhibit viral growth will be lower. At the end of the 90's, the possible use of live vaccines containing the avian influenza virus from waterfowl was reported in which the haemagglutinin of strain A / Malllard / Ohio / 556/1987 (H5N9) of low pathogenicity showed a sequence of amino acid identical to the amino acid sequences published for the H5 genes indicating its potential as an effective vaccine (Crawford, J. and Cois 1998). The vaccine was applied in the drinking water in chickens, observing protection against challenge and a decrease in viral content in sewage disposal. However, a great disadvantage is that the virus has a high capacity to mutate and therefore can become a strain of high pathogenicity for the same species or for others. There are no reports of the use of inactivated vaccines together with an adjuvant to stimulate the local immune system, applied by spraying for the control of Avian Influenza in the chicken.

OBJECTS OF THE PRESENT INVENTION An object of the present invention is to provide an inactivated vaccine effective against Avian Influenza, containing a strain of Avian Influenza virus of low pathogenicity. Another object of the invention is the application of this aqueous chemically inactivated vaccine by spray in fine droplet to activate the local immune response by production of secretory IgA antibodies in the mucosa of the upper respiratory tract.

Furthermore, within the invention, an inactivated vaccine is provided in aqueous solution that helps to provide a tool that does not interfere with the epidemiological follow-up of field outbreaks, since it does not generate measurable antibodies at the serum level, while at the same time providing possibility of preventing the virus that comes in contact with the vaccinated birds from replicating in them. Another one of the objects of the present is to provide a method of mass application for the prevention of Viral Influenza infection to improve the productive parameters of the animals.

DETAILED DESCRIPTION OF THE INVENTION The characteristic details of this novel invention are clearly shown in the following description. To obtain this vaccine, one or more strains of avian influenza viruses of low pathogenicity are used, preferably of the subtype H5N2, although it may be of any other virus that affects the birds, which can be isolated in the laboratory from animals. infected or acquired through authorized laboratories. Subsequently, the viruses are propagated by inoculating 0.2 ml of a solution containing 5,000 to 50,000 viral particles per ml in chicken embryos free of specific pathogens of 9-11 days of age. After 3 days of incubation, the allantoic fluid is collected under sterile conditions and an aliquot is taken to determine the content of viral particles (titre) and haemagglutinating units (UHA) obtained in the harvest, as well as the verification of the absence of bacterial contamination according to the Code of Federal Regulations 9 of the United States of America. Through various investigations, the applicant has found that in order to obtain a vaccine that provides adequate protection and avoids the problems of vaccines known to date, only allantoic fluid containing a titre between 10 8 5 and 0 9 5 DIE should be used. / ml and from 512 to 1024 UHA at least. Subsequently, the fluids containing the replicated avian influenza viruses are inactivated with an inactivating agent such as 0.1% formalin, 0.1% betapropiolactone or 1mM binary bromoethyleneamine. Tests are performed on these inactivated fluids to verify the absence of JDacterial contamination according to the Code of Federal Regulations 9 of the United States of America and to corroborate that the inactivation process was satisfactory by means of an inactivation test in chicken embryo. The applicant has concluded that the inactivation of the avian influenza virus by heat is not adequate because the sensitivity to relatively high temperatures and it is important to keep intact as much as possible the hemagglutinin protein as it is what confers the protection. The test to corroborate the inactivation consists of taking a sample of 0.2 ml of fluid containing the inactivated virus and inoculating at least 5 embryos of pathogen-free chickens via the allantoic cavity. The embryos are incubated and ovoleped daily. On the third day of inoculation, a sample of allantoic fluid is obtained and the haemagglutination test is performed to rule out the presence of influenza virus. The test is satisfactory when the fluids of the embryos do not present hemagglutination, that is to say that the virus has not reproduced due to being dead, which avoids the problems of some existing vaccines to date. The vaccine is finally obtained by agitating the allantoic fluid containing inactivated avian influenza virus to obtain from 1,000 to 10,000 UHA and from 10 to 12% inactivated E. coli cultures. It is complemented with sterile distilled water (cbp) and thimerosal at 0.001% as preservative, maintaining the agitation for a period of 1-2 hours or until achieving a homogeneity of each of the ingredients. Given that the route of entry of the virus is aerial, the applicant has investigated the best way to prevent the virus from colonizing the respiratory tract, because the parenteral or oral routes of the vaccines have not been able to provide adequate protection because the white organ is the trachea. That is why after several studies to determine an effective route of application, the applicant has determined that the direct application in the respiratory tract is the most effective to avoid the problems of lack of effectiveness of vaccines against Avian Influenza. existing to date. In case the user does not want to vaccinate the birds one by one, the application of the vaccine can be carried out by means of a fine drop sprinkler, this form of application allows the vaccination to be done massively to a relatively large group of animals , eliminating another of the disadvantages of having to vaccinate each animal parenterally. In preliminary studies a good response of the vaccine was observed, so to improve the results different aspects were investigated so that the protective results of the vaccine had greater impact and a constant response. Thus, it was determined that it was important to ensure that the vaccine penetrated to the lower respiratory tract, which is the place where it has been observed to house a large amount of virus, and the other aspect was that a better adherence of the vaccine in the respiratory tract was achieved. . To solve the first aspect, different particle sizes of the vaccine were used, observing better results with the vaccines applied to small size, preferably at a particle size of 20 to 50 microns, because they penetrate deeply. This can be done in vaccination cells consisting of relatively small spaces with no or minimal circulation of air where the sprinklers are placed in the upper part to supply the vaccine in micro droplets that form a cloud where the birds are placed. vaccinated for 2-15 minutes, enough time to achieve penetration in the respiratory tract of birds. In this way, significant amounts of vaccine are wasted. Of course, small spaces or the corners of the poultry houses can be used to carry out a massive vaccination of a flock of birds to avoid having to carry out the bird-by-bird vaccination. Subsequently, a mechanism was sought to retain the vaccine in the respiratory tract long enough to give an immune response, for this, different forms of fixation were evaluated by means of some compound or element, but no alternative was considered viable. Finally, it was concluded that when the respiratory system (eyes, nose, trachea and lungs) was immunosuppressed due to environmental or induced conditions, the conditions that allow an adequate permanence of the vaccine were presented. Of course, the induced conditions are the most suitable because they can be propitiated at the moment in which the vaccine of the present invention is desired to be applied. These controlled conditions can be created when the chickens are vaccinated against a virus that causes respiratory diseases such as Newcastle, Laryngotracheitis and / or Infectious Bronchitis. The inactivated vaccine subject of the present invention can be applied preferably around 5-7 days after the stimulation of the respiratory tract by the application of vicus virus vaccine against the mentioned diseases, to have a better permanence and obtain a better protection against the Influenza virus. To further improve the mucosal response, 10-12% mucosal adjuvant may be added to the vaccine, which may be inactivated cultures of E. coli or some B toxin of Vibrio cholerae or any other toxin. Sterility tests are performed on the inactivated vaccine to verify that the product is free from contamination by bacteria, fungi and yeast according to the Code of Federal Regulations 9 of the United States of America. To be effective, the vaccine is evaluated by a power-challenge test performed as detailed below. 100 one-day-old chickens are used that are free of avian influenza antibodies, preferably birds free of specific pathogens. They are divided into two groups of 50 chickens each and are housed in Horsfall-Buer type isolation units with negative pressure. One group is given a dose of vaccine (0.5 ml) with the aid of a fine-drop sprinkler of 20 microns in size, sprinkled at a distance of approximately 30 cm above the head of the animals and for approximately 1 minute . The other group is administered 0.5 ml of distilled water from the. same way as the vaccinated group. At 7 days of age, all animals are vaccinated with a live Newcastle vaccine by eye. At 12 days of age or 5 days after vaccination with Newcastle virus, a Second dose of influenza vaccine by spray in the same manner as described above in the vaccinated group only. Three weeks after the second vaccination with the influenza virus by spray, all birds are challenged with a minimum of 200,000 infective doses of a strain of low pathogenicity of avian influenza virus contained in 0.2 ml intranasally. After a week post challenge all the animals of each group are slaughtered. A tracheal swab is taken from each bird and placed in a tube containing brain heart infusion broth with a mixture of antibiotics (100 IU of penicillin sodium, 100 μg of Gentamicin and 100 μg of streptomycin sulfate per me). From each tracheal swab pathogen-free chicken embryos are inoculated with 0.2 ml per embryo via allantoic cavity and incubated for 72 hours at 37.0 C with 85% humidity. At the end of the test the presence of virus is determined by haemagglutination. The vaccine is viable when the challenge virus is recovered from the vaccinated group in no more than 20% of the chickens and not less than 80% of the chicks in the positive control group. Similarly, the ciliostasis should be between 75 -100% in the vaccinated group and no more than 25% in the positive control group. The ciliary movement under normal conditions (without infection) is 100%, while in animals infected by the avian influenza virus or infectious bronchitis, the cilia become infected which causes less movement and a greater amount of mucus secretion in the trachea which leads to infections by bacteria, so an additional way to measure the level of infection by the avian influenza virus is by observing the ciliary movement. Next, non-limiting examples of evidence are presented, which demonstrate the use of the aqueous vaccine against Avian Influenza object of this invention. Example 1. Four groups of 10 broilers of 22 days of age were formed from the same source and kept in separate units Hosfall Bauer. Two groups were given a dose of 0.5 ml of the vaccine prepared according to the aforementioned in the description of the present invention with the aid of a commercial fine drop sprayer (Biofogger). After 2 weeks of vaccination, a group of 10 vaccinated birds (group 1) and a group of 10 unvaccinated birds (group 2 positive control) were challenged with a strain of avian influenza virus of low pathogenicity by a dose of 0.03 mi in the eye. Ten chickens were maintained in a separate unit as a control without vaccination and without challenge (group 3) and another 10 chickens vaccinated and without challenge (group 4 negative control). All the animals were kept under observation for a week, then they were sacrificed and samples of tracheal swab, trachea wash and tracheal rings were taken to quantify ciliary movement. The results obtained are shown in table 1. It can be seen that the ciliary movement of group 1 shows an average of 3.98, taking as a reference that the rating of 4 is equivalent to 100% of ciliary movement that occurs in animals without disease, 3 is equivalent to 75% of ciliary movement, 2 to 50%, 1 to 25% and 0 to 0% of ciliary movement, ie absence of ciliary movement in highly infected and compromised animals, so in this trial group 1 vaccinated showed 99.5% of ciliary movement, compared to 1.6 (40%) of group 2 positive control and 4 (100%) of groups 3 and 4. This indicates that vaccination prevented the replication of the virus. Table 2 shows the percentages of viral reisolation of the same groups, finding 0% reisolation in vaccinated birds of group 1 and 100% in group 2 positive control. Groups 3 and 4 did not show viral isolation since they were not challenged. No antibodies of IgG type were presented in the blood stream in the birds immunized with the vaccine object of the present invention, so that the birds could be allowed in those geographic zones that have the use of emulsified vaccines, which generate detectable antibodies in the serum of vaccinated birds. Antibody measurement was carried out with a commercial Avian Influenza ELISA kit from IDEXX, which established that an S / P value less than or equal to 0.5 is considered negative and a higher value of 0. 5 as positive. The S / P value refers to the value that results from dividing the value of the optical density of the sample minus the value of the optical density of the positive control between the density value of the optical density of the positive control. Groups 3 and 4 showed negative S / P values in serum, as expected. Table 3 shows the levels of antibodies found in the tracheal washings measured by the Avian Influenza detection kit of IDEXX. On average, the S / P values of vaccinated group 1 (3,139) are almost 10-fold plus the high values with respect to those of group 2 positive control (0.270), which shows that there is presence of antibodies locally. All these results indicate that the sprinkling method of the vaccine in aqueous solution of avian influenza to dead virus, reason for the present invention, provides a protection to the challenge with a strain of low pathogenicity, by stimulating the local immune response, that is, the production of secretory IgA in such a way that the mucous membrane is prevented by the challenge virus and therefore Inactivated Avian Influenza vaccine applied by and a Positive Control group.

Example 2. 30 fattening chicks of 1 day of age were taken, divided into 3 groups A, B and C of 10 birds each and kept in isolation units type Hosfall Buer. Groups A and B were vaccinated at 7 days with a dose (0.03 ml) via the eye of a commercial live Newcastle virus virus vaccine. At 12 days of age, group A chickens were vaccinated with a dose (0.5 ml) of the inactivated vaccine against avian influenza of the present invention by spraying using a fine drop sprayer (drop size 45 microns), group B chickens received the same dose but sterile distilled water. Three weeks after the vaccination, the chickens of groups A and B were challenged by ocular route (0.03 ml) with virus of low pathogenicity of Avian influenza (same strain with which the inactivated vaccine was elaborated) with a titre of 10 60 DIE 50 / mi. Group C chickens remained as a negative control group, without vaccination and without challenge. All birds were sacrificed after 6 days post challenge, tracheal wash samples were taken for IgA and tracheal swabs for viral reisolation. Table 4 shows the results of another recognized method for said determination of IgA, in Optical Density Units (UDO), secreted in tracheal washings for the three groups at the end of the test. It is considered that there is presence of antibodies when UDO values are greater than 0.2 It can be seen that there is a positivity in group A vaccinated with inactivated vaccine against bird flu greater than that generated by group B with the virus of challenge alone without vaccine, group C that did not receive vaccine or challenge showed JgA values less than 0.2 Table 4. ELISA results for IgA in tracheal washings Table 5 shows the challenge protection of the treated and control groups measured by the percentage of viral reisolation in chicken embryos. The positive control group B (without vaccine treatment) showed viral reisolation in 100% of the chickens, at necropsy the animals were observed that the trachea presented hemorrhages and a high content of nasal secretion. In the case of the birds of the vaccinated group A, the challenge virus was not re-isolated and the tracheae did not show hemorrhages and the amount of mucus present was much lower and in some birds no traces of mucus were observed. Based on these results, it was concluded that the inactivated vaccine protects against a challenge of influenza with low pathogenicity. Table 5. Results of viral reisolation of the Avian Influenza challenge virus in chicken embryos ALPES.

Example 3 We used 130 lightweight male chickens of the 1-day-old Bovans breed from a commercial incubator. 120 animals were divided into groups of 40 chickens. Each group was placed separately in isolation units type Horsfall Bauer. They were fed Ad libitum initiation food. The remaining 10 chickens were sacrificed to quantify maternal antibody levels against avian influenza as measured by the hemagglutination inhibition test. The 1-day-old chicks had antibody titers against the Avian Influenza virus with a geometric mean of 28.3; it is well known that this relatively high amount of antibodies are transmitted to chickens by the mother during the embryonic stage. At 7 days of age, the animals of two groups were given a dose of live vaccine against Newcastie's disease by ocular route with the purpose of sensitizing the trachea and allowing a better absorption of the vaccine applied by spray against influenza. . At 12 days of age a group of chickens vaccinated against Newcastie was given the equivalent of 0.5 ml per chicken (one dose) of distilled water sprayed with the aid of a sprinkler with a drop size of no more than 20 microns ( Biofogger) for 5 minutes. This group was identified as a control group. The other group of animals vaccinated against Newcastie were given the equivalent of 0.5 ml per chicken (one dose) of the inactivated vaccine against Avian Influenza of the present invention by spraying in a similar manner to the previous group. This group was identified as a group with an aqueous vaccine. The chickens of the third group were vaccinated with a dose (0.5 ml) of a commercial emulsion vaccine against Avian Influenza, identified as a group with emulsified vaccine. At 4 weeks post-vaccination, blood samples were taken from the chickens of each group for antibody determination by haemagglutination (Hl) inhibition. Subsequently, the animals of the three groups were challenged by applying 0.03 ml of a solution containing 200,000 DIE of the Avian Influenza virus strain H5N2 of low pathogenicity by infra-ocular route. After 2 days of said challenge, all the birds were sacrificed for the reisolation of the challenge virus by taking trachea samples, tracheal washings, tracheal swabs and cloacal swabs and for the determination of antibodies by the haemagglutination inhibition (Hl) test confirming the presence of antibodies.; For the viral reisolation, all samples of tracheal and cloacal swabs from each group were processed, that is to say, one from the control group, another from the group vaccinated with the inactivated aqueous vaccine and another from the group vaccinated with the emulsified vaccine. 0.1 ml of the total sample of each group of chickens was inoculated into the allantoic fluid of pathogen-free chicken embryos 9-1 day old. The embryos were incubated for 3 days and after taking an aliquot of the allantoic fluid to perform the haemagglutination inhibition test. The results of the determination of antibody levels by Hl in chicken serum before the challenge for the group with the spray-applied aqueous vaccine were less than 0, value that is considered by experts in the technical field as negative, for the control group the antibody levels were less than 10 and for the group vaccinated with the emulsified vaccine the geometric mean measured by Hl was 274.4, which is considered an undesirable high response. The antibodies in tracheal washings of the chickens of the group with the aqueous vaccine after the challenge were of a geometric mean of 35, while in the control group they were kept below 10. Regarding the reisolation of the challenge virus from tracheal swabs , in the group vaccinated with the commercial vaccine, the virus was reisolated in 40% of the vaccinated birds, in the group vaccinated with the vaccine in aqueous solution applied by spray, the challenge virus was not reisolated, while in the control group the virus was challenged. reisolated in 80% of the birds. These findings confirm what was reported by Villarreal in 1995, where he mentions that vaccination with emulsified vaccine prevents the mortality caused by the Influenza virus but does not prevent the replication of the challenge virus.

However, with the application of an inactivated aqueous vaccine by aspersion if the replication of the challenge virus is avoided. Example 4. 40 fattening chicks of 1 day of age were taken, divided into 4 groups of 10 chickens and kept in isolation units type Hosfall Buer. Groups A and C were vaccinated at 7 days with a dose (0.03 ml) ocularly of a commercial live Newcastle virus virus vaccine. Subsequently, at 14 days of age, the chickens from both groups were vaccinated with a dose (0.5 ml) of the inactivated vaccine against avian influenza of the present invention by spraying using a fine drop sprinkler (drop size of 50 microns) . , The animals of groups A and B were challenged by applying a dose 104 of the Avian Influenza virus Asian strain H5N1 of high pathogenicity (Swan / Mongolia / 05) via infraocular, while the chickens of groups C and D received a dose similar to the Mexican strain of high pathogenicity H5N2 (CK / Queretaro / 95). The animals were observed during XX days and at the end of recorded the mortality observed in each group. Table 6 As observed, the vaccine prepared from low pathogenicity viruses provide good protection against highly pathogenic viruses.

Bibliography. 1. Brugh.M., C.W. Beard, and H.D. Stone.1979. Immunization of chickens and turkeys against Avian Influenza with monovalent and polyvalent Oil Emulsion Vaccines. Am.J.Vet.Res. 40: 165-169 2. Brugh.M. and H.D. Stone.1986. Immunization of Chickens Against Influenza with Hemagglutinin-Specific (H5) Oil Vaccine Emulsion. ln C.W. Beard and B.C. Eastday (Eds) .Proceedings of The Second International Symposium On Avian Influenza.U.S. Animal Health Association.Athens, Georgia.283-292. 1. 3. Crawford.J.W., Maricarmen García, H.Stone, D.Swayne, R.SIemons and M.L.Perdue. Molecular Characterization of the hamaglutinin gene and oral immunization with a waterfowl-origin avian influenza virus. 998. Avian. Dis. 42: 486-496. 4. Karunakaran, D., J.A. Newman.D.A. Halvorson, and A.Abraham.1987.Evaluation of Inactivated Influenza Vaccines in Market Turkeys. Avian Dis.31: 498-503 5. Ogra.P.L.et al. 2001. Vaccination strategies for mucosal immune * responses. Clin.Microbiol.Review. 14: 430-445 6. Stone, H.D. 1987. Efficacy of Avian Influenza Oil Emulsion Vaccines in Chickens of Various Ages. Avian Dis.483-490 7. Stone.H.D. 1988. Optimization of Hydrophile-lipophile Balance for Improve Efficacy of Newcastle Disease and Avian Influenza Oil Vaccine Emulsion. Avian Dis.32: 68-73. 8. Swayne.D.E.and David A. Halvoston. 2003. Influenza. In: Disease of Poultry. Saif, Y.M Eds. 11th Edition. Iowa State Press. 9. Swayne, D.E., J.R. Beck, and T.R. Mickle.1997. Efficacy of Recombinat Fowl Pox Vaccine in Protecting Chickens Against Highly Pathogenic Mexican- Origin H5N2. Avian Dis. 41: 910-922.

Villegas P and S.H.KIeven. 1976. Aerosol vaccination against Newcastle Disease. II. Effect of vaccine diluents. Avian Disease. 20: 260-267 Villarreal.C.L, and A.O. Flowers. The Mexican Avian Influenza (H5N2) outbreak.ln: Proceedings of the 4th International Symposium on Avian Influenza, 4th ed. FROM. Swayne and R.D. Slemons, eds U.S. Animal Health Association, Richmond, VA.pp 18-22.1997 Webster, R.G., J.Taylor, J. Pearson, E. Rivera, and E.Paoletti.1996. Immunity to Mexican H5N2 Avian Influenza Virus Induced by a Fowl Pox-H5 Recombinat. Avian Dis.40: 461-465. Weltzin, R. and Thomas P. Monath. 1999. Intranasal antibody prophylaxis for protection against Viral Disease. Clin.Microbiol Rev. 12 (3): 383-393.

Claims (6)

1. Claims Having described the present invention, this is considered as a novelty, for which the content of the following claims is claimed: 1. An inactivated vaccine for the prevention of Avian Influenza consisting of an aqueous solution containing Influenza virus. Avian inactivated at 100% equivalent to 1, 000 to 10,000 UHA, 10-12% adjuvant of the mucous membranes of the respiratory tract and 0.001 to 0.01 of thimerosal or any other suitable preservative.
2. 2. The vaccine of claim 1, wherein said inactivated Avian influenza virus is obtained by replication of one or more subtypes of Influenza virus. Avian, preferably of the H5N2 strain, in chicken embryos and the subsequent 100% inactivation of the viruses obtained with 0.1% formalin, 0.1% betapropiolactone or 1mM binary bromoethylamine.
3. 3. The vaccine of claim 1, wherein said inactivated avian influenza virus is selected from that which provides 80% or more of viral reisolation in challenge tests in unvaccinated animals and less than 20% in animals vaccinated with the vaccine of claim 1.
4. 4. The vaccine of the claim 1, wherein said mucosal adjuvant preferably consists of inactivated cultures of E. coli or some toxin B of Vibrio cholerae. The vaccine of claim 1, wherein the vaccine is massively administered by fine-drop spray to a group of birds with a droplet size not greater than 40 microns for 2-6 minutes at a dose of 0.2 ml to 0.
5. 5 ml per broiler chicken.
6. 6. The vaccine of claim 1, wherein the administration is carried out from the first day of age of the chickens, preferably 2 to 7 days after the application of live respiratory virus vaccines such as Newcastle Disease, Laryngotracheitis and / or Bronchitis. Infectious