| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | March 30, 2004 |
| PATENT TITLE |
Method of vaccination of newly hatched poultry |
| PATENT ABSTRACT | A method of vaccinating poultry by spraying the poultry with an effective amount of a live avirulent derivative of an enteropathogenic enterobacteria is disclosed |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | July 24, 1998 |
| PATENT REFERENCES CITED |
Benjamin. 1995. Live Salmonella Typhi Vaccine Strain, UABRF Dept. of Microbiology.* Coloe. 1992. PR-22: Use of Aro-Salmonella typhimurium as a vaccine in poultry. Victorian Dept. of Food and Agriculture.* Curtiss, III. 1990. Res. Microbiology 141:797-805.* Curtiss, III. 1991. Colonization Control of Human Bacterial Enteropathogens In Poultry. 169-198.* Curtiss, III. 1997. Live Attenuated Vaccines to Control Salmonella and Eschericha coli In Poultry, Dept. pf Biology, Washington Univ. and Megan Health, Inc. IVVDC First International Veterinary Vaccines and Diagnostics Conference, Jul. 27-31, 1997, Madison.* Garavax-T tm Escherichia coli Vaccine, Schering Plough Animal Health, Product Bulletin, 1995.* Grieve. 1997. Poultry Times, Sep. 22, 1997. pp. 18-19.* LaBudde. salmonella vaccine for poultry. FSNET. Jun. 4, 1997.* Stewart-Brown. Applying poutry vaccines via the aerosol route on the farm: technique and critique.Solvay Aniaml Health, Inc. 2 pages.* Al-Tarcha et al., Immunization Of Day-Old Chicks Having Maternally Derived Antibodies Against Infectious Bronchitis: Degree Of Protection As Monitored By Ciliary Activity After Intratracheal Challenge, Acta Veterinaria Hungarica, 39:83-93 (1991). Andreasen, Jr. et al., Studies Of Infectous Laryngotracheitis Vaccines: Immunity In Broilers, Avian Diseases, 33:516-523 (1989). Benjamin, Live Salmonella Typhi Vaccine Strain, UABRF Department of Microbiology, (1995). Clarke et al., Spray Vaccination Of Chickens Using Infectious Laryngotracheitis Virus, Australian Veterinary Journal, 56:424-428 (1980). Coloe, PR-22: Use Of Aro- Salmonella typhimurium As A Vaccine In Poultry, Victorian Department Of Food And Agriculture (1992). Curtiss, III et al., Stabilization Of Recombinant Avirulent Vaccine Strains In Vivo, Res. Microbiol., 141:797-805 (1990). Curtiss, III et al., Nonrecombinant And recombinant Avirulent Salmonella Live Vaccines For Poultry, Colonization Control Of Human Bacterial Enteropathogens In Poultry 169-198 (1991). Eidson et al., Application Of The Turkey Herpesvirus Vaccine By The Aerogenic Route For The Prevention Of Marek's Disease, Develop. Biol. Standard, 33:370-375 (1975). Folkers et al., Vaccination Against Avian Encephalomyelitis With Special Reference To The Spray Method, Develop. Biol. Standard, 33:364-369 (1975). Giambrone, Vaccination Methods In The Hatchery, Hatchery--World Poultry Misset, 13(7):19-23 (1997). Ibrahim et al., Spray Vaccination With An Improved F Newcastle Disease Vaccine. A Comparison Of Efficacy With the B1 And La Sota Vaccines, British Veterinary Journal, 139:213-219 (1983). Ibrahim et al., An Assessment Of The Australian V4 Strain Of Newcastle Disease Virus As A Vaccine By Spray, Aerosol And Drinking Water Administration, Australian Veterinary Journal, 57:277-279 (1981). Frazeur, Virogen Hatches New Poultry Vaccine, AG Innovation News, 6(4), 3 pages (1997). Grieve, Administration Of Live Vaccines To Poultry By Spray Is Widely Used, Poultry Times, Sep. 22, 1997. Gross, 4 Colibacillosis, Diseases Of Poultry, Iowa State University Press, 138-144 (1991). Kramer, Salmonella Vaccine For Poultry, New Vaccine Could Be Solution To Salmonella-Tainted Eggs, Iowa State University Press Release, Jun. 4, 1997. Lee, Scientists Testing New Poultry Vaccine Against "Bird Flu", What's New: Press Releases, Protein Sciences Corporation, Apr. 6, 1998. Ley et al., Transmissibility Of Live Mycoplasma Gallisepticum Vaccine Strains ts-11 and Jun. 1985 From Vaccinated Layer Pullets To Sentinel Poultry, Avian Diseases, 41:187-194 (1997). Promsopone et al., Evaluation Of An Avian-Specific Probiotic And Salmonella typhimurium--Specific Antibodies On The Colonization Of Salmonella typhimurium In Broilers, Journal Of Food Protection, 61(2):176-180 (1998). Stewart-Brown, Applying Poultry Vaccines Via The Aerosol Route On The Farm: Technique And Critique, Solvay Animal Health, Inc., 2 pages. Subramaniam et al., Study Of Immune Status Of Commercial Chicks Against Newcastle Disease Using Live-In-Oil Vaccines With Spray At Day Old, Indian J. Amin. Hlth., 36(1):31-36 (1997). Toro et al., Infectious Bronchitis: Effect Of Viral Doses And Routes On Specific Lacrimal And Serum Antibody Responses In Chickens, Avian Diseases, 41:379-387 (1997). Toth et al., Reaction Of The Avian Respiratory System To Intratracheally Administered Avirulent Salmonella typhimurium, Avian Diseases, 36:24-29 (1992). Voeten et al., Comparison Of The Effect Of Live Newcastle Disease Vaccine Clone 30 In Broilers Administered At Day 1 Or At Day 7 And The Effect Of H120 Vaccination At 17 Days Of Age: A Field Experiment, The Veterinary Quarterly, 38-48. Wallner-Pendleton et al., Respiratory Infections In Domestic Poultry Flocks, NebGuide, G91-1039A (1991). Yoder, Jr. Diseases Of Poultry Mycoplasm Gallisepticum, Mycoplasmosis, 8:208-212. Paratyphoid Vaccine, Salmonella Typhimurium Vaccination Injection For Pigeons, Vetafarm--Veterinary Articles, 1 page, Jun. 23, 1998. Chick Stim, Ostrich Chick Gut Immunostimulant, Chick Stim, 1 page, Jul. 1, 1998. Garavax.RTM.-T, Escherichia coli Vaccine (Avirulent Live Culture, Avian Isolate), Schering-Plough Animal Health, Product Bulletin, (1995). Spra-Vac, Rhone, Merieux, Equipment Manual. Jackwood, Current And Future Recombinant Viral Vaccines For Poultry, Poultry Diagnostic And Research Center, Department Of Avian Medicine, College Of Veterinary Medicine, IVVDC First International Veterinary Vaccines And Diagnostics Conference, Jul. 27-31, 1997, Madison, Wisconsin. Curtiss, III et al., Live Attenuated Vaccines To Control Salmonella And Escherichia coli In Poultry, Department of Biology, Washington University & Megan Health, Inc., IVVDC First International Veterinary Vaccines And Diagnostics Conference, Jul. 27-31, 1997, Madison, Wisconsin. |
| PATENT CLAIMS |
What is claimed is: 1. A method of vaccinating a domestic bird comprising administering by whole-body spray, an effective amount of a vaccine comprising a live avirulent derivative of an enteropathogenic bacteria to the bird, wherein said enteropathogenic bacteria is other than one that causes respiratory disease in birds. 2. The method according to claim 1 wherein the enteropathogenic bacteria is a Salmonella. 3. The method according to claim 2 wherein the spray is administered in a dose of from about 10.sup.5 to about 10.sup.8 colony forming units of the live avirulent derivative of a pathogenic bacteria. 4. The method according to claim 3 wherein the Salmonella is S. typhimurium. 5. The method according to claim 4 wherein the S. typhimurium is .chi.3985. 6. The method according to claim 3 wherein the bird is 3 weeks of age or less. 7. The method according to claim 6 wherein the bird is less than one day of age. 8. The method according to claim 7 wherein the bird is a chicken. 9. The method according to claim 7 wherein the administering by spray is followed by administering at least one booster dose of the vaccine. 10. The method according to claim 9 wherein the booster dose of the vaccine is administered in the drinking water. 11. The method according to claim 10 wherein a booster dose is administered 14 days after the administering by spray. 12. The method according to claim 1 wherein the spray is a coarse spray of droplets having diameters in the range of from 50 microns to 150 microns. 13. A method of reducing microbial contamination of poultry comprising immunizing the poultry against a microbial contaminant by whole-body spray administration of a vaccine comprising a live avirulent derivative of an enteropathogenic bacteria, wherein said enteropathogenic bacteria is other than one that causes respiratory disease in birds. 14. The method according to claim 13 wherein the enteropathogenic bacteria is a Salmonella. 15. The method according to claim 14 wherein the spray is administered in a dose of from about 10.sup.5 to about 10.sup.8 colony forming units of the live avirulent derivative of a pathogenic bacteria. 16. The method according to claim 15 wherein the Salmonella is a S. typhimurium. 17. The method according to claim 18 wherein the S. typhimurium is .chi.3985. 18. The method according to claim 16 wherein the poultry are less than 104 weeks of age. 19. The method according to claim 16 wherein the poultry are 3 weeks of age or less. 20. The method according to claim 19 wherein the poultry are less than one day of age. 21. The method according to claim 20 wherein the poultry are chickens. 22. The method according to claim 18 wherein the spray administration is followed by administration of at least one booster dose of the vaccine in the drinking water. 23. The method according to claim 22 wherein a booster dose is administered 14 days after the spray administration. 24. The method according to claim 13 wherein the spray administration comprises spraying droplets having diameters in the range of from 50 microns to 150 microns. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to poultry vaccines and, more particularly, to a novel method of vaccinating poultry involving spraying with a live avirulent derivative of a enteropathogenic bacteria. 2. Description of Related Art Contamination of poultry meat and eggs by enterobacterial human pathogens, such as Salmonella spp. is a well known cause of illness in humans when such contaminated products are consumed. The contamination occurs predominantly during processing of carcasses after slaughter by contact with intestinal contents that contain high levels of such enterobacteria. The enterobacteria colonize the intestinal tract, but do not normally cause disease in the poultry. In order to reduce the contamination of food with enteropathogens it would thus be desirable to diminish the amount of human enteropathogenic bacteria present in the intestinal tracts of market-age broilers. Efforts to reduce this contamination have focused on improved age broilers. Efforts to reduce this contamination have focused on improved sanitation during production and processing (Bailey, J. S., Poult. Sci., 72:1169-1173, 1993), but such techniques are time-consuming and expensive and are not totally effective in avoiding sporadic contamination. (See, e.g., Food Borne Disease Outlook Annual Summary, 1982; and Salmonella Surveillance Annual Survey 1992; both available from Center for Disease Control, U.S. Department of Health and Human Services, Atlanta, Ga.). Methods that depend upon sanitation during processing must be repeated frequently since processing equipment and personnel can be re-contaminated by each contaminated fowl that is processed. Methods that depend upon sanitation during production require constant vigilance due the high potential for contamination in the production environment. Therefore, a simple and inexpensive method to control enteropathogenic microbes in poultry during growth would be a key improvement in reducing carcass contamination during processing. Promosopone et al., J. Food Protect., 61(2): 176-180, 1998, have reported that S. typhimurium colonization of the intestinal tracts of poultry can be reduced by administration of an avian-specific probiotic combined with S. typhimurium specific antibodies. Lactobacillus acidophilus, Streptococcus faecium and S. typhimurium-specific antibodies were administered by spraying the chicks at one day of age followed by oral administration via the drinking water from day 1 to day 3. The chicks were challenged by oral administration of S. typhimurium on day 1 and significantly reduced amounts of S. typhimurium were recovered from the cecum and colon following probiotic-treatment at 31, 38 and 43 days. Although administration of probiotic and antibodies as early as 1 day of age may have been important in reducing colonization of the intestine by S. typhimurium, it is not clear from this report whether the initial spray administration of probiotic and antibodies or the more commonly used oral administration in the drinking water on days 1-3 was responsible for decreasing S. typhimurium colonization. Vaccines for use in preventing diseases in poultry have been reported and some of these vaccines are specific for Salmonella (See, e.g. U.S. Pat. Nos. 5,294,441, 5,389,368, 5,468,485 and 5,387,744). The methods for administration of vaccines in poultry vary, however, depending upon the target site of action of the active agent. In fact, it is commonly believed that the vaccination route should be tailored according to the preferential site of the microorganism for localization and replication. Thus, for Newcastle disease and infectious bronchitis viruses which multiply in the respiratory route, the vaccination methods of choice would be by eye drop into the eye, nasal passage and respiratory system of the chick or by the spray route. (Giambrone, World Poultry-Misset 13:19-23, 1997). Since many of the more important diseases of poultry occur in the respiratory tract, studies reporting on administration of spray vaccination for these diseases have used spray administration because an aerosol or spray is easily inhaled by the bird and thereby contacts the mucosal surfaces of the upper respiratory tract. Administration of vaccines for non-respiratory diseases, such as diseases of the tissues, circulatory system or gut, is usually by subcutaneous injection, or by oral administration, either by inoculation or by application in drinking water. References disclosing the use of the spray administration of vaccines have almost exclusively been directed to immunizing against viral agents that invade through the respiratory tract such as, for example, to prevent Newcastle disease, avian encephalomyelitis, Marek's disease, laryngotracheitis, infectious bronchitis and the like. Bacterial vaccines, in particular live attenuated mutants derived from highly virulent bacterial parent strains, have also been used in poultry (Roland, K. et al., Efficacy of Salmonella typhimurium vaccine strains expressing Escherichia coli 078 lipopolysaccharide to protect against E. coli challenge in chickens, Abstract of a presentation at Conf. Of Res. Workers in Animal Diseases, Chicago, Ill., Nov. 10, 1997). Derivation of the attenuated mutant strain from a highly virulent parent increases the likelihood that the attenuated mutant will not only colonize the intestinal tract but also colonize the gut associated lymphoid tissue (GALT) and, elicit protective immunity. (See, e.g., Curtiss III et al., in Colonization Control of Human Bacterial Enteropathogens in Poultry, Blankenship et al., eds, Academic Press, Inc., New York, 1991 169-198). In contrast, bacteria that colonize the intestine but do not invade and colonize the GALT may not elicit an immune reaction. For example, studies in mice have revealed that lipopolysaccharide (LPS) O-antigen repeats on the surface of S. typhimurium are important not only to withstand nonspecific host defense mechanisms (Microbial Toxins, Vol. V, Roantree et al., eds., Academic Press, New York, 1971), but also for effective invasion through the mucin and glycocalyx covering the intestinal tract. As a consequence, rough mutants lacking LPS O antigens, when given orally, are unable to invade and colonize the GALT (See, e.g. Curtiss et al., 1991, supra). Some references have reported on the administration of bacterial vaccines to poultry by oral or subcutaneous injection. For example, one commercial vaccine to prevent paratyphoid in pigeons comprises killed S. typhimurium administered by subcutaneous injection (Vetafarm Paratyphoid Vaccine, Vetafarm Pty. Ltd., Wagga Wagga, Australia). In addition, Curtiss et al, 1991, supra, report the use of an avirulent derivative of a pathogenic Salmonella as an orally administered vaccine in chicks. Spray vaccination has also been reported for bacterial vaccines that cause respiratory diseases. Hertman et al. report on oral and aerosol administration of a Pasteurella multocida vaccine to chickens and turkeys to prevent fowl cholera, which is a respiratory tract disease (U.S. Pat. No. 4,169,886). Ley et al. report on eye-drop and aerosol administration of a vaccine containing live Mycoplasma gallisepticum, which produces a respiratory tract disease (Ley et al., Avian Diseases 41:187-194, 1997). A commercially available vaccine recommends administration of a vaccine containing an avirulent strain of E. coli serotype 078 to immunize against the respiratory disease caused by the wild-type parent (see Product Bulletin for GARAVAX.RTM.-T, Schering-Plough Animal Health Corp., Omaha, Nebr.). The use of an aerosol administration for all of these vaccines would have been selected because the underlying disease for which the poultry were being vaccinated involved infection of the respiratory tract. Another reference reported that a vaccine containing a strain of the nonpathogenic E. coli K-12 lacking O-antigen could be administered as an aerosol (U.S. Pat. No. 4,404,186). Nevertheless, the K-12 strain is a laboratory-adapted strain and is not an enteropathogen and because this microbe has no ability to invade and colonize the gut associated lymphoid tissue, it is likely that any immunity elicited by this vaccine would have been due to immunization through the respiratory route. Localized spraying of bacterial vaccines such as by nasal spraying or ocular spraying had been suggested in some references (for example, see U.S. Pat. No. 5,294,441). Nevertheless, none of this earlier work suggested the use of whole body spray administration of enteropathogenic bacterial vaccines. Therefore, while spray-administered vaccines have been reported to be useful in controlling respiratory diseases in poultry, whole-body spray administration has not been suggested for vaccines in poultry for the control of human pathogens that are often present in and transmitted by poultry, but which are not the causative agents for respiratory disease in poultry. Accordingly, it would be desirable to provide a method of reducing the contamination of poultry by enteropathogenic microbes, especially Salmonella spp., that would be easy and inexpensive to administer under normal commercial poultry production conditions; which could be administered to newly hatched chicks without individual handling; and which would reduce or prevent infection of visceral and lymphatic tissues and the intestinal tract of poultry by enteropathogenic microbes. SUMMARY OF THE INVENTION In accordance with the present invention, it has been discovered that vaccines can be administered to domestic birds by whole-body spraying of the birds with the vaccine. The vaccines are administered by this whole-body spray route in an amount that is effective in eliciting an immune response, i.e. antibody and/or cellular immunity. While virtually any vaccine can be delivered by this method, whole-body spray administration is surprisingly effective for vaccines comprising a live avirulent derivative of an enteropathogenic bacteria. Such enteropathogenic bacteria are preferably Salmonella species. This spray administration of enteropathogenic bacteria avoids some of the disadvantages of other routes of administrations in that it does not require individual handling of chicks, it can be administered on day-of-hatch, and is easy to use under conditions normally found in commercial poultry production. The effective doses, which elicit an immune response, are unexpectedly low and roughly comparable to doses that are effective by the oral route of administration, such as administration in the drinking water. Typically, doses for administration of the live vaccines of the present invention are from about 10.sup.5 to about 10.sup.8 colony forming units. The spray route of administration of the vaccines is applicable to vaccination of birds, such as chickens, at any age at which they are susceptible to the beneficial effects of the vaccine, but is especially applicable to birds that are of an age of 3 weeks or less, and, preferably, to birds of less than 1 day of age. In some embodiments, the spray-administration can be followed by administration of the vaccine in at least one booster dose. Preferably such a booster dose can be administered orally by drinking water or by spray at about 14 days after administration by spray. Preferably, the spray is a coarse spray of droplets having diameters in the range of from about 50 microns to about 150 microns. In other embodiments the present invention is directed to a method for reducing microbial contamination of poultry. The method comprises immunizing the poultry against a microbial contaminant by whole-body spray administration of an immunogenic composition. The microbial contaminant may or may not be pathogenic to the poultry itself, however, when present in the poultry, such microbial contaminates can produce disease symptoms in humans consuming the meat or other food products produced from the poultry. The microbial contaminant can be any such contaminant, particularly, microbes that colonize the gastrointestinal tract of the poultry. The immunogenic composition is administered in an amount that is effective in eliciting an immune response, i.e. antibody and/or cellular immunity against the microbial contaminant. Preferably, the immunogenic composition comprises a live avirulent derivative of an enteropathogenic bacteria. Such enteropathogenic bacteria are preferably Salmonella species. The immunogenic composition is administered in doses, which are effective in eliciting an immune response. Such doses are roughly comparable to doses that are effective by the oral route of administration. Typically, doses for administration of the live vaccines of the present invention are from about 10.sup.5 to about 10.sup.8 colony forming units. The spray route of administration of the immunogenic composition of the present invention is applicable to vaccination of birds of any age at which they are susceptible to the beneficial effects of the vaccine, but is particularly applicable to birds, such as chickens, at an age of 3 weeks or less, and, preferably, to birds of less than 1 day of age. In some embodiments, the spray-administration can be followed by administration of the immunogenic composition in at least one booster dose by oral administration in the drinking water, preferably at about 14 days after administration by spray. The spray is, preferably, a coarse spray of droplets having diameters in the range of from about 50 microns to about 150 microns. Among the several advantages found to be achieved by the present invention, therefore, may be noted the provision of a new method for vaccinating a domestic bird using an enteropathogenic bacteria; the provision of a method for reducing the amount of colonization of the intestinal tract, lymphatic tissues and visceral tissues by enteropathogenic microbes, the provision of a method for reducing the microbial contamination of poultry destined for human consumption; the provision of a method that is easy and inexpensive to administer under normal commercial poultry production conditions; and the provision of a method that that allows administration to young, especially day-of-hatch chicks without individual handling. BRIEF DESCRIPTION OF THE DRAWINGS: FIG. 1 shows the recovery of S. typhimurium .chi.3985 vaccine strain from the spleen and bursal tissues, feces and cecal contents of white leghorn chicks 7 days after receiving (a) 10.sup.5 CFU, (b) 10.sup.7 CFU, or (c) 10.sup.9 CFU of .chi.3985 by coarse spray or by direct oral methods of delivery on day of hatch; FIG. 2 shows the recovery of S. typhimurium .chi.3985 vaccine strain from the spleen and bursal tissues, feces and cecal contents of white leghorn chicks 20 days after receiving (a) 10.sup.5 CFU, (b) 10.sup.7 CFU, or (c) 10.sup.9 CFU of .chi.3985 by coarse spray or by direct oral methods of delivery at days 1 and 14; and FIG. 3 shows the serum IgM, IgA and IgG responses at 20 days of age as detected by using purified S. typhimurium LPS in white leghorn chickens immunized and boosted with (a) 10.sup.5 CFU, (b) 10.sup.7 CFU, or (c) 10.sup.9 CFU of S. typhimurium .chi.3985 by coarse spray or by direct oral methods of delivery at days 1 and 14. DESCRIPTION OF THE PREFERRED EMBODIMENTS: The present invention is based upon the discovery that whole-body spray administration can be used to deliver vaccines or immunogenic compositions of live avirulent derivative of an enteropathogenic bacteria to domestic birds and effectively elicit an immune response. The whole-body spray administration of the present invention allows delivery of the vaccine or immunogenic compositions to the gastrointestinal tract of the poultry. Spray administration or spray vaccination as used herein is intended to mean the delivery of droplets of a liquid comprising a vaccine or immunogenic composition. Whole-body spray administration is intended to mean the delivery of such droplets of vaccine or immunogenic composition to a large portion of the entire body of the poultry. This in contradistinction to a localized spray administration such as is by intranasal spraying in humans in which administration is to only a specific, small, localized target area. The whole-body spray approach for administering enteropathogenic bacteria of the present invention indiscriminately delivers the vaccine microbe to a large portion of the body surface of the poultry constituting that portion of the entire body surface that is accessible to the spray device (see for example, U.S. Pat. Nos. 4,316,464 and 4,449,968 which are incorporated by reference). Such whole-body spray administration of the vaccines or immunogenic compositions of the present invention is particularly applicable for administration to large numbers of poultry at the same time. Spray administration in the present invention preferably involves delivery of a coarse spray containing the vaccine or immunogenic composition to the poultry. Although not wishing to be bound by a particular theory, it is believed that the administration of a vaccine or immunogenic composition as a coarse spray allows the spray droplets to contact the body surface while minimizing the amount of the vaccine that is inhaled into the lower respiratory system. This is to be distinguished from a spray of very fine droplets or mist, such as is commonly referred to as an aerosol in which droplets have a diameter of less than about 40 microns. Unlike the aerosol sprays, the coarse spray of the present invention is believed to not be deeply inhaled which assists in avoiding the development of respiratory infections seen with some spray vaccination (See for example, U.S. Pat. No. 4,449,968; Clarke et al, Austr. Vet. J. 56:424-428, 1980). A coarse spray as used herein is intended to mean a spray that is composed of liquid droplets having a diameter sufficient to substantially prevent the inhalation of the droplets into the lower respiratory system of the bird, but still causing the liquid droplets to contact the body surface of the bird. The consistency of such a coarse spray has been referred to as "misty rain", and it is preferred that the spray have less than about 1% of the droplets in a size range of less than about 12 microns. Preferably, the coarse spray is composed of droplets having a mean diameter of from about 40 to about 400 microns; more preferably from about 40 microns to about 200 microns, even more preferably from about 50 to about 150 microns and most preferably from about 50 to about 100 microns. Alternatively the coarse spray can have about 80% of droplets in a range of from about 90 to about 190 microns. The type of spray vaccination equipment that is used for the administration of the vaccine is not critical and almost any type of spray vaccination equipment capable of dispensing a coarse spray can be used (see for example, U.S. Pat. Nos. 4,316,464, 4,449,968, 4,674 and 5,312,353). The spray administration of the present invention delivers a vaccine comprising a live avirulent derivative of an enteropathogenic bacteria. The vaccine microbe is an enterobacteria capable of colonizing the intestinal tract and gut associated lymphoid tissues (GALT) of the poultry. Such microbes serve as the immunogenic component of the vaccine or immunogenic composition and include Enterobacteriaceae family members such as Escherichia, Klebsiella, Proteus, Yersinia, and Erwinia. In particular, Salmonella, Escherichia and Salmonella-Escherichia hybrids are useful in the present invention, including, preferably, E. coli and Salmonella such as S. typhimurium, S. typhi, S. paratyphi, S. enteritidis, S. dublin, S. gallinarum, S. pullorum, S. arizona, and S. choleraesuis. The avirulent derivative of an enteropathogenic bacteria can also serve as a carrier bacterium to deliver selected antigens to the GALT. Such carrier bacteria that contain and express a recombinant gene from a pathogenic organism so that antibodies and/or cellular immunity will be elicited against the antigenic gene product normally produced by the pathogenic organism. It is thus possible to use the avirulent derivative of an enteropathogenic bacteria, administered by spray, to deliver antigens to a wide variety of microbes and to elicit an immune response in the poultry against microbes that need not necessarily be able to colonize the gastrointestinal (GI) tract. The avirulent microbes can additionally be used as vectors for the synthesis of various proteins in the poultry. Because the avirulent microbes of this invention are able to traverse the GALT after spray administration and entry into the gastrointestinal tract of the poultry, the microbes can be used to make and deliver gene products such as, for example, growth factors or immunoregulatory products or substances that stimulate or suppress various physiological functions. Such microbes contain and express a recombinant gene that encodes the desired protein. The terms enteropathogenic bacteria are intended to mean microbes that are capable of colonizing the intestinal tract and the gut associated lymphoid system of the poultry. As used herein, pathogen is intended to mean a microbe that is capable of causing disease symptoms or impairing normal physiological functioning. The vaccines of the present invention contain avirulent derivatives of an enteropathogenic strain of bacteria. By derivative or derived strain reference is made to a strain that has been genetically modified from its parent from which it is descended. By pathogenic it is meant that the microbe is capable of causing disease or impairing normal physiological functioning. Reference to avirulence is intended to mean that a particular microbe strain is incapable of inducing a full suite of symptoms of the disease state that is normally associated with its virulent pathogenic counterpart. Thus, avirulence includes a state of diminished virulence or ability to produce disease conditions and the avirulent microorganisms are not necessarily completely absent of any ability to impair normal physiological functioning of the host. In addition, an avirulent microbe is not necessarily incapable of ever functioning as a pathogen, but the particular microbe being used is avirulent with respect to the particular individual being treated. Preferrably, the enteropathogenic bacteria from which the avirulent microbe is derived is pathogenic at least to day-of-hatch birds. In a preferred embodiment of the present invention, the live avirulent derivative of an enteropathogenic bacteria is an S. typhimurium, such as .chi.3985, which has .DELTA.cya-12/.DELTA.crp-11 mutations. This construction of this and other strains is described in detail in U.S. Pat. No. 5,294,441. An immunological response to a composition or vaccine is the development in the host of a cellular and/or antibody-mediated immune response to the composition or vaccine of interest. Usually, such a response consists of the subject producing antibodies, B cells, helper T cells, suppressor T cells, and/or cytotoxic T cells directed specifically to an antigen or antigens included in the composition or vaccine of interest. By vaccine is meant an agent used to stimulate the immune system of an individual so that protection is provided against an antigen not recognized as a self-antigen by the immune system. Immunization refers to the process of inducing a continuing high level of antibody and/or cellular immune response in which T-lymphocytes can either kill the invading microbe and/or activate other cells (e.g., phagocytes) to do so in an individual, which is directed against a microbe or antigen to which the organism has been previously exposed. The phrase immune system is intended to refer to the anatomical features and mechanisms by which an individual produces antibodies against an antigenic material which invades the cells of the individual or the extra-cellular fluid of the individual and is also intended to include cellular immune responses. In the case of antibody production, the antibody so produced can belong to any of the immunological classes, such as immunoglobulins, A, D, E, G or M. Of particular interest are vaccines which stimulate production of immunoglobulin A (IgA) since this is the principle immunoglobulin produced by the secretory system of warm-blooded animals, although vaccines of the invention are not limited to those which stimulate IgA production. For example, vaccines of the nature described herein are likely to produce a broad range of other immune responses in addition to IgA formation, for example cellular and humoral immunity. Immune responses to antigens are well studied and widely reported. A survey of immunology is provided in Elgert, Klaus D., Immunology, Wiley Liss, Inc., (1996); Stites et al., Basic & Clinical Immunology; 7th Ed., Appleton & Lange, (1991) the entirety of which are incorporated herein by reference. An individual treated with a vaccine of the present invention is intended to mean one of a species of birds, including domestic birds, particularly those of agricultural importance. Domestic birds or poultry as used herein includes any of a variety of domesticated avian species or individuals of that species, such as chickens, turkeys, ducks, geese, pigeons, guineas, ostriches, emus, and the like and, in particular, those domesticated avian species or individuals kept for the production of eggs or meat. The vaccine can be prepared by growing the vaccine strain in suitable growth media and then used as is or formed into a vaccine composition by combining the growing culture, or the cells therefrom, with a suitable diluent. Suitable diluents are preferably liquids and are more preferably a liquid that does not adversely effect the stability and vitality of the vaccine culture and which has a viscosity similar to water so that it will easily form droplets of a coarse spray. The diluent is preferably free of chlorine, antibiotics, antimicrobials, or any other agent that may be harmful to the live vaccine organisms. Vaccine should be dispersible in the diluent so that no solid lumps or chunks of vaccine remain and the diluent should be at a temperature that is not harmful to the live vaccine microbes. Examples of suitable diluents include water, distilled water, de-ionized water, skim milk, water containing Marek's vaccine stabilizer, buffered saline with gelatin, and similar compositions that are well-known to persons of skill in the art. The vaccine is preferably introduced into the diluent while the diluent is at a temperature of approximately room temperature or cooler more preferably from about 34.degree. C. to about 15.degree. C. In one embodiment, vaccine is prepared from S. typhimurium UK-1 .DELTA.cya .DELTA.crp .chi.3985. As used herein, this vaccine may be referred to as .chi.3985, or as Chi3985, or as .chi.3985, Production Code 19C1.01. The vaccine strain can be freshly prepared as described above, or may be recovered from a culture stored, for example, as a freeze-dried culture, in a frozen form (for example, as -70.degree. C. working seed stock), or otherwise. An inoculum from such culture is then grown to a late log-phase culture in Luria broth in 37.degree. C. By way of example, a -70.degree. C. seed stock can be used to inoculate 50 ml of Luria broth in a 250 ml sterile flask covered loosely with foil. The flask is incubated as a static culture at 37.degree. C. overnight. After about 12-24 hr., 50 ml of the static overnight culture is pipetted into 450 ml of prewarmed Luria broth in a 1 L nonbaffled flask at 37.degree. C. and placed in a New Brunswick incubator shaker at 150 rpm. After the culture reaches OD.sub.600.gtoreq.1.0, cells are pelleted by centrifugation (4400 rpm, 15 min in a Centra MP4 centrifuge, IEC swinging bucket 3224 rotor) at room temperature. Cells are resuspended in 40 ml of room temperature buffered saline with gelatin (BSG). The titer of the vaccine composition can be determined by serially diluting the cell suspension 10-fold in BSG and spreading 100 .mu.l of 10.sup.-6 and 10.sup.-7 dilutions onto MacConkey agar +1% maltose for plating. The titer of the vaccine strain is then determined by counting colonies that develop upon incubation of the plates. The titer is expressed in terms of colony forming units of the vaccine microbe (CFU) per unit volume of the vaccine composition. Vaccine for application to poultry is prepared as described above and the culture is diluted to the desired dose density, or titer, in a suitable diluent. The buffer of the diluent, if used, is adjusted to match the pH and ionic strength required to maintain the stability and vitality of the vaccine strain. The vaccine is then ready for loading into the sprayer and for administration to the poultry. Spray administration can also be performed in a manner to deliver a particular dosage per bird. One technique that can be used to deliver an accurate vaccine dosage is to spray birds in an enclosed space for a calculated period of time at a known volumetric delivery rate. By knowing the number of birds to be vaccinated, the desired dosage of the vaccine per bird, the titer of the vaccine and the delivery rate of the spray equipment, one skilled in the art can easily calculate the spraying time required to deliver the required dosage per bird. Furthermore, some models of commercially available spray equipment allow pre-selection of the volume of liquid to be delivered to a known number of birds. The vaccine or immunogenic composition of the present invention is administered in an effective dose or an effective amount. As used herein an effective amount is that quantity of vaccine or immunogenic composition which is sufficient to elicit an immune response against a target microbe or antigen for which the poultry is being vaccinated. Such immune response will involve the production of antibodies and/or cellular immunity. In one significant aspect of the present invention, the vaccine or immunogenic composition can be administered at a dose roughly equal to the dose effective upon oral administration, for example by administration in the drinking water. Preferably the spray administration is given to birds when they are less than one day old, i.e. on the day of hatch. It is often also desirable to administer one or more booster applications of the vaccine some time after the initial spray administration. Such booster applications can be administered at any time during the bird's life at which the bird is susceptible to the beneficial effects of the vaccine. Preferably, such booster applications are applied between 5 and 21 days of age, more preferably between 6 and 15 days of age and still more preferably between 7 and 14 days of age and most preferably at 7 days of age or 14 days of age or at both 7 and 14 days of age. The booster doses are typically administered orally in the drinking water although the booster dose can be administered by any route including by spray administration. Administration of the vaccine in the drinking water can be performed by any of a number of methods known in the art. By way of example only, administration in the drinking water can be performed using the following method. First all disinfectants, sanitizers and antimicrobials are removed from the drinking water being given to the birds 24 hours prior to vaccine administration. Such water free of disinfectants, sanitizers and antimicrobials is again given 24 hours after vaccination. The vaccine can then be mixed in the clean water that contains no sanitizing agents or antimicrobials. Fifty liters of vaccine-containing water can be used for 500 birds such as chickens and ample space should be provided for all birds to drink easily. Water containing vaccine should be consumed in 2 hours or less. To assure that all birds drink, water should be withheld for one to two hours prior to administration in the drinking water. Because the dosage amount for spray administration of the vaccine or immunogenic composition is approximately the same as the oral dose in the drinking water, both dosage amounts are, preferably, about the same. Thus, for example, if 10.sup.7 colony forming units are administered per bird by spray administration, then, preferably, about 10.sup.7 colony forming units are administered per bird in the drinking water. Preferably, the initial spray administration dose and any subsequent booster dose administered in the drinking water will differ by less than 100 fold, more preferably by less than 10 fold, even more preferable by less than three fold and still more preferably by less than 10%. It is preferred that the poultry to be vaccinated be of an age at which it is susceptible to the beneficial immunogenic effects of the vaccine. While this may vary with species, it has been found that, such beneficial effects are obtained in poultry that is of an age of from hatching to about 104 weeks of age. It is preferred that poultry be day-of-hatch to 52 weeks of age, more preferably from day-of-hatch to 3 weeks of age, even more preferably day-of-hatch to 2 weeks of age, still more preferably day-of-hatch to 1 week of age and most preferably that it be day-of-hatch. As used herein, the phrase "day-of-hatch" may be used interchangeably with the term "less than one day of age". One advantage of the present method is that it is amenable to application under conditions that normally occur in commercial poultry raising operations. Typically, large commercial chicken or turkey raising operations are characterized by large poultry houses having more or less automated feed and watering systems and housing over 1,000 birds per house; often over 5,000 birds per house and even over 20,000 birds per house. The present method can be used at the hatchery or at the poultry farm on newly hatched chicks by spraying the chicks in the chick boxes, or other trays or boxes, prior to their release into the brooder house or poultry house. Alternatively, either young or older poultry can be sprayed after release into the house. (See, e.g., Grieve, Poultry Times, p.18, Sep. 22,1997; and Giambrone, 1997, supra.) Because of the ease of application of the present method, the cost of poultry vaccination can be very low. The high cost of individual chick handling is avoided by the ability to vaccinate dozens of chicks at one time and in a matter of seconds. Moreover, the accurate administration of the dosage of the vaccine to each chick minimizes overdosing and inefficient application of the vaccine. The following examples describe preferred embodiments of the invention. Other embodiments within the scope of the claims herein will be apparent to one skilled in the art from consideration of the specification or practice of the invention as disclosed herein. It is intended that the specification, together with the examples, be considered exemplary only, with the scope and spirit of the invention being indicated by the claims which follow the examples. |
| PATENT EXAMPLES | available on request |
| PATENT PHOTOCOPY | available on request |
|
Want more information ? Interested in the hidden information ? Click here and do your request. |