Compositions and methods for ameliorating the clinical signs of an avian pneumovirus (APV) infection in a bird are disclosed. The compositions include immunologically effective amounts of an avirulent APV isolated from Canada geese. Methods for preparing avirulent Canada goose APV compositions and vaccines are also described.

SUMMARY OF THE INVENTION

In one aspect, the invention features an isolated avirulent Canada goose avian pneumovirus (aCg/APV). The invention also features a composition that includes an immunologically effective amount of an aCg/APV. In some embodiments, the composition further includes a pharmaceutically acceptable carrier. In other embodiments, aCg/APV isolates are designated APV/Canada goose/Minnesota/15a, APV/Canada goose/Minnesota/15b, APV/Canada goose/Minnesota/15c, APV/Canada goose/Minnesota/15d, and are assigned ATCC Accession Nos. PTA-4840, PTA-4841, PTA-4842, and PTA-4843, respectively.

These compositions containing an immunologically effective amount of an avirulent Canada geese APV are effective for lowering the risk of an APV infection in wild birds and domesticated birds. In particular, the compositions are useful for preventing an APV infection in poultry including turkeys, chickens, ducks, geese, pheasants, partridges, guinea fowl, and peacocks. In addition, the APV compositions are effective for ameliorating the clinical signs of an APV infection in a challenged bird.

In another aspect, the invention features methods for preparing an aCg/APV composition that includes the steps of providing a Canada goose APV (Cg/APV), and identifying avirulent Canada goose APV (aCg/APV) isolates. The Cg/APV can be cultured in and isolated from avian or non-avian cells, or a combination thereof. For example, the Cg/APV can be cultured in Vero cells, quail tumor cells (QT-35 cells) or chicken embryo fibroblasts (CEF). These methods result in the production of an aCg/APV that is effective for reducing or preventing the incidence of the clinical signs of an APV infection in poultry and, in particular, turkeys and chickens.

In another aspect, the invention features a method for reducing the risk of an APV infection in a bird by inoculating a bird with an immunologically effective amount of a composition including an aCg/APV. In some embodiments, the inoculated bird is allowed to become seropositive. Although many different dosages may be used to inoculate a bird, particularly useful dosages include at least 1.0×10¹ tissue culture infective dose (TCID₅₀) of an aCg/APV composition, at least 1.0×10² TCID₅₀ of an aCg/APV composition, at least 1.0×10³ TCID₅₀ of an aCg/APV composition, at least 1.0×10⁵ TCID₅₀ of an aCg/APV composition, or at least 1.0×10⁷ TCID₅₀ of an aCg/APV composition.

Any method of inoculation can be used including applying the composition to one or more eyes of a bird and/or one or more nostrils of a bird, aerosolizing the vaccine into the turkeys' habitat, supplying an avirulent Canada geese APV composition in the drinking water of a bird, or injecting an avirulent Canada geese APV composition into a bird subcutaneously or intramuscularly. Inoculated birds can be representative members of a flock of birds and the inoculated or vaccinated birds can cause a majority of the flock to become seropositive. Alternatively, all birds can be vaccinated simultaneously be aerosolization, by placing the vaccine in drinking water, or by instilling the vaccine in the eye and/or nostril of all birds. In some embodiments, the method is effective for reducing the incidence of the clinical signs of an APV infection in a challenged bird.

In another aspect, the invention features an inoculated bird, which is a bird containing an inoculant of an immunologically effective amount of an aCg/APV. In some embodiments, the bird is allowed to become or is seropositive for APV. In another aspect, the invention features a body part, such as a meat portion, of an inoculated or vaccinated bird. In particular, these birds can be turkeys.

In yet another aspect, the invention features compositions containing immunologically effective amounts of an aCg/APV that has been inactivated. In some embodiments, the composition further includes a pharmaceutically acceptable carrier or adjuvant. Compositions containing an inactivated, aCg/APV are also effective for lowering the risk of an APV infection in poultry, such as chickens or turkeys, and in other domesticated and wild birds. The compositions are also effective for ameliorating the clinical signs of an APV infection in a challenged bird. In some embodiments, inactivation is by formalin or β-propiolactone inactivation.

In another aspect, the invention features an article of manufacture containing an aCg/APV composition. The composition can be combined with packaging materials and instructions for their use. The instructions can describe how an aCg/APV vaccine is effective for preventing the incidence of an APV infection, preventing the occurrence of the clinical signs of an APV infection, ameliorating the clinical signs of an APV infection, lowering the risk of the clinical signs of an APV infection, lowering the occurrence of the clinical signs of an APV infection and/or lowering the spread of APV infections in birds. The articles of manufacture may combine one or more aCg/APV vaccines.

Articles of manufacture can also include diagnostic molecules (e.g., antibodies, indicator molecules, and/or other useful agents) that are effective for detecting the presence of APV or other avian infections in birds. Moreover, it is to be understood that aCg/APV vaccines themselves and/or articles of manufacture that include aCg/APV vaccines can include other components conventional to the art, for example sterile water, pharmaceutical carriers, vaccine carriers, and buffers that are useful for maintaining the viability of the APV vaccines. Conveniently, the aCg/APV vaccines of the invention may be provided in a pre-packaged form in quantities sufficient for a protective dose for a single bird or for a pre-specified number of birds in, for example, sealed ampoules, capsules or cartridges. The APV vaccines and/or articles of manufacture may also contain other virus strains, microorganisms, and antigens that protect the inoculated birds against other avian diseases.

It is to be further understood that each of the embodiments may be combined with any of the other embodiments described herein. For example, the methods disclosed herein are useful with any of the compositions, or articles of manufacture, and the dosages may be used in any of the various embodiments described herein.

Advantages of the invention include a safe and effective way to protect birds, especially turkeys, from an APV infection. Moreover, the invention can provide methods for raising antibodies to APV to be used in diagnostic kits and may be used in the diagnostic kits for detecting the presence of APV.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

DETAILED DESCRIPTION OF THE INVENTION

The invention features avirulent APV isolates obtained from Canada geese (aCg/APV), compositions containing one or more aCg/APVs, and methods for preparing an aCg/APV. An avian composition (e.g., a vaccine) can contain an immunologically effective dosage of an aCg/APV. These aCg/APV vaccines are both safe and immunogenic when administered to birds. Vaccinated birds are seropositive for anti-APV antibodies and resistant to infection by or challenge with a virulent APV. Using the compositions and/or vaccines described herein is an effective way for preventing, ameliorating, lowering the risk of, lowering the occurrence of and/or spread of APV infections in birds.

AcG APV Compositions and Their Uses

APV compositions of the invention contain an aCg/APV. A composition of the invention can be formulated in an immunologically effective dose and used, for example, as a vaccine. Vaccines of the invention are useful for any type of bird susceptible to APV infection including domesticated and wild birds. In particular, the vaccines described herein are useful for inoculating and/or treating birds living in flocks or other types of close living arrangements where an APV infection can rapidly spread from bird to bird. Domesticated birds that may benefit from receiving an aCg/APV vaccine include poultry such as turkeys, chickens, ducks, geese, pheasants, partridges, guinea fowl, peacocks, and any other type of domesticated bird. Wild birds that may benefit from receiving an aCg/APV vaccine include starlings, sparrows, turkeys, ducks, geese, pheasants, partridges, guinea fowl, peacocks, and any other type of wild bird that may contract an APV infection and/or transmit APV infection to another bird.

An immunologically effective dosage of an aCg/APV vaccine is a dosage that, when administered to a bird, elicits an immunological response in the bird but causes the bird to develop mild or no clinical signs of an APV infection. A bird that has received an immunologically effective dosage is an inoculated bird or a bird containing an inoculant of an immunologically effective amount of an aCg/APV. When an immunological response is produced in the bird, the bird is considered seropositive, i.e., the bird produces a detectable amount of anti-APV antibodies. Methods for detecting an immunological response in a bird are known, e.g., Chiang et al., 2000, J. Vet. Diag. Invest., 12:381-84.

Mild clinical signs can include watery eyes, nasal discharge or cough in a flock from about 5 to about 12 days post inoculation ("dpi"). The clinical signs of APV and other upper respiratory infections in birds are known. The clinical signs of an APV infection in turkeys include profuse ocular and nasal discharge, watery eyes, unilateral or bilateral sinus swelling, facial edema or swelling, depression, coughing, sinusitis, airsacculitis, respiratory distress, and mortality. In laying turkeys, a drop in egg production associated with respiratory distress may be seen.

As used herein, a vaccinated bird is an inoculated bird that is seropositive. Vaccinated birds can exhibit an immunological response following a challenge with a virulent APV. A vaccinated bird is resistant to or immune to an APV infection if it fails to develop clinical signs of APV infection or develops mild signs of APV infection after being challenged with a virulent APV. As a result, vaccinated birds that are subsequently challenged with a virulent APV may still pass slaughter inspections and continue to market. Methods and rating systems for passing or condemning birds destined for slaughter are known.

A vaccinated bird may shed the aCg/APV. Shedding is typically detectable from about 5 days to about 7 days post inoculation, and may range from 3 days to 21 days post inoculation. Methods for detecting the shedding of an APV are known. Useful methods include the methods described in Shin et al., 2000, Arch. Virol., 145:1239-46; and Goyal et al., 2000, J. Vet. Diagn. Invest., 12:166-68.

Virulent APV isolates are those isolates that cause a bird to develop clinical signs of an APV infection, to be unfit for market, and/or to die, provided the bird has not been previously exposed to APV and/or an APV vaccine. An APV isolate is virulent if it causes clinical signs of an APV infection in a bird at a dosage of at least 1.0×10² tissue culture infective dose (TCID₅₀). Avirulent APV isolates are those APV isolates that cause mild or no clinical signs of an APV infection in a bird at a dosage of about 1.0×10² TCID₅₀.

Methods for determining virus titers and TCID₅₀ are known. The TCID₅₀ is the reciprocal of the highest dilution of a virus that causes a specified reaction in 50% of the host cells inoculated with, or exposed to, that particular dilution of virus. Oftentimes with pathogenic viruses, the specified reaction is cytopathic effects such as cell rounding. See, e.g., Cook et al., 1989, Avian Pathology, 18:511-522.

Immunologically effective dosages can be determined experimentally and may vary according to the type, size, age, and health of the bird vaccinated. For example, an immunologically effective amount for a two-week-old turkey poult may include an APV vaccine dosage of about 200 μL of a 1.0×10¹ TCID₅₀/ml stock vaccine solution. Older turkeys may require larger dosages. For example, a dosage of at least about 200 μl of a 1.0×10³ TCID₅₀/bird stock vaccine solution can be administered to a bird, and doses as high as 200 μl of a 1.0×10⁵ TCID₅₀/bird or a 1.0×10⁷ TCID₅₀/bird stock vaccine solution can be administered. Dosages smaller than 200 μl of a 1.0×10¹ TCID₅₀/bird stock solution may result in ineffective vaccinations, and dosages larger than 200 μl of a 1.0×10⁷ TCID₅₀/bird stock vaccine solution may be less cost effective. The vaccination may include a single inoculation or multiple inoculations. Other dosage schedules and amounts including vaccine booster dosages may be useful.

The age of the bird receiving a vaccination may depend upon the type of bird and the purpose for which the bird is being kept. For example, it may be preferable to inoculate meat-producing birds in ovo, or at a young age, perhaps as newborns or hatchlings or when the birds are only a few weeks old. Alternatively, it may be useful to vaccinate egg-producing birds at other times, e.g., shortly before they are about to lay (perhaps with a vaccine booster dosage) so that maternal antibodies may be transmitted to the young. Of course, it may also be useful to inoculate egg-laying birds at an early age to prevent APV infection in the egg-laying flock.

The immunologically effective dosage of an aCg/APV composition may be given to a bird using any known method for inoculating birds including direct application intranasally or intraocularly, aerosolizing into a birds habitat, administering via a birds' water supply, and/or injecting subcutaneously or intramuscularly. The inoculation can be given to a single nostril or eye or divided between one or more nostrils/eyes. For example, a 200 μl dosage containing 1.0×10² TCID₅₀/ml can be evenly divided into four 50 μl dosages and administered to both nostrils and both eyes of a bird.

Alternatively, an immunologically effective dosage may be given to each member of a flock directly or the dosage can be applied to the food and/or water supply of a flock. For example, an immunologically effective dosage of an aCg/APV, e.g., about 1.0×10³ TCID₅₀/bird, can be aerosolized into the birds' habitat. Alternatively, an immunologically effective dosage can be dissolved in the water supply of a flock of birds. Most, if not all, of a flock should become vaccinated birds at approximately the same time when inoculating the flock through the food, water, or air supply. Dosages administered through the food or water supply can be easily computed by multiplying the amount a single bird eats or drinks per day by the number of birds to be inoculated to compute the unit of food or water consumed per day per bird. Then, the unit of food or water consumed per day is used to compute the vaccine dosage needed to dissolve in that unit of food or water so as to deliver, for example, at least 1.0×10² TCID₅₀/bird.

aCg/APV compositions or vaccines that are inactivated can be used in the same manner and under the same conditions used for aCg/APV compositions that are not inactivated. Inactivated APV vaccines are useful for any type of bird susceptible to APV infection including domesticated and wild birds. Methods for making inactivated virus vaccines are well known and include the use of formalin or β-propiolactone to make inactivated vaccines. Inactivated APV compositions or vaccines are typically administered by subcutaneous or intramuscular injection because they are no longer living. Such compositions may further include an adjuvant and/or pharmaceutical carrier.

Compositions containing an aCg/APV also have uses other than as a vaccine. These compositions may be used to induce a bird to raise antibodies to APV to be used in diagnostic tests for identifying one or more APV isolates. Further, the aCg/APV may be used in a diagnostic assay for detecting the presence of anti-APV antibodies in the sera of a bird. Methods for raising and purifying antibodies are known. Methods for preparing diagnostic kits for detecting antibodies in a serum source are known.

Methods for Obtaining Avirulent Canada Geese APV

An aCg/APV can be obtained by culturing APV from Canada geese in host cells, and determining the virulence of the Canada goose (Cg) APV. Methods for obtaining Canada geese APV first include the step of obtaining specimens from Cg. Specimens can be obtained using tracheal swabs, turbinates, or by isolating the trachea and/or lungs. Such birds may or may not be exhibiting clinical signs of an APV infection. The specimens are then used to inoculate hosts such as embryonated chicken eggs ("ECE"), embryonated turkey eggs ("ETE") or chicken embryo fibroblasts (CEF). APV can be isolated using any known method for isolating avian viruses. The methods described in Goyal et al., 2000, J. Vet. Diagn. Invest., 12:166-68 provide illustrative methods for isolating a turkey APV, and can be adapted for other avian viruses.

Subsequently, the APV can be grown in host cells such as CEF, quail tumor cell lines, Vero cells, or other avian or non-avian cells using known methods (see, for example, Pringle, In Virology: A Practical Approach, pp 95-117, IRL Press, Oxford, Washington, D.C.). A single type of cell or multiple types of cells can be used to isolate, identify, passage, and titrate APV. See Naylor et al., 1993, Vet. Bull., 63:439-49. Useful quail tumor cell lines include QT-35. Useful Vero cells include those available from the American Type Culture Collection (ATCC, 10801 University Blvd., Manassas, Va. 20110) having the designation CCL81. The infected host cells are incubated and passaged under standard conditions. It may be necessary for the infected host cells to undergo one or more blind passages before cytopathic effects, such as cell rounding, are observed. Cytopathic effects produced by various viruses in various cell cultures are well known. If no cytopathic effects are observed, the presence of the virus can be confirmed using, for example, PCR or immunofluorescence assays.

Host cells can be inoculated with a virus using any known method. To isolate virus particles from cells, the cells can be subjected to repeated freezing and thawing, or the cells can be sonicated. Cellular debris is removed by centrifugation and the supernatant containing virus particles then can be used to inoculate a different host cell. Although the length of each passage in a particular host cell may vary, each passage is typically 4-5 days in length. An aCg/APV is isolated when it has been removed from its original host, passaged in cell culture, and removed from the cells. The presence of a virus in cell culture can be confirmed using any known method. The presence of APV following isolation can be confirmed using RT-PCR as described in Dar et al., 1998, Proc. Annual Meeting Am. Assoc. Vet. Lab. Diagn., 41:18. Following removal from cell culture, a dosage of at least 1.0×10² TCID₅₀ of the aCg/APV can be administered to a bird that has not been exposed to APV and produces mild or no clinical signs of an APV infection in the bird.

Alternatively, APV may be directly isolated from an inoculation specimen using CEF, quail tumor cells, Vero cells, or other avian or non-avian cells. Briefly, tracheal swabs can be used to directly inoculate monolayers of, for example, CEF, quail tumor cells, or Vero cells. The host cells are passaged generally until cytopathic effects are observed and the virus isolated from cells by freezing and thawing.

Useful aCg/APV compositions were deposited with the ATCC on Dec. 4, 2002 and received ATCC Accession Nos. PTA-4840, PTA-4841, PTA-4842, and PTA-4843. In addition to the methods described herein, an aCg/APV composition or vaccine of the invention can be produced using any known method for producing viral compositions and vaccines. See, e.g., Williams et al., Avian Pathology, 20:585096 (1991). An aCg/APV vaccine of the invention is a vaccine that is safe, immunogenic, and avirulent in turkeys.

Article of Manufacture Containing APV Vaccines

A virulent Canada geese APV compositions or vaccines as described herein can be combined with packaging materials including instructions for their use to be sold as articles of manufacture or kits. Components and methods for producing articles of manufacture are well known. Instructions describing how an avirulent Canada geese APV vaccine is effective for preventing the incidence of an APV infection, preventing the occurrence of the clinical signs of an APV infection, ameliorating the clinical signs of an APV infection, lowering the risk of the clinical signs of an APV infection, lowering the occurrence of the clinical signs of an APV infection and/or spread of APV infections in birds may be included in such kits. The articles of manufacture may combine one or more avirulent Canada geese APV vaccines as described herein. In addition, the article of manufacture can also include diagnostic molecules (e.g., antibodies, indicator molecules, and/or other useful agents) that are effective for detecting the presence of APV or other avian infections in birds.

It is to be understood that avirulent Canada geese APV vaccines themselves and/or articles of manufacture that include a virulent Canada geese APV vaccines can include other components conventional to the art, for example sterile water, pharmaceutical carriers, vaccine carriers, and buffers that are useful for maintaining the viability of the APV vaccines. The APV vaccines and/or articles of manufacture may also contain other virus strains, microorganisms, and antigens that protect the inoculated birds against other avian diseases. Methods for producing such multi-effect vaccines are known. Conveniently, the a virulent Canada geese APV vaccines may be provided in a pre-packaged form in quantities sufficient for a protective dose for a single bird or for a pre-specified number of birds in, for example, sealed ampoules, capsules or cartridges. Avirulent Canada geese APV vaccines may also be supplied in a freeze-dried form.

Avirulent Canada geese APV vaccines may be combined with different vaccines or preventative methods directed to other avian diseases so as to produce birds that are relatively pathogen free, healthier, and/or resistant to more avian diseases than just APV. Other avian diseases include Ornithobacterium rhinotracheale, Bordetella avium, avian influenza, New Castle Disease, Mycoplasma spp., and Pasteurella multocida.

 

Claim 1 of 18 Claims

1. An isolated avirulent Canada goose avian pneumovirus (aCg/APV).

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.