MXPA06010631A - Method of vaccination against testicular bvdv infection - Google Patents

Abstract

The methods of the invention relate to methods for preventing testicular infection by bovine viral diarrhea virus by immunizing susceptible male animals against infection.

Description

METHOD OF VACCINATION AGAINST TESTICULAR INFECTION BY BOVINE VIRAL DIARRHEA VIRUS FIELD OF THE INVENTION The methods of the invention relate to methods for preventing testicular infection by bovine viral diarrhea virus by immunizing susceptible male animals against infection.

BACKGROUND OF THE INVENTION Bovine viral diarrhea virus (BVDV) is an economically significant pathogen of cattle and other susceptible animals that can be transmitted in the semen of persistently and acutely infected susceptible bulls and other susceptible male animals. This pathogen causes gastrointestinal, respiratory and reproductive disease in susceptible animals. Although gastrointestinal and respiratory disease due to highly pathogenic strains of BVDV are more clinically dramatic, the reproductive losses due to BVDV can be much more economically significant. The literature on the transmission of BVDV has established that the virus in bull semen can infect inseminated susceptible cows, causing reduced rates of pregnancy, early embryonic death, abortions and birth of calves persistently infected with BVDV.1"3 The persistently infected bulls infect susceptible cows susceptible in the most consistent way, because their semen contains a high concentration of virus (107'6 infectious doses of cell cultures (50%) / ml) (CCID50 / ml). 3 By comparison, the infected testicles of acutely infected immunocompetent bulls transmit lower concentrations of virus (5 to 75 CCID50 / ml) in the semen, but are also capable of transmitting BVDV.4 One study reported that 25 to 50 CCID50 / ml of virus in semen infected 5% of the inseminated heifers and the subsequent horizontal transmission of BVDV from the infected heifers to the pregnant herd mates, pro he voiced the persistent infection of his fetuses.5 Two teams of investigators have also reported that acute infection of post-pubescent bulls can cause persistent infection with BVDV that is located in the testicles.6,7 A single case occurred in a non-viraemic seropositive bull that was kept in a station of artificial insemination of New Zealand. The bull had been admitted to the artificial insemination center after attempts to isolate the BVDV from the blood were negative. Despite the presence of neutralizing antibodies against BVDV, the bull transmitted virus in the semen at low levels (2 x 103 CCID5Q / ml) continuously for 11 months until the animal was sacrificed. The source of the acute infection was unknown. In post-mortem examination, the virus was isolated only from the bull's testicles.6 Semen virus from this bull caused infection and subsequent seroconversion of 1 of 3 inseminated seronegative heifers.8 In a second artificially induced infection, the virus was detected. BVDV by reverse transcription polymerase chain reaction (RT-nPCR) in the semen of 2 of 3 non-viremic postpubertal bulls for up to 7 months, but the virus could not be isolated by standard tissue culture methods. The semen collected 5 months after the initial exposure caused BVDV infection in a seronegative heifer after intravenous administration. The semen collected on the day of exposure and 7 months after exposure did not cause infection in two additional seronegative heifers.7 Two studies have observed that acutely infected bulls can transmit BVDV in the semen with sperm with a concentration, motility and Acceptable morphology.4,5 Another study found a decrease in motility, an increase in diadema defects, small sperm heads and proximal droplets in acutely infected sires.9 Regardless of any uncertainty about the details in which refers to transmission capacity through natural breeding and the effect on the fertility of the bulls, it is clear that the testicular infection of susceptible male animals by BVDV has a significant economic impact by virtue of the reproductive losses due to the transmission of BVDV to the cows.

Viral testicular infections pose a significant challenge for treatment or prevention through immunization because it is known that the testicles are immunologically sequestered. Surprisingly, the inventors have shown that immunization is an effective means of controlling testicular infection by BVDV among susceptible animals.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a percentage of positive testicular biopsy samples for BVDV after exposure to type 2 BVDV. Legend: VI = virus isolation, PCR = polymerase chain reaction, IHC = immunohistochemistry, a 'b the percentages with different lowercase letters in superscript are significantly different (P <0.05).

BRIEF DESCRIPTION OF THE INVENTION The invention comprises a method for preventing or treating testicular infection by BVDV in a susceptible male animal comprising administering to the animal an effective amount of a vaccine which is selected from the group consisting of a vaccine with inactivated type 1 BVDV, a vaccine with inactivated type 2 BVDV, a live modified type 1 BVDV vaccine and a live modified type 2 BVDV vaccine.

The invention also comprises a vaccine which is selected from the group consisting of a vaccine with inactivated type 1 BVDV, a vaccine with inactivated type 2 BVDV, a modified live type 1 BVDV vaccine and a modified live type 2 BVDV vaccine for use as medicament to prevent testicular infection by BVDV in a susceptible male animal. The invention further comprises the use of a vaccine selected from the group consisting of an inactivated type 1 BVDV vaccine, an inactivated type 2 BVDV vaccine, a modified live type 1 BVDV vaccine and a modified live type 2 BVDV vaccine. to manufacture a medicament to prevent testicular infection by BVDV in a susceptible male animal. The invention further comprises a method for preventing testicular infection by BVDV in a susceptible male animal comprising identifying an animal with an increased risk of testicular infection by BVDV; and administering to the animal an effective amount of a vaccine selected from the group consisting of a vaccine with inactivated type 1 BVDV, a vaccine with inactivated type 2 BVDV, a modified live type 1 BVDV vaccine and a live type 2 BVDV vaccine modified. The invention also comprises a vaccine which is selected from the group consisting of a vaccine with inactivated type 1 BVDV, a vaccine with inactivated type 2 BVDV, a modified live type 1 BVDV vaccine and a modified live type 2 BVDV vaccine for use as medication to prevent testicular infection due to BVDV in a susceptible male animal with an increased risk of testicular infection due to BVDV. The invention further comprises the use of a vaccine selected from the group consisting of an inactivated type 1 BVDV vaccine, an inactivated type 2 BVDV vaccine, a modified live type 1 BVDV vaccine and a modified live type 2 BVDV vaccine. to manufacture a medicament to prevent testicular infection by BVDV in a susceptible male animal with an increased risk of testicular infection due to BVDV. The invention also comprises a manufactured article comprising a container or containers containing a BVDV vaccine which is selected from the group consisting of a vaccine with activated type 1 BVDV, a vaccine with inactivated type 2 BVDV, a vaccine with BVDV type 1 modified live and a modified live BVDV type 2 vaccine; and instructions for the use of the BVDV vaccine to prevent testicular infection by BVDV in a susceptible male animal. Susceptible male animals may include bulls, rams and boars. In a preferred embodiment the animal is a bull. The vaccines and articles of manufacture may comprise both a modified live BVDV type 1 vaccine and a live modified BVDV type 2 vaccine. Vaccines can be derived from a cytopathic or non-cytopathogenic virus.

Optionally, the vaccines and manufactured articles of the invention may comprise at least one additional antigen that is selected from the group consisting of bovine herpes virus (BHV-1); parainfluenza virus type 3 (PIV3); bovine respiratory syncytial virus (BRSV); Leptospira canicola, Leptospira grippotyphosa, Leptospira borgpetersenii hardio-prajitno, Leptospira icterohaemmorrhagia, Leptospira interrogans pomona, Leptospira borgpetersenii hardjo-bovis, Leptospira Bratislava, Campylobacter fetus, Mannheimia (Pasteurella) haemolytica, Pasteurella multocida, Mycobacterium bovis and Mycobacterium dispar. In a preferred embodiment the vaccines and manufactured articles of the invention may comprise at least one additional antigen that is selected from the group consisting of bovine herpes virus.

(BHV-1), parainfluenza virus type 3 (P1V3) and bovine respiratory syncytial virus (BRSV).

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for treating or preventing testicular infection by BVDV viruses, and the resulting transmission by semen, in a susceptible male animal. The method of the present invention is effective to prevent or reduce testicular infections caused by infections caused by BVDV types 1 and 2.

Definitions and abbreviations The "bovine viral diarrhea virus" ("BVDV") is a small positive, single-stranded RNA virus of the family Flaviviridae and the genus Pestivirus. Two biotypes of BVDV, cytopathic (CP) and non-cytopathic (NC) have been described based on the presence or absence of a cytopathic effect visible in vitro when monolayers of susceptible cells are infected. The non-cytopathic biotype is isolated from outbreaks in the field in a large majority of cases. Virus strains of bovine viral diarrhea can also be classified into 2 separate species (ie, genotypes), type 1 and type 2, based on substantial differences in viral RNA. The term "susceptible animal" refers to any animal that is susceptible to BVDV infections such as cattle, sheep and pigs. The term "susceptible male animal" means any male animal that is susceptible to testicular infections by BVDV, for example cattle, sheep and male pigs. Uncastrated male cattle are called "bulls". The uncastrated male sheep is called "rams". Uncastrated male pigs are called "boars". The term "prevent" or "control" with respect to testicular infection means reducing or eliminating the risk of BVDV infection. 1 and 2 virulent of the testes of a susceptible male animal; improve or alleviate the symptoms of an infection or accelerate the recovery of an infection. Vaccination is considered therapeutic if there is a reduction in the viral or bacterial load evaluated by testicular biopsy or presence of virus in the semen. The term "infection" can mean acute or persistent infection with BVDV. "Acute" or "transient" infection with BVDV occurs when an immunocompetent susceptible animal is exposed to a cytopathic or non-cytopathic strain of BVDV. Although subclinical infection is the most common, signs such as depression, inappetence, erosions and oral ulcerations, diarrhea and death can be observed. An acutely infected immunocompetent animal can transmit the virus to susceptible animals, but in a much less efficient way than persistently infected animals. An acute testicular infection refers to an acute or transient infection of the testes of a susceptible male animal as a result of a transient systemic infection. Some reports indicate that an acutely infected bull can transmit semen virus with acceptable concentration, motility and sperm morphology. However, in other reports, the authors have observed a decrease in sperm motility and an increase in the defects of the diadema., heads of small sperm and proximal droplets coinciding with acute infection. A persistent infection with BVDV occurs when a susceptible animal is infected with a non-cytopathic strain of BVDV before the development of immunocompetence at approximately 125 days of gestation. Persistently infected animals develop immunotolerance to the strain with which they have been infected, act as a reservoir of the pathogen and commonly transmit large amounts of virus in the urine, feces, semen, saliva, tears and nasal mucus throughout life. A persistent testicular infection refers to a persistent infection of the testes of a susceptible male animal as a result of an acute or persistent systemic infection.

Vaccines used in the invention A vaccine used in the invention comprises BVDV types 1 and / or 2 and a veterinarily acceptable vehicle. Traditionally, viral vaccines belong to two classes: Vaccines containing live pathogens contain live viruses that have been treated or cultured in such a way that they are less pathogenic (attenuated) and vaccines that contain dead (inactivated) virus particles. In the context of BVDV, the same viruses can be cytopathogenic or non-cytopathogenic. Virus strains of bovine viral diarrhea can also be classified into 2 separate species (ie, genotypes), type 1 and type 2, based on substantial differences in viral RNA. Thus, in principle, there could be eight main classes of BVDV vaccine, although the vast majority of commercial vaccines are based on cytopathogenic viruses. Among the BVDV vaccines that are currently commercially available are those in which the virus has been chemically inactivated (McClurkin et al., Arch. Virol. 58: 119 (1978); Fernelius, et al., Am. J. Vet. Res. 33: 1421-1431 (1972); and Kolar et al., Am. J. Vet. Res. 33: 1415-1420 (1972)). These vaccines have typically required the administration of multiple doses to achieve primary immunization, provide short-lived immunity and do not protect against fetal transmission (Bolin, Vet. Clin.No. Am. Food Anim. Pract. 11: 615-625 (nineteen ninety five)). In sheep, a subunit vaccine based on a purified E2 protein has been reported (Bruschke et al., Vaccine 15: 1940-1945 (1997)). Unfortunately, only one such vaccine seems to protect the fetuses from infection and this protection is limited to a strain of homologous viruses. In addition, live modified virus (MLV) vaccines have been produced using BVD virus that has been attenuated by repeated passage in bovine or porcine cells (Coggins et al., Cornell Vet. 51: 539 (1961); and Phillips et al. ., Am. J. Vet Res. 36: 135 (1975)) or by chemically induced mutations which confer a temperature-sensitive phenotype to the virus (Lobmann et al., Am. J. Vet. Res. 45: 2498 (1984 ), and Lobmann et al., Am. J. Vet. Res 47: 557-561 (1986)). It has been shown that a single dose of MLV vaccine is sufficient for immunization and the duration of immunity can be extended for years in vaccinated cattle (Coria et al., Can. J. Con. Med. 42: 239 (1978 )). In addition, cross protection has been reported in calves vaccinated with MLV type vaccines (Martin et al., In Proceedings of the Conference Res. Workers' Anim. Dis., 75: 183 (1994)). However, safety considerations, such as the possible fetal transmission of the virus, have been a major concern with respect to the use of these vaccines (Bolin, Vet., Clin. North Am. Food Anim. Pract. 11: 615-625 (1995 )). In a preferred embodiment, the component of BVDV type 1 is a modified live cyto-pathogen (strain cpBVD-1 NADL-National Animal Disease Center, United States, Dep. Of Agriculture, Ames, Iowa, ATCC VR-534). Preferred the BVDV type 2 component is modified live cytopathic (strain cp BVD-2 53637, ATCC No. PTA-4859) As described in co-pending U.S. Patent Application No. 60 / 490,834, filed on 3/29/03, both isolates contain an insertion in the NS2-3 region.The attenuated cpBVDV-1 contains an insertion of a DnaJ1 coding sequence of Bos taurus in 3 'position of thymidine at nucleotide position No. 4993 (NADL sequence numbering), which is the third nucleotide of the codon encoding the glycine residue at the position of amino acid 1536. The attenuated BVDV-2 cp contains an insertion of a DnaJ1 coding sequence of Bos taurus at the same site of the genome, in another pref mode However, the modified live antigens are dried, lyophilized or vitrified.

In one embodiment, the vaccine compositions of the present invention include an effective amount of one or more of the BVD viruses described above, preferably the strain cpBVD-1 NADL (strain cpBVD-1 NADL-National Animal Disease Center, United States). Department of Agriculture, Ames, Iowa, ATCC VR-534); strain cpBVD-2 53637 (ATCC No. PTA-4859), strain IBRV C-13 (Cutter Laboratories); the strain of PIV3 Reisinger (Univ. Nebraska); strain BRSV 375 (Veterinary Medical Research Institute, Ames, Iowa). The purified BVD viruses can be used directly in a vaccine composition, or preferably, the BVD viruses can be further modified by in vitro serial passages. Typically a vaccine contains between about 1 x 102 and about 1 x 1010 plaque-forming units or virus TCID50, with a veterinarily acceptable carrier and optionally an adjuvant, in a volume between 0.1 and 5 ml and preferably about 2 ml. The precise amount of a virus in a vaccine composition effective to provide a protective effect can be determined by an expert veterinarian. Veterinarily acceptable vehicles suitable for use in vaccine compositions can be any of those described below. Typically, a vaccine contains between approximately 1 x 102 and about 1 x 1010 plaque-forming units or virus colonies, with a veterinarily acceptable vehicle and an adjuvant, in a volume between 0.1 and 5 ml and preferably about 2 ml. The precise amount of a virus in a vaccine composition effective to provide a protective effect can be determined by an expert veterinarian. Veterinarily acceptable vehicles suitable for use in vaccine compositions can be any of those described below. The typical route of administration will be intramuscular or subcutaneous injection of between about 0.1 and about 5 ml of vaccine. The vaccine compositions of the present invention may also include additional active ingredients such as other BVDV vaccine compositions, for example those described in WO 95/12682, WO 99/55366, US Pat.

No. 6,060,457, United States Patent No. 6,015,795, the patent of United States No. 6,001, 613 and United States Patent No. 5,593,873. Vaccination will be achieved by a single inoculation or by multiple inoculations. If desired, serum can be collected from the inoculated animals and analyzed for the presence of antibodies against the BVD virus. In another embodiment of the present invention, the vaccine compositions are used to treat BVDV testicular infections. Accordingly, the present invention provides methods for controlling or preventing infections in animal subjects caused by BVD type 1 or 2 viruses, or a combination of type 1 and type 2, by administering to the animal an effective amount of a BVDV virus. of the present invention. In another embodiment, the vaccine compositions of the present invention are effective in improving herd fertility and in reducing the risk of testicular infections among susceptible male animals. In the practice of the present methods, a vaccine composition of the present invention is administered to cattle, preferably intramuscularly or subcutaneously, although other routes of administration, such as for example orally, intranasally, can also be used (e.g. aerosol or other forms of administration without needles), in lymph nodes, intradermal, intraperitoneal, rectal or vaginal administration or by a combination of routes. Intramuscular administration in the neck region of the animal is preferred. Reinforcements may be necessary and the dosage may be adjusted to provide optimal immunization. By "immunogenic" is meant the ability of a BVD virus to elicit an immune response in an animal against BVD type 1 or type 2 viruses or against both BVD type 1 and type 2 viruses. The immune response it can be a cellular immune response mediated mainly by cytotoxic T lymphocytes or a humoral immune response mediated mainly by T helper cells, which in turn activates the B lymphocytes that cause the production of antibodies. In accordance with the present invention, the viruses are preferably attenuated by serial passages in the cell culture before use in an immunogenic composition. Modification methods are well known to those skilled in the art.

The vaccine compositions that are used in the methods of the present invention may also include additional active ingredients such as other immunogenic compositions against BVDV, for example those described in co-pending patent application Serial No. 08 / 107,908. with the present, WO 95/12682, WO 99/55366, U.S. Patent No. 6,060,457, U.S. Patent No. 6,015,795, U.S. Patent No. 6,001, 613 and U.S. Pat. No. 5,593,873. In addition, the objectives of the present invention can be achieved by administering antigens other than BVDV types 1 and / or 2 (a "combined vaccine"). Such antigens include, but without limitation, BRSV, BHV-1, PIV3, Leptospira canicola, Leptospira grippotyphosa, Leptospira borgpetersenii hardio-prajitno, Leptospira icterohaemmorrhagia, Leptospira interrogans pomona, Leptospira borgpetersenii hardjo-bovis, Leptospira Bratislava, Campylobacter fetus, Mannheimia (Pasteurella) haemolytica, Pasteurella multocida, Mycobacterium bovis and Mycobacterium dispar. In several preferred embodiments, the source of the combined vaccine is Bovi-Shield® GOLD ™ IBR-BVD, Bovi-Shield® GOLD ™ 3, Bovi-Shield® GOLD ™ 5, Bovi-Shield® GOLD ™ IBR-BVD-BRSV- LP, Bovi-Shield® GOLD ™ FP 5 L5, Bovi-Shield® GOLD ™ FP 5 VL5 or Preguard® GOLD FP 10 (Pfizer, Inc.). In addition, the immunogenic and vaccine compositions that are employed in the methods of the present invention may include one or more veterinarily acceptable vehicles. As used herein, "a veterinarily acceptable carrier" includes any and all solvents, dispersion media, coatings, adjuvants, stabilizing agents, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption retarding agents. and similar. The diluents may include water, saline, dextrose, ethanol, glycerol and the like. Isotonic agents may include sodium chloride, dextrose, mannitol, sorbitol and lactose among others. The stabilizers include albumin, among others. Adjuvants include, but are not limited to, the RIBI adjuvant system (Ribi Inc.), alum, aluminum hydroxide gel, cholesterol, oil-in-water emulsions, water-in-oil emulsions, such as, for example, complete adjuvants. incomplete Freund, block copolymer (CytRx, Atlanta GA), SAF-M (Chiron, Emeryville CA), adjuvant AMPHIGEN®, saponin, Quil A, QS-21 (Cambridge Biotech Inc., Cambridge MA), GPI-0100 ( Galénica Pharmaceuticals, Inc., Birmingham, AL) or other fractions of saponins, monophosphoryl lipid A, Avridin lipid-amine adjuvant, thermolabile enterotoxin of E. coli (recombinant or not), cholera toxin or muramyl dipeptide, among many others. The vaccine compositions may also include one or more other immunomodulatory agents such as, for example, interleukins, interferons or other cytokines. The compositions of the vaccines that are employed in the methods of the present invention can also include gentamicin and merthiolate. Although the amounts and concentrations of adjuvants and additives useful in the context of the present invention can be readily determined by the person skilled in the art, the present invention contemplates compositions comprising from about 50 μg to about 2000 μg of adjuvant and preferably about 500 μg. μg / dose of 2 ml of the vaccine composition. In another preferred embodiment, the present invention contemplates compositions of vaccines comprising from about 1 μg / ml to about 60 μg / ml of antibiotic and, more preferably, less than about 30 μg / ml of antibiotic. The compositions of the vaccines that are employed in the methods of the present invention can be prepared in various forms depending on the route of administration. For example, vaccine compositions can be prepared in the form of sterile aqueous solutions or dispersions suitable for injectable use or prepared in lyophilized forms using lyophilization techniques. The compositions of the lyophilized vaccines are typically maintained at about 4 ° C and can be reconstituted in a stabilizing solution, for example saline or / and HEPES with or without adjuvant. The compositions of the vaccines of the present invention can be administered to animal subjects to induce an immune response against BVD type 1 or type 2 viruses or against both BVD type 1 and type 2 viruses. Consequently, another embodiment of the present invention provides methods for stimulating an immune response against BVD type 1 or type 2 viruses or against a combination of BVD type 1 and type 2 viruses by administering to an animal subject an effective amount of an immunogenic composition of the present invention. invention described above. "Animal subject" is intended to include any animal that is susceptible to BVDV infections, such as cattle, sheep and pigs. In accordance with the methods of the present invention, a preferred immunogenic composition for administration to an animal subject includes BVDV cp NADL virus and / or BVDV virus cp53637. An immunogenic composition containing a BVDV virus, preferably live modified by serial passage in culture, is administered to cattle preferably by the intramuscular or subcutaneous routes, although other routes of administration, such as for example orally, intranasal, can also be used. , in the lymph nodes, intradermal, intraperitoneal, rectal or vaginal administration or by a combination of routes. Immunization protocols can be optimized using procedures well known in the art. A single dose may be administered to animals or, alternatively, two or more inoculations may take place at intervals of two to ten weeks. Depending on the age of the animal, the immunogenic composition or the vaccine may be re-administered. For example, the present invention contemplates vaccination of healthy cattle before six months of age and revaccination at six months of age. In another example, the present invention contemplates vaccination of cattle before the breeding phase, approximately 5 weeks before the breeding phase (or before being added to a herd) and optionally again about 2 weeks before the breeding phase. breeding phase or during gestation to protect the fetus from infection caused by BVDV types 1 and 2. Single doses of the compositions of the present invention may also be administered approximately 3 to 4 weeks after a first dose. Semi-annual revaccination with a single dose of the combined vaccine to prevent fetal infection with BVDV is also contemplated. The degree and nature of the immune responses induced in cattle can be evaluated using a variety of techniques. For example, sera from the inoculated animals can be collected and analyzed for the presence of antibodies specific for BVDV viruses, for example in a conventional virus neutralization assay. The term "effective amount" refers to a quantity of combined vaccine sufficient to elicit an immune response in the animal to which it is administered. The immune response may comprise, without limitation, induction of cellular and / or humoral immunity. The amount of a vaccine that is therapeutically effective can vary depending on the particular virus being used, the condition of the cattle and / or the degree of infection and can be determined by a veterinarian.

Inactivated vaccines (parts of cells or whole cells) and vaccines with modified live pathogens Inactivated or modified live pathogen vaccines for use in the method of the present invention can be prepared using a variety of methods that are known in the art. For example, BVDV isolates can be obtained directly from the uterus of infected cows using known techniques. The BVDV isolates can be attenuated using a variety of known methods including serial passes, for example. In addition to the modified live virus isolates, a vaccine product employed in the methods of the present invention may also include an appropriate amount of one or more commonly used adjuvants. Suitable adjuvants may include, but are not limited to: mineral gels, for example aluminum hydroxide; surfactants such as lysolecithin; glycosides, for example saponin derivatives such as Quil A or GPI-0100; pluronic polyols; polyanions; nonionic block polymers, for example Pluronic F-127 (B.A.S.F., USA); peptides, mineral oils, for example Montanide ISA-50 (Seppic, Paris, France), carbopo !, Amphigen, Amphigen Mark II (Hydronics, USA), Alhydrogel, oil emulsions, for example a mineral oil emulsion such as BayolF / Arlacel A and water or a vegetable oil emulsion, water and an emulsifier such as lecithin; alum; bovine cytokines; cholesterol; and combinations of adjuvants. In a preferred embodiment, the oil-in-water emulsion containing saponin is conventionally microfluidized. A particularly preferred source of BVDV type 1 and 2, for use in the method of the present invention is the Bovi-Shield® GOLD ™ vaccine product line (PFIZER INC.), Which contains the NADL strain of BVDV (purchased from National Animal Disease Center (NADC), USDA, Ames, IA) and BVDV strain type 2 cp BVDV 53637 (Univ. Guelph, Guelph, Ont.) (ATCC No. PTA-4859). Preferably, strains NADL and 53637 are modified live strains. In accordance with the present invention, the strains of the present The invention may be adjuvanted with a commercially available adjuvant, preferably Quil A-Cholesterol-Amphigen (Hydronics, USA). A preferred dose of the immunogenic compositions and vaccines of the present invention is about 2.0 ml. Preservatives may be included in the compositions that are employed in the methods of the present invention. The preservatives contemplated by the present invention include gentamicin and merthiolate. A vehicle, preferably PBS, can also be added. The preparation of vaccines with living modified pathogens, such as by attenuating virulent strains by passage in cultures, is known in the art. The modified live BVDV isolates can also be combined with the following bacteria and viruses, including but not limited to bovine herpesvirus type 1 (BHV-1); bovine respiratory syncytial virus (BRSV); parainfluenza virus (PIV3); Leptospira canicola, Leptospira grippotyphosa, Leptospira borgpetersenii hardio-prajitno, Leptospira icterohaemmorrhagia, Leptospira interrogans pomona, Leptospira borgpetersenii hardjo-bovis, Leptospira Bratislava, Campylobacter fetus, Mannheimia (Pasteurella) haemolytica, Pasteurella multocida, Mycobacterium bovis and Mycobacterium dispar.

Dosage and modes of administration According to the present invention, an effective amount of a BVDV vaccine or combined vaccine administered to susceptible male animals provides effective immunity against testicular infection associated with BVDV type 1 and 2. In one embodiment, the vaccine it is administered to calves in two doses with an interval of approximately 3 to 4 weeks. For example, the first administration is performed when the animal is from about 1 to about 3 months of age. The second administration is performed from about 1 to about 4 weeks after the first administration of the combined vaccine. In another preferred modality, an administration is made approximately 4 to 5 weeks before the breeding phase of the animal or before admitting it in an artificial insemination facility. The administration of subsequent doses of vaccines is preferably performed annually. In another preferred embodiment, animals that are vaccinated before the age of about 6 months should be re-vaccinated after 6 months of age. Preferably the administration of doses of subsequent vaccines is carried out annually, although the present invention also contemplates the doses of subsequent biannual and bi-annual vaccines. The amount of vaccine that is effective depends on the ingredients of the vaccine and the administration schedule. Typically, when a modified live BVDV preparation is used in a vaccine, an amount of vaccine containing from about 102 to about 1010 units of TCID50 per dose of BVDV and preferably from about 104 to about 107 units of TCID50 per dose is effective. BVDV types 1 and 2 when administered once to susceptible animals. Preferably, a vaccine that provides effective immunity contains from about 104 to 107 units of TCID50 / doses of BVDV types 1 and 2 and more preferably, about 105 units of TCID50 / dose, when administered once to susceptible male animals. Preferably, the administration of doses of subsequent vaccines is performed annually. Animals that are vaccinated before the age of about 6 months should be re-vaccinated after 6 months of age. Preferably, the administration of doses of subsequent vaccines is performed annually. In accordance with the present invention, when the preferred product is administered, Bovi-Shield® GOLD ™ 5 (Pfizer, Inc.), the product is preferably administered once, in an amount of from about 0.1 ml to about 5.0 ml, preferably from about 1.5 ml to about 2.5 ml, and more preferably, about 2 ml. Preferably, the administration of doses of subsequent vaccines is performed annually. Animals that are vaccinated before the age of about 6 months should be re-vaccinated after 6 months of age. Preferably, the administration of doses of subsequent vaccines is performed annually. According to the present invention, administration can be achieved by known routes which include oral, intranasal, topical, transdermal and parenteral (eg, intravenous, intraperitoneal, intradermal, subcutaneous or intramuscular). A preferred route of administration is intramuscular or subcutaneous administration. The present invention also contemplates a single primary dose followed by annual revaccination, which eliminates the need to administer additional doses to calves before annual revaccination to generate and / or maintain immunity against infection. Vaccines that are administered according to the present invention can include additional components, such as an adjuvant (for example mineral gels, for example aluminum hydroxide); surfactants such as cholesterol, lysolecithin; glycosides, for example saponin derivatives such as Quil A, QS-21 or GPI-0100; pluronic polyols; polyanions; nonionic block polymers, for example Pluronic F-127; peptides, mineral oils, for example Montanide ISA-50, carbopol, Amphigen®, Alhydrogel, oil emulsions, for example a mineral oil emulsion such as BayolF / Arlacel A and water or a vegetable oil emulsion, water and an emulsifier such as lecithin; alum; bovine cytokines; and combinations of adjuvants). In accordance with the present invention, administration of an effective amount of a vaccine administered to susceptible male animals at approximately 3 months of age provides effective immunity against testicular infection. In a preferred embodiment, the vaccine is administered intramuscularly. In another preferred embodiment, the vaccine is administered subcutaneously. Furthermore, it is preferred that the dose of the vaccine comprises from about 1 ml to about 7 ml, and preferably about 2 ml, each ml containing from about 102 to about 1010 units of TCID50 / virus dose. Desirably, the combined vaccine is administered twice to the animal; once with about 1 to about 3 months of age and once about 5 to 3 weeks later. The present invention also contemplates annual revaccinations with a single dose.

Identification of animals with increased risk of BVDV infection The invention further comprises a method for preventing testicular infection by BVDV in a male animal susceptible to BVDV infection comprising: a) identifying an animal with an increased risk of testicular infection with BVDV; and b) administering to the animal an effective amount of a vaccine that is selected from the group consisting of a vaccine with inactivated type 1 BVDV, a vaccine with inactivated type 2 BVDV, a modified live type 1 BVDV vaccine and a type 2 BVDV vaccine Live modified. To identify the animal that has an increased risk of infection with BVDV, the expert recognizes that an animal suffers a high risk of infection when a BVDV infection is introduced into a herd that was previously not infected or in which a susceptible animal is in contact with another susceptible animal with a BVDV infection. BVDV is transmitted from one animal to another in a fecal-oral manner. The viral load necessary to cause the symptomatic infection correlates with the type and strain of BVD virus and this correlates with the rapidity of extension by the herd. The infected animals can be identified by the symptomatology. Common manifestations of BVDV infection may include: abortions, infertility, irregular heat cycles, early embryonic deaths, fetal mummification, immunosuppression, dysentery, thrombocytopenia and cerebral hypoplasia. The symptoms of the disease are usually preceded by leukopenia and experimental attempts to date have focused on the identification of this effect. Serological studies have shown that a high percentage of cattle infected with BVDV, including that which is considered to be persistently infected (PI), do not show clinical symptoms. Therefore, a preferred method to identify infected animals is to detect the presence of the virus itself instead of relying on the symptomatology. Several different analysis methods have been developed for the detection of BVDV and / or the detection of animals infected with BVDV. These methods of analysis include: reverse transcription - polymerase chain reaction, enzyme-linked immunoassay (ELISA), standard virus isolation techniques and immunohistochemistry (Haines et al., "Monoclonal Antibody-Based Immunohistochemical Detection of Bovine Viral Diarrhea Virus in Formalin-Fixed, Paraffin-Embedded Tissues, "Vet. Pathol., 29: 27-32 (1992)). Both the PCR technique and the virus isolation technique, due to their inherent sensitivity, are each capable of detecting very low levels of BVDV. Immunohistochemistry in tissue samples, such as samples of ear-notch biopsies, is an effective technique for detecting Pl animals, as well as ELISA technology, although somewhat less sensitive, is well suited as a basic diagnostic tool wide to detect BVDV infection in animals, because it is cheap, provides results in a short period of time and does not need highly trained technicians or highly specialized laboratory facilities. The ELISA methods for the detection of BVDV infection are described in the literature. See United States Patent No. 6,174,667 and WO 99/15900 of Huchzermeier et al., And United States patent application publication 20030143573 and have been compared with other methods, "Comparison of an Antigen Capture Enzyme- Linked Assay with Reverse Transcription-Polymerase Chain Reaction and Cell Culture Immunoperoxidase Tests for the Diagnosis of Ruminant Pestivirus Infections, "Vet. Microbiol., 43: 75-84 (1995)). The present invention is further illustrated, but not limited to the following examples.

EXAMPLE 1 Testicular protection General view of the study The Bovi-Shield® GOLD ™ 5 vaccine line, formulated with BVDV type 1 and BVDV type 2, was introduced in the cattle industry in November 2003 to optimize the level of fetal protection that vaccination provides against type 2 BVDV. Here the results of an efficacy study prior to obtaining the permit that assesses whether vaccination is reviewed prepubertal with Bovi-Shield® GOLD ™ 5 was effective in the prevention of testicular infection in the face of severe exposure to BVDV type 2, 10 compared with a placebo (Bovi-Shield® IBR-PI3-BRSV). All bull pups (n = 17) assigned to a more extensive type 2 BVDV exposure study were also evaluated to determine whether vaccination with Bovi-Shield® GOLD ™ 5 effectively prevented testicular infection with BVDV. All the offspring of the initial study were 3 to 4 month old calves for meat (males and females) deprived of colostrum. 28 days after vaccination with a formulation containing minimum doses of immunization (minimum dose levels of immunization are established before obtaining the vaccine permit and reflect a lower volume of antigen virus than is present in the product The determination of the minimum dose of immunization helps to ensure that when a product is used at the release levels it will stimulate adequate protection against the disease in a consistent manner.) of BVDV type 1 and BVDV type 2, all vaccinated females and Placebo controls were exposed by intratranasal route to the noncytopathogenic strain of BVDV type 2 24515. Strain 24515 was isolated in Canada in a severe outbreak of BVD that killed more than 40% of the cattle in the affected herds. After exposure, the 10 control calves developed a severe disease characterized by prolonged viraemia (9 to 14 days), fever that varied in the range of 40.9 ° C to 41.8 ° C for 4 to 9 days, leukopenia (1 to 9 days), Irombocytopenia (<; 100,000 per μl), morbidity (which varied from 4 to 9 days) and high mortality (7 of 10 (70%) of the controls died). In contrast, only 1 calf vaccinated developed viremia, 6 had fever for 1 or 2 days, 1 suffered leukopenia for 1 day, none suffered from thrombocytopenia and none died. Overall, 18 of the 20 vaccinated remained healthy throughout the study and only 2 subjects showed depression for 1 day. Two observations were not associated with viraemia, fever, leukopenia or previous or concurrent iobodopenia. 11 Evaluations to determine BVDV testicular infection began approximately 2 weeks after exposure. As shown in Table 1, testis samples were collected at the necropsy of 2 placebo controls on day 41 of the study and biopsy samples from each of the 5 Residen controls and 10 vaccinated with Bovi-Shield® GOLD ™ 5 on day 42. A second sample was also obluded from 7 of the 10 vaccinated with Bovi-Shield® GOLD ™ 5 which were still available at day 56. All the samples were analyzed to determine the presence of BVDV using methods of analysis of tissue cullive isolation, nucleic acid amplification (RT-nPCR) and immunohistochemical analysis. The personnel who collected and analyzed the individual samples did not have knowledge of the assignments of the age groups.TABLE 1 Study design: testicular protection after exposure to type 2 BVDV Vaccination Intranasal exposure * intramuscular Group of N ° Day of treatment day pups Dose Day Dose sampling bull Controls 7 0 2 ml 28 5 m! 41.42® with placebo Bovi-Shield® 10 0 2 ml 28 5 ml 42.56 # GOLD ™ 5 * Isolation 24515 was obtained from the University of Guelph, Guelph, Ontario, Canada.

® Samples from 2 of 7 bull calves vaccinated with placebo at necropsy were obtained on day 41 and samples from the remaining 5 calves were obtained on day 42. # Samples were obtained from the 10 bull calves on day 42 and a second sample of seven calves on day 56.

Virus isolation and PCR assays were performed at the Aubum University Veterinary Pathobiology and Clinical Sciences Laboratories and immunohistochemical analysis at the University of Nebraska-Llncoln Veterinary Diagnostic Center. The data was analyzed by a representative of Pfizer Animal Healíh, Veterinary Medicine and Research, Biomeirics, Technology and Qualiíy, with a caiegorical period (SAS / STAT software changes and improvements of version 6.12, SAS Instiíute, Cary, NC or SAS / STAT User Guide Version 8 and Method Guide for SAS Version 8). Fisher's exact analysis was used to compare the proportion of animals in each group with at least one positive result for BVDV. The descriptive statistics were calculated as considered appropriate.

Table 2 and Figure 1 summarize the percentages of the irradiation groups for the deviation of BVDV in the lesion biopsy samples. BVDV nucleic acids or antigens were determined in 6 of 7 (85.7% o) of the testicular samples collected in bulls vaccinated with placebo and exposed. No nucleic acids or BVDV aniigens were found in none (0.0%) of the ileal samples collected in the exposed exorcium pups with Bovi-Shield® GOLD ™, a significant difference (P <0.05).

TABLE 2 Overview of the results of the BVDV test for testicular samples No. of positives for BVDV Positive% of No. of calves Group of calves VI PCR IHC BVDV positive Placebo 7 5 6 4 6 85.7% to Bovi-Shield GOLD 10 0 0 0 0 0.0% b VI = virus isolation, PCR = polymerase chain reaction, IHC = immunohistochemistry, a 'b Percentages in a column with lowercase letters in different superscripts are significantly different (P <0.05).

Conclusion and discussion The results of the study demonstrated the safety as well as the effectiveness of bull vaccination with Bovi-Shield® GOLD ™ 5. The vaccine formulated with minimum dose levels of BVDV immunization, type 1 and 2 not only did not cause infection not only did they effectively protect prepuber bulls against ileal infection for a serious exposure to BVDV type 2. The use of Bovi-Shield® GOLD ™ 5 in prepubertal bulls can be an important component of conirol programs. of the BVD in the production of calves and milk. Timely vaccination can help protect bullflies against acute infections that have been associated with transient and persistent infection and the subsequent transmission of BVDV through semen to susceptible cows. In addition, vaccination of prepubertal bulls to prevent acute postpubertal infection with BVDV can help to maintain the quality of semen, which has been shown to be affected (decreased malfunction and morphological abnormalities) during the first 60 days after acute infection. by BVDV. 9 Although the present results demonstrate successful protection after exposure to type 2 BVDV, it would be expected that similar results would be observed for exposure to BVDV type 1.

EXAMPLE 2 Testicular protection against exposure to BVDV type 1 Study overview A study was conducted to evaluate The effectiveness of Bovi-Shield® GOLD ™ 5 in the prevention of ileal infection aids severe exposure to type 1 BVDV. The design of this study was a randomized generalized block design with a unidirectional irradiation structure. The information of the workshop was concealed from the staff of the laboratory of the study. Quarenia and five (45) bulls of entire peripúberes meat breed were assigned to the study. The calves were between 9 and 15 months old. All animals were seronegative for BVD viruses types 1 and 2, negative for the isolation of BVD virus in the serum and negative according to the analysis of the serum by reverse transcriptases polymerase chain reaction (RT-nPCR). On day 42, the means of least squares of the body weights of the turners in the placebo group (n = 23) and the experimental group (n = 22) were 283.8 ± 14.0 kg and 278.3 ± 16.3 kg, respectively. . Animals were vaccinated with placebo (Bovi-Shield® IBR-PI3-BRSV) or with Bovi-Shield® GOLD ™ 5 (IBR-BVDV1-BVDV2-PI3-BRSV). At 28 days after vaccination with a formulation that conferred minimum doses of immunization, all the vaccinated turners and the placebo controls were exposed intranasally to the BVDV strain type 1 to non-cytopathic strain SD-1 isolated in the Aubum Universiíy. The iníranasal exposure was performed by hyperventilating the bulls for 30 seconds placing a plastic bag over the nostrils and then instilling 5 ml of virus grown in MEM with Earle's salts supplemented with equine serum at 10% (vol / vol), sodium bicarbonate (0.75 mg / ml), L-glutamine (0.29 mg / ml) and antibiotics (100 units of penicillin G, 100 μg of stryptomycin and 0.25 μg of amphocyeric B / ml). Equine serum was free of BVD virus as determined by virus isolation and RT-nPCR. The evaluations of the tesicular infection by BVDV were made on day 42 and 93. The basic design of the study is described in Table 3 below.

TABLE 3 Study design: testicular protection after exposure to BVDV type 1 Vaccination Exposure intratransferase I * intramuscular Group of N ° of Day Dosage Day Dose Treatment day of bull sampling samples Controls 23 0 2 ml 28 5 ml 42.93 with placebo Bovi-Shield® 22 0 2 ml 28 5 ml 42.93 GOLD ™ 5 After the exposure, the serum of the animals of both groups was analyzed to determine the presence of virus. As analyzed by isolating the virus in serum, 18 calves in the placebo group suffered viremia on day 34. Nine of these 18 also suffered viremia on day 35. Five calves in the group with Bovi-Shield® GOLD ™ 5 suffered viremia only on day 34. No further analysis of virus isolation gave positive results to the calves of any of the breeding groups on any day of study. RT-nPCR analyzes were also used to determine if the calves suffered from viremia. For the group with placebo, there were 3, 9, 12 and 6 calves with positive results on days 34, 35, 36 and 37 respectively. In the group with Bovi-Shield® GOLD ™ 5, there were 1, 1, 2 and 1 calves with positive results on days 34, 35, 36 and 37, respectively. No other RT-nPCR analysis of the sera tested positive in the calves of any of the breeding groups on any day of the study. The medial mean values for least squares of both groups were measured. Only on day 36 were the means significantly different (40 ° C for placebo and 39 ° C for the group with Bovi-Shield GOLD 5) (P <0.05).

Testicular projection results The results of the analyzes performed on day 93 of the ileal biopsy samples are shown in Table 4. Samples from 6 of 23 calves (26.1%) in the placebo group gave positive results in the RT- analysis. nPCR, while all samples from the 22 calves in the group with Bovi-Shield® GOLD ™ 5 were negative. All samples from the biopsies of the groups with placebo and with Bovi-Shield® GOLD ™ 5 were negative in virus isolation. Biopsy samples from 5 of 23 calves (21.7%) from the placebo group and 0 from the 22 calves from the Bovi-Shield® GOLD ™ 5 group tested positive for IHC. All the semen samples of the calves in both groups of irradiation were negative in the RT-nPCR analysis to determine the presence of BVD virus on day 42 (Table 4). On day 93, 10 of 23 (43.5%) of the patients in the placebo group gave positive results in the RT-nPCR analysis and the 22 patients in the group with Bovi-Shield® GOLD ™ 5 gave negative results. No virus was isolated from any of the semen samples from any of the trawl groups on day 42 or day 93 (Table 4). In the placebo group 10 out of 23 calves tested positive in at least one analysis. Four of these ten authors gave positive results only in one analysis (RT-PCR of semen on day 93). One calf gave positive results in two analyzes (RT-nPCR of biopsy and semen RT-nPCR on day 93). Five authors gave positive results in the analysis (RT-nPCR of iaficular biopsy, RT-nPCR of semen and IHC of biopsy on day 93).

TABLE 4 Overview of the results of the BVDV test for testicular and semen samples Group N ° of positives N ° of N ° of positives% of calves for BVDV * positives positive for calves for BVDV "BVDV positive BVDV® VI PCR IHC VI PCR VI PCR Placebo 23 0 6 5 0 0 n 10 10 43.5 Bovi- 22 0 0 0 0 0 0 0 0 0% Shield® GOLD ™ 5 VI = virus isolation, PCR = polymerase chain reaction, IHC = immunohistochemistry, a 'b Percentages in a column with different superscript lowercase letters are significantly different (P <0.0002). * Day 93 Testicular biopsy specimens ® Day 42 Semen samples Day 93 Semen samples In each animal, the presence / absence of BVD virus was determined in semen or in tesiicular biopsies at any time point, were summarized by groups of were analyzed and analyzed using the exact Fisher code as described in Example 1.

Conclusion In this study, the vaccination of parrot pups with Bovi-Shield® GOLD ™ 5 prevented persistent testicular infection with type 1 BVD virus and the subsequent transmission of virus in the semen. The contrast between the animals with placebo and those vaccinated with at least one positive analysis by Fisher's exact bimodal test was significant (P = 0.0002). All patents, patent applications and publications cited above are incorporated herein by reference in their entirety insofar as they are not inconsistent with the description provided herein. The present invention is not limited in scope by the specific embodiments described, which are intended to be illustrations of individual aspects of the invention. The functionally equivalent compositions and methods are within the scope of the invention. In fact, various modifications of the invention, in addition to those shown and described herein, will be apparent to those skilled in the art from the foregoing description. It is intended that such modifications be within the scope of the annexes.

References 1. Meyling A, Jensen AM. Transmission of bovine virus diarrhea virus (BVDV) by artificial insemination (Al) with semen from a persistently- infected bull. 2. Kirkland PD, Mackintosh SG, Moyle A. The outcome of widespread use of semen from a bull persistently infected with pestivirus. Veterinary Record 1994; 135: 527-529. 3. McGowan MR, Kirkland PD. Early reproductive loss due to bovine pestivirus infection. British Vete nary Journal 1995; 151: 263-270. 4. Kirkland PD, Richards SG, Rothwell JT, et al. Replication of bovine viral diarrhea virus in the bovine reproductive tract and excretion of virus n semen during acute and chronic infections. Veterinary Record 1991; 128: 587-590. 5. Kirkland PD, McGowan MR, Mackintosh SG, et al. Insemination of cattle with semen from a bull transiently infected with pestivirus. Veterinary Record 1997; 140: 124-127. 6. Voges H, Horner GW, Rowe S, et al. Persistent bovine pestivirus infection localized in the lestes of an immuno-competent, non-viraemic bull. Veterinary Microbiology 1998; 61: 165-175. 7. Gives MD, Heath AM, Brock KV, et al. Detection of bovine viral diarrhea virus in semen obtained from inoculation of seronegative postpubertal bulls. AJVR 2003; 64: 428-434. 8. Niskanen R, Alenius S, Belak K, et al. Insemination of susceptible heifers with semen from a nonviraemic bull with persistent bovine virus diarrhea virus infection localized in the testes. Reproduction in Domestic Animáis 2002; 37: 171-175. 9. Patón DJ, Goodey R, Brockman S, et al. Evaluation of the quality and virological stamina of semen from bulls acutely infeeted with BVDV. Veterinary Record 1989; 124: 63-64.

Claims (9)

NOVELTY PE THE INVENTION CLAIMS

1. - The use of a vaccine that is selected from the group consisting of an inactivated type 1 BVDV vaccine, an inactivated type 2 BVDV vaccine, a modified live type 1 BVDV vaccine, and a modified live type 2 BVDV vaccine to manufacture a vaccine medicament to prevent testicular infection by BVDV in a susceptible male animal with an increased risk of tesicular infection by BVDV.

2. The use as claimed in claim 1, wherein the animal is selected from the group consisting of bulls, rams and boars.

3. The use as claimed in claim 2, wherein the animal is a bull.

4. The use as claimed in claim 1, wherein the vaccine comprises both a modified live BVDV type 1 vaccine and a live modified BVDV type 2 vaccine.

5. The use as claimed in claim 4, wherein at least one vaccine with modified live BVDV is derived from a cytopathic virus.

6. The use as claimed in claim 4, wherein at least one vaccine with modified live BVDV is derived from a non-cytopathogenic virus.

7. - The use as claimed in claim 4, wherein both live modified BVDV vaccines are derived from a cytopathogenic virus.

8. The use as claimed in claim 1-7, wherein the vaccine comprises a! minus one additional antigen that is selected from the group consisting of bovine herpes virus (BHV-1); parainfluenza virus type 3 (PIV3); bovine respiratory syncytial virus (BRSV); Leptospira canicola, Leptospira grippotyphosa, Leptospira borgpetersenii hardio-prajitno, Leptospira icterohaemmorrhagia, Leptospira interrogans pomona, Leptospira borgpetersenii hardjo-bovis, Leptospira Bratislava, Campylobacter fetus, Mannheimia (Pasteurella) haemolytica, Pasteurella multocida, Mycobacterium bovis and Mycobacterium dispar.

9. The use as claimed in claim 8, wherein said additional antigens comprise bovine herpes virus (BHV-1); parainfluenza virus type 3 (PIV3) and bovine respiratory syncytial virus (BRSV).