HK1078457B - Vaccine for respiratory and reproductive system infections in cattle - Google Patents

Description

Vaccines for the respiratory and reproductive systems of cattle

Technical Field

The present invention relates to the treatment or prevention of Bovine Viral Diarrhea Virus type 1 and type 2 (BVDV), Bovine herpes Virus type 1 (BHV-1), Bovine Respiratory Syncytial Virus (BRSV), Parainfluenza Virus (PI) in animals₃) A Campylobacter fetus (Campylobacter fetus), Leptospira canicola (Leptospira canicola), Leptospira influenzae (Leptospira grippotypha), Leptospira borgpetersenii hardjo-prajitno, Leptospira lutea haemorrhagi (leptospermocarrhagiae), Leptospira borgpetersenii hardjo-bovis, and Leptospira interrogans poma. The combination vaccine may be a live preparation of inactivated or modified whole cells or cell fractions.

Background

Five viral factors known to be associated with Bovine Respiratory Disease (BRD) complications are: bovine herpes virus type 1, also known as bovine papillitis virus (IBR); bovine viral diarrhea virus type 1 and type 2 (BVDV); bovine Respiratory Syncytial Virus (BRSV); and parainfluenza virus (PI)₃) It can lead to infections of the respiratory and reproductive systems of cows and the dairy industry of great economic importance on a global scale. BRD can cause a wide range of clinical symptoms, including acute episodes of respiratory disease and abortion. The respiratory form of BRD is characterized by inflammation, swelling, bleeding, and necrosis of the respiratory mucosa and may be accompanied by high fever, anorexia, depression, runny nose, dyspnea, and oronasal inflammation. The IBR and BVDV viruses can be pregnantIn all three trimesters, but most likely in the second half of pregnancy and often without other clinical signs (Ellis et al (1996)JAVMA208: 393-400; EllsWorth et al (1994) In: proceedings, 74^th Conference of Research Workers inAnimal Di sease：34)。

Bovine herpes virus type 1 (BHV-1) is a member of the subfamily of the alphaherpesviridae family and produces various forms of clinical disease in cattle, including: respiratory and reproductive infections, conjunctivitis, encephalitis, and abortion. Previously, vaccines have been used to control BHV-1 infections, which include attenuated live five-line cross-contamination (Gerber, J.D., et al, 1978, am. J.Vet. Res.39: 753-760; Mi tchell, D.1974, Can. Vet. Jour.15: 148-151), inactivated viruses (Frerichs, G.N., et al, 1982, Vet. Rec.111: 116-122), and subunits of viruses, such as one of the three major BHV-1 glycoproteins known in the art as gI, gIII and gIV (Babiuk, L.A., et al, 1987, Virology 159: 57-66; van Drunen, S.S., et al, 1993, Vaccine 11: 25-35). In addition, recombinant truncated forms of the BHV-1 gIV glycoprotein (referred to in the art as BHV-1 tgIV) have been demonstrated to have the ability to induce mucosal immunity against BHV-1 (van Drunen, S., et al, 1994, Vaccine, 12: 1295-BuSi 1302). However, this BHV-1 vaccine is banned from pregnant cows that are seropositive or seronegative, and also from pregnant cows that are lactating calves.

BVDV type 1 and type 2 have been implicated in various clinical symptoms. The study showed that: this virus can cause severe primary respiratory disease; persistently Infected (PI) cattle are the main source of infection for susceptible calves; moreover BVDV can infect leukocyte pools, causing profound and widespread defects in the immune system. Ellis et al (1996) JAVMA 208: 393-400; baum et al (1993) The compendium Collection: infectious diseases in Food Animal practice, Trenton, NJVinterary Learning Systems-113-; melying et al (1987) AgricPestivirus infection Rumin 225-231. Abortion or mummification may occur when pregnant cows become infected, especially during the first trimester. Bolin et al (1989) Am J.vet Res 52: 1033-1037. Mucosal disease is another frequently fatal manifestation of Bovine Viral Diarrhea (BVD) resulting from early fetal infection with a non-cytopathic BVDV biotype, immune tolerance to this virus, birth of a Persistently Infected (PI) calf, and subsequent reinfection with a cytopathic BVDV biotype. Bolin et al (1989) Am J.vet Res 52: BVDV types 1033-1037.2, once identified as a hemorrhagic BVDV isolate in cows, has been the predominant strain isolated from BVD-associated abortions and respiratory cases in most areas of the United states. Van Oirschot et al (1999) Vet Micro 64: 169-183.

BVDV is classified into pestivirus genus (pestivirus genus) and flaviviridae family. It is closely related to the virus responsible for a wide range of diseases and swine fever (classical swine fever) in sheep. Infected cattle show only "mucosal disease" characterized by elevated temperature, diarrhea, cough, and ulceration of the digestive mucosa (Olafson, et al, CornellVet.36: 205-. BVD virus is able to cross the placenta of pregnant cows and can lead to the delivery of PI calves ((Malmquist, J.am. ve.Med.Assoc.152: 763-768 (1968); Ross, et al, J.am. ve.med.Assoc.188: 618-619(1986)) which are immunologically resistant to the virus and persist in the blood in the rest of life. they are the source of outbreak of mucosal disease (Liess, et al, Dtsch. Tierttl. Wuarscr.81: 481-487(1974)) and are very susceptible to infection by microorganisms that cause diseases such as pneumonia or intestinal diseases (Barber, et al, ve.Rec.117: 459-464 (1985)).

According to studies of the growth of BVDV viruses in cultured cells, two viral biotypes can be isolated: viruses that induce cytopathic effects (cp) in infected cells and viruses that do not induce cytopathic effects (ncp) (Lee et al, am.J.Vet.Res.18: 952-953; Gillespie et al, CornellVet.50: 73-79, 1960). Cp variants may be derived from PI animals pre-infected with ncp virus (Howard et al, vet. Microbiol. 13: 361-369, 1987; Corapi et al, J. Virol. 62: 2823-2827, 1988). Based on the genetic diversity and antigenic differences of the 5' untranslated region (NTR) of the virion surface glycoprotein E2 of BVD virus, it is generally classified into two major genotypes: form I and form II. BVDV type 1 is a classical or classical viral strain that normally produces only mild diarrhea in immunocompetent animals, whereas BVDV type 2 is an emerging highly pathogenic virus that produces thrombocytopenia, hemorrhage and acute fatal disease (Corapi et al, J.Virol, 63: 3934-. BVDV viruses of type I and type II have different antigenicity as determined by cross-neutralization using a panel of monoclonal antibodies (Mabs) and using animal-generated virus-specific antisera (Corapi et al, am.J.vet.Res.51: 1388-1394, 1990). The virus of either genotype may be one of two biotypes, cp or ncp virus.

Investigation of BVD Virus infected animals showed that BVD virus induced B cell as well as T cell responses in the animals (Donis et al, Virology 158: 168-173, 1987; Larsson et al, Vet. Microbiol.31: 317-325, 1992; Howard et al, Vet. Immunol.32: 303-314, 1992; Lambot et al, J.Gen. virol.78: 1041-1047, 1997; Beer et al, Vet. Microbiol.58: 9-22, 1997).

A number of BVDV vaccines have now been developed using chemically inactivated BVD virus isolates (Fernelius et al, am. J. Vet. Res.33: 1421. sup. 1431, 1972; Kolar et al, am. J. Vet. Res.33: 1415. sup. 1420, 1972; McClurkin et al, Arch. Virol.58: 119, 1978). Inactivated virus vaccines require multiple doses to achieve primary immunization. Some inactivated BVDV vaccines can only provide protection against type I BVDV infection (Beer et al, vet. microbiology.77: 195-208, 2000). Inactivated BVDV vaccines do not protect the fetus because they provide only short-term immunity and are not efficient in providing cross-type protection (Bolin, Vet. Clin. North am. food animal. practice. 11: 615) 625, 1995).

On the other hand, Modified Live Virus (MLV) vaccines offer a higher level of protection. Currently, licensed BVDV MLV vaccines are made using attenuated viruses obtained by repeated subcultures in bovine or porcine cells (Coggins et al, Cornell vet.51: 539, 1961; Phillips et al, am.J.vet.Res.36: 135-, 1975), or chemically modified viruses exhibiting a temperature sensitive phenotype (Lobmann et al, am.J.vet.Res.45: 2498-, 1984; 47: 557-. A single dose of MLV vaccine is sufficient for immunization and the duration of immunization by vaccinated cattle can last for years. However, such vaccines were developed using BVDV type I virus strains and therefore only protect against type I viruses. Furthermore, such existing BVDV vaccines cannot be applied to pregnant cows or pregnant cows that are lactating calves.

At PI₃Viruses alone generally produce only moderate disease; however, this virus predisposes the respiratory tract to secondary infections caused by more pathogenic organisms (including the IBR virus, BRSV, and BVDV), resulting in the classic symptoms of shipping fever (shipping fever). Among the various viruses known to cause bovine respiratory disease, PI₃The virus is most widely spread. Ellis et al (1996) JAVMA 208: 393-400.

BRSV preferentially infects the lower respiratory tract, the severity of which is determined, for the most part, by the immune system's response to key viral proteins. Bolin et al (1990) Am J Vet Res 51: 703. infected cattle generally exhibit non-specific symptoms including nasal and ocular fluid discharge, mild (often biphasic) fever, and dry cough. More severely infected cattle develop severe coughing, difficult breathing in the mouth, and bubbling of saliva around the mouth, and refusal to eat and drink. Ellis et al (1996) JAVMA 208: 393-400.

Leptospirosis is a disease caused by leptospira, an animal infection of important economical livestock. Leptospira borgpetersenii serovar hardjo (Leptospira hardjo) and l. In the cattle survey in the United states, 29% reacted serologically with Leptospira hardjohnsonii and 23% reacted with Leptospira pomona. Leptospira invade the body via the mucosa or the ruptured skin, spreading via the blood. They tend to the kidneys and reproductive tract, and are less common in the vitreous humor and central nervous system of the eye. The most common method of infection is direct or indirect contact with infected urine, milk, or placental fluid, but also by sexual behavior and by ovarian infection. Cattle infected with leptospira develop acute fever, hypogalactia, abortion, or premature birth and produce weak infected calves, and can lead to reproductive failure and low conception rates. Infections can be treated with antibiotics, but antibiotic treatment is not evident only for non-lactating or non-pregnant cows. The cattle can develop acute or chronic kidney infections that cause the pathogenic organisms to be eliminated from the urinary tract and infect other animals or humans managing the cattle. Immunization with leptospira has serovar specificity and although vaccines have been available for many years, mostly poor and transient immunity can only be induced.

There is therefore a need to develop a combination vaccine against multiple antigens which is safe for pregnancy and lactating cows and their offspring and which meets the needs of the dairy and beef markets. The present invention therefore provides vaccines to treat and prevent the major infectious agents of respiratory and reproductive diseases in animals such as cows and calves. The invention further provides immunogenic compositions and methods to treat or prevent a disease or disorder in an animal.

Summary of The Invention

The present invention provides methods for treating or preventing a disease or condition in an animal caused by infection with at least one of the following agents: BVDV type 1 or 2, BHV-1, PI3, BRSV, Campylobacter fetus, Leptospira canicola, Leptospira typhosa, Leptospira borgpetersenii hardjo-prajitno, Leptospira luteo-tigrini, Leptospira borgpetersenii hardjo-bovis, and Leptospira interrogans pomona, the method comprising administering to said animal an effective amount of the combination vaccine.

The methods of the invention can protect animals, such as cattle (particularly cows), from respiratory infections and reproductive diseases. The methods of the invention protect animals such as pregnant cows from abortions caused by IBRs and persistent fetal infections caused by BVDV types 1 and 2. The methods of the invention also protect animals such as pregnant cows, lactating cows and lactating calves, from persistent infections caused by BVDV types 1 and 2. Thus, the methods of the invention can protect fertile, pregnant and lactating animals.

The combination vaccine used in the method of the invention may be a whole cell or a partial cell preparation (e.g. a modified live preparation). The combination vaccine administered according to the present invention may comprise further components, such as an adjuvant, and optionally a second or more antigens may be used in the combination vaccine. The second antigen is selected from, but not limited to, the following: bovine spore virus type 1 (BHV-1), bovine viral diarrhea virus (BVDV type 1 or 2), Bovine Respiratory Syncytial Virus (BRSV), parainfluenza virus (PI3), Leptospira canicola, Leptospira influenzae, Leptospira borgpetersenii hardio-prajitno, Leptospira lutescens, Leptospira interrogans pomona, Leptospira borgpetersenii hardjo-bovia, Leptospira bratisensis (Leptospira bratislava), Campylobacter fetus, Neosporosis canis (Neosporium caninum), fetal trichomonas (Trichostus), Mycoplasma bovis (Mycoplasma bovia), Haemophilus somnus, Mannheimia haemolyticus (Mannheimia Haemophilus) and Sarcodella multocida (Salmonella multocida).

Detailed Description

The present invention provides a method of treating or preventing a disease or condition in an animal caused by an infection with IBR, BVDV, PI3, BRSV, campylobacter fetus and/or leptospira comprising administering to said animal an effective amount of a combination vaccine.

In certain embodiments, the vaccines used in the methods of the invention comprise a modified live vaccine and a pharmaceutically acceptable carrier, or a modified live vaccine and an adjuvant.

For the purposes of clearly illustrating the invention, and not for the purposes of limiting the same, the detailed description of the invention may be divided into the following sections to describe or illustrate certain features, embodiments or uses of the invention.

Definitions and abbreviations

The term "treating or preventing" a disease or disorder herein refers to reducing or eliminating the risk of infection with a pathogenic type 1 and type 2 BVDV virus, IBR, PI3, BRSV, campylobacter and/or leptospira antigen, ameliorating or alleviating the symptoms of the infection, or accelerating the recovery of the infection. A therapeutic method is considered therapeutically effective if it reduces viral or bacterial load, reduces infection of the lungs, reduces rectal temperature, and/or increases food intake and/or growth. Treatment may also be considered therapeutically effective if it reduces fetal infections and urinary tract discharges caused by Leptospira species (e.g., Leptospira hardjo and Leptospira serovars pomona).

The methods of the invention are effective, for example, in preventing or reducing abortions caused by IBR and infections caused by BVDV type 1 and 2, and in reducing rectal temperature. The present invention thus protects the foetus against infection by IBR and BVDV types 1 and 2 and against Botrytis cinerea and Rinderpest viruses. The present invention may also provide protection of the fetus from persistent infections, such as persistent BVDV infections. By "persistent fetal infection" is meant an infection that occurs early in fetal development (e.g., 45-125 days of gestation) that results in a newborn animal being immune tolerant to BVDV and maintaining a high rate of BVDV replication and reproductive activity for months or years, becoming a permanent source of BVDV infection in the herd of cattle. Such persistently infected animals are also at risk of developing lethal mucosal disease if they are reinfected with a cytopathic viral biotype.

The term "combination vaccine" herein means a bivalent or multivalent combination of antigens, including modified live and/or inactivated antigens. The combination vaccine according to the invention comprises a modified live infectious IBR, PI3, BRSV and inactivated BVDV type 1 and type 2, one or more antigens such as, but not limited to, Leptospira canicola, Leptospira influenzae, Leptospira borgpetersenici iharihard-prajitno, Leptospira lutescens, Leptospira interrorangii spoonona), Leptospira borgpetersenii hardjo-bovis, Leptospira bradii, campylobacter foetidus, neospora canis, trichomonas foetida, mycoplasma bovis, haemophilus somni, mannheimia haemolytica and pasteurella multocida, a veterinarily acceptable carrier and an adjuvant. In a preferred embodiment, the modified live IBR component is a temperature sensitive IBR. In another preferred embodiment, the type 2 BVDV component is a cytopathic strain (cPVD-2 strain 53637-ATCC accession No. PTA-4859) and the type 1 BVDV component is a cytopathic 5960 strain (cPDV-1 strain 5960-National Animal Disease Center, United states department of Agriculture, Ames, Iowa). The invention also encompasses non-cytopathic type 1 and type 2 BVDV strains. In another preferred embodiment, the modified live antigen is a dried, freeze-dried or vitrified antigen.

The combination vaccine according to the invention may comprise inactivated BVDV type 1 and type 2, one or more antigens such as, but not limited to, Leptospira canicola, Leptospira influenzae, Leptospira borgpergpedentesis hardio-prajitno, Leptospira luteo-haemorrhagia, Leptospira interrogans poma, Leptospira borgpergpedentesis hardio-bovis, Leptospira bratislava, campylobacter fetalis, neospora caninum, trichomonas fetalis, mycoplasma bovis, haemophilus somni, mannheimia haemolytica and pasteurella multocida, a veterinarily acceptable carrier and an adjuvant. The term "combination vaccine" herein means a multi-component composition comprising at least one modified live antigen, at least one second antigen and an adjuvant, which may prevent or reduce the risk of infection and/or improve the symptoms of infection. In a preferred embodiment the second antigen is an inactivated antigen. In a preferred embodiment, the source of the combination vaccine is PregSure^®5(Pfizer，Inc.)、PregSure^®5-5L (Pfizer, Inc.) and PregSure^®5-VL5(Pfizer, Inc.). A particularly preferred source of combination vaccine is PregSure^®5-VL5。

The protective effect of combination vaccine compositions against pathogens is typically achieved by eliciting an immune response in a patient that is either cell-regulated or humoral or a combination of both. In general, elimination or reduction of the incidence of BVDV, IBR, and/or PI3 infection, amelioration of symptoms, or accelerated removal of virus from an infected subject may indicate a protective effect of the combination vaccine composition. The vaccine compositions provided by the present invention protect against infection by BVD virus type 1 and/or 2 and abortion caused by BHV-1(IBR) and respiratory infections caused by PI3 and BRSV.

The methods of the invention for treating or preventing a disease or disorder in an animal caused by an infection with an IBR, BVDV, PI3, BRSV, campylobacter fetus, and/or leptospira by administering a combination vaccine are also referred to herein as vaccination methods.

The term "combination vaccine" herein that may be used in the methods of the present invention includes, for example, inactivated whole cell or partial foetal Campylobacter and/or Leptospira cell preparations, inactivated type 1 and type 2 BVDV and/or one or more modified live antigens such as BHV-1, PI3 and/or BRSV.

In one embodiment, the vaccine composition of the present invention comprises an effective amount of one or more of the above mentioned BVDV viruses, preferably cpbvvd-2 strain 53537 (ATCC PTA-4859); cPBDD-1 strain 5960(cPBDV-1 strain 5960-National Animal Disease Center, United states department of agricultural future, Ames, Iowa); IBR ts mutant RBL 106(national institute of Veterimental Research, Brussels, Belgium); PI (proportional integral)₃ts mutant RBL 103(RIT, Rixensearch, Belgium); BRSV strain 375 (Veterimental medical research Institute, Ames, Iowa). The purified BVDV virus may be used directly in the vaccine composition or, preferably, the BVD virus may be chemically inactivated or serially passaged in vitroOne-step attenuation is carried out. Typically, vaccines contain about 1X 10³To about 1X 10¹⁰A plaque or colony forming unit lysing virus, and a veterinarily acceptable carrier and adjuvant in a volume of between 0.5 and 5 ml and preferably about 2 ml. The precise amount of virus required to provide an effective protective effect in a vaccine composition can be determined by the skilled veterinarian. A veterinarily acceptable carrier suitable for use in the vaccine composition may be any of the carriers described below.

A typical route of administration is intramuscular or subcutaneous injection of about 0.1 to about 5 ml of vaccine. The vaccine compositions of the invention may also comprise additional active ingredients, such as other vaccine compositions against BVDV, e.g. the vaccine compositions described in WO9512682, WO 9955366, U.S. patent No. 6,060,457, U.S. patent No. 6,015,795, U.S. patent No. 6,001,613, and U.S. patent No. 5,593,873.

Vaccination may be accomplished via a single vaccination injection or via multiple vaccination injections. Serum may be collected from the vaccinated animal and tested for the presence of anti-BVD virus antibodies if desired.

In another embodiment of the invention, the vaccine composition is for the treatment of BVDV infection. Accordingly, the present invention provides a method of treating an infection in an animal by a type 1 or type 2, or type 1 and type 2 BVD virus, comprising administering to the animal a therapeutically effective amount of a BVD virus of the present invention. In another embodiment, the vaccine composition of the present invention is effective in enhancing herd fertility and reducing the risk of disease transmission from cattle to humans.

By "animal subject" is meant a composition comprising any susceptible BVDV, BHV, PI₃BRSV or leptospira infected animals, for example: cattle, sheep and pigs.

In practicing the methods of the invention, the vaccine compositions of the invention are administered to cattle, preferably via the intramuscular or subcutaneous routes, although other routes may also be utilized, such as: oral, intranasal (e.g., aerosol or other routes of administration without needles), lymph node, cutaneous, intraperitoneal, rectal, or vaginal routes of administration, or a combination thereof. It may also be desirable to have a booster regimen, which may be adjusted to provide optimal immunization.

By "immunogenic" is meant the ability of the BVD virus to elicit an immune response in an animal against type 1 or type 2 BVD virus, or against both type 1 and type 2 BVD viruses. The immune response may be a cellular immune response mediated primarily by toxic T-cells, or a humoral immune response mediated primarily by helper T-cell activation, which in turn activates B cells, resulting in antibody production.

According to the invention, the virus is preferably attenuated by chemical inactivation or serial passage in a cell culture system prior to use in the immunogenic composition. Such attenuation methods are well known to those skilled in the art.

A preferred virus to be included in the immunogenic composition of the invention is BVDV cp53637 (atccno.pta-4859). Another preferred virus to be included in the immunogenic composition of the invention is BVDV 5960. A more preferred virus to be included in the immunogenic composition of the invention is the IBR strain ts mutant RBL 106. Another preferred virus to be included in the immunogenic composition of the invention is PI3 ts mutant RBL 103. Another preferred virus to be included in the immunogenic compositions of the invention is BRSV strain 375.

The immunogenic compositions of the invention may also comprise additional active ingredients, such as other immunogenic compositions against BVDV, such as those described in co-pending application serial No. 08/107,908, WO9512682, WO 9955366, U.S. patent No. 6,060,457, U.S. patent No. 6,015,795, U.S. patent No. 6,001,613, and U.S. patent No. 5,593,873.

In addition, the immunogenic and vaccine compositions of the invention can comprise one or more veterinarily acceptable carriers. As used herein, "veterinarily acceptable carrier" includes any and all solvents, dispersion media, coatings, adjuvants, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, absorption delaying agentsAnd the like. Diluents may include water, physiological saline, dextran, ethanol, glycerol, and the like. Isotonic agents may include sodium chloride, dextran, mannitol, sorbitol, lactose and the like. The stabilizer may include albumin, etc. Adjuvants include (but are not limited to): RIBI adjuvant system (RIBI Inc.), alum, aluminum hydroxide gel, cholesterol, oil-in-water emulsion, water-in-oil emulsion, such as: freund's complete and incomplete adjuvant, Block co-polymer (CytRx, Atlanta GA), SAF-M (Chiron, Emeryville CA), AMPHIGEN^®Adjuvants, saponins, Quil a, QS-21(Cambridge biotech inc., Cambridge MA), GPI-0100 (galenical Pharmaceuticals, inc., Birmingham, AL) or other saponin components, monophosphoryl lipid a, an avridine lipid-amine adjuvant, heat labile enterotoxin (recombinant or non-recombinant) from e. The immunogenic composition can further comprise one or more other immunomodulators, such as: interleukins, interferons, or other cytokines. The immunogenic composition can also include gentamicin and thimerosal. Although the amount and concentration of the adjuvant and additives of the present invention can be readily determined by one skilled in the art, the compositions of the present invention are contemplated to comprise about 50 micrograms to about 2000 micrograms of adjuvant and vaccine composition, preferably comprising a dose of about 500 micrograms per 2 milliliters of vaccine composition. In another preferred embodiment, the vaccine composition of the present invention is contemplated to contain from about 1 microgram/ml to about 60 microgram/ml of antibiotic, more preferably less than about 30 microgram/ml of antibiotic.

The immunogenic compositions of the invention can be prepared in a variety of different forms depending on the route of administration. For example, the immunogenic composition can be prepared in the form of a sterile aqueous solution or dispersion suitable for injection, or in a freeze-dried form using the freeze-drying technique. The lyophilized immunogenic composition is typically maintained at about 4 ℃, and may be reconstituted in a stable solution, such as saline or and HEPES, with or without adjuvant.

The immunogenic compositions of the invention may be administered to an animal subject to elicit an immune response against BVD viruses type 1 or type 2, or against both type 1 and type 2 BVD viruses. Accordingly, another embodiment of the present invention provides a method of stimulating an immune response against BVD virus type 1 or 2, or against a combination of BVD viruses type 1 and 2, comprising administering to an animal an effective amount of an immunogenic composition of the invention as described above. By "animal subject" is meant to include any animal susceptible to BVDV infection, for example: cattle, sheep and pigs.

Preferred immunogenic compositions for administration to an animal subject in accordance with the methods of the present invention include BVDV cp53637 virus and/or BVDV cp5960 virus. The immunogenic composition comprising BVDV virus (preferably attenuated by chemical inactivation or serial passage in culture) is administered to cattle, preferably via the intramuscular or subcutaneous routes, although other routes of administration may be utilized, for example: oral, intranasal, intraluminal, intralesional, intradermal, intraperitoneal, rectal, or vaginal routes of administration, or a combination of routes.

Immunization protocols can be optimized using methods known in the art. The animals may be administered a single dose, or two or more immunizations may be administered at intervals of two to ten weeks. Depending on the age of the animal, the immunogenic or vaccine composition may be administered again. For example, the present invention contemplates vaccination of healthy cattle six months prior to age, and vaccination at six months of age. In another embodiment, the present invention contemplates vaccinating a pre-natal cow about 5 weeks before birth and another about 2 weeks before birth to protect the fetus from infection by BVDV type 1 and type 2. Half-year revaccination with a single dose of the combination vaccine is also expected to prevent fetal infection with BVDV.

The extent and nature of the immune response induced in cattle can be assessed by various techniques. For example, sera may be collected from vaccinated animals and tested for the presence of specific antibodies against BVDV virus, e.g. using conventional virus neutralization assays.

The term "bovine" herein means a bovine animal, including (but not limited to): beef, bull, cow, and calf. As used herein, bovine means pregnant as well as lactating bovine. Preferably, the methods of the invention are applied to non-human mammals; preferably lactating or pregnant cows and their fetuses.

The term "therapeutically effective amount" or "effective amount" herein means an amount of the combination vaccine sufficient to elicit an immune response in the animal to which it is administered. The immune response may include (but is not limited to): induce cellular and/or humoral immunity. The therapeutically effective amount of the vaccine will vary depending on the particular virus used, the condition of the bovine, and/or the extent of infection, and can be determined by a veterinarian.

Inactivated (partial or whole cell) and modified live vaccines

Inactivated or modified live vaccines for use in the methods of the invention can be prepared using a variety of methods known in the art.

For example, BVDV isolates can be obtained directly from infected cow uteri using known techniques.

Isolates of BVDV may be inactivated using various known methods, such as treatment of the strain with Binary Ethyleneimine (BEI), as described in U.S. Pat. No. 5,565,205, or with formalin, glutaraldehyde, heat, radiation, BPL, or other inactivating agents known in the art.

In addition to the inactivated virus isolate, the vaccine product may also include appropriate amounts of one or more conventional adjuvants. Suitable adjuvants include (but are not limited to): inorganic gels, such as aluminum hydroxide; surface-active substances, such as lysolecithin; glycosides, e.g., saponin derivatives such as QuilA or GPI-0100; a polyol (pluronic polyol); a polyanion; non-ionic block polymers such as pluronic f-127(b.a.s.f., USA); peptides; mineral oils, such as Montanide ISA-50(Seppic, Paris, France), carbomer, ampheigen Mark II (Hydronics, USA), Alhydrogel, oily emulsions, such as mineral oil emulsions, such as emulsions of bayol f/Arlacel a and water, or emulsions of vegetable oils, water and emulsifiers, such as lecithin; alum; a bovine cytokine; cholesterol; and an adjuvant composition. In a preferred embodiment, the saponin-containing oil-in-water emulsion is microfluidized by conventional means.

A particularly preferred source of BVDV type 1 for use in the vaccines and methods of the present invention is PregSure^®(PFIZER INC.) containing BVDV strain 5960 (available from National Antibiotic Disease Center (NADC), USDA, Ames, IA). A particularly preferred source of BVDV type 2 for use in the vaccines and methods of the present invention is PregSure^®(PFIZER INC.) containing BVDV strain 53637(ATCC PTA-4859) available from University of Guelph, Guelph, Ontario.

Preferably, strains 5960 and 53637 are inactivated with BEI and adjuvanted with a commercially available adjuvant, preferably Quila-cholesterol-Amphigen (Hydronics, USA). The preferred dose of the immunogenic and vaccine compositions of the invention is about 2.0 ml. Preservatives may be included in the methods and compositions of the present invention. The present invention contemplates that the preservative may be gentamicin as well as thimerosal. A carrier, preferably PBS, may also be added. Methods for preparing modified live vaccines, e.g., subcultured via culture systems to render strains less virulent, are known in the art.

Inactivated BVDV isolates can also be combined with bacteria and viruses including, but not limited to, bovine herpes virus type 1 (BHV-1), Bovine Respiratory Syncytial Virus (BRSV), parainfluenza virus (PI3), Campylobacter fetus, Leptospira canicola, Leptospira influenzae, Leptospira borgpetersenii hardjo-prajitno, Leptospira luteo-haemorrhagica, Leptospira borgpetersenii hardjo-bovis, and Leptospira interrogans pomona.

Dosage and mode of administration

In accordance with the present invention, administration of an effective amount of the combination vaccine to cattle (pregnant cows including pregnant cows as well as lactating calves) provides effective immunity against disease and fetal infection associated with bovine viral diarrhea virus (type 1 and type 2). In one embodiment, two doses of the combination vaccine are administered to the calf with a time interval of about 3 to 4 weeks. For example, the first administration is performed at an animal age of about 1 to about 3 months. The second administration is performed about 1 to about 4 weeks after the first administration of the combination vaccine.

In a preferred embodiment, the first administration is performed about 5 weeks prior to animal breeding. The second administration is performed about 2 weeks before animal breeding. Administration of subsequent vaccine doses is preferably performed on an annual basis. In another preferred embodiment, the animals are vaccinated about 6 months prior to age and vaccinated 6 months after age. Administration of subsequent vaccine doses is preferably performed on an annual basis.

The effective amount of the combination vaccine depends on the components of the vaccine and the administration regimen. Typically, when inactivated bovine viral diarrhea virus preparations are used in the vaccine, each dose of BVDV vaccine (type 1 and type 2) will contain about 10³To about 10¹⁰Colony forming units, preferably containing about 10 BVDV (type 1 and type 2) vaccines per dose⁵To about 10⁸Colony forming units, are effective when administered to an animal twice between about 3 and 4 cycles. Preferably, the combination vaccine providing effective immunization contains about 10 BVDV (type 1 and type 2) per dose in case of two administrations to the animal between about 3 to 4 cycles⁵To 10⁸Colony forming units and more preferably about 10 per dose⁶A colony forming unit. The first administration is performed about 5 weeks before animal breeding. The second administration is performed about 2 weeks before animal breeding. Administration of subsequent vaccine doses is preferably performed on an annual basis. Animals were vaccinated approximately 6 months prior to age and vaccinated 6 months after age. Administration of subsequent vaccine doses is preferably performed on an annual basis.

In accordance with the present invention, the preferred product, PregSure, when applied^®5(Pfizer, Inc.), PregSure is preferably administered^®5 twice, each in an amount of about 0.5 ml to about 5.0 ml, preferably about 1.5 ml to about 2.5 ml, and more preferably about 2 ml. When the preferred product, PregSure, is administered^®5-L5 or PregSure^®5-VL5, PregSure is preferably administered^®5-L5 or PregSure^®5-VL5 is used twice, each at a level of about 0.5 ml to about 10.0 ml, preferably about 3 ml to about 7 ml, and more preferably about 5 ml. The first administration is performed about 5 weeks before animal breeding. The second administration is performed about 2 weeks before animal breeding. Administration of subsequent vaccine doses is preferably performed on an annual basis. If the animals are vaccinated about 6 months before age, they are vaccinated 6 months after age. Administration of subsequent vaccine doses is preferably performed on an annual basis.

According to the present invention, administration can be via known routes, including: oral, intranasal, topical, transdermal, and parenteral (e.g., intravenous, intraperitoneal, intradermal, subcutaneous, or intramuscular) routes. Preferred routes of administration are subcutaneous or intramuscular.

The present invention also contemplates a single dose initial administration followed by a one week re-inoculation of the cattle, so that additional doses of the calf one year ago need not be re-inoculated in order to develop and/or maintain immunity against infection.

The combination vaccine to be administered according to the invention can comprise additional ingredients such as adjuvants (e.g. inorganic gels such as aluminium hydroxide; surface active substances such as cholesterol, lysolecithin; glycosides such as saponin derivatives such as Quil a, QS-21 or GPI-0100; polyalcohols; polyanions; non-ionic block polymers such as Pluronic F-127; peptides; mineral oils such as montanide isa-50, carbomer, amphein, aloe @; oily emulsions such as emulsions of mineral oils such as bayol F/Arlacel a and water or emulsions of vegetable oils, water and emulsifiers such as lecithin; alum; bovine cytokines; and adjuvant combinations).

According to the present invention, administration of an effective amount of the combination vaccine to cattle aged about 3 months old provides effective immunity against respiratory infections and reproductive diseases, and reduces abortion.

The present invention also provides a method of immunizing cattle, including but not limited to cows, heifers, and pre-breeding heifers, against infection by BVDV (types 1 and 2) and respiratory diseases due to IBR, BVDV (types 1 and 2), PI3, BRSV, campylobacteriosis, and leptospirosis, the method comprising administering to the animal at least one dose, and preferably two doses, of a combination vaccine to immunize the animal against infection by BVD (types 1 and 2), IBR, PI3, BRSV, Leptospira canicola, Leptospira typhosa, Leptospira leptospermi-prajitno, Leptospira luteum, Leptospira endoprogans pomona, Leptospira leptospermi, Leptospira borgpetersenii jdovatis, Leptospira bratisb, and campylobacter fetalis.

In a preferred embodiment, the vaccine is administered subcutaneously. In another preferred embodiment, the vaccine is administered intramuscularly. In addition, preferred vaccine doses comprise about 2 ml to about 7 ml, and preferably about 5 ml, containing about 10 ml per ml³To about 10¹⁰Colony forming units per dose of virus. In another preferred embodiment, the vaccine comprises about 2 ml, containing about 10 per ml³To about 10¹⁰Colony forming units per dose of virus. The combination vaccine is preferably administered to the animal twice; once at about 1 to about 3 months of age and once after about 1 to 4 weeks. The present invention also contemplates revaccination within half a year as a single dose of vaccine as well as revaccination prior to birth.

The invention also provides a method of protecting a bovine fetus from fetal infection and persistent fetal infection comprising administering to the animal at least one dose, and preferably two doses, of a combination vaccine to immunize the fetus against infection by BVD (type 1 and type 2), IBR, PI3, BRSV, Leptospira canicola, Leptospira influenzae, Leptospira borgpe tersenii hardio-prajitno, Leptospira luteoviridae, Leptospira interrogans pomona, Leptospira borgpetersenii hardjo-bovis, Leptospira bratislava, and campylobacter fetus. The combination vaccine is preferably administered to the animal twice, once about five weeks prior to birth and once about two weeks prior to birth.

The invention also relates to the administration of an effective amount of the combination vaccine to an animal, preferably a bovine animal, to treat or prevent diseases in the animal including persistent fetal infections and reproductive disorders such as miscarriage.

The invention is further illustrated (but not limited) by the following examples.

Example 1

Materials and methods

Animal(s) productionFifty-six BVDV seronegatives suitable for fertility (i.e.they have a seroneutralization [ SN ]]Titer < 1: 2) cows were obtained from various sources and maintained in separate facilities of the study during the study. Each animal was confirmed with two ear tags, one tag per ear. If the animal loses the ear tag, a new tag is installed again. Prior to the study, test animals were vaccinated with commercial vaccines for leptospirosis, campylobacteriosis (vibriosis) and clostridium infection. The test animals were maintained under the supervision of a veterinarian who clinically monitored the test animals daily.

Test vaccines-the test vaccine is a multivalent, modified live Infectious Bovine Rhinotracheitis (IBR) secondary influenza virus 3(PI3) -Respiratory Syncytial Virus (RSV) vaccine, in dry form, rehydrated with an inactivated liquid adjuvant-mixed BVDV vaccine (Pfizer Inc, New York, NY.). The BVDV component contains minimal amounts of BVDV-1 and-2 immunizing doses, and is mixed with sterile adjuvant. The Geometric Mean Titer (GMT) of 8 replicate titrations of the bulk fluid used for the vaccine formulation was calculated to determine the potency of the BVDV immune antigen. After rehydration, a 2 ml dose of the IBR-PI3-BRSV-BVDV vaccine was administered by intramuscular Injection (IM) or subcutaneous injection (SC). The dry IBR-PI3-RSV vaccine reconstituted with sterile water was used as a placebo and IM injections were performed.

Viral attackUse of a non-cytopathic BVDV type 2 field isolate (line 94B-5359, available from Dr. Hana Van Campen, Wyoming State Veterinary laboratory, University oWyoming) as an attacking agent. Viral properties were confirmed by SN analysis and reverse transcriptase polymerase chain reaction (RT-PCR). RT-PCR analysis of the nucleotide sequences of the p125 protein and the 5-terminal untranslated region of type 2 BVDV was positive, while gp53 and p80 conserved sequences of type 1 BVDV were negative. 2 repeated titrations were performed immediately before and after challenge to determine the GMT of the efficacy of the challenge virus to be 10^3.2TCID₅₀Per milliliter. Challenge inoculation was performed intranasally in 4 ml aliquots (2 ml/nostril).

Serum assayThe seroneutralization titers of BVDV types 1 and 2 were determined in bovine cell culture using a fixed virus, reduced serum assay. Serial dilutions of serum were mixed with 50-300TCID₅₀Of a cytopathic type 1 BVDV strain 5960, or a similar amount of a cytopathic type 2 BVDV strain 125 c.

Virus isolation-isolation of BVDV in bovine cell culture from cow peripheral blood, amniotic fluid, foetal blood, and foetal tissue after challenge (PC). BVDV positive cell cultures were determined by indirect immunofluorescence using goat anti-BVDV polyclonal antibody. BVDV was attempted to be isolated from fetal tissue using the aforementioned immunohistochemical method. (Haines DM, Clark EG, and Dubovi EJ. vet Pathol 1992; 29: 27-32). 5-10 ml of whole blood samples were bled from the cow jugular vein and placed in tubes containing heparin to prepare buffy coat cells for virus isolation. Amniotic fluid was collected under local anesthesia with a left-adjacent laparotomy and 3-5 ml samples were aspirated from the uterus.

After cesarean production or spontaneous abortion, eyes, spleen, thymus, and 3 brain slices (brainstem/midbrain, brain, and cerebellum) were collected from the fetus under sterile conditions. The supernatant of the homogenized fetal tissue was used for cell culture to isolate the virus. For immunohistochemical evaluation, fetal tissues were embedded in paraffin and double retests were performed using 1: 800 and 1: 600 ascites dilutions of the anti-BVDV monoclonal antibody.

Biometric data analysisTo demonstrate protection after challenge, in vaccinationThe incidence of maternal and fetal type 2 BVDV infection should be demonstrated to be statistically significantly reduced in the groups (T2 and T3) relative to the placebo control group (T1).

Fisher's exact test was used to compare (1) the incidence of cow viremia on the first 14 days post challenge, (2) BVDV isolated from amniotic fluid, (3) BVDV isolated from fetal tissue and fetal blood after spontaneous abortion or cesarean section, and (4) immunohistochemistry for BVDV-positive fetal tissue. Serum neutralization reaction titers were analyzed using a mixed linear model and repeated measures. Geometric Mean Titers (GMTs) were calculated using the minimum root mean square of the variance analysis, which excluded SN data from non-challenged cows. Statistical significance was determined using a probability value P ≦ 0.05.

Study of fetal protection56 cows to be tested were randomly assigned to three test groups, IM placebo (T1), IM vaccinated (T2) and SC vaccinated (T3), as shown in Table 1. Cows were vaccinated or placebo on day 0 as well as day 21 of the study. In all cases, the day 0 immunization injection was administered on the left side of the neck, and the day 21 immunization injection was administered on the right side of the neck.

On day 1, the cows were given a diet covered with melengestrol acetate for 14 days. On day 32, all cows were injected with IM prostaglandin (Lutalyse, Pharmacia & Upjohn, kalamazo, MI) to synchronize estrus. Cows showing estrus were artificially inseminated with BVDV-negative semen. On day 100, approximately 65 days of conception, the cows were pregnant by rectal palpation. On day 105, 23 cows (7 control, 8 IM vaccinated, and 8 SC vaccinated) confirmed pregnancy were randomly selected from each test group, relocated to nearby isolation facilities, (Midwest Veterinary Services, Oaklank, NE) and mixed habitats. On day 119, the 23 cows to be tested were injected intranasally with the pathogenic BVDV. Blood samples were collected on the day of injection and at 8 PC intervals, i.e., on days 119, 121, 123, 125, 127, 129, 133, 140, and 147 (PC days 0, 2, 4, 6,8, 10, 14, 21, and 28) to isolate BVDV and serological analysis.

On day 147 (28 days post challenge), a laparotomy was performed from the left side of each cow and amniotic fluid was extracted from each cow. On day 297, approximately 7-14 days before parturition, the cows to be tested were transported to the university of Nebraska, Department of Veterinary and Biomedical Services for caesarean section. Before surgery, blood samples were collected from each cow for SN analysis. Blood samples were collected from each fetus after caesarean section (day 300-. The fetus was euthanized and the tissue was aseptically collected to isolate BVDV.

When natural flow occurred, blood samples were taken from the cattle pens at flow time and after two weeks. Paired blood samples were subjected to serum testing (University of Nebraska Veterinarydiagnosis Center, Lincoln, NE), BVDV isolation assessment (Pfizer Central Research, Lincoln, NE) and histopathological assessment of fetal tissues (Saskatoon Veterinary Biodiagnostics, Saskatoon, SK, Canada) were performed on the born fetus.

Results

No adverse effects were observed during or immediately after administration of 2 vaccine doses.

All cows were seronegative for BVDV type 1 and type 2 prior to vaccination (day 0), confirming that the test animals were immunologically naive to BVDV at the start of the study. The GMT values for BVDV type 1 are shown in table 2. Fifteen of the 16 vaccinated animals exhibited seroconversion after administration of 2 vaccine doses. The SN titers of type 1 BVDV in cows No. 61 (group T3) were < 1: 2 on day 0, 1: 19 on day 21, and < 1: 2 on days 33 and 119. The GMT values for BVDV type 2 are shown in table 3. All vaccinated animals (including cow # 61), seroconverted after the second dose. (SN titers of type 2 BVDV in cow 61 < 2 on day 0, 1: 19 on day 21, 1: 2 on day 33, 048, and 1: 431 on day 119). On breeding (days 32-41), the SN titer of BVDV type 1 in vaccinated cows (except cow 61) ranged from 1: 64 to 1: 13,777. The titer of BVDV type 2 on breeding of vaccinated cows ranged from 1: 64 to 1:6,889. Following vaccination, differences in GMT values for IM (T2) versus SC (T3) groups were not statistically significant at either the pre-challenge dosing interval or post-challenge. By challenge (day 119), all placebo cows (T1) remained seronegative for BVDV type 1 as well as type 2, indicating that this study was not disrupted by accidental exposure. All placebo group cows developed a serological response after challenge, confirming that each animal had received an effective challenge. The PC-GMT of BVDV type 1 in the placebo group was significantly lower than the immune memory response (anemincemedisplacement) obtained in either vaccine group (table 2, day 147). Type 2 BVDV was also lower in PC-GMT in cows of placebo group compared to either vaccine group, but this difference was only statistically significant for IM (T2) vaccinated animals.

Twenty-three cows of 3 test groups confirmed pregnancy, were challenged immunologically, and underwent amniocentesis (table 1). Between amniocentesis (day 147) and cesarean production (day 300-. In addition, 3 breeding cows (1 cow in the T1 placebo group and 2 cows in the T3 group) were found not to be pregnant at the time of cesarean delivery. The 3 cows were confirmed to be pregnant by rectal palpation on day 100 (approximately day 65 post-partum), indicating that undetected miscarriage or fetal reabsorption occurred later. These animals were removed from the study because fetal tissue from these 3 cows was not available for evaluation. On day 259, the T1 placebo group number 67 cow died, so its fetus was removed without BVDV isolation. Thus, in the end of the study, only 12 out of 23 challenged cows were subjected to caesarean section. BVDV isolation assessment was performed on these 12 fetuses derived from caesarean section plus 7 aborted fetuses and dead cows (20 total).

On day 156, cow 38 in group T2 aborted its fetus (day 123 of pregnancy, and 37 days after challenge). Paired serum samples were not evaluated, but BVDV isolation in peripheral blood and amniotic fluid of cow No. 38 after challenge appeared negative. The fetus is severely autolyzed. Histopathological and bacteriological evaluation of the fetus revealed purulent inflammation of the chorion and sub-chorionic connective tissue. Staphylococcus suis (Staphylococcus hyicus) was isolated from fluids of the lung, liver, and thoracic cavity. BVDV isolation and immunohistochemistry gave negative results indicating that the fetus was not infected by challenge.

Three T3 cows (numbers 21, 27, and 40) were aborted either on day 158 or on day 159 (day 125-. Since no miscarriage was observed, the fetus was not known to belong to that cow. They are called unknown fetuses 1, 2 or 3. Unknown fetal 1 is mummified and has histologically typical neosporosis (neosporisis) lesions. Unknown fetus 2 is autolysed, with multifocal suppurative and necrotic placentaitis and a mixture of large numbers of bacterial cocci with inflammatory exudates. Staphylococcus suis was isolated from lung, kidney, liver, stomach contents, and placental tissue. Unknown fetus 3 is softened and autolysed. Staphylococci are isolated from lung, liver, kidney, and stomach contents. BVDV isolation was obtained from all tissues of these 3 unknown fetuses as well as immunohistochemical negative results. All 3 cows showed negative BVDV isolation in peripheral blood after challenge. Similarly, amniotic fluid isolated from cows 21 and 40 was also BVDV negative, but amniotic fluid samples from cows 27 were BVDV positive. Matched serum samples of the cows were not evaluated.

On day 160, fetuses from group T2 cow No. 45 were aborted (day 128 of pregnancy, and day 41 post challenge). The results of the study clearly show extensive fetal autolysis. Staphylococci were isolated from lung, liver, kidney, stomach contents, and placenta. Placental inflammation with multifocal thrombosis and pyogenic angiitis appear. Pleural fluid serological assays were negative for IBR, Bovine Viral Diarrhea (BVD), and leptospirosis. BVDV isolation and immunohistochemical negative results were obtained for all fetal tissues.

On day 195, fetuses from group T2 cow 66 aborted (day 160 of pregnancy, and 76 post challenge). Significant purulent inflammation is observed extending from the fetal surface to the placenta lamina propria. Escherichia coli and Proteus vulgaris (Proteus vulgaris) were cultured from the stomach contents and placenta. Paired serum samples and fluid serum test results of the thoracic cavity showed that the etiology was not IBR, BVD, or leptospirosis. BVDV isolation and immunohistochemical negative results were obtained for all fetal tissues.

On day 295, group T2 of cow # 31 aborted its fetus (day 262 of pregnancy, and 176 days after challenge). Histopathological examination revealed diffuse necrotic suppurative placentaitis, chorioepithelial necrosis, and intense neutrophil inflammation. Gram-negative coccobacillus and bacilli were cultured from this inflamed lesion. Paired serological test results showed that the etiology was not IBR, BVD, or leptospirosis. BVDV isolation and immunohistochemical negative results were obtained for all fetal tissues.

PC peripheral blood samples from cows No. 67 of the T1 placebo group that died before the end of the study and amniotic fluid were positive for BVDV segregation. All fetal tissues from this cow were positive for BVDV isolation and immunohistochemistry.

SN titers of BVDV type 1 and type 2 were determined from blood samples collected from 12 cesarean fetuses. None of the 5 placebo or 7 vaccinated group cows exhibited type 1 or type 2 BVDV seropositivity in their cesarized fetuses.

The results of virus isolation after challenge are shown in table 4. All 7 cows of the T1 placebo group had BVDV viremia, confirming the serum outcome indicating that each non-vaccinated animal had a valid challenge. All blood samples from 16T 2 and T3 vaccinated groups (8 post challenge samples each) were negative for BVDV viremia. The difference in the ratio of PC viremia for the T2 and T3 vaccinated groups relative to the control was statistically significant (P.ltoreq.0.0001).

2 out of 16 vaccinated groups (12.5%) had positive BVDV, while 7 out of 7 placebo cows (100%) had positive results with significant statistical differences (P.ltoreq.0.0001). Amniotic fluid samples No. 27 and 60 in the T3 vaccinated group were positive. Virus isolation and immunohistochemical methods showed fetal analysis of cow 27 to be BVDV-negative.

Fetal tissue (7.1%) from 1 of the 14 vaccinated animals (i.e., cow # 60 in T3) was positive for BVDV segregation. BVDV was isolated with a statistically significant difference (P.ltoreq.0.0001) relative to 6 (100%) of the cow fetuses in the 6T 1 placebo group. The results of isolated BVDV in each fetal tissue were assessed as either fully positive or fully negative.

Immunohistochemistry for BVDV positive for fetal tissue was found in 1 of 14 animals from the T2 and T3 vaccinated groups (7.1%) of cows 60 from T3. 6 (100%) of the 6T 1 placebo cows were positive for BVDV-immunohistochemistry in fetal tissues, significantly higher than the incidence of vaccinated groups (P.ltoreq.0.0001). The results of BVDV immunohistochemical evaluation of each fetal tissue were either fully positive or fully negative.

Table 5 shows the origin of the virus isolation and the SN titers before challenge for 2 vaccinated cows with positive BVDV isolation results. Serum data showed that T3 vaccinated group nos. 27 and 60 had an immune response to vaccination. The SN titer of BVDV type 2 for cow No. 27 was lower than the GMT of T3 group at challenge, but this difference was not significant.

The intramuscular vaccination and SC vaccination together provided 92.9% efficacy against fetal BVDV-2 infection, with 13 out of 14 vaccinated cows showing negative BVDV isolation results (table 4). This corresponds to 88.9% protection of the same vaccine at an early stage against a fetal BVDV-1 challenge (see example 2) (16 out of 18 vaccinated animals were negative for BVDV-isolation). In both studies, 100% of fetal infections occurred in non-vaccinated placebo cows, confirming that vaccinated animals were exposed to severe immune challenge.

Challenge viruses were isolated from amniotic fluid of 2 vaccinated cows, No. 27 and 60 (table 5). Thus, both cows were considered positive for BVDV infection, although they were viremia negative at each of the 8 PC dosing intervals. No BVDV was detected in the aborted fetus of cow No. 27 by virus isolation and highly specific and sensitive immunohistochemical methods, so it was necessary to classify it as BVDV-negative. Fetal miscarriage may be due to a BVDV infection process in cows.

To confirm the results, two methods were used to assess protection of cows (viremia and isolation of virus from amniotic fluid) and their fetuses (immunohistochemistry and isolation of virus from tissue culture). The most conservative results were used to determine the protection ratio. Thus, the protective ratio of vaccinated cows was 87.5% (14 out of 16), which is the percentage of cows with amniotic fluid virus isolation negative, rather than 100% (viremia-percentage of negative cows). There are no BVDV challenge-immunization studies that result in 100% fetal protection in vaccinated animals under a challenge that results in 100% fetal infection in the non-vaccinated controls. In one study, even Modified Live Virus (MLV) vaccines (not commonly used to evaluate pregnant cows) were only able to provide no more than 83% protection against fetal BVDV-1 infection (cortex VS, Grooms, DL, Ellis J, et al (1998) Am J VetRs.59: 1409-.

TABLE 1 pregnancy status of test groups and final cows under bovine viral diarrhea Virus type 2 (BVDV) fetal challenge study

		Terminating pregnancy
									Group of	Treatment of	Number of cows vaccinated (day 0, 21)	Number of cow attacks (day 119)	(1) Cow deatha	(2) Abortionb	(3) Failure of pregnancyc	(4) Laparotomy production	Number of fetuses evaluated by BVDV isolation (1+2+4)
T1T2T3	Placebo (IM) vaccine group (SC)	181820	788	100	043	102	543	686

IM ═ intramuscular vaccination; SC ═ subcutaneous vaccination

^aCow 67 in T1 suffered a maternal death (day 259).

^bCow No. 38 (day 156), No. 45 (day 160), No. 66 (day 195), No. 31 (day 295) in T2 group, and cow No. 21, No. 27 in T3 group,And abortion occurred in number 40 (day 158 or 159).

^cAll cows that failed to conceive demonstrated pregnancy at day 100, i.e., approximately 65 days post-breeding.

TABLE 22 serological response of type BVDV challenged cows to type 1 Bovine Viral Diarrhea Virus (BVDV)

	Reciprocal of geometric mean titer of neutralization (SN) in selected test dosing interval type 1 BVDV sera
								Treatment group	Number of seropositive cows on the day of caesarean birtha	Vaccination (day 0)	Vaccination (day 21)	Propagation (day 32-41)	Attack (1 st)19 days)	Amniocentesis (day 147)	Laparotomy production (day 300-
T1(n＝7)T2(n＝8)T3(n＝8)T2&T3(n＝16 )	0/78/87/8(87.5％)15/16(93.8％)	＜2＜2＜2＜2	＜213.7b18.2c15.8c	＜22,383.1c1,116.6c1,631.3c	＜2480.7c570.4c523.6c	65.71,919.2c1,448.3c1,667.2c	833.6(n＝6)d762.7(n＝4)e691.7(n＝5)f726.3(n＝9)

^aThe reciprocal of SN titer is more than or equal to 8.

^bStatistically significant differences were vs. placebo (T1), P ≦ 0.0002.

^cStatistically significant differences were vs. placebo (T1), P ≦ 0.0001.

^d67 No. 2Cows died on day 259.

^eCows that develop abortion: no. 38 (day 156), No. 45 (day 160), No. 66 (day 195), and No. 31 (day 295); such cows were not bled at day 300-.

^fCows 21, 27, and 40 had miscarriages on days 158 and 159; such cows were not bled at day 300-.

TABLE 32 seroresponse of type BVDV challenged cows to type 2 Bovine Viral Diarrhea Virus (BVDV)

	Reciprocal of geometric mean titer of neutralization (SN) in selected test dosing interval type 2 BVDV sera
								Treatment groups (number)	Number of seropositive cows on day of breedinga	Vaccination (day 0)	Vaccination (day 21)	Propagation (day 32-41)	Attack (day 119)	Amniocentesis (day 147)	Laparotomy production (day 300-
T1(n＝7)T2(n＝8)T3(n＝8)T2&T3(n＝16)	0/78/88/816/16	＜2＜2＜2＜2	＜27.0b12.8c9.5c	＜21,217.7d1,837.6d1,495.9d	＜2285.2d261.7d273.2d	402.62,598.6e1,217.51,778.7f	2,823.8(n＝6)g922.2b(n＝4)h621.3(n＝5)I756.9b(n＝9)

^aThe reciprocal of SN titer is more than or equal to 8.

^bStatistically significant differences were vs. placebo (T1), P.ltoreq.0.0064.

^cStatistically significant differences were vs. placebo (T1), P ≦ 0.0005.

^dStatistically significant differences were vs. placebo (T1), P ≦ 0.0001.

^eStatistically significant difference vs. placebo (T1), P ═ 0.0128.

^fStatistically significant difference vs. placebo (T1), P ═ 0.0023.

^gCow 67 died on day 259.

^hCows that develop abortion: no. 38 (day 156), No. 45 (day 160), No. 66 (day 195), and No. 31 (day 295); such cows were not bled at day 300-.

^ICows 21, 27, and 40 had miscarriages on days 158 and 159; such cows were not bled at day 300-.

TABLE 4 summary of isolation of cow and fetal Bovine Viral Diarrhea Virus (BVDV) after challenge

	Post-attack virus isolation method and incidence
					Treatment group	Cow with viremiaa	Isolation of amniotic fluid virus	Fetal tissue virus isolationb	Immunohistochemical analysis of fetal tissuesb
T1 placebo (IM) T2 vaccine (IM) T3 vaccine (SC) T2&T3	7/7(100％)0/8(0％)d0/8(0％)d0/16(0％)d	7/7(100％)0/8(0％)d2/8(25％)d2/16(12.5％)d	6/6(100％)c0/8(0％)d1/6(16.7％)c，d1/14(7.1％)d	6/6(100％)c0/8(0％)d1/6(16.7％)c，d1/14(7.1％)d

^aSamples were collected from 9 dosing intervals from day 119 (challenge) to day 147 (amniocentesis) and buffy coat cells were prepared for virus isolation. If any blood sample is positive for BVDV, it is a viremic cow.

^bFetal tissue is collected following abortion or laparotomy.If any tissue is positive, the fetus is positive for BVDV.

^cOne T1 cow and two T3 cows were excluded from the study because they were found not to be pregnant at the time of caesarean section and no abortion was observed.

^dStatistically significant differences were vs. placebo (T1), P ≦ 0.0001.

TABLE 5 results of maternal Serum Neutralization (SN) titers and challenge in case of isolation of Bovine Viral Diarrhea Virus (BVDV) from vaccinated cows or their fetuses

			BVDV serotype and SN titer reciprocal at challenge
						Test group	Cow number	Sources of viral isolation	Termination of pregnancy	BVDV1	BVDV2
T3T3T3	2760 groups of GMTs	AFAF，FT，IHCN/A	Abortive cesarean production of N/A	512181570	91152262
						AF is amniotic fluid; FT ═ fetal tissue; IHC — immunohistochemistry of fetal tissue; GMT is geometric mean titer; NA is not applicable

Example 2

Materials and methods

Animal(s) productionFifty-nine BVDV seronegatives (i.e., they have seroneutralization [ SN ]]Titer < 1: 2) healthy cows of fertile age were obtained from a variety of sources and maintained in the isolated research facility during the study period in Nebraska. Each animal was confirmed with two ear tags, one tag per ear. If the animal loses the ear tag, a new tag is installed again. Prior to the study, test animals were vaccinated with commercial vaccines for leptospirosis, campylobacteriosis (vibriosis), and clostridium infection. The test animals were under the supervision of a veterinarian who clinically monitored the test animals daily.

Test vaccinesThe test vaccine is a multivalent, modified live Infectious Bovine Rhinotracheitis (IBR) -parainfluenza virus 3(PI3) -Respiratory Syncytial Virus (RSV) vaccine in dry form, rehydrated with an inactivated liquid BVDV vaccine (cottlemaster/PregaSure 5, Pfizer Inc, New York, NY). BVDV ingredients were mixed with sterile adjuvants. The efficacy of BVDV immune antigens was determined by calculating the geometric mean titer of 8 repeated titrations of the bulk fluid used for the vaccine preparation. After rehydration, a 2 ml dose of the IBR-PI3-BRSV-BVDV vaccine was administered by Intramuscular (IM) or Subcutaneous (SC) injection. Dried IBR-PI3-RSV vaccine reconstituted with sterile water was used as placebo.

Attacking virusesUse of a non-cytopathic BVDV type 1 field isolate (line 816317, available from Dr.E.J.Dubovi, New York State College of Veterinary medicine, Cornell Universal series) as an attacking agent. Viral properties were confirmed by SN analysis and reverse transcriptase polymerase chain reaction (RT-PCR). RT-PCR analysis of the nucleotide sequences of the gp53 and p80 proteins and the 5-terminal untranslated region of type 1 BVDV gave a positive result, while the p125 sequence of type 2 BVDV gave a negative result. 2 repeated titrations immediately before and after challenge were performed to determine the GMT for efficacy against the virus to be 10^4.3 TCID₅₀Per milliliter. Challenge inoculum was administered intranasally in 4 ml aliquots (2 ml/nostril).

Serum assayThe type 1 and type 2 BVDV serum neutralization titers were determined in bovine cell culture using fixed virus, reduced serum assays. Serial dilutions of serum were mixed with 50-300TCID₅₀Is selected from the group consisting of a cytopathic type 1 BVDV strain 5960, or a similar amount of a cytopathic type 2 BVDV strain 125 c.

Virus isolationPost challenge BVDV isolation in bovine cell culture from cow peripheral blood, amniotic fluid, and fetal tissue. BVDV positive cell cultures were determined by indirect immunofluorescence using goat anti-BVDV polyclonal antibody. Isolation of BVDV from fetal tissue was also attempted using the aforementioned immunohistochemical methods. Haines DM, Dlark EG, and Dubovi ej. vetpathol 1992; 29: 27-32. 5-10 ml of whole blood samples were withdrawn from the cow jugular vein and placed in tubes containing heparin to prepare buffy coat cells for virus isolation. Amniotic fluid was collected under local anesthesia with a left-sided laparotomy and 3-5 ml samples were aspirated from the uterus. After cesarean production or spontaneous abortion, eyes, 3 brain slices, spleen, and thymus were collected from each fetus under sterile conditions. The supernatant of the homogenized fetal tissue was attempted for virus isolation in cell culture. For immunohistochemical evaluation, fetal tissues were embedded in paraffin and double-replicate tests were performed using 1: 800 and 1: 600 ascites dilutions of the anti-BVDV monoclonal antibody.

Biometric data analysisIn the vaccinated groups (T2 and T3) the incidence of maternal and fetal infections was statistically significantly reduced relative to the placebo control group (T1), showing protection after challenge. Fisher's exact test was used to compare the incidence of cow viremia and isolation of BVDV from amniotic fluid, fetal tissue, and immunohistochemistry of fetal tissue. Serum neutralization reaction titers were analyzed using a mixed linear model and repeated measures. Geometric Mean Titers (GMTs) were calculated using the minimum root mean square of the variance analysis, excluding SN data of non-challenged cows.

Study of fetal protection-59 test animals were randomly assigned to one of the three test groups, IM placebo (T1), IM vaccinated (T2), and SC vaccinated (T3), as shown in Table 6. Cows were vaccinated or placebo on day 0 and day 21 of the study. In all cases, the day 0 immunization injection was administered on the left neck, and the day 21 immunization injection was administered on the right neck.

On day 32, all cows were injected with IM prostaglandin (Lutalyse, Pharmacia & Upjohn, kalamazo, MI) to synchronize estrus. Cows showing estrus were artificially inseminated with BVDV-negative semen. On day 96, approximately 60 days of conception, the pregnancy of the cows was determined by rectal palpation. On day 103, 10 cows randomly selected from each test group, which confirmed pregnancy, were relocated to nearby isolation facilities (Midwest Veterinary Services, Oakland, NE). On day 117, the 30 cows were injected intranasally with the pathogenic BVDV. Blood samples were collected on the day of challenge and at 8 post-challenge intervals, i.e., on days 119, 121, 123, 125, 127, 131, 138, and 145 for BVDV isolation.

On day 145 (28 days post challenge), a left laparotomy was performed and amniotic fluid was withdrawn from each cow. On day 295, approximately 7-14 days before expected parturition, test cows were shipped to the University of Nebraska, Department of Veter intake and biomedicalServices for caesarean section. Before surgery, blood samples were collected from each cow for SN analysis. Blood samples were collected from each fetus after caesarean section (day 298-300). Fetuses were euthanized and tissues were aseptically collected for BVDV isolation.

If spontaneous abortion occurred, blood samples were taken from the cows at parturition and after two weeks. Paired blood samples and aborted fetuses were subjected to serum testing of blood samples (university of Nebraska Veterimental diagnostic Center, Lincoln, NE), isolation of viruses from fetal tissues, (Pfizer Central Research, Lincoln, NE) and assessment of histopathology of fetal tissues. (Saskatoon Veterinary Biodiagnostics, Saskatoon, SK, Canada).

Results

Individual SN values for 30 cows tested for fetal protection were negative for BVDV type 1 and type 2 on day 0, confirming that these test animals were immunologically naive to BVDV challenge at the start of the study. GMT values (tables 7 and 8) indicate that both IM (T2 group) and SC (T3 group) vaccination elicited a serum response after administration of both doses. All cows in groups T2 and T3 switched (SN Titers ≧ 1: 8) to type 1 BVDV and type 2 BVDV after administration of the second vaccine dose. On propagation (days 34-37), the SN titres of BVDV type 1 ranged from 1: 27 to 1: 2,900, and BVDV type 2 ranged from 1:609 to 1: 13,777. After vaccination, GMT values for SC groups were slightly elevated relative to IM groups at each pre-challenge interval, but the differences were not statistically significant. Twenty-eight days post challenge (day 145), GMT values for BVDV type 1 (table 7) showed statistically significant differences in SC vaccinated groups (T3 group) versus IM (T2) group.

By challenge (day 117), all placebo cows (T1) remained seronegative for BVDV type 1 as well as type 2, indicating that the study was not compromised by incidental exposure. Cows in the placebo group responded serologically to challenge, but their GMT response to BVDV type 1 and type 2 on day 145 was significantly lower than the immunological memory response of either vaccine group (tables 7 and 8).

Between amniocentesis (day 145) and cesarean production (day 298-300), 2 cows aborted, 1 being the T1 group and the other being the T3 group. In addition, 4 breeding cows (2 cows in the T1 placebo group and 1 cow in each of the T2 and T3 groups) were found not to be pregnant at the time of cesarean delivery. The 4 cows were confirmed to be pregnant by rectal palpation on day 96 (approximately day 60 post-partum), indicating that undetected miscarriage or fetal reabsorption had occurred. These animals were removed from the study because fetal tissue from these 4 failed-pregnant cows was not available for evaluation. Thus, at the end of the study, 24 out of 30 challenged cows were subjected to caesarean section and a total of 26 fetuses either caesarean section or aborted were evaluated for BVDV isolation (table 6).

Fetuses of cow 1317 of group T1 aborted at day 238 (day 201 of pregnancy, and day 121 post challenge). Histopathological and bacteriological assessment of the fetus revealed pneumonia, necrosis of chorionic epithelium, and isolation of corynebacterium species from the stomach and placenta. Paired serum samples from the cows do not support IBR, BVD, or leptospirosis as the etiology of abortion. Cow peripheral blood collected at 6,8, and 10 days post challenge; amniotic fluid; and fetal brain, eye, and thymus, but not spleen, which are positive for BVDV isolation in cell culture. BVDV immunohistochemistry positive for fetal brain, eye, thymus, and spleen. Isolation of the virus and serum evidence in this case shows that cows experiencing viremia after challenge aborts a fetus infected with BVDV.

Fetuses of cow 1331 of the T3 vaccine group aborted on day 249 (day 212 of pregnancy, and day 132 post challenge). Histopathological and bacteriological evaluation of the fetus revealed pneumonia with extensive suppuration. The stomach contents and lung cultures showed a large growth of coliform bacteria. These matched serum samples of cows do not support the etiology of abortion of IBR, BVD, or leptospirosis. BVDV isolation was negative after cell culture of cow peripheral blood, amniotic fluid, and fetal brain, eye, spleen, and thymus collected at 9 intervals after challenge. Immunohistochemical results of fetal tissues were negative. However, BVDV isolation was positive in pooled fetal tissue cell cultures. This contradictory result suggests that fetal tissue may be contaminated by exposure to pastures containing BVD challenge virus or that necropsy equipment previously isolating BVDV is contaminated with vehicle. The lack of post-challenge viremia in cows, their positive seroconversion status, and negative BVDV isolation of specific fetal organs support the conclusion that the fetus is not infected with BVDV due to the challenge. SN titers of BVDV type 1 and type 2 were determined in blood samples collected from each cesarean fetus. None of the 7 fetuses from the cesarean section of the cows of the T1 placebo group were seropositive for BVDV type 1 or type 2. Five of the 17 fetuses from the caesarean section of the T2 and T3 vaccinated group of cows were seropositive for BVDV type 1. The type 1 SN titer of four fetuses was 1: 2 or 1: 4, the type 1 SN titer of a T2 cow 1421 fetus was 1: 181, and the type 2 SN titer was 1: 512.

The results of virus isolation after challenge are shown in table 9. Nine of the 10T 1 placebo cows experienced BVDV viremia, indicating that a potent challenge occurred. Each blood sample of 19 of the 8 post-challenge samples from 20T 2 and T3 vaccinated animals was negative for BVDV viremia. On day 123 (6 days post challenge), T2 vaccinated group 1421 was positive for BVDV, and the born fetuses were seropositive as described above, but virus-isolates were negative. The incidence of BVDV viremia after 5% challenge in both T2 and T3 vaccinated groups was significantly less than the 90% incidence (P.ltoreq.0.0001) of controls.

Fetal tissues (11.1%) of 2 of 18 vaccinated animals (i.e., T2 cows nos. 1301 and 1335) were positive for BVDV segregation. BVDV was isolated with a statistically significant difference (P.ltoreq.0.0001) relative to 8 (100%) of the cow fetuses in the 8T 1 placebo group. BVDV isolates were BVDV-positive or-negative for all fetal tissues evaluated, with the exception of T1 cow 1317, which was positive for 3 out of 4 fetal tissue samples.

2 out of 20 vaccinated animals (10%) had positive BVDV, which was statistically significantly different (P.ltoreq.0.0001) compared to 10 out of 10 placebo cows (100%) with positive results. Amniotic fluid samples numbered 1301 and 1335 of the T2 vaccinated animals were positive.

Fetal tissue (11.1%) of 2 of 18 vaccinated animals (i.e., T2 cows nos. 1301 and 1335) was positive for BVDV immunohistochemistry. All fetal tissue assessments of these vaccinated cows were positive. This result is in concert with the results of BVDV isolation from amniotic fluid and fetal tissue using cell culture methods in two identical cows. 8 (100%) of 8 cows in the T1 placebo group were positive for BVDV in fetal tissues, significantly higher than the incidence in the vaccinated group (P.ltoreq.0.0001).

The pre-challenge serum status of these three vaccinated cows with positive BVDV isolation results is shown in table 10. Cows 1301 and 1335, which produced BVDV-positive fetuses, and cows 1421, which have a viral disease, all responded immunologically to vaccination.

Sixteen out of 18 fetuses vaccinated with cows (88.9%) were resistant to challenge that produced 100% of fetuses in the non-vaccinated controls. Intra-nasal attacks not only mimic the natural route of infection, but also dose much larger than the expected field exposure. The challenge efficacy also exceeded that previously found to consistently achieve experimental BVDV type 1 viremia and fetal infection. See Ficken M, Jeevararera thnam S, Wan Welch SK, et al: BVDV featalinfections with selected isolates. in: proceedings of the International Symposium on bone visual Diarrhea Virus, aFifty-Yeast review. I thaca, NY, 1996; 110-112. Non-cytopathic challenge lines were used because this biotype was associated with persistent infection as well as immune-tolerant fetal infection. See cortex VS: bone virus diarrrea virus and mucosaldease. in: current vectoriary Therapy 4, Food animal practice, philiadelphia, PA: WB Saunders, 1999; 286-291.

Serum data confirmed the antigenicity of the vaccine under IM as well as SC route administration. All vaccinated cows sera were switched to these two BVDV types and a memory response clearly occurred after challenge (tables 7 and 8), with GMT titers continuing to the end of the study (6 months later). Seroconversion also occurred in three vaccinated cows associated with positive virus isolation after vaccination (table 10). Calves produced by caesarean section from seropositive cows 1301 and 1335 were positive for BVDV. Results of virus isolation in seropositive cows or their fetuses suggest that humoral antibodies are involved in protection, but not the only determinant thereof. Cellular or mucosal mechanisms may also be involved.

TABLE 6 Final pregnancy status of test groups and cows under bovine viral diarrhea Virus type 1 (BVDV) fetal challenge study

		Terminating pregnancy
									Group of	Treatment of	Number of cows inoculated (day 0, 21)	Number of cow attacks (day 119)	(1) Cow deatha	(2) Abortionb	(3) Failure of pregnancyc	(4) Laparotomy production	Number of fetuses evaluated by BVDV isolation (1+2+4)
T1T2T3	Placebo (IM) vaccine group (SC)	181820	788	100	043	102	543	686

IM ═ intramuscular vaccination; SC ═ subcutaneous vaccination

^aMiscarriage occurred on day 238 (T1 cows) and on day 249 (T3 cows).

^bAll cows that failed pregnancy demonstrated pregnancy at day 96, approximately 60 days after reproduction.

TABLE 71 serological response of type BVDV challenged cows to type 1 Bovine Viral Diarrhea Virus (BVDV)

	Reciprocal of geometric mean titer of neutralization (SN) in selected test interval type 1 BVDV sera
								Treatment group	Number of seropositive cows on day of breedinga	Vaccination (day 0)	Vaccination (day 21)	Propagation (days 34-37)	Attack (day 117)	Amniocentesis (145 th day)	Laparotomy (298-
T1(n＝10)T2(n＝10)T3(n＝10)T2&T3(n＝20)	0/1010/1010/1020/20	＜2＜2＜2＜2	＜25.5b7.3b6.3b	＜2414.1b630.2b510.8b	＜2177.4b281.2b223.3b	118.010,380.1b20,169.2b，c14,469.2b	808.2(n＝9)d2,233.2b(n＝10)3,804.6b(n＝9)e2,914.9b(n＝19)

^aThe reciprocal of SN titer is more than or equal to 8.

^bStatistically significant differences were vs. placebo (T1), P ≦ 0.0003.

^cIm vaccine group (T2), P0.0446.

^dOne cow is inAbortions on day 238.

^eOne cow aborted on day 249.

TABLE 81 seroresponse of type BVDV challenged cows to type 2 Bovine Viral Diarrhea Virus (BVDV)

	Reciprocal of geometric mean titer of neutralization (SN) in selected test interval type 2 BVDV sera
								Treatment group	Number of seropositive cows on day of breedinga	Vaccination (day 0)	Vaccination (day 21)	Propagation (days 34-37)	Attack (day 117)	Amniocentesis (145 th day)	Laparotomy (298-
T1(n＝10)	0/10	＜1.4	＜1.4	＜1.4	＜1.4	48.5	604.9(n＝9)d
								T2(n＝10)	10/10	＜1.4	7.6b	1,782.9b	174.8b	3,756.0b	1,634.9b(n＝10)
T3(n＝10)	10/10	＜1.4	17.7b，c	2,749.6b	309.7b	4,24 0.4b	1,879.9b(n＝9)e
								T2&T3(n＝20)	20/20	＜1.4	11.6b c	2,214.1b	232.7b	3,99 0.9b	1,753.1b(n＝19)

^aThe reciprocal of SN titer is more than or equal to 8.

^bStatistically significant differences were vs. placebo (T1), P ≦ 0.02.

^cIm vaccine group (T2), P0.0415.

^dOne cow was aborted on day 238.

^eOne cow aborted on day 249.

TABLE 9 summary of isolation of cow and fetal Bovine Viral Diarrhea Virus (BVDV) after challenge

	Post-attack virus isolation method and incidence
					Treatment group	Cow with viremiaa	Isolation of amniotic fluid virus	Fetal tissue virus isolationb	Immunohistochemical analysis of fetal tissuesb
T1 placebo (IM) T2 vaccine (IM) T3 vaccine (SC) T2&T3	9/10(90％)1/10(10％)d0/10(0％)d1/20(5％)d	10/10(100％)2/10(20％)d0/10(0％)d2/20(10％)d	8/8(100％)c2/9(22.2％)c，d0/9(0％)d2/18(11.1％)d	8/8(100％)c2/9(22.2％)c，d0/9(0％)c，d2/18(11.1％)d

^aSamples were collected at 9 intervals from day 117 (challenge) to day 145 to make buffy coat cells and virus isolation was performed. If any blood sample is positive for BVDV, it is a viremic cow.

^bFetal tissue is collected following abortion or laparotomy. If any tissue is positive, the fetus is positive for BVDV.

^c2T 1 cows, 1T 2 cows, and 1T 3 cows were excluded from the study because they were found not to be pregnant at the time of cesarean production and no abortion was observed.

^dStatistically significant difference was vs. placebo (T1), P ≦ 0.008.

TABLE 10 Serum Neutralization (SN) titers of preinstalled cows when Bovine Viral Diarrhea Virus (BVDV) is isolated from vaccinated cows or their fetuses

			BVDV serotype and SN titer reciprocal at challenge
					Test group	Cow number	Source of virus isolation	BVDV1	BVDV2
T2	1301	FT，AF，IHC	128	181
					T2	1335	FT，AF，IHC	109	91
T2	1421	CV	64a	27a
					T2	GMT group	N/A	177.4	174.8

FT ═ fetal tissue; AF is amniotic fluid; IHC — fetal tissue immunohistochemistry; CV-cow viremia; NA is not applicable; GMT-geometric mean potency

Example 3

A total of 16 calfs were inoculated twice with 2 ml of the l.hardjo/bordetella combination vaccine prepared from two adjuvant formulations at 3 week intervals: 1) 2.5% Amphigen with QuilA/cholesterol 250mcg/mL each, and 2) Amphigen/A1-gel. The vaccine consists of leptospira deadly with the culture broth removed, so the free endotoxin content is low. Recording body temperature, injection site response, and general health status after two injectionsThe method is described. No systemic effects were observed, with only mild local reactions, clinically acceptable. Sixteen additional cattle were injected with saline as a control group. Instillation in the eye and vagina for 3 consecutive days at 5X 10 times four weeks after vaccination⁶Leptospira to attack cattle. Half of each treatment group was challenged with the serovariant hardjo and the other half with leptospira pomona. For unrelated reasons, the two boluses of the control group were removed from the study, leaving only 6 animals in this group. Urine was collected once a week, and kidney samples were collected at necropsy (8 weeks post challenge) and evaluated by culture, PCR, and fluorescent antibody microscopy (FA).

After the l.hardjo challenge, urine and/or kidney cultures were detected with viable organisms from 100% of the uninoculated control group (8/8), and no positive cultures could be obtained from any of the vaccinated animals (0/16). After challenge with leptospira pomona 67% (4/6) of the control group that had not been vaccinated showed infection at urine/kidney culture, but none of the vaccinated animals were kidney or urine culture positive (0/16).

Since leptospirosis can be transmitted through contaminated urine, a useful measure of vaccine efficacy is the ability to prevent or reduce urinary tract discharge. Both vaccine formulations reduced leptospira excretion from the urethra by a statistically significant amount relative to the control group. This data shows that vaccination with bivalent l.hardjo/pomonella vaccine with either adjuvant had a significant effect; inoculation with formalin-killed combination vaccine was demonstrated to protect cattle from leptospira infection.

Example 4

Materials and methods

Animal(s) production36 BVDV and Leptospira seronegative (i.e. it has a seroneutralization reaction [ SN)]Potency < 1: 2, [ MAT ] of Leptospira serovar Hardjo and Leptospira pomona]Titer < 1: 20) from various sources,maintained in the isolated research facility during the study in Nebraska. Each animal was confirmed with two ear tags, one tag per ear. If the animal loses the ear tag, a new tag is installed again. Prior to the study, test animals were vaccinated against clostridium disease and bovine respiratory disease agents (except BVD virus). The test animals were maintained under the surveillance of a veterinarian who clinically monitored the test animals daily.

Test vaccines-the experimental test vaccine is a liquid vaccine containing formalin inactivated l.hardjo-bovis or leptospira pomona, or both, and inactivated BVD type 1 and type 2 viruses. BVDV ingredients were mixed with sterile adjuvants. Geometric Mean Titers (GMTs) of 8 replicate titrations of the large fluid used for vaccine formulations were calculated to determine the potency of BVDV immune antigens. The potency of leptospira to immunize antigens was determined according to the hamster lethality model protocol. Adjuvants for the experimental test vaccines include: 2.5% Amphigen (v/v) and Quil A/cholesterol/, each 100mcg/mL, with or without 2% (v/v) aluminum hydroxide; or 2.5% Amphigen (v/v) and QuilA/dimethyldioctadecylammonium bromide (DDA) each at 100mcg/mL, with or without 2% (v/v) aluminum hydroxide. Experimental test vaccines were administered by Subcutaneous (SC) injection at a dose of 5 ml. Monovalent l.hardjo-bovis bacterins were used as positive controls according to the manufacturer's instructions. The placebo vaccine containing saline was used as a negative control.

Attacking bacteria-using Leptospira borgpetetersenii serovar hardjo-bovis strain 203(National Animal disease center, Ames, Iowa) as an attacking agent. The l.hard jo-bovis challenge material was prepared as a first passage organism isolated from the urine of l.hard jo-bovis infected experimental cattle. Challenge material was applied once daily for three consecutive days. On each day of challenge, about 2.5X 10 will be included⁶A total of two milliliters of challenge material per milliliter of l.hardjo-bovis organism was applied to three separate anatomical sites. The challenge route was instilled into the conjunctival sac (1/2 ml each) and vagina (1 ml) of each eye.

Serum assayThe type 1 and type 2 BVDV serum neutralization titers were determined in bovine cell culture using fixed virus, reduced serum assays. Serial dilutions of serum were mixed with 50-300TCID₅₀5960, or a similar amount of a cytopathic type 2 BVDV strain 125 c. L.hardjo-bovis and Leptospira pomonensis serum microaggregation titers (MAT) were tested at a qualified Veterinary clinical laboratory using standard assays.

Separation of leptospiraExamination of urine and renal tissue homogenates (kidneys concentrated to the left and right) for the presence of leptospira. Urine and leptospira in kidney culture were examined weekly for 8 weeks using standard procedures. Leptospira Fluorescent Antibody (FA) technology was performed at a qualified veterinary clinical Laboratory (Cornell University College of veterinary Medicine Diagnostic Laboratory) using standard assays.

Biometric data analysisVaccination group (table 11) (T02, T03, T04 and T05) showed a statistically significant reduction in incidence of leptospira infection relative to placebo control group (T1), showing protection following challenge. Data for nephrocolonization and urine output are summarized by treatment and time points. The percentage of animals with leptospira detected in the kidney was compared between treatments. The percentage of animals with leptospira detected in urine was compared between treatments. Fisher's exact test was used for the above analysis. The time of the leptospira excretion from the urine was compared using a common linear mixing model. Statistical significance was determined using a probability value P of 0.05 or less.

Leptospira protection study-randomly assigning 36 test animals to one of six test groups as shown in table 11. On days 0 and 21, each animal from T01-T05 received a 5 ml SC dose of the appropriate experimental test vaccine or placebo vaccine. On days 0 and 28, positive controls of 2 ml SC dose were obtained per T06 animalA vaccine. On days 57-59, all animals were challenged with L.hardjo-bovis strain 203, supra.

On days 0, 21, 35, 56, 84, and 111, blood samples were collected from each animal to determine type 1 and type 2 BVDV titers.

On days 1, 56, 70, 77, 84, 91, 98 and 105, urine samples (approximately 45 ml) were collected from each animal for leptospira separation as described above.

Animals were euthanized on days 112 and 113 and the kidneys were assessed for the presence of leptospira as described above.

Results

GMT values (table 12) show that all animals receiving BVDV-leptospira combination vaccine (T02, T03, T04 and T05) had sero-response after administration of two vaccine doses. All animals in groups T02, T03, T04, and T05 switched sera to type 1 BVDV (SN Titers ≧ 1: 8) after administration of the second vaccine dose. All animals in the T02, T04, and T05 groups switched sera to type 2 BVDV (SN Titers ≧ 1: 8) after administration of the second vaccine dose. All cows in the placebo group (T01) or the experimental group receiving the monovalent l.hardjo-bovis vaccine remained seronegative for BVDV type 1 as well as type 2, indicating that this study was not compromised by accidental exposure. Overall, BVDV serology data show for the first time: combination vaccines comprising inactivated BVDV types 1 and 2 and inactivated L.hardjobovis and Leptospira pomona formulated in four different adjuvants were able to elicit a protective response against bovine BVDV disease, since it is known in the art that a cow BVDV SN titre of ≧ 1: 8 indicates protection against BVDV disease.

The urinary and renal leptospira results (table 13) indicate that all animals receiving BVDV-leptospira combination vaccine (T02, T03, T04, and T05) were urine Culture (CX) negative (table 13, column 2) at all eight test time points and kidney culture negative (table 13, column 8) at necropsy (day 112 or 113). Cows that received a monovalent vaccine against leptospira (T06, positive control group) were also protected against leptospira infection. In contrast, cows receiving placebo vaccine (T01, negative control group) showed to be infected based on urine (table 13, column 2) and kidney culture (table 13, column 8), indicating that the vaccine challenge study was effective. Overall, the l.hardjobovis separation data shows for the first time: combination vaccines comprising inactivated BVDV type 1 and 2 and inactivated l.hardjobovis and leptospira pomona formulated in four different adjuvants were able to induce a protective response against leptospira disease in cattle.

TABLE 11 Calf test group in BVDV-Leptospira combination vaccine study

Treatment of	Vaccine	Number of animals	Volume of dose	Route of administration	Number of doses/animal	Dose spacing
							T01	Saline placebo	6	5 ml of	SC	2	3 weeks
T02	L. hardjobovis-Leptospira pomona-BVDV-1-BVDV-2, dissolved in QAC	6	5 ml of	SC	2	3 weeks
							T03	L. hardjobovi s-Bordetella pomona-BVDV-1-BVDV-2, dissolved in QAC/AIOH	6	5 ml of	SC	2	3 weeks
T04	L. hardjobovis-Leptospira pomona-BVDV-1-BVDV-2, dissolved in DDA	6	5 ml of	SC	2	3 weeks
							T05	L. hardjobovi s-Bordetella pomona-BVDV-1-BVDV-2, dissolved in DDA/AIOH	6	5 ml of	SC	2	3 weeks
T06	Monovalent l.hardjobovis	6	2 ml of	SC	2	4 weeks

TABLE 12-reciprocal geometric mean titer and range (# - #) of type 1 and type 2 BVDV serum neutralization on day 35

Treatment of	Reciprocal geometric mean titer and range (# - #) of serum virus neutralization on day 35
			Type 1 BVD virus	Type 2 BVD virus
	T01	＜2(＜2-＜2)			＜2(＜2-＜2)
T02			1084.9(609-2435)	20.8(16-54)
	T03	148.0(＜2-1218)			5.8(＜2-19)
T04			1877.9(1024-2896)	34.0(19-91)
	T05	1084.6(152-3444)			36.1(10-91)
T06			＜2(＜2-＜2)	＜2(＜2-＜2)

TABLE 13 efficacy results of BVDV-Leptospira combination vaccine against Leptospira hardjo-bovis challenge

	Percentage of calves whose urine was once positive for leptospira			Minimum root mean square percentage of leptospira positive urine days			Percentage of calves whose kidneys were once positive for the leptospira
										Treatment of	CX	FA	PCR	CX	FA	PCR	CX	FA	PCR
T01n＝6	100a	83.3a	83.3a	50.9a	29.1a	37.1a	83.3a	0a	16.7a
										T02n＝6	0b	0b	0b	0b	0b	0b	0b	0a	0a
T03n＝6	0b	0b	0b	0b	0b	0b	0b	50.0a	0a
										T04n＝6	0b	0b	0b	0b	0b	0b	0b	0a	0a
T05n＝6	0b	16.7ab	16.7ab	0b	0.3b	0.4b	0b	16.7a	0a
										T06n＝6	0b	0b	33.3ab	0b	0b	1.6b	0b	0a	0a

Values within the column that do not share common superscripts are significantly different (P < 0.05).

Claims (12)

1. An immunogenic composition comprising:

modified live bovine herpes virus (BHV-1);

modified live parainfluenza virus type 3(PI 3);

modified live Bovine Respiratory Syncytial Virus (BRSV);

inactivated bovine viral diarrhea virus type 1 (BVDV-1);

inactivated bovine viral diarrhea type 2 virus (BVDV-2); and

a veterinarily acceptable carrier.

2. The immunogenic composition of claim 1, wherein the carrier comprises: a saponin; an oil-in-water emulsion comprising saponin; and/or QuilA, Amphigen, and cholesterol.

3. The immunogenic composition of claim 2, wherein the carrier is microfluidized.

4. The immunogenic composition of claim 1 wherein the bovine viral diarrhea virus type 1 (BVDV-1) and bovine viral diarrhea virus type 2 (BVDV-2) are cytopathic.

5. The immunogenic composition of claim 4, characterized in that said bovine viral diarrhea virus type 1 (BVDV-1) is BVDV strain 5960(National Animal disease center, US department of Agriculture, Ames, Iowa).

6. The immunogenic composition of claim 4, characterized in that the bovine viral diarrhea virus type 2 (BVDV-2) is BVDV strain 53637(ATCC PTA-4859).

7. The immunogenic composition according to any one of the preceding claims, characterized in that it further comprises at least one antigen selected from the group consisting of: leptospira canicola, Leptospira influenzae, Leptospira borgpetersenii hardio-prajitno, Leptospira luteo-haemorrhagica, Leptospira interrogans pomona, Leptospira borgpetersenii hardjo-bovis, and Campylobacter fetus.

8. Use of an immunologically effective amount of a composition as claimed in any one of claims 1-6 in the preparation of a vaccine for inducing an immune response in an animal subject against at least one of:

(a) bovine herpes virus type 1;

(b) bovine viral diarrhea virus type 1;

(c) bovine viral diarrhea virus type 2;

(d) parainfluenza virus type 3(PI 3); or

(e) Bovine Respiratory Syncytial Virus (BRSV).

9. Use of an immunologically effective amount of a composition of claim 7 in the preparation of a vaccine for inducing an immune response in an animal subject against at least one of:

(a) bovine herpes virus type 1;

(b) bovine viral diarrhea virus type 1;

(c) bovine viral diarrhea virus type 2;

(d) parainfluenza virus type 3(PI 3);

(e) bovine Respiratory Syncytial Virus (BRSV);

(f) (ii) campylobacter fetus; or

(g) Leptospira canicola, Leptospira influenzae, Leptospira borgpetersenii hardio-prajitno, Leptospira luteo-haemorrhagi, Leptospira interrogans poma, Leptospira borgpetersenii hardjo-bovis, Leptospira bratislava, Neosporosis canis, fetal trichomonas, Mycoplasma bovis, Haemophilus somnadensis, Mannheimia haemolytica, and Pasteurella multocida.

10. Use of an effective amount of the immunogenic composition of any one of claims 1-7 in the manufacture of a medicament for preventing a persistent fetal infection in an animal subject.

11. Use of an effective amount of an immunogenic composition according to any one of claims 1-7 in the manufacture of a medicament for the prevention of persistent BVDV infection in an animal subject.

12. The use of claim 10 or 11, wherein the animal is a cow, calf or heifer.