Bovine respiratory disease vaccine

Abstract

The present invention relates to vaccines for treating bovine respiratory disease. Such vaccines contain a combination of bovine influenza D virus and Mannheimia haemolytica antigens. An upper respiratory infection with an IDV leads to an increased potential for M. haemolytica pathology in the lungs. The vaccines may contain further antigens from other bovine respiratory pathogens.

Claims

1. An immunogenic composition comprising a bovine influenza D virus antigen and a Mannheimia haemolytica antigen.

2. The immunogenic composition of claim 1, wherein the Mannheimia haemolytica antigen is selected from the group of an inactivated bacterium, an extract of outer membrane proteins, and a recombinant leukotoxin, and any combination thereof.

3. The immunogenic composition of claim 1, wherein the bovine influenza D virus antigen is an inactivated virus.

4. The immunogenic composition of claim 1, wherein the bovine influenza D virus antigen is a modified, live virus.

5. The immunogenic composition of claim 4, wherein the modified, live bovine influenza D virus antigen is a modified, live virus modified by codon deoptimization.

6. The immunogenic composition of claim 4, wherein the modified, live bovine influenza D virus antigen comprises at least one codon-deoptimized genomic segment having a cDNA sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19.

7. A vaccine for bovine respiratory disease comprising a bovine influenza D virus antigen and a Mannheimia haemolytica antigen, and a pharmaceutically acceptable excipient, stabilizer, solubilizer, or diluent, and optionally an adjuvant.

8. The vaccine of claim 7, further comprising at least one antigen from an additional bovine pathogen.

9. The vaccine of claim 8, wherein the additional bovine pathogen is selected from the group of bovine viral diarrhea virus (BVDV), bovine respiratory syncytia virus (BRSV), bovine herpesvirus (BHV), parainfluenza virus 3 (PB), Pasteurella multocida, Leptospira species, and Histophilus somni, and any combination thereof.

10. A method of treating or preventing bovine respiratory disease, comprising administering to a bovine the immunogenic composition of claim 1.

11. The method of claim 10, wherein the administering is done orally, intranasally, intratracheally, or by injection.

12. A method of treating or preventing bovine respiratory disease, comprising administering to a bovine the vaccine of claim 7.

13. The method of claim 12, wherein the administering is done orally, intranasally, intratracheally, or by injection.

Description

EXAMPLE 1

(1) This study evaluated if respiratory pathogenesis develops in 6-9 month old cattle when administered a high titer influenza D virus (IDV) and if that pathogenesis can be mitigated by a homologous inactivated vaccine.

(2) A total of 83 Holstein or Holstein cross cattle approximately 6 months old were used for this study. The animals were allowed to acclimate for a minimum of 5 days to their new environment at the testing facility. Housing consisted of an ABSL-2 containment room, with common feed line bunk and open water tank system in each room. The animals met the following inclusion criteria: BVDV-PI negative via ear notch and antigen capture ELISA (ACE); in good health based on physical examination conducted on Day 0; and seronegative to IDV. Animals were randomly allocated to treatment groups (Table 2) using a random allocation plan. Study personnel conducting clinical observations, collecting samples and analyzing samples were masked to treatment identity. Following administration of each treatment (e.g. vaccination), animals were housed by treatment group such that each group was in an isolated pen with no physical contact between each treatment group, with the exception of calves from treatment group four (T04), which were co-housed with challenged cattle (groups T02 and T03).

(3) TABLE-US-00002 TABLE 2 Experimental design. Serial slaughter Groups Treat- Vaccina- Chal- Serol- (post-challenge days) ment tion lenge ogy 37 d 39 d 41 d 43 d 45 d groups day 35 d 14-96 d (2) (4) (6) (8) (10) T01 0, 14 No 10 head 3 hd 3 hd 3 hd 3 hd 3 hd (hd) T02 No IDV — 5 hd 5 hd 5 hd 5 hd 5 hd T03 0, 14 IDV — 5 hd 5 hd 5 hd 5 hd 5 hd T04 No No — — — — 3 hd 4 hd

(4) The experimental vaccine comprised beta-propriolactone (BPL) inactivated IDV strain D/Bovine/KS/14-22/2012. Vaccine was standardized to 640 hemagglutinatin units (HAU) per dose. Antigen was batched and formulated/adjuvanted with 30% EMULSIGEN® D. The vaccine underwent a modified sterility test consisting of final product being plated on blood agar, incubated at 37° C. and examination of colonies formed approximately 48 hours later. No colonies were identified and product considered sterile. Calves were vaccinated (2.0 mL) by subcutaneous (SQ) injection on the right side of the neck (20-gauge 1-inch needle) on Day 0 and Day 14. Animals were observed at approximately one (1) hour post-vaccination to assess response to vaccination. No abnormal events were noted.

(5) Challenge occurred on Day 35 (˜7 months of age). On Day 35 (35 d) each calf was challenged intranasally (5.0 ml; 2.5 mL/nostril) with IDV strain D/Bovine/KS/14-22/2012 at 10.sup.6 TCID.sub.50.

(6) All animals were evaluated for depression, body condition and respiratory distress on Days 35 through necropsy and scored. Animals were evaluated and scores recorded prior to movement or feeding. Rectal body temperatures were recorded daily during the challenge period (Day 35-45). Temperatures were recorded at approximately the same time each day. One blood sample was collected at least 7 days prior to arrival at the facility and the rest of the blood samples for the determination of IDV hemagglutination inhibition were collected on Days 0, 14, 21, 28, 35, 42, 49, 56, 63, 70, 84, 98, 112. One nasal swab sample was collected at least 7 days prior to arrival at the facility and the rest of the nasal swabs were collected from all animals on Days 35 (pre-challenge), 37, 39, 41, 43, and 45. Swabs were placed into a tube containing transport medium. Tubes were labeled with date of collection, animal identification (ear tag number), sample type (nasal), and study number. Tubes were cooled immediately by placing them into a cooler containing ice packs. The tubes were stored at −70° C.

(7) On Days 35, 37, 39, 41, 43, and 45, the respective animals were humanely euthanized for lung lesion scoring, collection of bronchoalveolar lavage (BAL) fluid, and the respective tissue. Lungs were removed from each animal. BAL fluid was collected aseptically from the excised lungs. Sterile phosphate buffered saline (PBS) was pipetted into the lungs and pipetted back as the lungs are gently massaged. The BAL fluid extracted was placed into a tube kept on ice. Each sample of BAL fluid was divided into two aliquots and labeled with animal ID and date of sample collection. Both set of aliquots were stored frozen (≤−20° C.).

(8) Each of the seven pulmonary lobes was examined both visually and by palpation for gross characteristic lesions attributable to IDV. The amount of lesion/consolidation in each pulmonary lobe was scored and recorded as an actual value between 0 and 100% of the lobe. The amount of lesion (score) in each lobe was inserted in a weighted formula in order to calculate the percentage of lung with lesions. The weight assigned to each of the seven lobes (according to the relative weight of the lung lobes) is as follows: left apical lobe=10%; left cardiac lobe=10%; left diaphragmatic lobe=25%; right apical lobe=10%; right cardiac lobe=10%; right diaphragmatic lobe=25%; and accessory lobe=10%. The addition of the weighted percentages resulted in a composite lung lesions score.

(9) Lung, turbinate, and tracheal tissues were examined at necropsy for lesions and a section was excised for immunohistochemistry. The tracheobronchial lymph node was excised for immunohistochemistry evaluation as well. All samples were stained with hematoxylin and eosin for structural evaluation as well as stained for IDV virus using standard immunohistochemistry techniques.

(10) Nasal swabs were evaluated for IDV virus via RT-PCR using a specific primer set developed by the Kansas State University (KSU) veterinary diagnostic laboratory. The mean with standard deviation of the threshold cycle (CT) value of the tested nasal swabs were extrapolated using a standard curve of known tissue culture infectious dose (TCID.sub.50) IDV virus to produce a viral titer value of the nasal swabs. The CT value was extrapolated using a standard curve of known tissue culture infectious dose (TCID.sub.50) IDV virus to produce a viral titer value (Table 3).

(11) Bronchioalveolar lavage fluid (Table 4) and tracheal swabs (from days 41, 43, and 45) (Table 5) were also evaluated for IDV virus via RT-PCR, and the CT value was extrapolated using a standard curve of known tissue culture infectious dose (TCID.sub.50) IDV virus to produce a viral titer value for each sample group.

(12) TABLE-US-00003 TABLE 3 Nasal Swab Viral Titer (TCID.sub.50). At 2 4 6 8 10 Treatment Group challenge DPC.sup.1 DPC DPC DPC DPC T01- Vaccination+ 0 0 0 0 0 0 Challenge− T02- Vaccination− 0 2.64 5.68 3.765 0.24 0.275 Challenge+ T03- Vaccination+ 0 1.52 3.98 2.01 1.23 0 Challenge+ T04- Contact 0 0 0.65 0.78 3.34 4.95 Controls .sup.1Days post challenge (DPC).

(13) TABLE-US-00004 TABLE 4 Tracheal Swab Viral Titer (TCID.sub.50). Treatment Group 6 DPC 8 DPC 10 DPC T01- Vaccination+ 0 0 0 Challenge− T02- Vaccination− 5.34 0.64 0.18 Challenge+ T03- Vaccination+ 1.72 0.64 0 Challenge+ T04- Contact NA 4.63 3.3 Controls

(14) Assessments of the upper respiratory tract and lung lesion scoring as outlined above were taken by the blinded study investigator. Composite lung lesions scores are outlined in Table 6 as mean composite scores/number of animals per group at each time point.

(15) TABLE-US-00005 TABLE 5 BALF Viral Titer (TCID.sub.50). Treatment Group 2 DPC 4 DPC 6 DPC 8 DPC 10 DPC T01- Vaccination+ 0 0 0 0 0 Challenge− T02- Vaccination− 0.54 2.68 4.98 1.52 1.26 Challenge+ T03- Vaccination+ 0 1.22 0 0 0 Challenge+ T04- Contact NA NA NA 1.73 3.15 Controls

(16) TABLE-US-00006 TABLE 6 Mean Composite Lung Lesion Scores Treatment Group 2 DPC 4 DPC 6 DPC 8 DPC 10 DPC T01- Vaccination+ 0.59/3 0.00/3 0.24/3 0.06/3 0.04/3 Challenge− T02- Vaccination− 0.29/5 1.01/5 2.46/5 0.64/5 1.70/5 Challenge+ T03- Vaccination+ 0.18/5 0.72/5 1.34/5 0.58/5 1.04/5 Challenge+ T04- Contact NA NA NA 0.59/3 0.15/2 Controls

(17) Mild lung lesion scores were observed with slight trending differences between the vaccinates versus challenged animals. Stronger statistical power could be added as well as refinement of the challenge model to further explore the gross lung pathology caused by IDV. Gross pathological examinations of the upper respiratory tract, exclusively the trachea demonstrated a mucoid exudate with peak observations at 4 and 6 days post challenge.

(18) Fixed tissue samples from the nasal turbinate and the trachea were processed, hematoxylin and eosin stained, and then stained with antibody specific for IDV virus. The number of positive samples out of total number of animals per group for nasal turbinate samples and for trachea samples are represented in Tables 7 and 8, respectively.

(19) TABLE-US-00007 TABLE 7 Immunohistochemistry Results (number positive) of Nasal Turbinate Samples 2 Days Post Challenge Treatment Group (DPC) 4 DPC 6 DPC 8 DPC 10 DPC T01- Vaccination+ 0/3 0/3 0/3 0/3 0/3 Challenge− T02- Vaccination− 1/5 1/5 5/5 1/5 0/5 Challenge+ T03- Vaccination+ 0/5 0/5 0/5 0/5 0/5 Challenge+ T04- Contact NA NA NA 1/3 2/2 Controls

(20) TABLE-US-00008 TABLE 8 Immunohistochemistry Results (number positive) of Trachea Samples 2 Days Post Challenge Treatment Group (DPC) 4 DPC 6 DPC 8 DPC 10 DPC T01- Vaccination+ 0/3 0/3 0/3 0/3 0/3 Challenge− T02- Vaccination− 0/5 4/5 5/5 0/5 0/5 Challenge+ T03- Vaccination+ 0/5 2/5 0/5 0/5 0/5 Challenge+ T04- Contact NA NA NA 1/3 0/2 Controls

(21) Serological antibody titers were assessed at study day (SD) 0, 14, 21, 28, 35, and 42 for serological conversion to the vaccine. Geometric mean titer (GMT) of hemagglutination inhibition (HI) titer of the serum from each group is depicted in Table 9. Serological evaluation shows there was an increase in HI titer against IDV in the vaccinated groups compared to the non-vaccinates. Routine dose and vaccine optimization could be evaluated further to possibly increase the antibody titer. For comparison, in humans a HI titer of 40 (5.3 log.sub.2) is generally considered protective against influenzavirus A infection but does not fully alleviate disease symptoms.

(22) TABLE-US-00009 TABLE 9 Hemagglutination Inhibition (HI) Serum Titer (log.sub.2). At 14 21 28 35 42 Treatment Group challenge DPC DPC DPC DPC DPC T01- Vaccination+ 28 58.7 154.7 210.7 160 160 Challenge− T02- Vaccination− 28.8 37.6 24 32 34.4 40 Challenge+ T03- Vaccination+ 28.8 49.6 132.8 201.6 208 328 Challenge+ T04- Contact 32 48 32 36 40 36 Controls

(23) Clinical observations of depression and body condition scoring were mostly unremarkable. Respiratory distress was demonstrated in the challenged animals with peak clinical signs at 6 days post challenge. No differences in respiratory distress between vaccinated and unvaccinated animals were observed.

(24) The primary objective of this study was to assess if bovine influenza D virus (IDV) was capable of eliciting clinical pathogenesis and discover if a homologous inactivated vaccine preparation was able to prevent the clinical pathogenesis. In animals challenged with IDV, replication of the virus took place in the upper respiratory tract based on RT-PCR. The kinetics of viral replication indicate that the highest viral titers were shown at 6 days post challenge. The amount of virus was trending towards being higher in the nasal swabs, tracheal swabs, and the BALF of treatment group 2 (T02) as compared to the vaccinated groups with the greatest differences in the tracheal swabs and BALF samples. Additionally, immunohistochemistry staining of IDV was performed on the fixed excised tissues of the upper respiratory tract (turbinate and trachea). Challenged animals were positive for IDV virus via staining with five of five cattle being positive at 6 days post challenge. No animals receiving vaccine were positive for IDV via IHC staining. It can be hypothesized that immunization may limit the amount of viral spread by reducing the viral load in the upper respiratory tract. Further, the contact control group (T04) showed that the virus was passed from the challenged animals (T02) to co-housed naïve animals.

(25) Based on the results of this study IDV appears to replicate in the upper respiratory tract and elicit a mild inflammation based on immunohistochemical observations. This viral infection may be an important contributor to the bovine respiratory disease complex in which preventive intervention like vaccines could increase overall cattle health.

EXAMPLE 2

(26) This study evaluated the hypothesis that the upper respiratory infection caused by Influenza D virus is a precursor to, or exacerbator of, a more severe secondary Mannheimia haemolytica infection.

(27) A total of 70 Holstein or Holstein cross cattle 5-9 months old were used for this study. The animals were allowed to acclimate for a minimum of 5 days to their new environment at the testing facility. Housing consisted of an ABSL-2 containment room, with common feed line bunk and open water tank system in each room. The animals met the following inclusion criteria: BVDV-PI negative via ear notch and antigen capture ELISA (ACE); in good health based on physical examination conducted on Day 0; seronegative to IDV; and seronegative to leukotoxin (LKT) of M. haemolytica.

(28) The IDV experimental vaccine comprised BPL inactivated IDV strain D/Bovine/NE/103795/2012. The strain of influenza virus used for immunizations and challenge was changed from D/Bovine/KS/14-22/2012 to D/Bovine/NE/103795/2012 due to the latter virus growing better for scale up. Vaccines were standardized to 1280 HAU per dose. Antigen was batched and formulated/adjuvanted 1:1 weight by weight with SEPPIC MONTANIDE ISA 201 VG. The vaccine underwent a 7-day media bottle sterility test in Fluid Thioglycollate Media (FTM) and Tryptic Soy Broth (TSB) broth prior to use.

(29) The M. haemolytica vaccine comprised an inactivated outer membrane proteins (OMP) extract and recombinant LKT (rLKT). Vaccines were standardized to 250 μg of OMP and 125 μg of LKT per dose. Antigen was batched and formulated/adjuvanted 1:1 weight by weight with SEPPIC MONTANIDE ISA 201 VG. The vaccine underwent a 7-day media bottle sterility test in FTM and TSB broths prior to use.

(30) TABLE-US-00010 TABLE 10 Treatment groups. IDV M. haemolytica Necropsy and Treatment Vaccine Animals/ Dose/ Challenge Challenge Lung Scores Group (SD 0 & 14) Treatment Route (SD 35) (SD 41) (SD 48) T01 Adjuvanted 10 2 mL/SC + +* + Saline T02 Saline 10 2 mL/SC + −  + T03 Saline 9 2 mL/SC − +* + T04 Adjuvanted 10 2 mL/SC −  +** + Saline T05 IDV vaccine 10 2 mL/SC + +* + T06 M. haemolytica 10 2 mL/SC + +* + vaccine T07 M. haemolytica + 10 2 mL/SC + +* + IDV vaccine *Mannheimia haemolytica challenge administered intranasally with disposable atomizer (5.0 mL; 2.5 mL/nostril) and 5.0 mL intratracheally with endoscope to be delivered at the larynx/trachea **Mannheimia haemolytica challenge administered intratracheally by locating the bifurcation of the trachea and pulling back approximately 10 cm and delivering 60 mL with M. haemolytica strain

(31) The combination vaccine comprised BPL inactivated IDV strain D/Bovine/NE/103795/2012 plus inactivated outer membrane proteins (OMP) extract and rLKT. Vaccines were standardized to 1280 HAU per dose of IDV, 250 μs of OMP, and 125 of LKT per dose. Antigens were batched and formulated/adjuvanted 1:1 weight by weight with SEPPIC MONTANIDE ISA 201 VG. The vaccine underwent a 7-day media bottle sterility test in FTM and TSB broth prior to use.

(32) All candidate animals that met selection criteria and inclusion/exclusion criteria were eligible for enrollment. Animals were randomly allocated to treatment groups using a random allocation plan. Study personnel conducting clinical observations, collecting samples and analyzing samples were masked to treatment identity. The study investigator who performed the lung lesion scoring was blinded to the specific calf ID during scoring.

(33) Following arrival at the study site, animals were housed within three ABSL-2 containment rooms. Groups T01, T02, T05, T06 and T07 were housed in two rooms (25 animals per room) with equal representation of treatment groups between rooms. Groups T03 and T04 were housed in a separate third room to prevent shedding of IDV from challenged animals to cross contaminate IDV naïve animals. Animals were comingled within the three rooms. Following M. haemolytica challenge on study day 41 (SD41) the entire herd was resorted to equally distribute treatment groups (T01-T07) between the three ABSL-2 containment rooms with 23-24 animals per room. Equal representation of each treatment group (3-4 animals per group per room) was obtained following the resort. Calves were vaccinated (2.0 mL) by subcutaneous (SQ) injection on the right side of the neck (18-gauge 1-inch needle) on Day 0 and left side of the neck on Day 14. Animals were observed at approximately one (1) hour post-vaccination to assess response to vaccination. Study animals were observed daily to determine general health status beginning on Day −5 and continuing through Day 48. Observations were made at approximately the same time each day. Animal health observations consisted of pen-side visual assessments of animals for indicators of animal health.

(34) Influenza D Virus challenge occurred on Day 35 (˜6-10 months of age) for Groups T01, T02, T05, T06, and T07. On Day 35, each calf was challenged intranasally with disposable PREVAL® can atomizer (10.0 mL; 5 mL/nostril) with IDV strain D/Bovine/NE/103795/2012 at 10.sup.5.7 TCID.sub.50.

(35) IDV was produced as follows. Three 850 cm.sup.2 tissue culture polystyrene roller bottles were planted with ST-C cells at a target density of 2×10.sup.7 cells/bottle (i.e. 23529 cells/cm.sup.2) in EMEM media with 7.5% FBS and 4 mM L-glutamine. The roller bottles rotated at 0.25 rpm at 37° C. After 7 days, the cells were infected. To infect, the used media was first replaced with pre-warmed EMEM media only, and IDV (D/Bovine/NE/103795/2012) was added at a multiplicity of infection (MOI) of 0.05. After an additional 3 days of incubation at 0.25 rpm and 37° C., the entire roller bottle and contents were harvested by freezing at −80° C., thawing and dispensed aseptically.

(36) At time of harvest, samples were taken for virus titer assay. To assay, ST-C cells were planted at 15,000 cells/well in 96-well plates. 3 days post-planting, when the cells were ˜95% confluent, the wells were washed with PBS two times before 100 μL EMEM with 0.2 mL/L gentamicin was added to each well. Next, the harvested IDV samples were serially diluted 1:10 before 100 μL of each dilution was added to each well. After an additional 3 days of incubation at 37° C. and 5% CO.sub.2, the plates were fixed with 80% acetone at −20° C. for at least 20 minutes, before being dried at 37° C. For staining, the primary antibody was diluted in D-PBS, added to each well, and incubated at 37° C. for 1 hour. Following three washes in PBS, the secondary antibody was diluted in D-PBS, added to each well, and incubated at 37° C. for 75 minutes. After two washes in PBS, the results were obtained visually under a fluorescent microscope and calculated via the Spearman-Karber equation.

(37) Mannheimia haemolytica Challenge occurred on Day 41 (˜6-10 months of age) for Groups T01, T03, T04, T05, T06, and T07. On Day 41, each calf from Groups T01, T03, T05, T06, and T07 was challenged intranasally with a disposable atomizer (5.0 mL; 2.5 mL/nostril) and 5.0 mL intratracheally with an endoscope at the larynx/trachea bifurcation with M. haemolytica strain at 10.sup.9 CFU/mL. On Day 41, each calf from Group T04 was challenged intratracheally by locating the bifurcation of the trachea and pulling back approximately 10 cm and delivering 60 mL of M. haemolytica strain at 10.sup.9 CFU/mL with endoscope.

(38) Rectal body temperatures were recorded daily during the challenge period (Day 35-48). Temperatures were recorded at approximately the same time each day.

(39) One blood sample was collected at a minimum of 7 days prior to arrival at the facility and the rest of the blood samples for the determination of IDV hemagglutination inhibition and anti-M. haemolytica LKT antibody were collected on Days −2, 14, 21, 28, 35, 41, and 48.

(40) One nasal swab sample was collected at a minimum of 7 days prior to arrival at the facility and the rest of the nasal swabs were collected from all animals on Days 35 (pre-challenge), 37, 39, 41, 43, 45 and 47. In addition, swabs of the trachea were obtained at necropsy on Day 48.

(41) On Day 48, the indicated animals were humanely euthanized for lung lesion scoring, collection of bronchoalveolar lavage (BAL) fluid, and tissue collection. Lungs were removed from each animal, and BAL fluid was collected aseptically from the excised lungs. Sterile phosphate buffered saline (PBS) was pipetted into the lungs and collected by pipette as the lungs were gently massaged. The BAL fluid extract was placed into a tube kept on ice. Each sample of BAL fluid was divided into two aliquots and labeled with animal ID and date of sample collection. Both set of aliquots were stored frozen (≤−20° C.).

(42) Each of the seven pulmonary lobes was examined both visually and by palpation for gross characteristic lesions attributable to M. haemolytica and/or IDV. The amount of lesion/consolidation in each pulmonary lobe was scored and recorded as an actual value between 0 and 100% of the lobe. The amount of lesion (score) in each lobe was inserted in a weighted formula in order to calculate the percentage of lung with lesions. The weight assigned to each of the seven lobes (according to the relative weight of the lung lobes) is as follows: left apical lobe=10%; left cardiac lobe=10%; left diaphragmatic lobe=25%; right apical lobe=10%; right cardiac lobe=10%; right diaphragmatic lobe=25%; and accessory lobe=10%.

(43) Nasal turbinates, trachea, tracheobronchial lymph node, and lung tissues were examined at necropsy for lesions and a section of each was excised. The section was divided so that one half was placed in fixative (buffered formalin or equivalent) for immunohistochemistry evaluation as well as stained with hematoxylin and eosin for structural evaluation, and the other half will be placed in RNALATER® or frozen at −70° C. for cytokine and inflammatory gene PCR analysis.

(44) The primary variable, total percent lung lesion score on SD48, was calculated and analyzed as in Example 1. Secondary variables were summarized or analyzed including daily depression scores, daily body condition score, daily respiratory score and daily rectal body temperature from SD 35 to SD48 was well as mortality and removals from SD 35 to SD48.

(45) Data from the seven treatments T01, T02, T03, T04, T05, T06 and T07 were pooled and analyzed. For each variable, calculations were done on a per animal basis because the animal was the experimental unit. All testing was conducted at the 5% significance level unless otherwise stated.

(46) Total percent lung lesion scores on Day 48 were transformed using arsin (sqrt(Total Percent Lung Score)) and were analyzed using a linear mixed model analysis (SAS® PROC MIXED v 9.4 or higher), with fixed effect, per treatment. Linear contrasts were conducted using the following pairwise comparisons:

(47) T01 vs. T02, T03, T04;

(48) T02 vs. T03, T04;

(49) T03 vs. T04; and

(50) T04 vs. T05, T06, and T07.

(51) Least squares means (LSMEANs) and standard errors as well as arithmetic means and standard deviations were calculated for each treatment group and back-transformed results are presented.

(52) In addition, summary statistics were presented for the mitigated fractions (MF) (R version 3.0.3 (2014 Mar. 6)). Frequency distributions of daily depression scores, daily body condition score and daily respiratory scores were obtained for each time by treatment.

(53) There were two mortalities due to morbidity, both in Treatment Group 4. No other groups had mortalities.

(54) The primary objective of this study was to evaluate the mortality and lung lesion scores of the different treatment groups and to evaluate the hypothesis that the upper respiratory infection caused by Influenza D virus is a precursor to a more severe secondary Mannheimia haemolytica infection. Mean lung lesion scores of animals challenged intranasally with M. haemolytica following an Influenza D virus challenge showed a greater than fourfold increase compared to animals intranasally challenged with M. haemolytica alone (Group T01=5.6 and Group T03=1.2 respectively). This supports the hypothesis that an upper respiratory infection with an IDV leads to an increased potential for M. haemolytica to migrate from the upper respiratory tract where it typically colonizes to the lower respiratory tract and lungs where pathology starts to be exhibited.

(55) The mean lung lesion score in the IDV and M. haemolytica challenged cattle was lower than what is observed in the traditional model of M. haemolytica infection in which a larger volume of bacteria (60 mL compared to 10 mL intranasal/intratracheal) is delivered directly to the lungs. The differences in the severity can be explained by the decreased dose and the anatomical and mechanical immunological defenses between the upper respiratory tract and the lungs.

(56) TABLE-US-00011 TABLE 11 Mean Lung Lesion Scores IDV M. haemolytica Mean Treatment Challenge Challenge Lung Group Vaccine (SD 0 & 14) (SD 35) (SD 41) Scores T01 Adjuvanted Saline + +* 5.6 T02 Saline + −  0.5 T03 Saline − +* 1.2 T04 Adjuvanted Saline −  +** 23.9 T05 IDV vaccine + +* 2.5 T06 M. haemolytica + +* 0.9 vaccine T07 M. haemolytica + + +* 0.1 IDV vaccine *Mannheimia haemolytica challenge administered intranasally with disposable atomizer (5.0 mL; 2.5 mL/nostril) and 5.0 mL intratracheally with endoscope was delivered at the larynx/trachea **Mannheimia haemolytica challenge administered intratracheally by locating the bifurcation of the trachea and pulling back approximately 10 cm and delivering 60 mL with M. haemolytica strain

(57) TABLE-US-00012 TABLE 12 Mitigated Fraction Statistical Analysis of Lung Lesion Scores Group versus Group MF Lower Bound Upper Bound T04 T05 0.90 0.66 1.00 T04 T06 0.92 0.70 1.00 T04 T07 1.00 1.00 1.00 T01 T05 0.42 −0.06 0.83 T01 T06 0.44 −0.06 0.86 T01 T07 0.70 0.28 1.00

(58) Additionally, a combination inactivated vaccine containing both IDV and M. haemolytica antigens provided statistically significant protection (as measured by MF) to the dual challenge of lung lesion scoring that would be considered efficacious as the current statistical criteria outlines for M. haemolytica models used to license vaccines (>0.4 MF with positive upper and lower bounds). Monovalent vaccines of IDV (mean lung lesion score of 2.5) and M. haemolytica (mean lung lesion score of 0.9) provided a MF above the criteria but the upper and lower confidence bounds did not meet the satisfactory criteria. Therefore, a combination vaccine of IDV and M. haemolytica is an effective approach to controlling bovine respiratory disease.