Onset of Protection from Experimental Infection with Type 2 Bovine Viral Diarrhea Virus Following Vaccination with a Modified-Live Vaccine*

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1 Veterinary Therapeutics Vol. 8, No. 1, Spring 27 Onset of Protection from Experimental Infection with Type 2 Bovine Viral Diarrhea Virus Following Vaccination with a Modified-Live Vaccine* K. V. Brock, DVM, MS, PhD, DACVM a P. Widel, DVM, MS b P. Walz, DVM, MS, PhD, DACVIM c H. L. Walz, DVM, DACVP a a Department of Pathobiology College of Veterinary Medicine Auburn University, AL c Department of Clinical Sciences College of Veterinary Medicine Auburn University, AL b Boehringer Ingelheim, Inc. 556 Corporate Drive, Suite 16 St. Joseph, MO CLINICAL RELEVANCE The onset of protection after the administration of a modified-live bovine viral diarrhea virus (BVDV) vaccine was determined. Protection was determined following experimental infection with a virulent type 2 BVDV (strain 1373) in cattle vaccinated 3, 5, or 7 days before BVDV infection. Protection, as measured by reduced virus shedding, lack of leukopenia, reduction in viremia, and reduced mortality, was present as early as 3 days after vaccination with a single dose of modified-live BVDV vaccine. Complete protection was obtained in cattle vaccinated 5 or 7 days before BVDV experimental infection. INTRODUCTION Humoral and cell-mediated immune responses are known to be important in the protection against viral infections. In addition, although pathogen- and antigen-specific humoral and cell-mediated responses occur, the general innate immune response is also very important, especially during the acute or initial phase of viral replication and pathogenesis. 1 The innate immune response and the development *This work was supported by Boehringer Ingelheim, Inc., St. Joseph, MO. of a specific immune response following vaccination can be measured using several methods. Humoral response to vaccination against viruses is most commonly measured through the determination of serum virus neutralization antibody titers after vaccination. 2 4 The development of antibody titers can generally be detected as early as 7 to 1 days after vaccination. 5 Cell-mediated responses are more difficult to measure. Methods such as lymphocyte proliferation assays, T cell cytotoxicity assays, and cytokine assays can provide measurements of the 88

2 K. V. Brock, P. Widel, P. Walz, and H. L. Walz A significant level of protection was obtained in animals that were vaccinated 5 or 7 days before experimental challenge. specific components of the humoral and cellmediated response, 6,7 which can correlate to protection. Experimental studies comparing the effects of virus infection in different groups of vaccinated and nonvaccinated animals are an ideal way to measure the ability of a vaccine to elicit an effective immune response. Previous studies have characterized the response of animals immediately after vaccination against bovine herpesvirus-1. 8 These studies have shown that a cell-mediated response becomes effective within 72 hours after vaccination. 8 This response is most likely from the innate immune response and the early development of the cell-mediated immune response to vaccination. 8 Bovine viral diarrhea virus (BVDV) is a common pathogen of cattle, causing reproductive, enteric, and respiratory disease problems in cow calf production units and stocker and feedlot cattle. 9 The disease syndrome in cow calf production units is recognized primarily by reproductive disease and the subsequent problems associated with the birth of calves persistently infected with BVDV. Controlling BVDV in such situations involves the efficacious vaccination of cows, bulls, and heifers before breeding; using adequate biosecurity to ensure that no BVDV exposure occurs during the first trimester of gestation; and appropriate testing of newborn calves, if required. 1 When cattle leave the farm of origin, BVDV continues to cause disease as animals from different backgrounds are commingled. BVDV is recognized as a significant pathogen causing bovine respiratory tract disease in such situations. BVDV-associated disease primarily occurs when weaned calves are marketed and commingled as stocker cattle and into the feedyards. 11 In these environments, the ability to provide rapid, protective immune responses is essential. Modified-live vaccines generally provide faster immune responses than killed virus vaccines through virus replication that stimulates the innate immune response. 8 The objective of this experimental study was to characterize the response to modified-live BVDV vaccine immediately after administration. The purpose of the study was to determine whether a modified-live BVDV vaccine can elicit a protective response to BVDV infection within 72 hours after vaccination. This was accomplished by measuring specific responses to vaccination and by using an experimental BVDV challenge to determine the ability of vaccination to protect against BVDV infection at various postvaccination intervals. MATERIALS AND METHODS Animals Forty bulls (8 to 9 months of age) were used in the study. All animals were determined not to be persistently infected with BVDV by immunohistochemistry of ear notch biopsies. In addition, all animals were seronegative against BVDV (<1:5) as determined by serum neutralization assay to both type 1 and type 2 BVDV reference viruses. All animals were clinically normal at the onset of the study. Animals were randomly assigned to four treatment groups of 1 animals each using a random number generator (Research Randomizer, 89

3 Veterinary Therapeutics Vol. 8, No. 1, Spring 27 Study Design A group of 1 animals consisted of a treatment group. Vaccinations (Vx) were administered as follows: Day 3 Vx Group: Vaccinated 3 days before BVDV experimental challenge Day 5 Vx Group: Vaccinated 5 days before BVDV experimental challenge Day 7 Vx Group: Vaccinated 7 days before BVDV experimental challenge Nontreated Control Group: Animals were not vaccinated Vaccination The vaccine administered was a USDA-licensed stock material released for sale (Express 5, Boehringer Ingelheim). The Express 5 vaccine is a modified-live combination viral vaccine containing BVD type 1a (Singer strain) and type 2a (296 strain), bovine rhinotracheitis (IBR), parainfluenza 3 (PI 3 ), and bovine respiratory syncytial virus (BRSV). A single 2- ml dose was administered subcutaneously using a sterile 18-guage 1-inch needle for each animal according to manufacturers recommendations. Only one dose of vaccine was given at the designated time according to treatment group before BVDV experimental challenge. Virus Challenge All animals were experimentally infected with a noncytopathic type 2 BVDV isolate 1373 obtained from J. Ridpath, National Animal Disease Center (Ames, IA). Challenge inoculum consisted of an infected cell culture supernatant at cell culture 5% infective doses (CCID 5 )/ml of virus. The experimental intranasal viral infection was done by aerosolization of 5 ml of inoculum using a DeVilbiss aerosolizer (DeVilbiss, Somerset, PA) and a vacuum pump. Sampling Samples collected on postchallenge days, 4, 6, 7, 8, 1, 12, and 14 included whole blood, nasal swabs, and clotted blood for serum. Body temperatures were also recorded on postchallenge days, 4, 6, 7, 8, 1, 12, and 14. Individual body weights were obtained for all calves on postchallenge days, 6, 7, and 14 using an electronic scale. Observation Animals were observed daily; clinical scores and rectal temperatures were recorded for each calf on days and 4 through 14 (no data was collected on days 1 to 3). Clinical signs depression, dyspnea, nasal discharge, ocular discharge, appetite, coughing, and diarrhea were assigned a numeric value based on severity and were recorded on a clinical assessment form. Individuals observing the daily clinical signs after challenge were blinded to the group assignments of individual animals. Laboratory Analysis Total leukocyte counts and platelet counts were conducted using an automatic Coulter counter at the Auburn University College of Veterinary Medicine clinical pathology service laboratory. Leukocyte and platelet counts were recorded as numbers/cc. γ-interferon levels were determined by ELISA on serum collected on postchallenge days, 4, 7, and 12 (IFN- Gamma ELISA, BioSource [Invitrogen], Carlsbad, CA). Virus Isolation Virus isolation was done on nasal swabs, serum, and buffy coat cell preparations. Virus isolation was conducted using Marden Darby bovine kidney cells and 25 cm 2 cell culture flasks or 96-well tissue culture plates supplemented with 1% horse serum. Virus isolation was determined by detection of viral antigen 9

4 K. V. Brock, P. Widel, P. Walz, and H. L. Walz Mean Body Weight (lb) Significant Differences in Mean Body Weight Were Seen in the 3 Vx and 5 Vx Groups vs. Controls Day Day 14 Control Day 3 Vx Day 5 Vx Day 7 Vx Treatment Groups Figure 1. Body weights of animals in the various treatment groups determined on day and postchallenge day 14. Asterisks indicate treatment groups that were significantly different from the nonvaccinated control group (P <.5). by immunoperoxidase staining using a BVDVspecific monoclonal antibody. Necropsy Complete necropsies, including histopathologic examination of H&E fixed tissues, were performed on all animals that died or were euthanized during the course of the study. Gross examinations were performed within 12 hours of death. Statistical Analysis Statistical analysis was conducted using JMP 5.1 Statistical Discovery Software, (SAS Institute, Cary, NC). Analysis of variance and mean comparisons for all pairs was accomplished using Tukey Kramer honestly significant difference (HSD). Statistical significance was determined at P <.5. RESULTS Postchallenge determination of virus titers in * the challenge inoculum indicated that the virus titer of the inoculum was CCID 5 /ml. * During challenge, 5. ml of BVDV strain 1373 was inoculated into each animal on day. There were no observed postvaccination complications or evidence of clinical signs in any of the 3 vaccinated and 1 nonvaccinated animals. Body temperature varied among treatment groups, but the difference was not measurable. Clinical scores were higher in the nonvaccinated control animals but were not significantly increased (P >.5). Day and postchallenge day 14 body weights were compared between treatment groups (Figure 1). The mean differences in weights of the nonvaccinated, day 3 Vx, day 5 Vx, and day 7 Vx groups from day to day 14 were 34, 1, 25, and 2 lb, respectively. There was a significant difference in mean weights between the nonvaccinated control group and the 5 Vx and 7 Vx groups (P <.5). There was no significant difference between the nonvaccinated control group and the day 3 Vx group (P >.5). BVDV was isolated from serum, nasal swabs, and leukocytes after experimental infection. Virus was isolated from at least one sample type (serum, leukocytes, or nasal swab) from all animals in the nonvaccinated control group on at least one day during the 14 days after challenge (Figures 2 and 3). The ability to isolate virus from the animals vaccinated 3, 5, or 7 days before experimental infection was significantly reduced compared with the nonvaccinated controls for days 6, 7, and 8 (P <.5; Figure 3). BVDV was isolated from nasal 91

5 Veterinary Therapeutics Vol. 8, No. 1, Spring 27 swabs collected from all nonvaccinated control animals (Figure 2). Animals in the 3Vx group had limited virus isolation positive samples during the sampling period: Only four of 1 animals had a positive nasal swab during the sampling period. Mean total leukocyte counts were compared between treatment groups during the postchallenge sampling period (Figure 4). On postchallenge day 6, a transient leukopenia, which is characteristic for acute BVDV infection, was observed in the nonvaccinated control group. The characteristic leukopenia was not observed in any of the vaccinated groups, with the exception of two animals vaccinated on day 3 (these animals died); in addition, virus was isolated from nasal swabs and leukocytes from these same two animals. On postchallenge day 6, there was a significant difference in the mean leukocyte counts between the nonvaccinated animals and all the vaccinated treatment groups (Figure 4). On postchallenge days 7 and 8, there was a significant difference in mean leukocyte counts between the nonvaccinated treatment group and 5 Vx and 7 Vx groups. A significant difference in mean leukocyte count between the nonvaccinated treatment group and the 3 Vx No. Positive Figure 2. Virus isolation from nasal swabs collected after challenge. No. Positive Vx Group 5 Vx Group 3 Vx Group Control Group Virus Isolation from Nasal Swabs Virus Isolation from Buffy Coat Cell Preparations Vx Group 5 Vx Group 3 Vx Group Control Group Figure 3. Virus isolation from buffy coat cell preparations collected after challenge. group occurred only on postchallenge day 6. Platelet counts were compared between treatment groups during the postchallenge sampling period (Figure 5). There were no significant differences between the mean platelet counts among the different treatment groups. γ-interferon levels in serum samples were determined by ELISA and recorded as optical 92

6 K. V. Brock, P. Widel, P. Walz, and H. L. Walz No./ml 8, 7, 6, 5, 4, Leukopenia Occurred at Day 6 in the Nonvaccinated Control Group 3, 2, 1, * 3 Vx Group 5 Vx Group 7 Vx Group Control Group Figure 4. Comparison of mean leukocyte counts for the different treatment groups during the postchallenge sampling period. Note leukopenia occurring at day 6 in the nonvaccinated control group. Asterisk indicates significant difference between mean counts at day 6 (P <.5). density values using a 45 nm filter (OD 45nm ). Duplicate values were determined for each sample, and an average value for each sample was obtained for postchallenge sample collection days, 4, 7, and 12. Mean OD 45nm values were compared between the four different treatment groups (Figure 6); no trends or significant changes in values between treatment groups were detected. By postchallenge day 14, six of 1 nonvaccinated control animals died (n = 5) or required euthanasia (n = 1). By postchallenge day 14, two of 1 animals in the 3 Vx group died (n = 1) or required euthanasia (n = 1). None of the animals in the 5 Vx and 7 Vx groups died or were euthanized. Necropsies were performed on all animals that died or were euthanized. Gross lesions included oral mucosal ulcerations and mild ulcerations of the mucosa of the gastrointestinal tract, including the esophagus. Lungs were normal to mildly congested in appearance, with no evidence of bacterial pneumonia. No other gross lesions were evident. Histopathologic examination indicated lymphoid depletion in Peyer s patches, mesenteric lymph nodes, and the spleen. In two of the six calves, the lungs had minimal to mild multifocal infiltrates of small numbers of neutrophils, lymphocytes, and macrophages within the alveolar septa. BVDV antigen specific immunohistochemistry staining of formalinfixed tissues identified the presence of BVDV antigen in association with microscopic lesions in Peyer s patches. DISCUSSION The clinical and laboratory observations following experimental inoculation with type 2 BVDV isolate 1373 indicated that severe, acute BVDV infection occurred after experimental infection of seronegative animals. Based on the investigator s (K. V. B.) previous experience with other BVDV isolates using the same experimental challenge methods, the 1373 isolate induces more severe clinical disease with a significant level of postinfection mortality than seen with more typical BVDV infections. This is consistent with previous reports of experimental infection with the 1373 BVDV isolate. 12,13 In addition, severe disease was induced in normal weaned bulls approximately 8 to 9 months old. Previous reports of experimental infection using the 1373 isolate have been done in colostrum-deprived calves, with a similar pattern of disease severity and high mortality. 12 In the present study, a mortality rate of 6% (six of 1 bulls) was observed in nonvaccinated seronegative animals. This is consistent with clinical reports of severe outbreaks associated with similar BVDV isolates

7 Veterinary Therapeutics Vol. 8, No. 1, Spring 27 In the present study, intranasal aerosolization of the 1373 isolate induced acute disease and a resulting systemic BVDV infection. Gross and histopathologic examination did not reveal secondary pneumonia. Death losses were primarily due to BVDV systemic infection. Evaluation of clinical pathology data indicated that a transient leukopenia typical of acute BVDV infection occurred. However, in the present study, the type 2 isolate did not induce thrombocytopenia as has been reported previously, generally in No./ml 9, 8, 7, 6, 5, 4, 3, 2, 1, younger animals. 15 Experimental reproduction of thrombocytopenia has been achieved in colostrumdeprived calves. 15 It is interesting to note that the severe clinical disease and mortality that occurred following the experimental infection did not begin until approximately 12 days after infection. Laboratory data, such as viremia (detected in leukocytes, nasal swabs, and serum) and leukopenia (as determined by leukocyte counts), suggest that the disease peaked approximately 6 to 8 days after experimental infection. This type of pattern is typical of a bi-phasic febrile response of viral diseases. This delayed effect has not been the typical pattern of BVDV-induced diseases previously reported. It may be reasonable to speculate that the initial leukopenia was related to the primary phase of BVDV replication and that the subsequent demand created the severe lymphoid depletion seen histologically in most lymphoid tissues. Lymphoid depletion has been reported to occur in severe acute BVDV infections and has also been reported in classical mucosal disease. 16 It is likely that severe lymphoid depletion is associated with acute death. Ridpath and No Significant Difference in Mean Platelet Counts Occurred between Treatment Groups 3 Vx Group 5 Vx Group 7 Vx Group Control Group Figure 5. Comparison of mean platelet counts for the different treatment groups during the postchallenge sampling period. No significant difference in mean counts occurred between treatment groups. associates have reported that the lymphocytopathogenicity of BVDV isolates in vitro correlates with virulence. 16 Further characterization of the pathogenesis of the lymphoid depletion following BVDV infection is needed. To determine the effect of modified-live vaccination, various types of data were compared, including virus isolation results, clinical assessment, total leukocyte counts, and mortality. It is clear that vaccination had a positive effect on protecting animals from disease following experimental infection with the type 2 BVDV isolate From statistical evaluation of data between treatment groups, a significant level of protection was obtained in animals that were vaccinated 5 or 7 days before experimental challenge. Although there was some protection in animals vaccinated 3 days before the challenge, the difference cannot be considered significant based on mortality. From evaluation of virus isolation from nasal swabs, vaccination completely reduced nasal 94

8 K. V. Brock, P. Widel, P. Walz, and H. L. Walz Absorbance (45 nm) γ-interferon Levels 3 Vx Group 7 Vx Group 5 Vx Group Control Group Figure 6. Comparison of mean optical density values specific for γ-interferon in serum collected on the sample days as determined by ELISA assay. shedding of BVDV following experimental infection when animals were vaccinated 5 or 7 days before infection (Figure 3). Nasal shedding occurred in animals vaccinated 3 days before infection but was reduced compared with the nonvaccinated controls (four of 1 versus 1 of 1, respectively). The ability of vaccination to reduce nasal shedding in infected animals within days following vaccination can provide a benefit in reducing the spread of BVDV in commingled cattle. Therefore, modified-live vaccination of high-risk cattle may immediately reduce the potential for BVDV transmission within groups of cattle at risk of developing acute BVDV infections because of commingling or potential exposure to animals persistently infected with BVDV. Current requirements, as determined by Title 9 of the Code of Federal Regulations (9 CFR), for BVDV protection are based on preventing leukopenia and the ability to isolate BVDV from blood after challenge. These requirements are based on the fact that severe clinical disease does not typically occur after experimental BVDV challenge with most isolates. The subclinical nature of BVDV infections has made it difficult to observe and quantitate the level of protection afforded by various methods of vaccination and types of vaccines. Using a challenge strain such as the type isolate may provide an improved method of virus challenge to demonstrate protection as a result of an increased ability to measure differences in treatment groups following experimental challenge. It is likely that the protection observed in the 5 Vx and 7 Vx groups would not have been noted if a less-virulent BVDV challenge strain had been used. In addition, it is noteworthy that the animals that died in the 3 Vx group developed severe leukopenia that did not start to rebound until approximately 1 to 11 days after infection. These two animals were the only animals in the 3 Vx group in which BVDV was isolated from leukocytes and nasal swabs. Viremia was detected only once in two other animals in the 3 Vx group, and that was in the serum on day 6. Therefore, prevention of viremia and leukopenia may be important factors associated with the ability of vaccination to provide protection from BVDV infection. CONCLUSION Based on the results of the present study, it can be concluded that vaccination with modified-live virus vaccine immediately before virus exposure can provide benefits. Vaccination of animals with the modified-live BVDV vaccine (Express 5) 5 or 7 days before experimental infection induced rapid protection against the 95

9 Veterinary Therapeutics Vol. 8, No. 1, Spring 27 type 2 BVDV strain that resulted in severe mortality in unvaccinated animals. Vaccination 3 days before challenge provided measurable protection by preventing BVDV replication through the reduction of the level of viremia and the development of leukopenia after experimental infection. In addition, vaccination reduced the level of nasal shedding of virus following experimental inoculation. REFERENCES 1. Peterhans E, Jungi TW, Schweizer M: BVD virus and innate immunity. Biologicals 31:17 112, Jones L, Van Campen H, Zu ZC, Schnackel JA: Comparison of neutralizing antibodies to type 1a, 1b and 2 bovine viral diarrhea virus from experimentally infected and vaccinated cattle. Bovine Pract 35:137 14, Fulton RW, Ridpath JF, Saliki JT, et al: Bovine viral diarrhea virus (BVDV) 1b: Predominant BVDV subtype in calves with respiratory disease. Can J Vet Res 66:18, Hamers C, di Valentin E, Lecomte C, et al: Virus neutralizing antibodies against a 22 bovine isolates in vaccinated calves. Vet J 163:61 67, Cortese VS, Whittaker R, Ellis J, et al: Specificity and duration of neutralizing antibodies induced in healthy cattle after administration of a modified-live virus vaccine against bovine viral diarrhea. Am J Vet Res 59: , Lambot M, Douart A, Joris E, et al: Characterization of the immune response of cattle against non-cytopathic and cytopathic biotypes of bovine viral diarrhoea virus. J Gen Virol 78: , Endsley JJ, Quade MJ, Terhaar B, Roth JA: Bovine viral diarrhea virus type 1- and type 2-specific bovine T lymphocyte-subset responses following modified-live virus vaccination. Vet Ther 3: , Fairbanks KF, Campbell J, Chase CCL: Rapid onset of protection against infectious bovine rhinotracheitis with a modified-live virus multivalent vaccine. Vet Ther 5(1):17 25, Brock KV: The many faces of bovine viral diarrhea virus. Vet Clin North Am Food Anim Pract 2(1):1 3, Brock KV: Strategies for the control and prevention of bovine viral diarrhea virus. Vet Clin North Am Food Anim Pract 2(1):171 18, Brock KV, Grooms D: Reproductive consequences of bovine viral diarrhea virus infections, in Goyal SM, Ridpath JF (eds): Bovine Viral Diarrhea Virus: Diagnosis, Management and Control, Ames, IA, Blackwell Publishing, Liebler-Tenoria E, Ridpath JE, Neill JD: Distribution of viral antigen and development of lesions after experimental infection with highly virulent bovine viral diarrhea virus in calves. Am J Vet Res 63(11): , Stoffregen B, Bolin SR, Ridpath JF, Pohlenz J: Morphologic lesions in type 2 BVDV infections experimentally induced by strain BVDV recovered from a field case. Vet Microbiol 77: , Ridpath JF, Neill JD, Vilcek S, et al: Multiple outbreaks of severe acute BVDV in North America occurring between 1993 and 1995 linked to the same BVDV2 strain. Vet Micro 114:196 24, Corapi WV, Elliott RD, French TW, et al: Thrombocytopenia in young calves experimentally infected with noncytopathic bovine viral diarrhea virus. JAVMA 196(4): , Ridpath JF, Bendfeldt S, Neill JD, Liebler-Tenorio E: Lymphocytopathogenic activity in vitro correlates with high virulence in vivo for BVDV type 2 strains: Criteria for a third biotype of BVDV. Virus Res 118: 62 69,