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Vaccination with a modified-live bovine viral diarrhea virus (BVDV) type 1a vaccine completely protected calves against challenge with BVDV type 1b strains
Vaccine 29 (2011) 70–76
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Vaccination with a modified-live bovine viral diarrhea virus (BVDV) type 1a vaccine completely protected calves against challenge with BVDV type 1b strains Wenzhi Xue ∗ , Debra Mattick, Linda Smith, Jerry Umbaugh, Emilio Trigo Intervet/Schering-Plough Animal Health, 35500 W. 91st Street, De Soto, KS 66018, USA
a r t i c l e
i n f o
Article history: Received 24 June 2010 Received in revised form 24 September 2010 Accepted 6 October 2010 Available online 27 October 2010 Keywords: Modified-live virus vaccine Bovine viral diarrhea virus BVDV-1a BVDV-1b Cross protection Duration of immunity Vaccination/challenge study
a b s t r a c t Vaccination plays a significant role in the control of bovine viral diarrhea virus (BVDV) infection and spread. Recent studies revealed that type 1b is the predominant BVDV type 1 subgenotype, representing more than 75% of field isolates of BVDV-1. However, nearly all current, commercially available BVDV type 1 vaccines contain BVDV-1a strains. Previous studies have indicated that anti-BVDV sera, induced by BVDV-1a viruses, show less neutralization activity to BVDV-1b isolates than type 1a. Therefore, it is critically important to evaluate BVDV-1a vaccines in their ability to prevent BVDV-1b infection in calves. In current studies, calves were vaccinated subcutaneously, intradermally or intranasally with a single dose of a multivalent, modified-live viral vaccine containing a BVDV-1a strain, and were challenged with differing BVDV-1b strains to determine the efficacy and duration of immunity of the vaccine against these heterologous virus strains. Vaccinated calves, in all administration routes, were protected from respiratory disease caused by the BVDV-1b viruses, as indicated by significantly fewer clinical signs, lower rectal temperatures, reduced viral shedding and greater white blood cell counts than non-vaccinated control animals. The BVDV-1a vaccine elicited efficacious protection in calves against each BVDV-1b challenge strain, with a duration of immunity of at least 6 months. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Bovine viral diarrhea virus (BVDV), a pestivirus of the family Flaviviridae, is an important pathogen to the cattle industry, often resulting in severe economic losses [1,2]. BVDV-associated diseases can range from clinically inapparent to severe, and involve the respiratory, enteric, reproductive, immune and endocrine systems [3–6]. Infection with BVDV poses a major threat to the cattle industry, even though commercial BVDV vaccines are available. Antigenic diversity represents a major characteristic for BVDV, and is a primary contributor to the failure of the vaccination program in some cases. Genetically, BVDV isolates belong to one of the two genotypes: type 1 (BVDV-1) and type 2 (BVDV-2), currently considered as two distinct species of pestivirus [7]. Within the two distinct species of BVDV, the virus isolates have been further grouped into subgenotypes by phylogenetic analysis. At present, at least 13 BVDV-1 subgenotypes (BVDV-1a to BVDV-1l and an unassigned) and two BVDV-2 subgenotypes (BVDV-2a and BVDV-2b) have been differentiated [8–10]. Even prior to the establishment of BVDV-1 and BVDV-2 genotypes, antigenic differences were found among BVDV field isolates, and were considered as one reason for the occasional failure of the vaccination program [11,12]. BVDV vac-
cines, in use since the mid-to-late 1960s, have played a significant role in the control of various clinical diseases associated with BVDV infections, as well as the spread of the virus [13]. Before 2000, nearly all commercial BVDV vaccines contained only BVDV-1 strains. The recognition of the BVDV-2 genotype, due to its differences in gene sequence and antigenicity from BVDV-1, convinced the vaccine regulation agency and manufacturers to add BVDV-2 strains to BVDV vaccines for broader protection against BVDV. Recent studies have shown that although there are many subgenotypes of BVDV-1, BVDV-1b is the predominant subgenotype in BVDV field isolates, especially in North and South America [10,14,15]. Serum crossneutralization studies indicated differences between BVDV-1a and BVDV-1b strains when tested against homologous or heterologous sera [9,10]. It is not known if the variation among subgenotypes BVDV-1a and BVDV-1b is significant enough to impact the protection afforded by current BVDV vaccines, nearly all of which contain BVDV-1a strains, but no BVDV-1b strain. Therefore, the purpose of this study was to demonstrate the efficacy of a modified live viral (MLV) vaccine, containing BVDV-1a and BVDV-2 strains, administered either subcutaneously, intradermally or intranasally in calves, against infection with one of two BVDV-1b strains. 2. Materials and methods
∗ Corresponding author. Tel.: +1 913 422 6889; fax: +1 913 422 6074. E-mail address: [email protected] (W. Xue). 0264-410X/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2010.10.012
To determine the efficacy of a BVDV-1a MLV vaccine against infection by BVDV-1b strains, four separate immunogenicity stud-
W. Xue et al. / Vaccine 29 (2011) 70–76
ies were designed and conducted in colostrum-deprived (CD) calves of different ages. For each study, calves were vaccinated subcutaneously, intradermally or intranasally, and challenged with a BVDV-1b strain three to four weeks, or six months, postvaccination. 2.1. Calves Colostrum-deprived (CD) calves (purchased from J & R Livestock, Lake Mills, IA) were used in each of the studies. The calves were removed from dams at birth before taking colostrum, fed milk replacer and were housed in individual hutches before vaccination and in separated pens after vaccination. All calves were serum neutralizing (SN) antibody-free to BVDV at vaccination and free of BVDV persistent infection. 2.2. Vaccine and administration A combination vaccine,1 containing modified-live BVDV-1a and BVDV-2, infectious bovine rhinotracheitis (IBR) virus, bovine parainfluenza type 3 (PI-3) virus and bovine respiratory syncytial virus (BRSV), was used in each study. The BVDV-1a antigen in the vaccine was batched at its minimum protective dose level. In all studies, control animals received a placebo vaccine containing only sterile water diluent. Experiment #1. A total of 34 calves, ranging in age from 10 to 12 weeks, were enrolled in this study. Calves in the vaccinate group (n = 22) received a single 2 mL dose of the MLV vaccine administrated subcutaneously, and calves in the control group (n = 12) received a 2 mL dose of sterile water diluent. On day 28 post-vaccination, the calves were commingled and intranasally challenged with an aerosolized virulent BVDV-1b strain NY-1. Experiment #2. In this study, 14 3–8 day-old CD calves were intradermally vaccinated with a single 1 mL dose of the MLV vaccine, and 14 control calves received a 1 mL dose of sterile water diluent. Three weeks post-vaccination, the calves were commingled and intranasally challenged with an aerosolized virulent BVDV-1b strain NY-1. Experiment #3. This study was designed to measure the duration of immunity of intranasal vaccination with the BVDV-1a vaccine against BVDV-1b challenge. Fourteen CD calves, 6–8 weeks of age, were intranasally vaccinated with a single 2 mL dose of the MLV vaccine, and 14 control calves received a 2 mL dose of sterile water diluent. Six months post-vaccination, the calves were commingled and intranasally challenged with an aerosolized virulent BVDV-1b strain T1186a. Experiment #4. This experiment evaluated the duration of immunity of subcutaneous vaccination with the BVDV-1a vaccine against BVDV-1b challenge. Thirty calves, about 3 months of age, were registered in this study. Twenty calves were subcutaneously vaccinated with a single 2 mL dose of the MLV vaccine, and 10 control calves received a 2 mL dose of sterile water diluent. At day 183 post-vaccination, the calves were commingled and intranasally challenged with an aerosolized virulent BVDV-1b strain NY-1. 2.3. Challenge viruses BVDV strain NY-1, a non-cytopathic (ncp) BVDV-1b strain, was used as the challenge virus in the subcutaneous and intradermal immunogenicity experiments. It was also used for the duration of immunity trial of subcutaneous administration. BVDV strain T1186a [16], an ncp BVDV-1b strain, was used in the duration
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Vista 5TM SQ, Intervet Inc. Millsboro, DE.
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of immunity experiment for intranasal vaccination. These two BVDV-1b challenge strains were obtained from National Veterinary Services Laboratories (NVSL, Ames, IA) and used according to NVSL instructions. The challenge was performed by spraying 2 mL of virus into each nostril, using a Devilbiss Atomizer (Devilbiss, Somerset, PA). Each animal received approximately 1–5 × 106 50% tissue culture infectious dose (TCID50 ) of challenge virus in a total 4 mL volume.
2.4. Clinical assessment Calves were observed daily for clinical signs on days −1 and 0, prior to challenge and days 1 through 14 after challenge, as described previously [17]. Clinical assessments were made at the same time each morning by investigators who were blinded to the treatment groups in each study. Clinical signs monitored included depression, nasal and ocular discharges, diarrhea, coughing and rectal temperatures.
2.5. Sample collection Blood samples were collected for serum at the day of vaccination, challenge and 14 days post-challenge, and virus neutralizing (VN) antibody titers were determined to BVDV-1a, BVDV-1b and BVDV-2 strains. Deep nasal swab specimens were obtained from both nares from one day prior to challenge through 10 days post-challenge. After collection, swabs were placed in 3 mL of cold transport medium consisting of Dulbecco’s Modified Eagle Medium (HyClone, Logan, UT) supplemented with 10% horse serum (Sigma, St. Louis, MO), 1% gelatin (Fisher Scientific, Fair Lawn, NJ), 1× antibiotic antimycotic solution (Sigma, St. Louis, MO) and 30 g/mL gentamicin (Sigma, St. Louis, MO). All swab specimens were stored at −70 ◦ C or below until they were cultured for quantitative virus isolation. Blood was also collected from each calf using EDTA-coated tubes from day 1 or 2 pre-challenge through day 8 post-challenge, and white blood cell (WBC) and platelet counts were conducted by a clinical pathology laboratory (Physicians Reference Laboratory, Overland Park, KS).
2.6. Virus neutralizing antibody analysis The VN antibody titers to BVDV-1a, BVDV-1b and BVDV-2 were measured by use of a standard microtiter plate assay. BVDV cytopathic strains Singer (BVDV-1a), Illc (BVDV-1b) and 125cp (BVDV-2) were used as neutralizing viruses in the assay. Briefly, two-fold dilutions of each serum sample were made in 96-well U-bottom plates, in duplicate, and approximately 100–200 TCID50 (50% tissue culture infectious dose) of each respective virus was added to each serum dilution. The mixture was incubated for 1 h at 37 ◦ C and then was transferred to a 96-well cell culture plate with confluent monolayers of bovine kidney cells (MDBK cell lines purchased from ATCC). After 3 days of incubation at 37 ◦ C, with 5% CO2 , the plates were observed for cytopathic effect (CPE) of BVDV. The neutralizing antibody titer of each sample was determined using the Reed–Muench method. The endpoint dilutions reflected the highest dilution of serum that inhibited the replication of virus in cell culture. The serologic relatedness was simply expressed as percentage (P) of the heterologous VN value as compared with the homologous VN value using the formula, P = 100 × (BA/AA), where BA is the VN titer of serum from vaccinated calves against BVDV-1b or BVDV-2, and AA is the VN titer of serum from vaccinated calves against BVDV-1a [10].
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Table 1 Virus neutralizing antibody titers (GMTs) against BVDV strains in vaccinate groups.a Experiment
#1 #2 #3 #4
Day of challenge
14 days post-challenge
>BVDV-1a
>BVDV-1b
>BVDV-2
>BVDV-1a
>BVDV-1b
>BVDV-2
1651 42 86 1566
333 31 44 397
83 2 11 135
4291 3488 540 5226
1799 3210 779 7167
259 62 34 640
Experiment #1 = subcutaneous vaccination against challenge with BVDV-1b NY-1 strain. Experiment #2 = intradermal vaccination against challenge with BVDV-1b NY-1 strain. Experiment #3 = intranasal vaccination against BVDV-1b T1186a strain for duration of immunity. Experiment #4 = subcutaneous vaccination against BVDV-1b NY-1 strain for duration of immunity. a GMT = geometric mean titer (the reciprocal of the endpoint dilution); BVDV = bovine viral diarrhea virus.
2.7. Quantitative virus isolation Virus isolation from nasal swabs was conducted using bovine kidney cell monolayers in 96-well tissue culture plates. Briefly, following centrifugation of samples, the supernatants were used to infect cell monolayers for virus quantitation. After 3 days of incubation at 37 ◦ C, with 5% CO2 , titers (TCID50 /mL) of the recovered BVDV-1b viruses from nasal swabs were calculated by immunofluorescence of cell cultures stained by BVDV-1 typespecific monoclonal antibodies 4e1, a murine Mab was prepared as described previously [11].
Table 2. Based on a 100% VN value to the homologous BVDV-1a subgenotype, the average VN value to the heterologous BVDV1b subgenotype was 43% post-vaccination, and prior to challenge, ranged from 20% to 74% in the different experiments. Fourteen days after challenge with the BVDV-1b strain, the VN antibody titers against BVDV-1b increased dramatically, and the average VN value for the four studies was 104%, compared to the average VN value against the BVDV-1a strains. Meanwhile, the average VN value against the BVDV-2 strain remained the same before and after challenge. 3.2. Efficacy of subcutaneous vaccination against BVDV-1b NY-1
2.8. Data analysis In each study, the two treatment groups (vaccinate and control) were analyzed and compared with respect to the primary clinical signs including rectal temperature, nasal and ocular discharges, diarrhea, leukopenia and virus shedding, using PROC NPAR1WAY of SAS® Version 9.1.3 to perform the Fisher Exact test. The prevented fraction and associated confidence intervals were estimated. 3. Results 3.1. Serological response and serologic relatedness All calves were free of VN antibodies to both BVDV-1a and BVDV-1b at the time of vaccination, and control calves were VN antibody negative to the viruses prior to the challenge. Following vaccination with the BVDV-1a MLV vaccine, the highest geometric mean titer (GMT) of VN antibodies was elicited against the homologous BVDV1a strain, followed by the BVDV-1b and BVDV-2 strains, respectively (Table 1). Two weeks post-challenge, VN antibody titers were boosted in MLV vaccinates in all experiments, with VN titers to the BVDV-1b subgenotype similar to, or greater than, BVDV-1a VN titers. The average VN value for vaccinated calves from each experiment against BVDV-1a, BVDV-1b and BVDV-2 is shown in
All control calves (12/12) developed mild disease symptoms associated with BVDV-1b infection, including nasal discharge, coughing, diarrhea and depression. In comparison, fewer vaccinated calves (4/22) developed clinical signs from the challenge. Although the clinical signs were mild, the clinical scores of the control group were significantly higher (P < 0.05) than the vaccinated group at days 4–5 and 7–13 post-challenge (Fig. 1A). Post-challenge, all control calves showed elevated rectal temperatures, with peak temperatures at days 7–8 post-challenge (Fig. 1B). In contrast, the vaccinated calves did not develop fever at any time post-challenge. Additionally, WBC counts of control calves started decreasing from day 2 post-challenge. The mean WBC counts for the control group dropped significantly from 15.3 (1000 per mm3 ) at day −1 to 9.4 (1000 per mm3 ) at day 6 post-challenge, a decrease of approximately 40% (Fig. 1C). The mean WBC counts in the vaccinate group did not decrease during the challenge period, and were significantly higher (P < 0.05) than those of the control group at days 3–7 post-challenge. Nasal swab samples were collected from challenged calves from day −1 to day 10 post-challenge for the detection of virus shedding. All control calves shed BVDV on multiple days during the challenge period, with high titers of virus shed compared to MLVvaccinated calves, which shed virus for a much shorter period after
Table 2 Cross-neutralization as percentage of VN values against BVDV-1a.a Experiment
#1 #2 #3 #4 Average
Day of challenge
14 days post-challenge
>BVDV-1a
>BVDV-1b
>BVDV-2
>BVDV-1a
>BVDV-1b
>BVDV-2
100 100 100 100 100
20 74 52 25 43
5 6 13 9 8
100 100 100 100 100
42 92 144 137 104
6 2 6 12 7
Experiment #1 = subcutaneous vaccination against challenge with BVDV-1b NY-1 strain. Experiment #2 = intradermal vaccination against challenge with BVDV-1b NY-1 strain. Experiment #3 = intranasal vaccination against BVDV-1b T1186a strain for duration of immunity. Experiment #4 = subcutaneous vaccination against BVDV-1b NY-1 strain for duration of immunity. a VN = virus neutralization; BVDV = bovine viral diarrhea virus.
W. Xue et al. / Vaccine 29 (2011) 70–76
1A. Clinical Scores Vaccinate
Mean Scores
3
**
Control
2.5
**
2
* *
1.5
*
*
*
1
2
3
4
5
6
7
8
40
39 38.5
9 10 11 12 13 14
-1 0 1 2
12
* *
*
*
10 8
Vaccinate
6
Control
4
1D. Virus Shedding
2.5
Virus Titer (Logs)
WBC (x1000/mm3)
*
14
3 4 5 6 7 8 9 10 11 12 13 14
Days Post-challenge
1C. White Blood Cell Counts
16
*
39.5
Days Post-Challenge
18
*
Control
40.5
38
0 1
Vaccinate
41
** **
0.5 -1 0
1B. Rectal Temperatures
41.5
Temperature (C)
3.5
73
Vaccinate
2
*
Control
*
*
1.5
*
1 0.5 0
-1
0
1
2
3
4
5
6
7
8
Days Post-challenge
-1
0
1
2
3
4
5
6
7
8
9
10
0
Days Post-Challenge
Fig. 1. Subcutaneous vaccination with a BVDV-1a MLV vaccine protected calves from challenge with BVDV-1b strain NY-1. Specifically, the vaccinated calves were protected from clinical disease (A); elevated rectal temperatures (B); decrease of white blood cell counts (C); and virus shedding (D). Statistical values are *P < 0.05 and **P < 0.01.
challenge. Statistical analysis of the virus isolation results demonstrated that there were significantly (P < 0.01) less vaccinates that shed BVDV than controls. Furthermore, the BVDV titers from control animals were significantly (P < 0.05) higher than vaccinates at days 6–9 post-challenge, with peak viral shedding occurring at days 6–8 post-challenge (Fig. 1D).
Virus isolation from nasal swab samples demonstrated that all control calves (14/14) shed BVDV on various days post-challenge. In contrast, none (0/14) of the vaccinated calves shed virus. The prevention of virus shedding by the MLV vaccine was 100%. The titers of isolated viruses from controls peaked at days 6–8 postchallenge, and they were significantly (P < 0.05) higher than the vaccinate group at days 5–9 post-challenge (Fig. 2D).
3.3. Efficacy of intradermal vaccination against BVDV-1b NY-1
3.4. Duration of immunity of the vaccine against BVDV-1b T1186a
After challenge, all control calves (14/14) developed clinical disease associated with BVDV infection, including nasal discharge, coughing, diarrhea and depression on various days. Specifically, 13 of 14 control calves developed diarrhea, and only 1 of 14 vaccinated calves displayed diarrhea, which occurred on a single day (day 9 post-challenge). The prevented fraction for the diarrhea was 92%. Eight of 14 control calves showed nasal discharge compared to none of the vaccinated calves, indicating a 100% prevented fraction for nasal discharge. Total clinical scores of depression, diarrhea, nasal discharge and coughing were significantly higher (P < 0.05) in control animals than in vaccinated calves at days 6–10 post-challenge (Fig. 2A). The rectal temperatures of calves were measured daily from days −1 to 14 post-challenge. All control calves (14/14) had rectal temperatures higher than 40 ◦ C on several days, while none (0/14) of the vaccinated calves had rectal temperatures greater than 40 ◦ C. The prevention of fever was 100% by the MLV vaccine, with the vaccinate group having significantly (P < 0.01) lower rectal temperatures than the control calves post-challenge. Moreover, the mean rectal temperatures of control calves peaked at days 7–8 postchallenge and were significantly (P < 0.05) higher than the vaccinate group (Fig. 2B). WBC counts of control calves began decreasing from day 2 postchallenge. The mean WBC counts for the control group dropped significantly from 8.6 (1000 per mm3 ) at day −1 to 5.4 (1000 per mm3 ) at day 4 post-challenge (Fig. 2C). The mean WBC counts in the vaccinate group did not decrease during the challenge period, and were significantly higher (P < 0.05) than the control group at days 3–4 post-challenge.
Six months after intranasal vaccination with the BVDV-1a MLV vaccine, the calves were challenged with BVDV-1b strain T1186a, a challenge strain recommended by the USDA, which resulted in severe leukopenia compared to BVDV-1b challenge strain NY-1 [16]. After challenge, 14 of 14 control calves developed moderate clinical signs associated with BVDV infection, including mild leukopenia, decrease in platelet counts, nasal discharge and fever. Following challenge, 13 of 14 control calves displayed nasal discharge on differing days, compared to only one calf from the vaccinate group (P < 0.01), with a prevented fraction of 0.92 with a 95% confidence interval (Fig. 3A). All control calves displayed elevated rectal temperatures (>40 ◦ C), with several animals showing fever for multiple days after challenge. The temperatures of controls were slightly elevated at day 3 post-challenge and peaked at day 8 post-challenge. In comparison, only 3 of the 14 MLV-vaccinated calves developed temperatures >40 ◦ C. The proportion of calves with fever (>40 ◦ C) was significantly higher (P < 0.001) in controls compared to the vaccinate group. A prevention fraction of 0.786 was obtained for the vaccine group with a 95% confidence interval. Moreover, mean rectal temperatures of the control group were significantly higher (P < 0.05) than the vaccinate group at days 8–9 post-challenge (Fig. 3B). White blood cell counts of control calves started decreasing from day 3 post-challenge. The mean WBC counts for the control group dropped significantly from 14.8 (1000 per mm3 ) at day 0 to 7.2 (1000 per mm3 ) at day 5 post-challenge (Fig. 3C). The mean
W. Xue et al. / Vaccine 29 (2011) 70–76
2A. Clinical Scores
5 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0
Vaccinate
*
2 3
40.5
**
Control
-1 0 1
2B. Rectal Temperatures
**
4 5
*
*
6 7
Temperature (C)
Mean Scores
74
8 9 10 11 12 13 14
0
* *
40 39.5 39 38.5 Vaccinate
38
Control
37.5 -1 0
Days Post-Challenge
1
2 3
4
5 6
7 8
9 10 11 12 13 14
Days Post-challenge 2C. White Blood Cell Counts
9
*
8 7
*
6 5
Vaccinate
4
Control
3 -2
-1
0
1
2
3
4
5
6
2D. Virus Shedding
3.5
Virus Titer ( Logs)
WBC (x1000/mm3)
10
7
8
Days Post-challenge
Vaccinate
3
**
Control
2.5 2
*
1.5
**
*
** *
1 0.5 0 -1
0
1
2
3
4
5
6
7
8
9
10
Days Post-challenge
Fig. 2. Intradermal vaccination with a BVDV type 1a MLV vaccine protected calves from challenge with BVDV type 1b strain NY-1. Specifically, the vaccinated calves were protected from clinical disease (A); elevated rectal temperatures (B); decrease of white blood cell counts (C); and virus shedding (D). Statistical values are *P < 0.05 and **P < 0.01.
WBC count in the vaccinate group decreased less than 15% during the challenge period, and the WBC count was significantly higher (P < 0.05) than that of the control group at days 3–6 post-challenge. Platelet counts demonstrated a decrease of greater than 30% in the control group at day 8 post-challenge. In contrast, for vaccinated calves, the platelet counts decreased less than 10%. All animals in the control group shed virus at various days; however, only two vaccinated calves shed BVDV, at a single time point for each. The proportion of calves shedding virus was significantly greater (P < 0.001) in control animals than in the vaccinate group,
with an estimated prevented fraction for virus shedding of 0.857 in the MLV-vaccinated calves. The BVDV nasal swab titers from the control group peaked at day 7 post-challenge, and were significantly (P < 0.05) higher than the vaccinate group at days 5–9 post-challenge (Fig. 3D). 3.5. Duration of immunity of the vaccine against BVDV-1b NY-1 Six months following subcutaneous vaccination, the calves were challenged with BVDV-1b strain NY-1 to determine the duration of
Fig. 3. Intranasal vaccination with a BVDV type 1a MLV vaccine protected calves from challenge with BVDV type 1b strain T1186a, six months post-vaccination. Specifically, the vaccinated calves were protected from nasal discharge (A); elevated rectal temperatures (B); leukopenia (C); and virus shedding (D). Statistical values are *P < 0.05 and **P < 0.01.
W. Xue et al. / Vaccine 29 (2011) 70–76
4A. Clinical Scores
Mean Scores
2.5
Control
*
2 1.5
** * * *
*
1 0.5 0
4B. Rectal Temperatures
42
Vaccinate
41.5
Temperature (C)
3
75
Vaccinate
41
*
Control
40.5 39.5 39 38.5 38
-1 0 1
2 3 4
5 6
7 8
-1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
9 10 11 12 13 14
Days Post-challenge
Days Post-challenge
4C. White Blood Cell Counts 12
*
10
*
*
Virus Titer (Logs)
14
WBC (x1000 mm3 )
*
40
*
8 6
Vaccinate
4
Control
2 -1
0
1
2
3
4
5
6
7
8
Days Post-challenge
4D. Virus Shedding
2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0
**
Vaccinate
**
Control
-1
0
1
2
3
4
*
*
*
5
6
7
8
9
10
Days Post-challenge
Fig. 4. Subcutaneous vaccination with a BVDV type 1a MLV vaccine protected calves from challenge with BVDV type 1b strain NY-1, six months post-vaccination. Specifically, the vaccinated calves were protected from clinical disease (A); elevated rectal temperatures (B); leukopenia (C); and virus shedding (D). Statistical values are *P < 0.05 and **P < 0.01.
immunity. All 10 control calves developed moderate clinical signs associated with BVDV infection, including nasal discharge, coughing, diarrhea and depression. In contrast, the vaccinated calves developed few clinical signs from the challenge. The clinical scores of the control group were significantly higher (P < 0.05) than the vaccinated group at days 6–11 post-challenge (Fig. 4A). All control calves (10/10) developed fever, with elevated rectal temperatures >40 ◦ C, and several animals had fever for multiple days following challenge. In contrast, none of the vaccinated calves developed elevated temperatures of >40 ◦ C. Mean rectal temperatures of the control group were significantly higher (P < 0.05) than the vaccinate group at days 8–9 post-challenge (Fig. 4B). Two days after challenge, WBC counts of control calves started decreasing, reaching the lowest point at day 4 post-challenge, an approximate 45% of decrease. Furthermore, the mean WBC counts for the control group were significantly lower (P < 0.05) than the vaccinate group at days 4–7 post-challenge (Fig. 4C). All animals in the control group shed virus on various days; however, only 8 out of 20 vaccinated calves shed virus on a single day for each. The titers of isolated viruses from the control group were significantly (P < 0.05) higher than the vaccinate group at day 4 and days 6–9 post-challenge (Fig. 4D). 4. Discussion Currently available parenteral combination MLV vaccines against BVDV types 1 and 2 can clearly induce a protective response against BVDV infection and spread. However, BVDV vaccination programs occasionally fail, and this is believed to be the result of diversified BVDV antigenicity among strains and isolates, including variations between BVDV-1 and BVDV-2 genotypes, and even between subgenotypes of the BVDV-1 species [9,10,14]. For decades, majority of BVDV vaccines (both MLV and KV) only included BVDV-1a strains, such as Singer, NADL or Oregon C24V cytopathic strains, and very few vaccines contained non-cytopathic strains. The recognition of severe disease and fetal losses in vaccinated cows, associated with BVDV-2, led to the inclusion of
cytopathic BVDV-2 strains in most current, commercially available BVDV vaccines [18,19]. At present, there is an increasing concern pertaining to BVDV antigenic variation among BVDV-1 isolates. Recent studies revealed that most BVDV-1 field isolates (>75%) were of the BVDV-1b subgenotype, predominant to BVDV-1a in North America [10,14]. Cross-neutralization results showed that anti-BVDV antibodies induced by BVDV-1a strains have less neutralization power to heterologous BVDV-1b strains, with only 36% of the VN value to BVDV-1b, compared to the homologous BVDV-1a strains [10]. The studies in this paper demonstrated similar results as previously described with respect to VN antibody titers and relatedness of neutralizing antibody response to different BVDV-1 subgenotypes [9,10]. Interestingly, the BVDV-1a vaccine strain induced considerably high VN antibody titers to BVDV-1b in all experiments included in this study. Compared to the homologous BVDV-1a virus (100% VN value), calves in all four experiments displayed a lower overall average VN value (43%) to the BVDV-1b strains prior to challenge. This value is similar to the previously reported diversities between BVDV-1a and BVDV-1b subgenotypes [9,10]. Noticeably, the VN value against BVDV-1b in each experiment was different, ranging from 20% to 74%, and several factors may be attributable. First, the MLV vaccine contains only one BVDV-1a strain and the serum antibody titers induced by the vaccine may not have a broad neutralizing power to BVDV-1b strains, as indicated in a previous study where multiple BVDV-1a strains were used to create antiserum [10]. Secondly, different vaccine administration routes were involved in the studies, and the serological response might be different to the various routes. The third attributable factor may be the different ages of the calves at vaccination, as each might have responded differently to the antigen. Most interestingly, the BVDV-1a vaccinated calves boosted their VN antibody titers after challenge with BVDV-1b strains, with the BVDV-1b VN value increasing from 43% at challenge to 104% two weeks post-challenge. Therefore, it is likely that the BVDV-1a vaccine stimulates immune memory against the BVDV-1b subgenotype, as it does against the BVDV-1a subgenotype, although the primary
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serological response appears to be lower. As a result, this is sufficient enough for the BVDV-1a vaccine strain to protect calves against clinical diseases caused by BVDV-1b strains. In the current study, four separate experiments were conducted to measure the efficacy of a BVDV-1a MLV vaccine in the prevention of infection and clinical disease caused by BVDV-1b strains. Regardless of the route of immunization, vaccinated calves in all experiments were significantly spared of clinical disease following challenge with BVDV-1b strain, NY-1 or T1186a. Generally, when WBC counts decrease less than 40% following BVDV infection, the infecting virus is considered low-virulent [16,20]. Moreover, if a BVDV strain can induce a WBC decrease greater than 40%, the virus strain is considered a high-virulent strain [21,22]. BVDV-1b strains NY-1 and T1186a were used as challenge viruses in these experiments, because they are the most commonly used BVDV-1 challenge virus strains for BVDV vaccine development in the U.S., and can be obtained from the National Animal Diseases Laboratories (NADL, USDA). As a result, the ability of the BVDV-1a vaccine to prevent calves from developing leukopenia against these strains further demonstrated the efficacy of the MLV vaccine against BVDV1b infection. These studies demonstrated the efficacy of the MLV vaccine when it was administrated subcutaneously or intradermally. A previous study indicated that the vaccine is equally efficacious when administered to 3–8-day-old young calves intranasally [17]. Additionally, the duration of immunity experiment showed that six months after intranasal or subcutaneous vaccination, the vaccine was still able to provide protection against BVDV-1b challenge, indicating that the BVDV-1a vaccine can protect against BVDV-1b infection at least six months beyond the date of primary vaccination. Currently, more than 75% of BVDV-1 isolates belong to the BVDV-1b subgenotype in North America [10,14]. We assume there might be slight antigenic diversity among the BVDV-1b strains, although there is no data to support this conclusion. However, two different BVDV-1b challenge virus strains were used in the duration of immunity experiments, and vaccinates demonstrated similar efficacies against each of the different challenge strains, confirming protection provided by the BVDV-1a vaccine against the BVDV-1b subgenotype. A feature of BVDV is that it can cause immunosuppression in the infected animal. The majority of current, commercially available BVDV vaccines contain both BVDV-1a and BVDV-2 strains. There is a concern that immunosuppression may be a byproduct of vaccination. As a result, there must be careful consideration taken to the possible addition of BVDV-1b strains to existing vaccines, because of the assumption that the more BVDV strains in a MLV vaccine, the greater the risk the vaccine may have in causing immunosuppression in the vaccinated animals [10,14]. Acknowledgements All host animal studies in this manuscript were conducted at the Intervet, Inc. animal research facility, following all animal treat-
ment regulations from USA Registrations. The authors thank all technicians involved in animal care and sample collection. The authors also thank David Whiteman for statistical data analysis and Ryan Brady for help in the manuscript preparation. References [1] Duffell SJ, Harkness JW. Bovine viral diarrhoea-mucosal disease infection in cattle. Vet Rec 1985;117:240–5. [2] Houe H. Epidemiological features and economical importance of bovine virus diarrhea virus (BVDV) infections. Vet Microbiol 1999;64:89–107. [3] Baker JC. Bovine viral diarrhea virus: a review. J Am Vet Med Assoc 1987;190:1449–58. [4] Baker JC. The clinical manifestations of bovine viral diarrhea infection. Vet Clin North Am Food Anim Pract 1995;11:425–45. [5] Bolin SR. Control of bovine viral diarrhea infection by use of vaccination. Vet Clin North Am 1995;11:616–25. [6] Radostits OM, Littlejohns IR. New concepts in the pathogenesis, diagnosis and control of diseases caused by the bovine viral diarrhea virus. Can Vet J 1988;29:513–28. [7] Thiel HJ, Collett MS, Gould EA. Family Flaviviridae. In: Fauquet CM, Mayo MA, Maniloff J, editors. Eighth report of the international committee on taxonomy of viruses. San Diego, CA: Elsevier Academic Press; 2005. p. 981–98. [8] Vilcek S, Paton DJ, Durkovic B, Strojny L, Ibata G, Moussa A, et al. Bovine viral diarrhoea virus genotype 1 can be separated into at least eleven genetic groups. Arch Virol 2001;146:99–115. [9] Bachofen C, Stalder H, Braun U, Hilbe M, Ehrensperger F, Peterhans E. Coexistence of genetically and antigenically diverse bovine viral diarrhoea viruses in an endemic situation. Vet Microbiol 2008;131:93–102. [10] Ridpath JF, Fulton RW, Kirkland PD, Neil JD. Prevalence and antigenic differences observed between bovine viral diarrhea virus subgenotypes isolated from cattle in Australia and feedlots in the southwestern United States. J Vet Diagn Invest 2010;22:184–91. [11] Corapi WV, Donis RO, Dubovi EJ. Characterization of a panel of monoclonal antibodies and their use in the study of the antigenic diversity of bovine viral diarrhea virus. Am J Vet Res 1990;51:1388–94. [12] Xue W, Blecha F, Minocha HC. Antigenic variations in bovine viral diarrhea viruses detected by monoclonal antibodies. J Clin Microbiol 1990;28:1688–93. [13] Deregt D. Introduction and history. In: Goyal SM, Ridpath JF, editors. Bovine viral diarrhea virus: diagnosis, management and control. Ames, IA: Blackwell Publishing; 2005. p. 3–34. [14] Fulton RW, Ridpath JF, Saliki JT, Briggs RE, Confer AW, Burge LJ, et al. Bovine viral diarrhea virus (BVDV) 1b: predominant BVDV subtype in calves with respiratory disease. Can J Vet Res 2002;66:181–90. [15] Stahl K, Benito F, Felmer R, Zuniga J, Reinhardt G, Rivera H, et al. Genetic diversity of bovine viral diarrhoea virus (BVDV) from Peru and Chile. Pesq Vet Bras 2009;29:41–4. [16] Ridpath JF, Neill JD, Peterhans E. Impact of variation in acute virulence of BVDV1 strains on design of better vaccine efficacy challenge models. Vaccine 2007;25:8058–66. [17] Xue W, Ellis J, Mattick D, Smith L, Brady R, Trigo E. Immunogenicity of a modified-live virus vaccine against bovine viral diarrhea virus types 1 and 2, infectious bovine rhinotracheitis virus, bovine parainfluenza-3 virus, and bovine respiratory syncytial virus when administered intranasally in young calves. Vaccine 2010;28:3784–92. [18] Van Campen H, Vorpahl P, Huzurbazar S, Edwards J, Cavender J. A case report: evidence for BVDV type 2-associated disease in beef herds vaccinated with a MLV-BVDV type 1 vaccine. J Vet Diagn Invest 2000;12:263–5. [19] Van Campen H. Epidemiology and control of BVD in the U.S. Vet Microbiol 2010;142:94–8. [20] Liebler-Tenorio EM, Ridpath JF, Neill JD. Distribution of viral antigen and development of lessons after experimental infection of calves a BVDV 2 strain of low virulence. J Vet Diagn Invest 2003;15:221–32. [21] Bolin SR, Ridpath JF. Differences in virulence between two non-cytopathic bovine viral diarrhea viruses in calves. Am J Vet Res 1992;53:2163–75. [22] Liebler-Tenorio EM, Ridpath JF, Neill JD. Distribution of viral antigen and development of lessons after experimental infection with highly virulent bovine viral diarrhea virus type 2 in calves. Am J Vet Res 2002;63:1575–84.
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Vaccination with a modified-live bovine viral diarrhea virus (BVDV) type 1a vaccine completely protected calves against challenge with BVDV type 1b strains Wenzhi Xue ∗ , Debra Mattick, Linda Smith, Jerry Umbaugh, Emilio Trigo Intervet/Schering-Plough Animal Health, 35500 W. 91st Street, De Soto, KS 66018, USA
a r t i c l e
i n f o
Article history: Received 24 June 2010 Received in revised form 24 September 2010 Accepted 6 October 2010 Available online 27 October 2010 Keywords: Modified-live virus vaccine Bovine viral diarrhea virus BVDV-1a BVDV-1b Cross protection Duration of immunity Vaccination/challenge study
a b s t r a c t Vaccination plays a significant role in the control of bovine viral diarrhea virus (BVDV) infection and spread. Recent studies revealed that type 1b is the predominant BVDV type 1 subgenotype, representing more than 75% of field isolates of BVDV-1. However, nearly all current, commercially available BVDV type 1 vaccines contain BVDV-1a strains. Previous studies have indicated that anti-BVDV sera, induced by BVDV-1a viruses, show less neutralization activity to BVDV-1b isolates than type 1a. Therefore, it is critically important to evaluate BVDV-1a vaccines in their ability to prevent BVDV-1b infection in calves. In current studies, calves were vaccinated subcutaneously, intradermally or intranasally with a single dose of a multivalent, modified-live viral vaccine containing a BVDV-1a strain, and were challenged with differing BVDV-1b strains to determine the efficacy and duration of immunity of the vaccine against these heterologous virus strains. Vaccinated calves, in all administration routes, were protected from respiratory disease caused by the BVDV-1b viruses, as indicated by significantly fewer clinical signs, lower rectal temperatures, reduced viral shedding and greater white blood cell counts than non-vaccinated control animals. The BVDV-1a vaccine elicited efficacious protection in calves against each BVDV-1b challenge strain, with a duration of immunity of at least 6 months. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Bovine viral diarrhea virus (BVDV), a pestivirus of the family Flaviviridae, is an important pathogen to the cattle industry, often resulting in severe economic losses [1,2]. BVDV-associated diseases can range from clinically inapparent to severe, and involve the respiratory, enteric, reproductive, immune and endocrine systems [3–6]. Infection with BVDV poses a major threat to the cattle industry, even though commercial BVDV vaccines are available. Antigenic diversity represents a major characteristic for BVDV, and is a primary contributor to the failure of the vaccination program in some cases. Genetically, BVDV isolates belong to one of the two genotypes: type 1 (BVDV-1) and type 2 (BVDV-2), currently considered as two distinct species of pestivirus [7]. Within the two distinct species of BVDV, the virus isolates have been further grouped into subgenotypes by phylogenetic analysis. At present, at least 13 BVDV-1 subgenotypes (BVDV-1a to BVDV-1l and an unassigned) and two BVDV-2 subgenotypes (BVDV-2a and BVDV-2b) have been differentiated [8–10]. Even prior to the establishment of BVDV-1 and BVDV-2 genotypes, antigenic differences were found among BVDV field isolates, and were considered as one reason for the occasional failure of the vaccination program [11,12]. BVDV vac-
cines, in use since the mid-to-late 1960s, have played a significant role in the control of various clinical diseases associated with BVDV infections, as well as the spread of the virus [13]. Before 2000, nearly all commercial BVDV vaccines contained only BVDV-1 strains. The recognition of the BVDV-2 genotype, due to its differences in gene sequence and antigenicity from BVDV-1, convinced the vaccine regulation agency and manufacturers to add BVDV-2 strains to BVDV vaccines for broader protection against BVDV. Recent studies have shown that although there are many subgenotypes of BVDV-1, BVDV-1b is the predominant subgenotype in BVDV field isolates, especially in North and South America [10,14,15]. Serum crossneutralization studies indicated differences between BVDV-1a and BVDV-1b strains when tested against homologous or heterologous sera [9,10]. It is not known if the variation among subgenotypes BVDV-1a and BVDV-1b is significant enough to impact the protection afforded by current BVDV vaccines, nearly all of which contain BVDV-1a strains, but no BVDV-1b strain. Therefore, the purpose of this study was to demonstrate the efficacy of a modified live viral (MLV) vaccine, containing BVDV-1a and BVDV-2 strains, administered either subcutaneously, intradermally or intranasally in calves, against infection with one of two BVDV-1b strains. 2. Materials and methods
∗ Corresponding author. Tel.: +1 913 422 6889; fax: +1 913 422 6074. E-mail address: [email protected] (W. Xue). 0264-410X/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2010.10.012
To determine the efficacy of a BVDV-1a MLV vaccine against infection by BVDV-1b strains, four separate immunogenicity stud-
W. Xue et al. / Vaccine 29 (2011) 70–76
ies were designed and conducted in colostrum-deprived (CD) calves of different ages. For each study, calves were vaccinated subcutaneously, intradermally or intranasally, and challenged with a BVDV-1b strain three to four weeks, or six months, postvaccination. 2.1. Calves Colostrum-deprived (CD) calves (purchased from J & R Livestock, Lake Mills, IA) were used in each of the studies. The calves were removed from dams at birth before taking colostrum, fed milk replacer and were housed in individual hutches before vaccination and in separated pens after vaccination. All calves were serum neutralizing (SN) antibody-free to BVDV at vaccination and free of BVDV persistent infection. 2.2. Vaccine and administration A combination vaccine,1 containing modified-live BVDV-1a and BVDV-2, infectious bovine rhinotracheitis (IBR) virus, bovine parainfluenza type 3 (PI-3) virus and bovine respiratory syncytial virus (BRSV), was used in each study. The BVDV-1a antigen in the vaccine was batched at its minimum protective dose level. In all studies, control animals received a placebo vaccine containing only sterile water diluent. Experiment #1. A total of 34 calves, ranging in age from 10 to 12 weeks, were enrolled in this study. Calves in the vaccinate group (n = 22) received a single 2 mL dose of the MLV vaccine administrated subcutaneously, and calves in the control group (n = 12) received a 2 mL dose of sterile water diluent. On day 28 post-vaccination, the calves were commingled and intranasally challenged with an aerosolized virulent BVDV-1b strain NY-1. Experiment #2. In this study, 14 3–8 day-old CD calves were intradermally vaccinated with a single 1 mL dose of the MLV vaccine, and 14 control calves received a 1 mL dose of sterile water diluent. Three weeks post-vaccination, the calves were commingled and intranasally challenged with an aerosolized virulent BVDV-1b strain NY-1. Experiment #3. This study was designed to measure the duration of immunity of intranasal vaccination with the BVDV-1a vaccine against BVDV-1b challenge. Fourteen CD calves, 6–8 weeks of age, were intranasally vaccinated with a single 2 mL dose of the MLV vaccine, and 14 control calves received a 2 mL dose of sterile water diluent. Six months post-vaccination, the calves were commingled and intranasally challenged with an aerosolized virulent BVDV-1b strain T1186a. Experiment #4. This experiment evaluated the duration of immunity of subcutaneous vaccination with the BVDV-1a vaccine against BVDV-1b challenge. Thirty calves, about 3 months of age, were registered in this study. Twenty calves were subcutaneously vaccinated with a single 2 mL dose of the MLV vaccine, and 10 control calves received a 2 mL dose of sterile water diluent. At day 183 post-vaccination, the calves were commingled and intranasally challenged with an aerosolized virulent BVDV-1b strain NY-1. 2.3. Challenge viruses BVDV strain NY-1, a non-cytopathic (ncp) BVDV-1b strain, was used as the challenge virus in the subcutaneous and intradermal immunogenicity experiments. It was also used for the duration of immunity trial of subcutaneous administration. BVDV strain T1186a [16], an ncp BVDV-1b strain, was used in the duration
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of immunity experiment for intranasal vaccination. These two BVDV-1b challenge strains were obtained from National Veterinary Services Laboratories (NVSL, Ames, IA) and used according to NVSL instructions. The challenge was performed by spraying 2 mL of virus into each nostril, using a Devilbiss Atomizer (Devilbiss, Somerset, PA). Each animal received approximately 1–5 × 106 50% tissue culture infectious dose (TCID50 ) of challenge virus in a total 4 mL volume.
2.4. Clinical assessment Calves were observed daily for clinical signs on days −1 and 0, prior to challenge and days 1 through 14 after challenge, as described previously [17]. Clinical assessments were made at the same time each morning by investigators who were blinded to the treatment groups in each study. Clinical signs monitored included depression, nasal and ocular discharges, diarrhea, coughing and rectal temperatures.
2.5. Sample collection Blood samples were collected for serum at the day of vaccination, challenge and 14 days post-challenge, and virus neutralizing (VN) antibody titers were determined to BVDV-1a, BVDV-1b and BVDV-2 strains. Deep nasal swab specimens were obtained from both nares from one day prior to challenge through 10 days post-challenge. After collection, swabs were placed in 3 mL of cold transport medium consisting of Dulbecco’s Modified Eagle Medium (HyClone, Logan, UT) supplemented with 10% horse serum (Sigma, St. Louis, MO), 1% gelatin (Fisher Scientific, Fair Lawn, NJ), 1× antibiotic antimycotic solution (Sigma, St. Louis, MO) and 30 g/mL gentamicin (Sigma, St. Louis, MO). All swab specimens were stored at −70 ◦ C or below until they were cultured for quantitative virus isolation. Blood was also collected from each calf using EDTA-coated tubes from day 1 or 2 pre-challenge through day 8 post-challenge, and white blood cell (WBC) and platelet counts were conducted by a clinical pathology laboratory (Physicians Reference Laboratory, Overland Park, KS).
2.6. Virus neutralizing antibody analysis The VN antibody titers to BVDV-1a, BVDV-1b and BVDV-2 were measured by use of a standard microtiter plate assay. BVDV cytopathic strains Singer (BVDV-1a), Illc (BVDV-1b) and 125cp (BVDV-2) were used as neutralizing viruses in the assay. Briefly, two-fold dilutions of each serum sample were made in 96-well U-bottom plates, in duplicate, and approximately 100–200 TCID50 (50% tissue culture infectious dose) of each respective virus was added to each serum dilution. The mixture was incubated for 1 h at 37 ◦ C and then was transferred to a 96-well cell culture plate with confluent monolayers of bovine kidney cells (MDBK cell lines purchased from ATCC). After 3 days of incubation at 37 ◦ C, with 5% CO2 , the plates were observed for cytopathic effect (CPE) of BVDV. The neutralizing antibody titer of each sample was determined using the Reed–Muench method. The endpoint dilutions reflected the highest dilution of serum that inhibited the replication of virus in cell culture. The serologic relatedness was simply expressed as percentage (P) of the heterologous VN value as compared with the homologous VN value using the formula, P = 100 × (BA/AA), where BA is the VN titer of serum from vaccinated calves against BVDV-1b or BVDV-2, and AA is the VN titer of serum from vaccinated calves against BVDV-1a [10].
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Table 1 Virus neutralizing antibody titers (GMTs) against BVDV strains in vaccinate groups.a Experiment
#1 #2 #3 #4
Day of challenge
14 days post-challenge
>BVDV-1a
>BVDV-1b
>BVDV-2
>BVDV-1a
>BVDV-1b
>BVDV-2
1651 42 86 1566
333 31 44 397
83 2 11 135
4291 3488 540 5226
1799 3210 779 7167
259 62 34 640
Experiment #1 = subcutaneous vaccination against challenge with BVDV-1b NY-1 strain. Experiment #2 = intradermal vaccination against challenge with BVDV-1b NY-1 strain. Experiment #3 = intranasal vaccination against BVDV-1b T1186a strain for duration of immunity. Experiment #4 = subcutaneous vaccination against BVDV-1b NY-1 strain for duration of immunity. a GMT = geometric mean titer (the reciprocal of the endpoint dilution); BVDV = bovine viral diarrhea virus.
2.7. Quantitative virus isolation Virus isolation from nasal swabs was conducted using bovine kidney cell monolayers in 96-well tissue culture plates. Briefly, following centrifugation of samples, the supernatants were used to infect cell monolayers for virus quantitation. After 3 days of incubation at 37 ◦ C, with 5% CO2 , titers (TCID50 /mL) of the recovered BVDV-1b viruses from nasal swabs were calculated by immunofluorescence of cell cultures stained by BVDV-1 typespecific monoclonal antibodies 4e1, a murine Mab was prepared as described previously [11].
Table 2. Based on a 100% VN value to the homologous BVDV-1a subgenotype, the average VN value to the heterologous BVDV1b subgenotype was 43% post-vaccination, and prior to challenge, ranged from 20% to 74% in the different experiments. Fourteen days after challenge with the BVDV-1b strain, the VN antibody titers against BVDV-1b increased dramatically, and the average VN value for the four studies was 104%, compared to the average VN value against the BVDV-1a strains. Meanwhile, the average VN value against the BVDV-2 strain remained the same before and after challenge. 3.2. Efficacy of subcutaneous vaccination against BVDV-1b NY-1
2.8. Data analysis In each study, the two treatment groups (vaccinate and control) were analyzed and compared with respect to the primary clinical signs including rectal temperature, nasal and ocular discharges, diarrhea, leukopenia and virus shedding, using PROC NPAR1WAY of SAS® Version 9.1.3 to perform the Fisher Exact test. The prevented fraction and associated confidence intervals were estimated. 3. Results 3.1. Serological response and serologic relatedness All calves were free of VN antibodies to both BVDV-1a and BVDV-1b at the time of vaccination, and control calves were VN antibody negative to the viruses prior to the challenge. Following vaccination with the BVDV-1a MLV vaccine, the highest geometric mean titer (GMT) of VN antibodies was elicited against the homologous BVDV1a strain, followed by the BVDV-1b and BVDV-2 strains, respectively (Table 1). Two weeks post-challenge, VN antibody titers were boosted in MLV vaccinates in all experiments, with VN titers to the BVDV-1b subgenotype similar to, or greater than, BVDV-1a VN titers. The average VN value for vaccinated calves from each experiment against BVDV-1a, BVDV-1b and BVDV-2 is shown in
All control calves (12/12) developed mild disease symptoms associated with BVDV-1b infection, including nasal discharge, coughing, diarrhea and depression. In comparison, fewer vaccinated calves (4/22) developed clinical signs from the challenge. Although the clinical signs were mild, the clinical scores of the control group were significantly higher (P < 0.05) than the vaccinated group at days 4–5 and 7–13 post-challenge (Fig. 1A). Post-challenge, all control calves showed elevated rectal temperatures, with peak temperatures at days 7–8 post-challenge (Fig. 1B). In contrast, the vaccinated calves did not develop fever at any time post-challenge. Additionally, WBC counts of control calves started decreasing from day 2 post-challenge. The mean WBC counts for the control group dropped significantly from 15.3 (1000 per mm3 ) at day −1 to 9.4 (1000 per mm3 ) at day 6 post-challenge, a decrease of approximately 40% (Fig. 1C). The mean WBC counts in the vaccinate group did not decrease during the challenge period, and were significantly higher (P < 0.05) than those of the control group at days 3–7 post-challenge. Nasal swab samples were collected from challenged calves from day −1 to day 10 post-challenge for the detection of virus shedding. All control calves shed BVDV on multiple days during the challenge period, with high titers of virus shed compared to MLVvaccinated calves, which shed virus for a much shorter period after
Table 2 Cross-neutralization as percentage of VN values against BVDV-1a.a Experiment
#1 #2 #3 #4 Average
Day of challenge
14 days post-challenge
>BVDV-1a
>BVDV-1b
>BVDV-2
>BVDV-1a
>BVDV-1b
>BVDV-2
100 100 100 100 100
20 74 52 25 43
5 6 13 9 8
100 100 100 100 100
42 92 144 137 104
6 2 6 12 7
Experiment #1 = subcutaneous vaccination against challenge with BVDV-1b NY-1 strain. Experiment #2 = intradermal vaccination against challenge with BVDV-1b NY-1 strain. Experiment #3 = intranasal vaccination against BVDV-1b T1186a strain for duration of immunity. Experiment #4 = subcutaneous vaccination against BVDV-1b NY-1 strain for duration of immunity. a VN = virus neutralization; BVDV = bovine viral diarrhea virus.
W. Xue et al. / Vaccine 29 (2011) 70–76
1A. Clinical Scores Vaccinate
Mean Scores
3
**
Control
2.5
**
2
* *
1.5
*
*
*
1
2
3
4
5
6
7
8
40
39 38.5
9 10 11 12 13 14
-1 0 1 2
12
* *
*
*
10 8
Vaccinate
6
Control
4
1D. Virus Shedding
2.5
Virus Titer (Logs)
WBC (x1000/mm3)
*
14
3 4 5 6 7 8 9 10 11 12 13 14
Days Post-challenge
1C. White Blood Cell Counts
16
*
39.5
Days Post-Challenge
18
*
Control
40.5
38
0 1
Vaccinate
41
** **
0.5 -1 0
1B. Rectal Temperatures
41.5
Temperature (C)
3.5
73
Vaccinate
2
*
Control
*
*
1.5
*
1 0.5 0
-1
0
1
2
3
4
5
6
7
8
Days Post-challenge
-1
0
1
2
3
4
5
6
7
8
9
10
0
Days Post-Challenge
Fig. 1. Subcutaneous vaccination with a BVDV-1a MLV vaccine protected calves from challenge with BVDV-1b strain NY-1. Specifically, the vaccinated calves were protected from clinical disease (A); elevated rectal temperatures (B); decrease of white blood cell counts (C); and virus shedding (D). Statistical values are *P < 0.05 and **P < 0.01.
challenge. Statistical analysis of the virus isolation results demonstrated that there were significantly (P < 0.01) less vaccinates that shed BVDV than controls. Furthermore, the BVDV titers from control animals were significantly (P < 0.05) higher than vaccinates at days 6–9 post-challenge, with peak viral shedding occurring at days 6–8 post-challenge (Fig. 1D).
Virus isolation from nasal swab samples demonstrated that all control calves (14/14) shed BVDV on various days post-challenge. In contrast, none (0/14) of the vaccinated calves shed virus. The prevention of virus shedding by the MLV vaccine was 100%. The titers of isolated viruses from controls peaked at days 6–8 postchallenge, and they were significantly (P < 0.05) higher than the vaccinate group at days 5–9 post-challenge (Fig. 2D).
3.3. Efficacy of intradermal vaccination against BVDV-1b NY-1
3.4. Duration of immunity of the vaccine against BVDV-1b T1186a
After challenge, all control calves (14/14) developed clinical disease associated with BVDV infection, including nasal discharge, coughing, diarrhea and depression on various days. Specifically, 13 of 14 control calves developed diarrhea, and only 1 of 14 vaccinated calves displayed diarrhea, which occurred on a single day (day 9 post-challenge). The prevented fraction for the diarrhea was 92%. Eight of 14 control calves showed nasal discharge compared to none of the vaccinated calves, indicating a 100% prevented fraction for nasal discharge. Total clinical scores of depression, diarrhea, nasal discharge and coughing were significantly higher (P < 0.05) in control animals than in vaccinated calves at days 6–10 post-challenge (Fig. 2A). The rectal temperatures of calves were measured daily from days −1 to 14 post-challenge. All control calves (14/14) had rectal temperatures higher than 40 ◦ C on several days, while none (0/14) of the vaccinated calves had rectal temperatures greater than 40 ◦ C. The prevention of fever was 100% by the MLV vaccine, with the vaccinate group having significantly (P < 0.01) lower rectal temperatures than the control calves post-challenge. Moreover, the mean rectal temperatures of control calves peaked at days 7–8 postchallenge and were significantly (P < 0.05) higher than the vaccinate group (Fig. 2B). WBC counts of control calves began decreasing from day 2 postchallenge. The mean WBC counts for the control group dropped significantly from 8.6 (1000 per mm3 ) at day −1 to 5.4 (1000 per mm3 ) at day 4 post-challenge (Fig. 2C). The mean WBC counts in the vaccinate group did not decrease during the challenge period, and were significantly higher (P < 0.05) than the control group at days 3–4 post-challenge.
Six months after intranasal vaccination with the BVDV-1a MLV vaccine, the calves were challenged with BVDV-1b strain T1186a, a challenge strain recommended by the USDA, which resulted in severe leukopenia compared to BVDV-1b challenge strain NY-1 [16]. After challenge, 14 of 14 control calves developed moderate clinical signs associated with BVDV infection, including mild leukopenia, decrease in platelet counts, nasal discharge and fever. Following challenge, 13 of 14 control calves displayed nasal discharge on differing days, compared to only one calf from the vaccinate group (P < 0.01), with a prevented fraction of 0.92 with a 95% confidence interval (Fig. 3A). All control calves displayed elevated rectal temperatures (>40 ◦ C), with several animals showing fever for multiple days after challenge. The temperatures of controls were slightly elevated at day 3 post-challenge and peaked at day 8 post-challenge. In comparison, only 3 of the 14 MLV-vaccinated calves developed temperatures >40 ◦ C. The proportion of calves with fever (>40 ◦ C) was significantly higher (P < 0.001) in controls compared to the vaccinate group. A prevention fraction of 0.786 was obtained for the vaccine group with a 95% confidence interval. Moreover, mean rectal temperatures of the control group were significantly higher (P < 0.05) than the vaccinate group at days 8–9 post-challenge (Fig. 3B). White blood cell counts of control calves started decreasing from day 3 post-challenge. The mean WBC counts for the control group dropped significantly from 14.8 (1000 per mm3 ) at day 0 to 7.2 (1000 per mm3 ) at day 5 post-challenge (Fig. 3C). The mean
W. Xue et al. / Vaccine 29 (2011) 70–76
2A. Clinical Scores
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Fig. 2. Intradermal vaccination with a BVDV type 1a MLV vaccine protected calves from challenge with BVDV type 1b strain NY-1. Specifically, the vaccinated calves were protected from clinical disease (A); elevated rectal temperatures (B); decrease of white blood cell counts (C); and virus shedding (D). Statistical values are *P < 0.05 and **P < 0.01.
WBC count in the vaccinate group decreased less than 15% during the challenge period, and the WBC count was significantly higher (P < 0.05) than that of the control group at days 3–6 post-challenge. Platelet counts demonstrated a decrease of greater than 30% in the control group at day 8 post-challenge. In contrast, for vaccinated calves, the platelet counts decreased less than 10%. All animals in the control group shed virus at various days; however, only two vaccinated calves shed BVDV, at a single time point for each. The proportion of calves shedding virus was significantly greater (P < 0.001) in control animals than in the vaccinate group,
with an estimated prevented fraction for virus shedding of 0.857 in the MLV-vaccinated calves. The BVDV nasal swab titers from the control group peaked at day 7 post-challenge, and were significantly (P < 0.05) higher than the vaccinate group at days 5–9 post-challenge (Fig. 3D). 3.5. Duration of immunity of the vaccine against BVDV-1b NY-1 Six months following subcutaneous vaccination, the calves were challenged with BVDV-1b strain NY-1 to determine the duration of
Fig. 3. Intranasal vaccination with a BVDV type 1a MLV vaccine protected calves from challenge with BVDV type 1b strain T1186a, six months post-vaccination. Specifically, the vaccinated calves were protected from nasal discharge (A); elevated rectal temperatures (B); leukopenia (C); and virus shedding (D). Statistical values are *P < 0.05 and **P < 0.01.
W. Xue et al. / Vaccine 29 (2011) 70–76
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Fig. 4. Subcutaneous vaccination with a BVDV type 1a MLV vaccine protected calves from challenge with BVDV type 1b strain NY-1, six months post-vaccination. Specifically, the vaccinated calves were protected from clinical disease (A); elevated rectal temperatures (B); leukopenia (C); and virus shedding (D). Statistical values are *P < 0.05 and **P < 0.01.
immunity. All 10 control calves developed moderate clinical signs associated with BVDV infection, including nasal discharge, coughing, diarrhea and depression. In contrast, the vaccinated calves developed few clinical signs from the challenge. The clinical scores of the control group were significantly higher (P < 0.05) than the vaccinated group at days 6–11 post-challenge (Fig. 4A). All control calves (10/10) developed fever, with elevated rectal temperatures >40 ◦ C, and several animals had fever for multiple days following challenge. In contrast, none of the vaccinated calves developed elevated temperatures of >40 ◦ C. Mean rectal temperatures of the control group were significantly higher (P < 0.05) than the vaccinate group at days 8–9 post-challenge (Fig. 4B). Two days after challenge, WBC counts of control calves started decreasing, reaching the lowest point at day 4 post-challenge, an approximate 45% of decrease. Furthermore, the mean WBC counts for the control group were significantly lower (P < 0.05) than the vaccinate group at days 4–7 post-challenge (Fig. 4C). All animals in the control group shed virus on various days; however, only 8 out of 20 vaccinated calves shed virus on a single day for each. The titers of isolated viruses from the control group were significantly (P < 0.05) higher than the vaccinate group at day 4 and days 6–9 post-challenge (Fig. 4D). 4. Discussion Currently available parenteral combination MLV vaccines against BVDV types 1 and 2 can clearly induce a protective response against BVDV infection and spread. However, BVDV vaccination programs occasionally fail, and this is believed to be the result of diversified BVDV antigenicity among strains and isolates, including variations between BVDV-1 and BVDV-2 genotypes, and even between subgenotypes of the BVDV-1 species [9,10,14]. For decades, majority of BVDV vaccines (both MLV and KV) only included BVDV-1a strains, such as Singer, NADL or Oregon C24V cytopathic strains, and very few vaccines contained non-cytopathic strains. The recognition of severe disease and fetal losses in vaccinated cows, associated with BVDV-2, led to the inclusion of
cytopathic BVDV-2 strains in most current, commercially available BVDV vaccines [18,19]. At present, there is an increasing concern pertaining to BVDV antigenic variation among BVDV-1 isolates. Recent studies revealed that most BVDV-1 field isolates (>75%) were of the BVDV-1b subgenotype, predominant to BVDV-1a in North America [10,14]. Cross-neutralization results showed that anti-BVDV antibodies induced by BVDV-1a strains have less neutralization power to heterologous BVDV-1b strains, with only 36% of the VN value to BVDV-1b, compared to the homologous BVDV-1a strains [10]. The studies in this paper demonstrated similar results as previously described with respect to VN antibody titers and relatedness of neutralizing antibody response to different BVDV-1 subgenotypes [9,10]. Interestingly, the BVDV-1a vaccine strain induced considerably high VN antibody titers to BVDV-1b in all experiments included in this study. Compared to the homologous BVDV-1a virus (100% VN value), calves in all four experiments displayed a lower overall average VN value (43%) to the BVDV-1b strains prior to challenge. This value is similar to the previously reported diversities between BVDV-1a and BVDV-1b subgenotypes [9,10]. Noticeably, the VN value against BVDV-1b in each experiment was different, ranging from 20% to 74%, and several factors may be attributable. First, the MLV vaccine contains only one BVDV-1a strain and the serum antibody titers induced by the vaccine may not have a broad neutralizing power to BVDV-1b strains, as indicated in a previous study where multiple BVDV-1a strains were used to create antiserum [10]. Secondly, different vaccine administration routes were involved in the studies, and the serological response might be different to the various routes. The third attributable factor may be the different ages of the calves at vaccination, as each might have responded differently to the antigen. Most interestingly, the BVDV-1a vaccinated calves boosted their VN antibody titers after challenge with BVDV-1b strains, with the BVDV-1b VN value increasing from 43% at challenge to 104% two weeks post-challenge. Therefore, it is likely that the BVDV-1a vaccine stimulates immune memory against the BVDV-1b subgenotype, as it does against the BVDV-1a subgenotype, although the primary
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serological response appears to be lower. As a result, this is sufficient enough for the BVDV-1a vaccine strain to protect calves against clinical diseases caused by BVDV-1b strains. In the current study, four separate experiments were conducted to measure the efficacy of a BVDV-1a MLV vaccine in the prevention of infection and clinical disease caused by BVDV-1b strains. Regardless of the route of immunization, vaccinated calves in all experiments were significantly spared of clinical disease following challenge with BVDV-1b strain, NY-1 or T1186a. Generally, when WBC counts decrease less than 40% following BVDV infection, the infecting virus is considered low-virulent [16,20]. Moreover, if a BVDV strain can induce a WBC decrease greater than 40%, the virus strain is considered a high-virulent strain [21,22]. BVDV-1b strains NY-1 and T1186a were used as challenge viruses in these experiments, because they are the most commonly used BVDV-1 challenge virus strains for BVDV vaccine development in the U.S., and can be obtained from the National Animal Diseases Laboratories (NADL, USDA). As a result, the ability of the BVDV-1a vaccine to prevent calves from developing leukopenia against these strains further demonstrated the efficacy of the MLV vaccine against BVDV1b infection. These studies demonstrated the efficacy of the MLV vaccine when it was administrated subcutaneously or intradermally. A previous study indicated that the vaccine is equally efficacious when administered to 3–8-day-old young calves intranasally [17]. Additionally, the duration of immunity experiment showed that six months after intranasal or subcutaneous vaccination, the vaccine was still able to provide protection against BVDV-1b challenge, indicating that the BVDV-1a vaccine can protect against BVDV-1b infection at least six months beyond the date of primary vaccination. Currently, more than 75% of BVDV-1 isolates belong to the BVDV-1b subgenotype in North America [10,14]. We assume there might be slight antigenic diversity among the BVDV-1b strains, although there is no data to support this conclusion. However, two different BVDV-1b challenge virus strains were used in the duration of immunity experiments, and vaccinates demonstrated similar efficacies against each of the different challenge strains, confirming protection provided by the BVDV-1a vaccine against the BVDV-1b subgenotype. A feature of BVDV is that it can cause immunosuppression in the infected animal. The majority of current, commercially available BVDV vaccines contain both BVDV-1a and BVDV-2 strains. There is a concern that immunosuppression may be a byproduct of vaccination. As a result, there must be careful consideration taken to the possible addition of BVDV-1b strains to existing vaccines, because of the assumption that the more BVDV strains in a MLV vaccine, the greater the risk the vaccine may have in causing immunosuppression in the vaccinated animals [10,14]. Acknowledgements All host animal studies in this manuscript were conducted at the Intervet, Inc. animal research facility, following all animal treat-
ment regulations from USA Registrations. The authors thank all technicians involved in animal care and sample collection. The authors also thank David Whiteman for statistical data analysis and Ryan Brady for help in the manuscript preparation. References [1] Duffell SJ, Harkness JW. Bovine viral diarrhoea-mucosal disease infection in cattle. Vet Rec 1985;117:240–5. [2] Houe H. Epidemiological features and economical importance of bovine virus diarrhea virus (BVDV) infections. Vet Microbiol 1999;64:89–107. [3] Baker JC. Bovine viral diarrhea virus: a review. J Am Vet Med Assoc 1987;190:1449–58. [4] Baker JC. The clinical manifestations of bovine viral diarrhea infection. Vet Clin North Am Food Anim Pract 1995;11:425–45. [5] Bolin SR. Control of bovine viral diarrhea infection by use of vaccination. Vet Clin North Am 1995;11:616–25. [6] Radostits OM, Littlejohns IR. New concepts in the pathogenesis, diagnosis and control of diseases caused by the bovine viral diarrhea virus. Can Vet J 1988;29:513–28. [7] Thiel HJ, Collett MS, Gould EA. Family Flaviviridae. In: Fauquet CM, Mayo MA, Maniloff J, editors. Eighth report of the international committee on taxonomy of viruses. San Diego, CA: Elsevier Academic Press; 2005. p. 981–98. [8] Vilcek S, Paton DJ, Durkovic B, Strojny L, Ibata G, Moussa A, et al. Bovine viral diarrhoea virus genotype 1 can be separated into at least eleven genetic groups. Arch Virol 2001;146:99–115. [9] Bachofen C, Stalder H, Braun U, Hilbe M, Ehrensperger F, Peterhans E. Coexistence of genetically and antigenically diverse bovine viral diarrhoea viruses in an endemic situation. Vet Microbiol 2008;131:93–102. [10] Ridpath JF, Fulton RW, Kirkland PD, Neil JD. Prevalence and antigenic differences observed between bovine viral diarrhea virus subgenotypes isolated from cattle in Australia and feedlots in the southwestern United States. J Vet Diagn Invest 2010;22:184–91. [11] Corapi WV, Donis RO, Dubovi EJ. Characterization of a panel of monoclonal antibodies and their use in the study of the antigenic diversity of bovine viral diarrhea virus. Am J Vet Res 1990;51:1388–94. [12] Xue W, Blecha F, Minocha HC. Antigenic variations in bovine viral diarrhea viruses detected by monoclonal antibodies. J Clin Microbiol 1990;28:1688–93. [13] Deregt D. Introduction and history. In: Goyal SM, Ridpath JF, editors. Bovine viral diarrhea virus: diagnosis, management and control. Ames, IA: Blackwell Publishing; 2005. p. 3–34. [14] Fulton RW, Ridpath JF, Saliki JT, Briggs RE, Confer AW, Burge LJ, et al. Bovine viral diarrhea virus (BVDV) 1b: predominant BVDV subtype in calves with respiratory disease. Can J Vet Res 2002;66:181–90. [15] Stahl K, Benito F, Felmer R, Zuniga J, Reinhardt G, Rivera H, et al. Genetic diversity of bovine viral diarrhoea virus (BVDV) from Peru and Chile. Pesq Vet Bras 2009;29:41–4. [16] Ridpath JF, Neill JD, Peterhans E. Impact of variation in acute virulence of BVDV1 strains on design of better vaccine efficacy challenge models. Vaccine 2007;25:8058–66. [17] Xue W, Ellis J, Mattick D, Smith L, Brady R, Trigo E. Immunogenicity of a modified-live virus vaccine against bovine viral diarrhea virus types 1 and 2, infectious bovine rhinotracheitis virus, bovine parainfluenza-3 virus, and bovine respiratory syncytial virus when administered intranasally in young calves. Vaccine 2010;28:3784–92. [18] Van Campen H, Vorpahl P, Huzurbazar S, Edwards J, Cavender J. A case report: evidence for BVDV type 2-associated disease in beef herds vaccinated with a MLV-BVDV type 1 vaccine. J Vet Diagn Invest 2000;12:263–5. [19] Van Campen H. Epidemiology and control of BVD in the U.S. Vet Microbiol 2010;142:94–8. [20] Liebler-Tenorio EM, Ridpath JF, Neill JD. Distribution of viral antigen and development of lessons after experimental infection of calves a BVDV 2 strain of low virulence. J Vet Diagn Invest 2003;15:221–32. [21] Bolin SR, Ridpath JF. Differences in virulence between two non-cytopathic bovine viral diarrhea viruses in calves. Am J Vet Res 1992;53:2163–75. [22] Liebler-Tenorio EM, Ridpath JF, Neill JD. Distribution of viral antigen and development of lessons after experimental infection with highly virulent bovine viral diarrhea virus type 2 in calves. Am J Vet Res 2002;63:1575–84.