Comparison of humoral immune responses in dairy heifers vaccinated with 3 different commercial vaccines against bovine viral diarrhea virus and bovine herpesvirus-1

Abstract

A randomized clinical trial was conducted to compare the humoral immune response to 3 different commercial vaccines in dairy heifers housed in 3 different dairy farms in Quebec. All heifers were seronegative to type 1 bovine viral diarrhea virus (BVDV) (Singer strain), type 2 BVDV (NVSL 125c strain), and bovine herpesvirus-1 (BHV-1) at the beginning of the trial. In addition, control heifers in group 1 remained seronegative to the 2 viruses till the end of the trial. Significant differences in humoral immune responses occurred among the 3 commercial vaccines at 4 weeks and 6 months following vaccination. The vaccine in group 2 elicited higher mean antibody titers and seroconversion rates to both type 1 and type 2 BVDV than that in groups 3 or 4. Vaccines in groups 2 and 3 induced higher mean antibody titers to BHV-1 than did the vaccine in group 4.

Introduction

Differences in the efficacy of commercial vaccines, as shown through the use of challenge trials, have been reported in veterinary medicine (1,2,3,4,5,6,7). Unfortu nately, challenge trials are expensive and concerns for animal rights prevent their widespread use. Nevertheless, alternative methods can be used to compare immune responses to vaccines. Serology, for instance, has been used in the past to quantify the immune stimulation by different vaccines (8).

Previous challenge studies have shown that protection against some viral infections is correlated with antibody titers present at the time of challenge. Bolin et al (9) reported that calves with seroneutralizing (SN) titers of 1/16 or higher, as a result of passive immunization, were protected against severe disease following challenge with a type 2 bovine viral diarrhea virus (BVDV) strain, although some clinical signs were still present in calves with SN titers higher than 1/16. The severity and duration of these clinical signs were negatively correlated with seroneutralization titers. Beer et al (10) recently vaccinated cattle with 3 different killed-BVDV vaccines and reported that the postchallenge reduction of leukopenia, viral shedding, and viremia were highly correlated with SN titers. In cattle vaccinated with a killed-infectious bovine rhinotracheitis virus (BHV-1) vaccine, a negative correlation was found between SN titers and clinical scores (r = -0.80; P < 0.001) (11).

The objective of this study was to compare the humoral immune response to type 1 and 2 BVDV and to BHV-1 in dairy heifers from 3 different farms in Québec vaccinated with 3 different commercial vaccines.

Materials and methods

Animals and trial site

The study was carried out on 3 commercial dairy farms (farms 1, 2, and 3) located in southern Québec. Fifty-two Holstein heifers, aged between 6 and 13 mo, were selected for the trial. All heifers were seronegative for antibodies to type 1 and 2 BVDV and BHV-1 at the beginning of the trial. A blood metabolic profile was done in all herds to check for any nutritional imbalance and to verify the integrity of the immune system in yearling heifers. Any abnormal values detected for glucose, total protein, globulins, vitamin E, selenium, gamma glutamyl transferase, and copper were addressed by appropriate modification of the ration, and a 2nd metabolic profile was done 2 wk after the changes to confirm normal values.

Experimental design

The 52 heifers were blocked by farm and age and were randomly allocated to 1 of 4 groups. Heifers in group 1 (n = 13) served as controls and received no vaccine. Heifers in group 2 (n = 13) were vaccinated with 5 mL of vaccine against BHV-1, parainfluenza-3 (PI3), bovine respiratory syncytial virus (BRSV), type 1 and 2 BVDV, and 5 serovars of Leptospira (Triangle 9 + Type 2 BVD; Wyeth Animal Health, Guelph, Ontario) on days 0 and 15. Heifers in group 3 (n = 13) were vaccinated with 5 mL of BHV-1, PI3, BRSV, type 1 BVDV, and 5 serovars of Leptospira (Cattlemaster 4 + L5; Pfizer Canada, London, Ontario) on days 0 and 15. Heifers in group 4 (n = 13) were vaccinated with 5 mL of BHV-1, PI3, BRSV, type 1 BVDV, and 5 serovars Leptospira (Sentry 9; Boehringer Ingelheim, Burlington, Ontario) on days 0 and 15. Heifers were vaccinated IM. The duration of intervals between vaccinations followed guidelines provided by each manufacturer. For each commercial vaccine line, the type of viral component and adjuvant are summarized in Table 1 (12,13).

Table 1.

Serology

Blood was drawn into an evacuated tube (Red top Vacutainer; BD, Franklin Lakes, New Jersey, USA) on days 0 and 28 after vaccination in spring 2001. All sera were kept frozen until the last bleeding. Sera were sent as a batch to the Animal Health Laboratory, University of Guelph.

All heifers were bled anew in late fall 2001, about 6 mo after the initial vaccination. Because control heifers on farm 2 were vaccinated before the last bleeding in the fall, 4 unvaccinated sentinel heifers older than 6 mo and in contact with the vaccinated heifers on trial were bled and used as controls for this farm. Sera were sent as a 2nd batch to the same laboratory at the University of Guelph.

Antibody titers to type 1 and type 2 BVDV and BHV-1 were assayed by the microtiter SN test. For type 1 BVDV, the Singer strain was used in the assay, while for type 2 BVD, the NVSL 125 strain was used. Methods are described elsewhere (14,15).

Statistical analysis

All statistical analyses were carried out using computer software (Statistical Analysis Systems (SAS), version 8.2; Cary, North Carolina, USA). Variance components analyses revealed little contribution of herd (0%) to the percentage of explained variation in titers among treatment groups. Therefore, individual heifers were used as statistical units of analysis. A repeated measures linear model, with time as the crossover factor, was used to study the effects of treatment group on mean log₂- transformed type 1 and type 2 BVDV and BHV-1 titers over time (16). Tukey's post-hoc tests were used to compare pairs of means. Estimates from the model are reported in the anti-log format. The G-test of independence followed by post-hoc tests with an adjusted P-value was used to study the effects of treatment groups on seroconversion rate and on the proportion of heifers with antibody titer greater than, or equal to, a given SN titer. The results were considered significant at P < 0.05. Seroconversion rate was defined as the proportion of heifers showing a 4-fold antibody titer increase.

Results

Type 1 BVDV serology

All control heifers were seronegative to type 1 BVDV at the onset of the study and remained seronegative throughout the trial. Sentinel heifers used as controls on farm 2 for the bleeding at 6 mo postinitial vaccination were also seronegative. On day 28, mean postvaccinal SN titers for type 1 BVDV differed significantly among treatment groups (Table 2).

Table 2.

The seroconversion rates were 100%, 69%, and 38% for groups 2, 3, and 4, respectively. Seroconversion rate was statistically higher in group 2 than in groups 3 and 4, while no statistical difference was found between groups 3 and 4. The percentage of heifers with a titer greater than, or equal to, a given titer was tested for each group, and the groups that were statistically different at each titer threshold are given in Figure 1.

Figure 1. Percentage of heifers with a 28-day postvaccinal titer to type 1 bovine viral diarrhea virus (BVDV) greater than or equal to a given titer cutoff (black bars: group 2; white bars: group 3; grey bars: group 4).

^abcValues with different superscripts at the same titer cutoff are statistically different (P < 0.05)

At 6 mo postinitial vaccination, mean postvaccinal SN titers for type 1 BVDV differed significantly among treatment groups (Table 3). Seroconversion rates were 100%, 77%, and 42% for groups 2, 3, and 4, respectively. The seroconversion rate was statistically higher in group 2 than in group 4, while no statistical difference was found between groups 2 and 3, and between groups 3 and 4. At 6 mo postinitial vaccination, the percentage of heifers with a titer greater than, or equal to, a given titer was also tested for each group, and statistically different groups are shown on Figure 2.

Table 3.

Figure 2. Percentage of heifers with a 6-month postvaccinal titer to type 1 bovine viral diarrhea virus (BVDV) greater than or equal to a given titer cutoff (black bars: group 2; white bars: group 3; grey bars: group 4).

^abcValues with different superscripts at the same titer cutoff are statistically different (P < 0.05)

Type 2 BVDV serology

All control heifers were seronegative to type 2 BVDV at the onset of the study and remained seronegative throughout the trial. Sentinel heifers used as controls on farm 2 for the bleeding at 6 mo postinitial vaccination were also seronegative. On day 28, mean postvaccinal SN titers for type 2 BVDV differed significantly among treatment groups (Table 2).

Seroconversion rates were 100%, 46%, and 0% for groups 2, 3, and 4, respectively. The seroconversion rate was statistically higher in group 2 than in groups 3 and 4, while the seroconversion rate was statistically higher in group 3 than in group 4. On day 28, the percentage of heifers with a titer greater than, or equal to, a given titer cutoff was also tested for each group and statistically different groups are shown on Figure 3.

Figure 3. Percentage of heifers with a 28-day postvaccinal titer to type 2 bovine viral diarrhea virus (BVDV) greater than or equal to a given titer cutoff (black bars: group 2; white bars: group 3; grey bars: group 4).

^abcValues with different superscripts at the same titer cutoff are statistically different (P < 0.05)

At 6 mo postinitial vaccination, mean postvaccinal SN titers for type 2 BVDV differed significantly among treatment groups (Table 3). Seroconversion rates were 100%, 38%, and 0% for groups 2, 3, and 4, respectively. The seroconversion rate differences between treatment groups were the same as those on day 28 postinitial vaccination, with group 2 showing a higher seroconversion rate than groups 3 and 4, while the seroconversion rate was statistically higher in group 3 than in group 4. The percentage of heifers with a titer greater than, or equal to, a given titer cutoff was also tested for each treatment group at 6 mo postinitial vaccination, and statistically significant differences are presented on Figure 4.

Figure 4. Percentage of heifers with a 6-month postvaccinal titer to type 2 bovine viral diarrhea virus (BVDV) greater than or equal to a given titer cutoff (black bars: group 2; white bars: group 3; grey bars: group 4).

^abcValues with different superscripts at the same titer cutoff are statistically different (P < 0.05)

Bovine rhinotracheitis virus-1 serology

All control heifers were seronegative to BHV-1 at the onset of the study and remained seronegative throughout the trial. Sentinel heifers used as controls on farm 2 for the bleeding at 6 mo postinitial vaccination were also seronegative. On day 28, mean postvaccinal SN titers for BHV-1 differed significantly among treatment groups (Table 2).

Seroconversion rates were 100%, 100%, and 92% for groups 2, 3, and 4, respectively, and no statistical difference occurred between any groups. The percentage of heifers with a titer greater than, or equal to, a given titer was also tested for each group by using the same cut-off titers as before (Figure 5).

Figure 5. Percentage of heifers with a 28-day postvaccinal titer to bovine rhinotracheitis virus-1 (BHV) greater than or equal to a given titer cutoff (black bars: group 2; white bars: group 3; grey bars: group 4).

^abcValues with different superscripts at the same titer cutoff are statistically different (P < 0.05)

At 6 mo postinitial vaccination, mean postvaccinal SN titers for BHV-1 also differed significantly among treatment groups (Table 3). Seroconversion rates at 6 mo were 85%, 92%, and 17% for groups 2, 3, and 4, respectively. The seroconversion rate was statistically higher in group 2 and group 3 than in group 4, while no statistical difference occurred between groups 2 and 3. The percentage of heifers with a titer greater than, or equal to, a given titer threshold were also tested for each treatment group by using the same cut-off titers as before (Figure 6).

Figure 6. Percentage of heifers with a 6-month postvaccinal titer to bovine rhinotracheitis virus-1 (BHV) greater than or equal to a given titer cutoff (black bars: group 2; white bars: group 3; grey bars: group 4).

^abcValues with different superscripts at the same titer cutoff are statistically different (P < 0.05)

Discussion

Type 1 and 2 BVDV serology

Differences in the humoral immune response to the 3 commercial vaccines were observed for some fractions. The only inactivated vaccine with a type 2 BVDV fraction (group 2) elicited by far the highest titer to a heterologous type 2 BVDV strain at 2 wk and 6 mo after the recommended vaccination schedule of 0 and 14 d. This finding is in accordance with a recent study where the inactivated vaccine containing a type 2 BVDV strain stimulated the highest geometric mean titer to heterologous type 2 BVDV strains (8). In addition, differences in the geometric mean titers to different type 1 BVDV strains were also reported in that study. In fact, the BHV-1, P13, BRSV, and type 1 BVDV, plus Pasteurella haemolytica, vaccine combination (Triangle 4PH) used in this previous study stimulated higher SN titers to 12 cytopathic and 2 noncytopathic type 1 BVDV strains than did the other BHV-1, P13, BRSV, and type 1 BVDV vaccine combination (Cattlemaster 4) (8). The choice of the Singer strain for the SN test in our study may have favored the vaccines used in groups 2 and 4 (both of which contain the Singer strain) compared with the vaccine used in group 3 (which contains the 5960 and 6309 strains). However, a previous study counters this possible disadvantage for the vaccine used in group 3, since the homologous strain contained in the group 3 vaccine gave a higher titer for the vaccine in group 2 than for the vaccine in group 3 (8).

A 10-year-old Canadian study designed to compare 8 commercial vaccines found intermediate seroconversion rates to 3 different strains of type 1 BVDV for the BHV-1, P13, and type 1 BVDV vaccine combinations (Triangle 3; Wyeth Animal Health, and Cattlemaster 3; Pfizer Canada) and a low seroconversion rate for the BHV-1, P13, type 1 BVDV, and Haemophilus somnus vaccine combination (Sentry 1 + BHV-1/PI3/Somnugen; Boehringer Ingelheim) (15). The lower seroconversion rate observed in that study compared with our study may be related to the presence of antibodies at the onset of the study, which might have prevented seroconversion. By contrast, in our study, all heifers were seronegative at the time of vaccination. Another reason might be that heifers in our study came from nutritionally well- managed herds under a nutritional monitoring program. It is also conceivable that the vaccine potency might have increased over the last decade, resulting in higher seroconversion rates. For example, with the group 2 vaccine, a BVDV protein stabilizer was added in 1992 and a type 2 BVDV strain in 1999.

Several studies have shown that differences in vaccine technology can translate into differences in vaccine immunogenicity. Choice of vaccine strains, virus titers, and adjuvant can all be important factors (1,5,17,18,19). Although the virus titers of the 3 commercial vaccine lines used in our study are unknown, viral strains and adjuvant are different. The vaccine in group 2 is the only one to contain a type 2 BVDV strain and the saponin adjuvant. These particular characteristics might explain, in part, the higher humoral immune response elicited by this vaccine against type 1 and type 2 BVDV.

Several studies have reported a correlation between SN titers and protection against BVDV. In a challenge study comparing 3 different inactivated BVDV vaccines, the reduction in leucocytes, virus shedding, and viremia was strongly dependent on the titer of neutralizing antibodies on the day of challenge infection (10). In passively immunized calves, some authors found that severity and duration of disease decreased as serologic titers of viral neutralizing antibody increased (9). In another challenge study performed in pregnant cattle, the cows that gave birth to persistently infected calves had a lower mean SN titer than protected cows (20). The spectrum of cross-neutralization can also play a role in protection. In a challenge trial done in pregnant cattle with multiple BVDV strains, BVDV infected calves were born to dams with humoral antibody titers to a low number of challenge viruses, while dams of normal calves usually had antibody to a much greater variety of strains (21). Based on those previous studies, it can be speculated that a better humoral immune response, as measured by postvaccinal SN titers, might translate into a better protection against BVDV. A comparative BVDV challenge study would be of great interest in comparing the efficacy of these commercial vaccines.

Bovine rhinotracheitis virus-1 serology

The results of our study for the BHV-1 fraction differ from those in the Canadian study reported in 1991 (15) and in the study reported in 1995 (22). In these studies, mean postvaccination SN titers were higher with the BHV-1, P13, and type 1 BVDV vaccine combination (Cattlemaster 3 or 4) than for the other BHV-1, P13, and type 1 BVDV vaccine (Triangle 3 or 3PH), while our study found no statistical difference between vaccines from groups 2 and 3 on the BHV-1 fraction, 28 d and 6 mo after initial vaccination. Part of this discrepancy can be explained by the 6-fold increase of the BHV-1 antigenic mass in the group 2 vaccine line, which occurred in 1993. No published comparative data were found for the inactivated BHV-1 fraction of the group 4 vaccine combination.

One study reported a correlation between SN titers to BHV-1 and protection (10). In this challenge trial that was done on calves vaccinated with an inactivated BHV-1 vaccine, a strong negative correlation was found between virus shedding and SN titers and between sick scores and SN titers (10). Others did not find any correlation, either because intranasal vaccines were used or because the animals were challenged as early as 7 d postvaccination, which did not allow time for the appearance of SN titers (23,24). An evaluation of the cytotoxic T-lymphocyte stimulation would be interesting to perform in order to compare the potency of the BHV-1 fractions of the group 2 and 3 vaccines. Based on our results and on the data correlating SN titers and protection, it can be speculated that both group 2 and group 3 vaccines should provide a better protection against BHV-1 than the group 4 vaccine.

In conclusion, the vaccine combination used in group 2 stimulated the highest postvaccinal titers to both type 1 and type 2 BVDV, compared with the other 2 tested commercial vaccines, and produced a humoral immune response similar to that of group 3 for BHV-1. A comparative challenge study would be necessary in order to confirm any efficacy differences among these commercial vaccines. CVJ