Impact of the implementation of a Bovine Viral Diarrhea Virus vaccination program in dairy farms: Longitudinal analysis

1. Introduction

Bovine Viral Diarrhea Virus (BVDV), one of the most important bovine pathogens worldwide and is responsible for enormous production losses in beef and dairy herds [1,2,3]. In this work we focused on the impact of the virus in dairy farms. BVDV belongs to the genus Pestivirus within the family Flaviviridae and includes three species, BVDV-1 (Pestivirus A), BVDV-2 (Pestivirus B), and Hobi-like pestivirus (HoBiPeV; Pestivirus H) [4]. The genome consists of a single-stranded, positive-sense RNA of 12.3–13 kb encoding a single open reading frame (ORF), which is flanked by 5′- and 3′-untranslated regions (UTRs) [4]. BVDV can affect both the reproductive tract of cows and bulls, or the semen in artificial insemination programs, at different levels that could leads to low fertility rates or even infertility, embryonic death, fetal loses and abortion as is reviewed by Oguejio, in 2019 [5]. BVDV infection during gestation can also lead to persistent fetal infection if dams are infected with a non citopatic virus biotype during the period of fetal immunocompetence development [6,7,8,9]. Moreover, BVDV infection can also result in fetal malformations in dams infected during the period of fetal organ formation, most probably due to viral-induced lesions, and disruption of embryogenesis that can lead to the development of congenital defects of several organ systems including cerebellar hypoplasia, hydrocephalus, ocular degeneration, thymic hypoplasia, pulmonary hypoplasia, brachygnathism, arthrogryposis, and growth retardation [9,10,11]. These congenital deformities lead to significant reproductive losses in the form of fetal losses, decreased calf yield, decreased availability of replacement heifers, dystocia and cows culled for reproductive problems [5].

Another important consequence of BVDV infection and the trademark of the BDV-viruses is immunosuppression that leads to a decrease in the number of white cells and platelets and a misfunction of immune cells in BVDV-infected cattle [12,13,14,15,16]. It also leads to the occurrence of increased disease and pathology severity when BVDV-infected cattle are coinfected with other pathogens such as Mannheimia haemolytica, bovine herpesvirus-1, bovine respiratory syncytial virus, and bovine Coronavirus [17,18,19,20,21,22,23]. Bovine respiratory disease may cause the greatest economic impact in the cattle feeding industry (feed lot) and dairy farms because of increased health related costs from morbidity and fatalities as well as decreased performance [24,25]. Vaccination against BVDV is an important component of prevention and control programs since it can prevent clinical signs of BRD, reduce viral spread and the birth of new persistently infected (PI) animals. In most countries, only modified live vaccines (MLV) and inactivated vaccines are used in vaccination programs. Both have historical disadvantages; MLV in terms of safety and inactivated vaccines in terms of immune protection. Over the last decade, our research group developed and optimized the first targeted subunit BVDV vaccine (BVD-TV). The core of the BVD TV is the E2 protein of the virus (Singer strain, BVDV 1a) fused to single chain antibody called APCH, that acts as a targeting molecule [26]. The APCH single chain antibody is directed against an invariant MHC II epitope. It was first developed and tested in swine but has now been shown to cross react with several species, including bovines [27,28,29]. The vaccine is produced in SF9 cells utilizing the baculovirus production system and was first released to the market in 2018. Vaccine immunogenicity tested in guinea pigs and cattle and efficacy results in feedlot cattle had been previously published [30,31]. In the current work we present new results regarding the improvement of herd reproductive and productive parameters collected during a ten-year period in six commercial dairy farms from three different provinces of Argentina that incorporated the BVD-TV to their routine vaccination schedules. A total of 256627 inseminations were studied, leading to 105175 pregnant dams and 77051 calves born alive, which represents one of the greatest BVDV-vaccine studies ever conducted and reported.

2. Materials and Methods

Dairy Farms: The longitudinal study includes six dairy farms (Dairy Farm 1 to 6) from 3 different provinces of Argentina: Dairy Farms 1 to 3 are located in Santa Fe, Dairy Farms 4 and 5 in Entre Ríos and Dairy Farm 6 in Buenos Aires provinces, respectively. All farms tested positive to BVDV infection. Information of the dairy farms is presented in Table 1.

Vaccines and Vaccine protocol: the different dairy farms have similar standard vaccination protocols:

Respiratory vaccine: a combined vaccine containing inactivated cultures of Bovine Alpha herpesvirus 1 (BoAHV1), bovine viral diarrhea virus (BVDV) and parainfluenza-3 virus (PI3) and bacterins of Mannheimia haemolytica, Pasteurella multocida and Histophilus somni. Calves received the first dose of respiratory vaccine between 45 and 60 days of age and the second dose 21 days later. The third dose was applied when the animals reach 180 kg, around 6 months of age.

Reproductive vaccine: a combined vaccine containing inactivated cultures of Bovine Alpha herpesvirus 1 (BoAHV1), bovine viral diarrhea virus (BVDV) and bacterins of Histophilus somni, Campylobacter fetus fetus, Campylobacter fetus veneralis, Leptospira interrogans and Leptospira borgpetersenii. Heifers received the first dose of reproductive vaccine at the eleventh months of age, the second dose 30 days later and the third dose at pregnancy confirmation. Cows received two doses, the first when they were cleared to breeding (50 to 60 days post-partum) and the second at pregnancy confirmation.

Targeted vaccine against BVDV (BVD-TV): Vedevax Block^® (Bioinnovo S.A.) targeted subunit vaccine against BVDV [30]. Heifers received the first dose of the BVD-TV at the eleventh month of age, the second dose 30 days later and the third 30 days pre-partum. Cows received two doses, the first when they were cleared to breeding (50 to 60 days post-partum) and the second at confirmation of pregnancy (Figure 1).

Data collection: Data from all dairy farms were collected using the DairyComp 305^® software (Valley Ag. Software). Data was collected individually from each dairy farm and then analyzed individually for each dairy farm or as the average of all farms for each parameter evaluated.

Parameters Evaluated: In this study all the reproductive, mortality rates and productive parameters were annually estimated and divided in three stages: i) the years before the introduction of the BV- TV (Pre-BVD TV), ii) year 0, when the BVD-TV was introduced, and iii) years after the introduction of the BVD-TV (Post-BVD TV). The different parameters evaluated were:

(a)

Conception Rate: defined as the number of pregnant out of all the inseminated dams. In the dairy farms included in this study gestation is diagnosed between days 35 and 45 post insemination.

(b)

Abortion Rate: defined as the number of pregnant dams that lost the pregnancy at any gestation point after being diagnosed pregnant.

(c)

Days Open: refers to the interval between calving and conception (DO).

(d)

Calves per Insemination ratio: defined as the ratio between the number of calves born that live more than 24 h and the number of inseminations performed.

(e)

Neonatal mortality: defined as the death of a live-born calf within the first 24 hours of life.

(f)

Overall Mortality: defined as the ratio between the number of death and the average number of animals present in the dairy farm. It does not include neonatal mortality.

(g)

Milk Production: defined as the average milk production per cow per day.

BVDV Infection Analysis

Viral circulation on the dairy farms was evaluated using a commercial anti-P80 ELISA (CIVTEST BOVIS BVD/BD P80, HIPRA) following the manufacturer’s instructions. Serum samples were taken between February and April 2024. Data from 97 farms (beef and dairy) of the same region collected during 2023 and 2024 were used as controls.

Statistical Analysis

Three different statistical analyses were conducted. The first one included the comparison of all the parameter rates two-year pre-vaccination compared with the rate obtained two years post-vaccination, excluding year 0 of vaccine introduction (2YPP). The second analysis compared the parameter rates included the same numbers of years pre and post-vaccination excluding year 0 (SYPP). And the third one, included all the years available for each dairy farm over a 10-years span from 1/1/2014 to 12/31/2023 (10YS) (Figure 2).

In all cases the Chi-square test was used for comparison of proportions. However, when the Chi-square test assumptions were not met, the Fisher’s exact test was applied to ensure the accuracy of the results. Additionally, a contingency analysis was conducted pre- and post-vaccination, odds ratios were calculated using the Baptista-Pike method [32].

Mean rates pre and post-vaccination in each farm and as a whole was compared using a paired Student T test, using the Welch correction when homoscedasticity assumption was not met. A repeated measures ANOVA was conducted to determine the evolution of each mean parameter through time. The different parameters on the farms were analyzed by a general linear mixed statistical model (GLMM). The model included two main fixed factors: farms (Dairy farms 1 to 6) and time (years pre and post vaccination). Animals were included in the model as a random variable. The GLMM analysis was conducted by using the Glimmer function (lme4 package, R Development Core Team, 2014). The analysis was performed with R 3.0.3 (R Development Core Team, 2014). Statistical significance was assessed at p < 0.05 for all comparisons.

Finally, a linear regression analysis was conducted pre- and post-BVD.TV incorporation to evaluate the evolution trend of the indexes.

Figure 2: Years evaluated for each dairy farm.

3. Results

The viral circulation analysis revealed a significant decrease (Fisher Exact Test, p < 0.0001) in BVDV circulation measured and the proportion of animal seropositive for antibodies against P80 viral protein in the dairy farms that included the BVD-TV at the end of the study period compared with proportion of seropositive cattle in control farms located the same region (Figure 3). The comparison of P80 antibody prevalences in the farms under study with other farms from the same region tested in 2024, was performed because there was no data available from all the farms under study in the pre-BVD TV period.

Figure 3: Viral Circulation Analysis

To analyze the effect of the BVD-TV on the dairy farms different parameters were evaluated both annually and as the summation the years before and after the application of the BVD-TV. The parameters can be grouped in three categories: i) reproductive parameters: conception and abortion rate, open days, and calves born per insemination; ii) mortality rates: neonatal and overall herd mortality; and iii) productive parameters: milk production.

The results of the comparison of reproductive, mortality rates and productive rates over the same number of years before and after BVD-TV application (SYPP) are shown in Table 2. The SYPP analysis demonstrated a significant improvement in all the parameters evaluated which were like those observed in the preliminary 2YPP analysis (data not shown, available upon request).

Table 2: Bold types with asterisks indicate significant differences. OR: Odds Ratio. IC95: 95% confidence interval.

Reproductive parameters:

Conception rate: the SYPP analysis shows this index increased significantly 4.4 points from 39.1% the years before to 43.5% the years after vaccine introduction. Odd ratios analysis shows that after the introduction of the BVD-TV cows have 22% more chances of getting pregnant when the is applied (Table 2).

The 10YS analysis, farm by farm, shows a significant increase in dairy farms 1, 3, 4 and 5 as well as a non-significant increase in farms 2 and 6 (Figure 4 and Table S1).

Figure 4: Conception Rate by farm

The average conception rate of the six dairy farms in the 10YS analysis indicated a significant increase of 5.4%, from 37.9% to 43.3% (Figure 5).

Figure 5: Conception Rate, average

Abortion Rate: The SYPP analysis shows a significant decrease in the dairy farms evaluated (Table 2). The 10YS analysis farm by farm showed a significant decrease in dairy farms 1 and 6 as well as a non-significant decrease in farms 2, 3, 4 and 5 (Figure 6 and Figure 1 SM & Table S2).

Figure 6: Abortion Rate

After the introduction of the BVD-TV a significant trend to reduce the abortion rate was observed (Figure 7). As examples, after 6 year of systematic vaccination Farm 1 reduced the abortion rate from 21% to 11% and Farm 2 went from 16% to 7% in three years Figure 1 SM). The overall result went from no trend in the years pre-vaccination to a significant linear reduction of 0.77 percentual points per year in the post vaccination period (R2=0.76, p-value=0.02).

Figure 7: Abortion Rate Tendency

Days Open: The SYPP analysis shows a significant decrease in 11 days open from 131 days open the years pre-BVD TV to 120 days open the years post-BVD TV (Table 2). The 10YS analysis farm by farm showed a significant decrease in dairy farms 1, 2, 4 and 6 as well as a non-significant decrease in farms 3 and 5 (Figure 8 and Table S3).

Figure 8: Days Open by farm

The Days open average of the six dairy farms in the years before and after the application of the BVD-TV indicated a significant decrease of 11 days, from 130 to 119 days (Figure 9).

Figure 9: Days open, average

Calves born per Insemination ratio: the final parameter among the reproductive parameters, encompassing all other reproductive parameters, is the Calves born per Insemination ratio. The SYPP analysis shows a significant increase of 5% from 28% the years pre-BVD TV to 33% the years pre-BVD TV calves born per insemination. OR analysis indicates that the chances of an inseminated cow delivering a live calf were increased in a 22% (Table 2). The 10YS analysis shows a significant increase in dairy farms 1, 2 and 4 as well as a non-significant increase in farms 5 and 6. On the other hand, dairy farm 3 showed a non-significant decrease, primarily because in year pre -3, the first year with data from this farm, the index was exceptionally high at 42% (Figure 10 and Table S4).

Figure 10: Calves per insemination ratio by farm

The overall mean rates of calves born per insemination of the six dairy farms in the years before and after the application of the BVD-TV indicated a significant increase of 6%, from 27% to 33% (Figure 11).

Figure 11: Calves per insemination ratio.

The kinetics of the reproductive parameters are summarized in Figure 12A and Figure 12B. In both graphs, greater separation between the lines and bars indicates better reproductive performance.

Figure 12: Kinetics of reproductive parameters, average

Mortality Rates:

Neonatal mortality: The SYPP analysis shows a significant decrease of 1,7%, from 6% the years pre-BVD TV to 4.3% days open the years post-BVD TV. The OR analysis for neonatal mortality revealed that a calf has 61% more chance to survive the first 24 h after the application of the BVD-TV against BVDV than before (Table 2). The 10YS analysis farm by farm shows a significant decrease in dairy farms 1 and 2 as well as a non-significant decrease in farms 3 to 6 (Figure 13 and Table S5).

Figure 13: Neonatal Mortality by farm

The neonatal mortality average of the six dairy farms in the years before and after the application of the BVD-TV presented a significant reduction of 2.8%, from 7% to 4.2% days (Figure 14)

Figure 14: Neonatal Mortality, average

Overall mortality: The SYPP analysis shows a significant decrease of 1.4%, from 9.0% the years pre-BVD TV to 7.6% the years post-BVD TV. The OR analysis for overall mortality revealed that bovines in the dairy farms evaluated have 26% more chances to survive after the application of the BVD-TV against BVDV than before (Table 2). The 10YS analysis farm by farm reveals a significant decrease in dairy farms 1, 2 and 5 as well as a non-significant decrease in farms 4 and 6. On the other hand, dairy farm 3 showed a non-significant increase, primarily influenced by the dry weather that affect the area in the years after the introduction of the vaccine (Figure 15 and Table S6).

Figure 15: Overall Mortality by farm

The overall mortality average of the six dairy farms in the years before and after the application of the BVD-TV presented a significant reduction of 2,6%, from 10.1% to 7.5% (Figure 16)

Figure 16: Overall Mortality

In the Figure 17 the kinetic of the mortality rates is represented.

Figure 17: Mortality Rates kinetic in the 6 dairy farms, average

Productive parameters

Milk production: The SYPP analysis shows a significant increase of the mean daily cow production of 2,34 L, from 25,85 the years pre-BVD TV to 28,19 L the years post-BVD TV (Table 2). The 10YS analysis farm by farm shows a significant increase in dairy farms 1 and 5 as well as a non-significant increase in farms 2, 3, 4 and 6 (Table 3).

Table 3: Bold type with asterisks indicates significant differences between means pre- vs post vaccination period within each farm (paired Student T Test).

The average daily milk production per cow of the six dairy farms in the years before and after the application of the BVD-TV presented a significant increase of 2 L, from 25.9 L to 27.9 L pre- vs post- vaccine introduction in the farms (Figure 18).

Figure 18: Milk production, average

The kinetic of the milk production is summarized in Figure 19.

Figure 19: Kinetic of milk production, average

4. Discussion

BVDV have been eradicated or is in the path to eradication in some European countries due to an extended campaign focused on the detection an elimination of PI animals with or without vaccination [33]. But on developing countries like Argentina, with large herds and extensive production system, this control strategy focused on PI elimination is not feasibly to reach at country level. It could be reached at individual farm level, but in the current economical and productive situation it is not feasibly to achieve at regional, province or country level. Therefore, an alternative approach to control and minimize the negative impact of the BVDV infection should be implemented. The strategy to minimize the impact of the virus was to add a targeted subunit BVD-TV to the current vaccination programs, vaccinating all animals in the herd, starting at 6 months of age. There is a previous work applying a similar strategy showing the impact of a live-attenuated vaccine (BVDV-MLV) in four dairy farms in Europe [34]. But in Argentina we cannot use this strategy since MLV are forbidden and only non-replicative vaccines are allowed. Vedevax Block^® is the first recombinant targeted subunit vaccine approved to be used in bovines worldwide [35]. In the dairy farms under study combined killed vaccines were also used, before and after the introduction of the recombinant vaccine, with mild effect against BVDV. The synergistic effect of traditional killed vaccines and the BVD-TV was previously discussed. Although it cannot be completely ruled out, its influence in the improvement of the reproductive and production parameters evaluated appears to be significant [31].

To measure the impact of the BVD-TV introduction into the vaccine calendar data from more than 100,000 animals on 6 farms for a ten-year period was evaluated. Reproductive, mortality rates and production parameters were tested using different statistical approaches and significant improvements in all the parameters were observed after vaccine introduction. All farms tested positive for BVDV which was expected since the virus is endemic in the country [36] and almost 100% of the dairy farms tested positive for infection-related antibodies in milk tanks [37], but the average of animals showing signs of infection was significantly reduced in the farms under study after the incorporation of the vaccine, revealing the protective effect of the BVD-TV on the field and the massive impact that this virus may have in the dairy farms.

An important issue is that to a greater or lesser extent in the six farms there was an improvement in all the seven parameters evaluated (conception and abortion rates, days open, calves born per service, neonatal and overall mortality, and average daily milk production) after the introduction of the BVD-TV.

The 6% increment in the rate of calves born per insemination achieved post versus pre-BVD TV from 27% to 33%, resulted in the birth of a total of 2463 more calves post-BVD TV introduction in the vaccine calendar of the farms. This improvement was achieved together with a reduction of services in 12,861. The improvement in reproductive efficiency has a significant impact on dairy farms, as it affects not only the number of animals but also the milk production and heifer replacements.

To analyze the incidence of factors other than the BVD-TV, such as annual differences due to climate or external factors or a trend toward improved productivity on dairy farms, national data were evaluated from two databases. Different databases presented data in different manners, so we chose the two parameters with year-by-year data to compare: conception rate and milk production.

The conception rate outcome of the dairy farms in the study was compared with the outcome of almost 200 Argentinian dairy farms users of the DairyComp 305 software (DC 305 users) (Apendix B). At the national level the conception rate maintained stable from 2014 to 2020 between 31% and 32% and then improved to 35% in 2021. This behavior contrasts to the one observed in the analyzed dairy farms where a clear trend to the improvement was observed after the incorporation of the BVD-TV. It is important to note that there was not such a trend before the incorporation of the BVD-TV in these farms. Furthermore, the improvement in reproductive performance places the dairy farms in the top 15% of DC 305 users in Argentina, both in terms of conception and abortion rates.

To analyze milk production data from the National Minister of Agriculture was used (https://www.magyp.gob.ar/sitio/areas/ss_lecheria/estadisticas/_01_primaria/index.php). Data shows a peak of production in 2015 and then a decline from to 2016 to 2019, a slight recovery from 2020 to 2022 and again a drop in the milk production in 2023. Again, at national level milk production does not follow a tendency to improve in the period evaluated as it does in the six dairy farms after the incorporation of the BVD-TV.

Furthermore, in terms on annual or climate factors as it is a longitudinal analysis which not started at the same time, all the years between 2018 and 2021 could be year 0, pre or post vaccination depending on the farms. For example, 2020 is year 0 in farm 4, post-BVD-TV in farms 1 to 3 and pre-BVD-TV in farms 5 and 6, so positive or negative annual variances would affect both the pre and post vaccination periods depending on the farm.

Considering all these facts it cannot be discarded the influence of external variable in the observed results, but the influence of such factors would be minor and cannot explain the significant results that were consistently observed in the different seven parameters tested in six different farms located in 3 different provinces and with vaccination program starting at different time points.

Two critical aspects of BVDV infection are immunosuppression and foetal infections, both factors contributing to calf mortality. Interestingly, a recent study reported a reduction in the calves’ mortality index from 7.45% to 4.38% after implementing a control program based on the detection and elimination of PI animals [38]. These results are comparable to those obtained in the dairy farms included in this study, which implemented a BVD control program based on vaccination with a BVD-TV.

5. Conclusions

The addition of a subunit BVD-TV against BVDV into the vaccination programs of dairy farms contributed to a reduction in the viral circulation and an improvement in productive, reproductive and health parameters such as abortion and conceptions rates, days open, neonatal and general mortality and daily milk production. These findings highlight the importance of incorporating a BVD-TV with an excellent safety profile and DIVA capacity into comprehensive BVDV control strategies to enhance overall herd health and productivity.