Introduction

Foot-and-mouth disease (FMD) is a highly contagious transboundary viral disease of cloven-hoofed animals caused by the foot-and-mouth disease virus (FMDV), a member of Aphthovirus genus within the family Picornaviridae. The virus has seven immunologically distinct serotypes: A, O, C, South African Territories (SAT-1, SAT-2, SAT-3), and Asia11,2. FMD remains endemic in many countries in most parts of Asia, Africa, and the Middle East. In areas where diseases like FMD are endemic, control and prevention mainly depend on repeated vaccination, controlling animal movement, and physically separating wildlife from livestock3. The vaccine is one of the main tools proven to better manage the disease when properly applied, with desirable quality and composition4.

Most FMD vaccines used globally are inactivated, for prophylactic or emergency use, and generally manufactured using the same basic methodology outlined in the World Organization for Animal Health2. Although the pathogen inactivation process is essential in the production of potent and safe inactivated vaccines, recommendations in international guidelines regarding the inactivation process require the establishment of optimal conditions by the manufacturers5. For FMD vaccines in particular, guaranteed safety is essential because any occurrence of the disease will have great economic consequences6. The virus particles need to be completely inactivated to be safe, instead, the inactivation method should only have a minor effect on the viral antigenic properties by maintaining the integrity of 146 s particles of FMDV, as the immune system of the host has to recognize the neutralizing epitopes to produce neutralizing antibodies against the antigen7. It is recommended that virus inactivation methods for vaccines should not compromise the integrity of viral proteins8,9.

Vaccine quality is a critical requirement for the successful control of FMD through immunization. When uncertainty in vaccine performance arises, assessing its quality is essential. Poor-quality vaccines not only fail to protect from disease but also decrease farmers’ confidence and participation in vaccination programs. In Ethiopia, vaccination is the main strategy to combat the huge economic damage due to the occurrence of repeated FMD outbreaks. The country has produced chemically inactivated trivalent vaccines from local isolates of three serotypes namely; serotype O (ETH/38/2005), A (ETH/6/2000), and SAT-2 (ETH/64/2009)10.

Although FMD vaccines have been used in Ethiopia for many years, their efficacy has not been extensively evaluated. Reports showed that the currently available FMD vaccine does not reliably provide full protection, which has been evidenced by the occurrence of FMD outbreaks despite regular vaccination practices11. Moreover, a randomized controlled trial by Jemberu et al.12 found that the overall vaccine effectiveness in preventing clinical FMD infection in affected herds was only 31%, significantly lower than the internationally recommended 75% expected percentage of protection for a standard potency FMD vaccine2. This highlights the need to investigate factors contributing to the limited effectiveness of the FMD vaccine produced in Ethiopia. One potential concern is the use of formalin as an inactivant during production. Therefore, this study aimed to compare three methods of FMDV inactivation; binary ethyleneimine (BEI), formaldehyde, and a combined approach, focusing on their inactivation kinetics and the immune responses elicited by the resulting vaccine.

Materials and methods

Virus propagation

The FMDV serotype O FMDV (O-ETH/38/2005) was obtained from the National Veterinary Institute (NVI) of Ethiopia pathogen bank and propagated in confluent baby hamster kidney cell line 21 (BHK-21) (ECACC, UK) according to the recommended protocol2.

Identification and titration of the virus

FMDV vaccine strain

FMDV vaccine strain (O-ETH/38/2005) identity test was performed using conventional reverse transcription polymerase chain reaction (RT-PCR). Total RNA was extracted using Qiagen (RNeasy® mini kit, Germany). A one-step RT-PCR was used using FMDV serotype O specific primers targeting 600 bp; Forward-5′-CTGCCACCGTCGAGAACTAC-3′ and Reverse-5′-CAGGCGCCACTATCTTCTGT-3′. The final PCR product was visualized using agarose gel electrophoresis.

Virus infectivity titer was determined in tissue culture infective dose 50 (TCID50/ml) by 50% endpoint dilution using the flat bottom 96-well tissue culture plates. Titration was carried out three times and the average titer for the virus was expressed as log10 TCID50/ml13.

Inactivation of virus

Formaldehyde inactivation

Formaldehyde (FA) inactivation of FMDV was performed by adding 40% formalin to the virus suspension at a 1:10 formalin in distilled water, achieving a final concentration of 0.06% v/v formalin14. The mixture was incubated at 26 °C for 48 h with constant agitation on a magnetic stirrer. Then, the pH was adjusted to 8.015, and any excess formaldehyde remaining after the inactivation process was neutralized with sodium bisulfite (Na2S2O5, molecular weight = 190.1) as described by Mahdy et al.16.

BEI inactivation

The BEI inactivation of FMDV was performed in accordance with Wu et al.17 and WOAH2. Briefly, by dissolving 0.21 g of Bromoethylamine Hydrobromide (BEA) into 10 ml of pre-warmed 0.2 N NaOH solution (0.8 g pellets/100 ml distilled water). The solution was then incubated in a water bath at 37 °C for 1 h, mixed by inverting the tube every 15 min to facilitate BEA conversion to BEI. The formation of BEI was indirectly visualized by the color change with the addition of 1% 2-naphthol violet (pH indicator) that was changed from violet to orange upon formation of BEI. Finally, the fresh BEI (0.1 M) was filter sterilized using a 0.2 µm pore size filter pad and added to the virus suspension at a final concentration of 1.5 mM (1.5% v/v). Hence, the inactivation was carried out at 37 °C for 24 h with constant agitation and an intermittent change of the flask at 12 h intervals. The BEI inactivation was stopped by neutralizing the excess of BEA by the addition of 2% (final concentration) sodium thiosulphate pentahydrate, Hi-AR™ (Na2S2O3. 5H2O, molecular weight = 248.18, HiMedia®, India), which prepared as 20% in double distilled water and sterilized by autoclaving as described by Wu et al.17.

The BEI and FA combination inactivation

Inactivation using a combination of BEI and FA was carried out according to the general methods of Barteling and Cassim6 and Sarkar et al.18. The virus was inactivated at 30 °C for 24 h in such a way that the 0.1 M BEI and 40% FA were simultaneously added into the virus suspension at a final concentration of 1 mM and 0.04%, respectively. The inactivation was completed by maintaining pH at 7.5–7.6, with constant agitation and intermittent change of the flask at 12 h intervals. Both sodium thiosulfate and sodium bisulfite were prepared separately in a 20% solution used to end the inactivation16.

Inactivation kinetics and innocuity testing

Inactivation kinetics and innocuity tests were determined based on the WOAH (2022) manual. The inactivation kinetics for each inactivation method of FMDV serotype O was performed in a timed series of samples (3 ml/hour) collected every hour up to 8 h, then at 16 h and 24 h post-inactivation according to Sarkar et al.18. For each sample collected, the reaction stopped and the pH was maintained. The titer of the virus for each sample was calculated by the improved Spearman–Kärber method13. The in vitro innocuity tests were carried out according to Wu et al.17. Samples with no cytopathic effect (CPE) in virus titration plates were checked to detect residual infectivity and ensure the complete inactivation of the virus.

Preparation and safety of monovalent vaccine

The monovalent FMD vaccines of the O ETH/38/2005 strain were prepared following the manufacturing method of conventional vaccine for the formulation of aqueous type of FMD vaccine as described on WOAH2. The inactivated virus suspension was purified for NSP with chloroform at a final concentration of 2.5% and incubated at + 4 °C overnight with slow agitation, following the methods described by Park et al.19. Subsequently, the aqueous vaccines were formulated using aluminum hydroxide gel and saponin adjuvant. The prepared vaccines were kept at + 4 °C until used.

The safety of the vaccine antigens was assessed for the presence of live FMDV both in vitro and in vivo. The infectivity of the inactivated virus, using various inactivants, was evaluated by inoculating samples on BHK-21 cell monolayers for three serial blind passages as described by Mahdy et al.16. Then, final safety testing of the vaccine was conducted on the target animals to identify any abnormal local or systemic adverse reactions, following the WOAH manual2. Specifically, a newly developed monovalent vaccine for each inactivation method was administered subcutaneously in two doses (4 ml per dose) to FMDV seronegative calves and monitored for 14 days.

Animal vaccination and experimental approach

In this experimental study, a total of 20 male calves, with an age of 6–9 months, were used. These calves had not received prior FMD vaccination and were confirmed to be seronegative for FMD-specific non-structural protein antibodies. All animals were ear-tagged and an identity number was given individually. These calves were randomly allocated into 4 groups; 3 vaccinated (each with 5 calves) and one control (non-vaccinated) group. Consequently, the vaccinated groups were again randomly subjected to receive the monovalent vaccines prepared using various inactivants.

Immunization and serum preparation

Each vaccine group was immunized with 4 ml vaccines subcutaneously and a booster dose was given 15 days from initial immunization. About 10 ml of blood was collected at 0, 7, 14, 21, 28, and 42 days post-immunization to evaluate the humoral immune response20. The sera were harvested aseptically and stored at − 20 °C until testing.

Evaluation of vaccine-induced immune response

Serum samples collected from the vaccinated and non-vaccinated (control) groups were evaluated for the humoral immune response. The antibody titer produced against the structural protein of FMDV specific to serotype O was evaluated using the quantitative solid phase competitive ELISA kits (IZSLER, Brescia, Italy) according to the manufacturer’s protocol.

Statistical analysis

The inactivation kinetics under the three inactivation methods were analyzed and plotted by using the regression function in the Microsoft Excel data analysis package (Microsoft excel® 2016). The extrapolation of individual kinetics was drawn as the trend analysis for each inactivation method. Data from SPCE results were recorded on a Microsoft Excel spreadsheet. The statistical analyses were performed using Statistical Package for the Social Sciences (SPSS) software, version 25 (IBM Corp. ©). The one-way repeated measures analysis of variance (RM-ANOVA) was used to determine the statistical significance of variations among the antibody response levels of the three different inactivant-treated vaccines followed by Tukey’s post hoc test. Continuous variables were expressed as the mean and 95% confidence interval. All analyses were based on two-sided p values, p < 0.05 was considered statistically significant.

Results

Confirmation of the virus strain

The FMDV strain O-ETH/38/2005 was identified using a one-step RT-PCR using specific primers targeting the 600 bp region and the result was visualized using agarose gel electrophoresis as shown in Fig. 1.

Fig. 1
figure 1

Agarose gel electrophoresis of RT-PCR products for identification of serotype “O” FMDV. “M” and “L” indicate the molecular ladder and lane numbers, respectively. Samples of Lane 1–4 are positive for FMDV “O” which is approximately 600 bp. Lanes 5–7 are extraction control, negative control, and positive control, respectively.

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Infectivity titration for timely sampling and innocuity test

The initial titer of the virus for all inactivation methods before inactivation was 6.9 log10 TCID50/ml. The result of the residual live virus titer and innocuity test for the time course samples treated with BEI, and FA, and combined BEI and FA inactivants are shown in Table 1. The finding indicated that the combination treatment completed inactivation, with no evidence of CPE observed in the virus titration plates within 5 h. This was followed by the BEI treatment, which required 6 h for complete inactivation. In contrast, samples treated solely with FA did not achieve complete inactivation even after 8 h. In all cases, no CPE was observed in the virus-negative control wells as well as virus-inactivated samples while a significant CPE was noted in positive control virus.

Table 1 Infectivity titers and innocuity test of the vaccine strain of FMDV serotype O with different methods of chemical inactivants over time.
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Comparison of the inactivation kinetics for BEI + FA, BEI, and FA methods

The reduction in log10 infectivity titer of the hourly samples of FMDV strain O-ETH/38/2005 revealed linear patterns of inactivation kinetics for the combination and BEI inactivants. In contrast, the plot was curvilinear with immediate decline at the initial time and later became linear without showing the endpoint in FA inactivation (Fig. 2). Thus, the linear patterns fitted a model (y = mx + b). Where “y” is the virus titer (log10 TCID50/ml), “x” is the inactivants contact time, “m” is the slope (inactivation rate) in log10 TCID50/hour, and “b” is the Y-intercept that estimates the original virus titer (log10 TCID50/ml). In the plots, the “R2” represents the coefficient of determination of the regressions functions, which determine the proportion of variance in virus titer reductions that is predictable from inactivant contact time.

Fig. 2
figure 2

Plots for inactivation kinetics and trend analysis of serotype O FMDV (ETH/38/2005) with BEI + FA (A), BEI only (B), and FA (C). In each inactivation plot, the intersection of the arrow end of the trend line with the dotted line at a 10–7 virus titer indicates the predicted time required to ensure the absence of one infectious particle per 104 L of virus suspension.

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The results indicated that the inactivation rate of FMDV strain O-ETH/38/2005 with BEI + FA, BEI, and FA were 1.27, 1.05, and 0.34 log10 TCID50/hr, respectively. Using these rates, the time required to ensure the absence of one infectious unit from 104 L of virus suspension was determined by extrapolating the trend line below the x-axis for the three inactivation methods (Fig. 2). The minimum endpoint recommended by WOAH2 was lower than one log10 TCID50 per 104 L of virus culture used for inactivation. Accordingly, the minimum endpoint for a 104-L volume would be 10–7. Hence, the linear regression analysis results of this study demonstrated that the minimum inactivant contact time required for ensuring less than one infectious particle per 107 ml of virus suspension ranged from 10 to 12 h with combination, 13–15 h with BEI, and 38–41 h with FA inactivation methods.

Safety and purity test result

The vaccine’s safety was assessed both in vitro using BHK-21 cell culture and in vivo by vaccinating target animals. The result showed that CPE was not observed after three blind passages of the virus and no local and/or systemic signs were recorded 14 days after vaccination. Similarly, the purity test revealed negative results. Thus, the formulated monovalent vaccine was considered safe and pure for animal experimentation as per the WOAH requirements for vaccine preparation.

Antibody responses to FMD vaccines inactivated with BEI + FA, BEI, and FA

In the present study, the humoral immune responses, measured by antibody titer records expressed as percent inhibition (PI), showed consistent patterns across all groups of vaccinated animals over time post-vaccination (Fig. 3). The pairwise comparisons (adjusted Bonferroni) of the total antibody titers revealed a significant increase (p < 0.031) from day 0 to its maximum value at day 21. This was followed by a slight decrease in the titers, which remained at low levels until the end of the experiment.

Fig. 3
figure 3

Mean inhibition (%) of antibody responses in vaccinated groups (Days 0–42).

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In this study, a significant difference exists between the total antibody titers of the four experimental groups (p = 0.000). Tukey’s post hoc multiple comparisons disclosed that the antibody responses of a combined-treated vaccine have significant differences (p = 0.006) with FA-treated vaccines. In contrast, there was no statistically significant difference (p = 0.696) between the antibody responses of combined and BEI-inactivated vaccines. The BEI treatment showed a marginally non-significant difference (p = 0.051) with FA. Although the mean antibody level was not statistically significant, the average antibody titer was higher for the combined approach followed by BEI and FA-inactivated vaccines (Table 2). The antibody titers in the unvaccinated calves were consistent with no significant increase or decrease throughout the experiment period.

Table 2 Mean values of the antibody titers in percent inhibition (PI) for one to ten serum dilutions of experimental animals along with time after initial vaccination.
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The SPCE was performed using four serum dilutions (threefold) of the calves’ serum collected between 0 and 42 days post-vaccination. The mean antibody titers in vaccinated calves showed a significant increase in the PI values. At one to ten serum dilutions, all vaccinated groups showed PI values exceeding the cut-off value of 70% from day 21 to day 42 post-vaccination. However, at one to 30 serum dilutions, only the groups that received the combined-treated vaccine were able to maintain the PI value above the cut-off from day 14 to 42 after vaccination (Fig. 4).

Fig. 4
figure 4

Mean antibody inhibition (%) quantified by SPCE for each experimental group over time. (A) Displays the data for one to ten serum dilutions, while (B) demonstrates the data for one to thirty serum dilutions. The dotted line at 70% indicates the cut-off value for SPCE, above which results are considered positive.

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Discussion

The findings of the present study indicated that efficient inactivation of FMDV serotype O ETH/38/2005 can be achieved by combining BEI and FA methods. The combined method was the fastest with an inactivation rate of (1.27 log10 TCID50/hr) followed by BEI inactivation (1.05 log10 TCID50/hr). When compared with FA, the inactivation kinetics of BEI and combined methods increased the inactivation rate by 3 to 4 folds, respectively. Consequently, 5 and 6 h of inactivation were sufficient to obtain acceptable safety for combined and BEI methods, respectively. Notably, the rate of inactivation of FMDV serotype O ETH/38/2005 was slower when inactivated with FA (0.34 log10 TCID50/hr), suggesting potential safety risks associated with this method. The FA method required 38–41 h of exposure to achieve complete inactivation, which is by far longer than the hours suggested as the standard protocol. Thus, vaccine producers that rely on formaldehyde inactivation methods should consider longer contact hours to ensure the maximum safety of the vaccine.

Earlier studies on FMDV commonly accepted that the virus 146 s particles, containing viral antigens, are key components in FMD vaccines. The immunogenicity of FMD vaccines is dependent on the presence of the intact capsid preserved after safe inactivation15. Although the antigenicity of the virus was not assessed before and after inactivation in this study, the inactivated virus showed no residual viable virus as confirmed in tissue cultures and experimental animals. Similar findings were reported elsewhere in other studies on the inactivation of FMDV, which assessed the antigenicity of FMDV specifically the integrity of 146 s particles, before and after treatment with different inactivants, indicating that the combined and BEI methods do not adversely affect the antigenic mass. However, the downstream procedures during inactivation could lead to a relative loss of the antigen7,16,17,18,21,22,23.

In this study, significantly higher antibody titer was observed in vaccines that used a combined and BEI inactivated compared to those inactivated with FA. This suggests that, for the O-ETH/38/2005 strain, the use of BEI alone or in combination with FA was more effective in eliciting immunogenicity in local calves than the use of FA inactivant. Although the protective status and the duration of immune response were not evaluated, the findings align with those of Mahdy et al.16, who reported on the duration of immune response to inactivated vaccine in calves for serotype O FMD, noted that a combination treatment-induced higher antibody titer than BEI alone. The significantly higher antibody titers observed in calves that received vaccines inactivated by combination followed by BEImight be attributed to the superior preservation of the virus’s antigenic structure compared to FA treated vaccine. The short inactivation times help to minimize proteolytic destruction of the 146 s antigen, thereby enhancing antigen yields. Moreover, the synergistic effects of combined (BEI and FA) are anticipated to favorably influence the endurance of the immune response6. Although the exact mechanism of this synergistic effect remains unclear, FA is recognized for its ability to cross-link the property of the viral capsid proteins, thereby stabilizing the 146 s particles and enabling the BEIto easily access the nucleic acid24. On the contrary, the lower antibody titer observed in calves that received the FA-inactivated vaccine might be due to the destructive effect of FA on the virus’s antigenic site. Previous studies have demonstrated treatment with FA alters the structure of the virion and reduces the vaccine’s immunizing activity25,26.

In conclusion, the inactivation methods, utilizing a combination of BEI and FA, as well as BEI alone, showed linear inactivation kinetics. Both approaches achieved a comparable and even higher rate of virus titer reduction, resulting in complete inactivation with no residual live virus detected after three cell culture passages within 5 to 6 h. In contrast, the formalin inactivation method exhibited curvilinear kinetics with a slower rate of virus reduction, failing to achieve complete inactivation by the 8th hour and showing residual live virus at the 16th hrs during the innocuity test. Trend analysis indicated that formalin required a longer minimum contact time to ensure the absence of less than 1 TCID50 virus particle per 104 L of virus suspensions compared to the combination and BEI inactivation methods. Additionally, the SPCE result showed that the combination and BEI inactivation methods produce higher and comparable antibody levels compared to the formalin methods. Therefore, it is recommended that the use of formalin as an inactivant be replaced with a combination inactivant, following optimization and validation of the appropriate inactivation procedures for each vaccinal strain to maximize FMD vaccine effectiveness.