Assessment of tetanus revaccination regimens in horses not vaccinated in the previous year

Abstract

A two-dose revaccination against tetanus is recommended for horses over 2 years old in Japan with no history of vaccination in the previous year. Here, the need for two-dose revaccination was evaluated in terms of antibody titers for each vaccine type, namely monovalent or multivalent. There was no difference in antibody titers between one- and two-dose regimens for up to 1 year, except at 8 weeks with the multivalent vaccine, and all horses had sufficient antibody titers for 1 year of tetanus prophylaxis. These results suggest that one-dose revaccination, regardless of the vaccine type, is as effective as two-dose in preventing tetanus for at least 1 year in horses not vaccinated in the previous year.

Tetanus is a common neurological disease of humans and animals worldwide and is characterized by spastic paralysis [2, 10, 11]. The causative agent is the tetanus neurotoxin produced by Clostridium tetani, a gram-positive, spore-forming anaerobic bacterium found in soil. The disease commonly develops as a result of wound contamination with soil containing C. tetani spores [7]. Horses are considered highly susceptible to tetanus [7]; the main clinical signs in this species are hyperesthesia, trismus, prolapse of the third eyelid, dyspnea, recumbency, and dysphagia [4, 11]. Once equine tetanus develops, mortality rates have been reported to be as high as 23.5% (in 17 horses [2]), 68% (in 31 equids, namely 30 horses and 1 donkey [11]), or 75% (in 20 horses [4]). Vaccination against tetanus can suppress the onset of the disease in most cases; even in horses that have developed the disease, survival rates are high in those that have been vaccinated [4]. Therefore, prevention through proper vaccination is considered very important in this species [3, 6].

Long-lasting humoral immunity induced by tetanus toxoid vaccine has been demonstrated in horses [8], but booster vaccination intervals after an initial series of two or three doses vary widely among countries. In some countries, including Japan and the United States, annual booster shots are recommended, whereas in others multi-year intervals are recommended, such as every 2 years in the United Kingdom, every 3 years in Sweden, and every 5 years in New Zealand [3, 5]. In addition, the guidelines of the Japanese Council of Equine Health recommend revaccination, starting with a two-dose primary immunization, if there is no history of vaccination in the previous year. Despite differences in the vaccines used and the adjuvants included, given the long duration of humoral immunity to tetanus toxoid vaccine in horses and the fact that booster shots are recommended at multi-year intervals in some countries, it is questionable whether the process of two-dose primary reimmunization in horses needs to be repeated, especially if a horse has only missed one annual vaccination.

Here, we examined the effect of the number of revaccination doses (one or two) on the degree of humoral immunity in horses vaccinated with tetanus toxoid vaccine after an interval of approximately 2 years. We also examined whether or not the number of revaccination doses required in horses differed depending on the type of vaccine used in Japan, namely monovalent or multivalent.

All experiments were conducted in accordance with ethical and welfare regulations of the animal care committee of the Equine Research Institute and ethics regarding this study were approved by the committee under the identification number 21–29.

An iELISA (indirect enzyme-linked immunosorbent assay) was prepared by using a commercial tetanus toxoid vaccine (Nisseiken Co., Ltd., Tokyo, Japan) as the antigen directly bound to the solid phase. After the toxoid antigen had been mixed by vortexing, 5 mL of the suspension was centrifuged (13,000 g, 10 min, 4°C). The supernatant was discarded and 5 mL of phosphate-buffered saline (PBS) was added. The same procedure was then repeated. The toxoid antigen in PBS was added to a 96-well microtiter plate at 50 µL per well and incubated overnight at 4°C. After the antigen solution had been discarded, the plate was blocked for 15 min at room temperature with 250 µL/well of a commercially available blocking buffer (ImmunoBlock, KAC Co., Ltd., Tokyo, Japan) diluted five-fold with distilled water. Test sera diluted 1:300 in a LowCross Buffer (CANDOR Bioscience, Wangen, Germany) were added in triplicate at 50 µL per well and incubated at 37°C for 1 hr. The wells were then washed four times with 300 µL PBS with 0.05% Tween 20 (PBST). Then 50 µL of horseradish peroxidase (HRP)-conjugated recombinant protein G (Invitrogen, Waltham, MA, USA) diluted 1:10,000 in LowCross Buffer was added to the wells, which were then incubated at 37°C for 1 hr. After being washed four times with PBST, the plate was developed with 50 µL of a substrate solution (TMB Extended Range HRP Microwell Substrate (TMBX), Surmodics, Eden Prairie, MN, USA) for 15 min in the dark. The reaction was stopped by the addition of 50 µL of stop buffer (450 nm Liquid Stop Solution for TMB Microwell Substrates, Surmodics), and the absorbance was measured at a wavelength of 450 nm. Antibody titers (UI/mL) for each sample were calculated from a calibration curve for each assay by using a commercially available tetanus immune globulin (Takeda Pharmaceutical Co., Ltd., Osaka, Japan).

As of November 2021, ninety-seven horses approximately 2 to 2.5 years post-tetanus vaccination were enrolled in the study. The 97 horses were allocated to the following four groups: A) 18 horses in the one-dose monovalent vaccine group; B) 18 horses in the two-dose monovalent vaccine group; C) 30 horses in the one-dose multivalent vaccine group; and D) 31 horses in the two-dose multivalent vaccine group. The vaccines and volumes used were 3 mL of tetanus toxoid monovalent vaccine (Nisseiken Co., Ltd., Tokyo, Japan) and 1 mL of equine influenza, Japanese encephalitis, and tetanus multicomponent vaccine (KM Biologics Co., Ltd., Kumamoto, Japan). The initial study vaccination was administered to horses in all groups in November 2021 in accordance with the manufacturers’ instructions. An additional booster vaccination was given to groups B and D 4 weeks after the initial vaccination. Blood samples were collected from all horses according to the following schedule: at the time of the initial vaccination (pre-), and 1, 2, 4, 8, 26, and 52 weeks later. Sera were stored at −20°C. Twenty-four horses were excluded from the investigation for reasons unrelated to the study. The remaining 73 horses (14 in group A, 14 in group B, 24 in group C, and 21 in group D) were able to participate for the entire study period. The following analyses were performed by using data from these 73 horses. Detailed information on the 73 horses is provided in Supplementary Table 1.

A combination of the Friedman test and the Wilcoxon signed rank test (false discovery rate (FDR) P-value-adjusted by the Benjamini-Hochberg method) was used for statistical analysis to compare antibody titers in the same group between the time of initial vaccination and each sampling day. For each monovalent and multivalent vaccine, the Wilcoxon rank-sum exact test (FDR P-values adjusted by using the Benjamini-Hochberg method) was used to compare antibody titers between the one-dose and two-dose groups on each sampling day. All statistical analyses were performed by using R software version 4.2.1 [9], and a P-value of <0.05 was defined as statistically significant. The ggplot2 package in R software was used to visualize the results [12].

Antibody titers at the time of pre-vaccination ranged from 0.07 to 5.51 UI/mL (median 0.69 UI/mL). These initial vaccination values were higher in all samples than the antibody titer required for tetanus prophylaxis (0.01 UI/mL or greater [3, 5]), indicating that the protective effect of antibodies produced by the tetanus booster vaccine could persist for at least 2 years in horses, consistent with a previous finding [8]. Tetanus vaccination significantly increased the antibody titers in all groups compared with in the pre-vaccination samples for up to 8 weeks in group A, 52 weeks in group B, 26 weeks in group C, and 8 weeks in group D (Fig. 1). At the vaccination doses used in this study, the monovalent vaccine contained approximately three times the amount of toxoid (approximately 15 Lf) as was in the multivalent vaccine (approximately 5 Lf). In addition, multivalent vaccines against multiple pathogens—even when they contain the same amount of antigen as in monovalent vaccines—have been shown to have a potentially substantial detrimental effect on antibody production when compared with monovalent vaccines [1]. These reasons may explain why higher antibody titers than at pre-vaccination were maintained for longer in the two-dose monovalent vaccine group than in the multivalent groups.

Fig. 1.

Changes in antibody titer after tetanus vaccination. The upper panel shows values for the monovalent vaccine and the lower panel shows values for the multivalent vaccines. Antibody titers on the first day of vaccination (pre-) and on each sampling day in the same group were compared, and significant differences (P<0.05) were found between each of a vs. aʹ, b vs. bʹ, c vs. cʹ, and d vs. dʹ. Comparison between the one-dose and two-dose vaccine regimens at each sampling date indicated that there was a significant difference in titer only at 8 weeks and only with the multivalent vaccine.

Comparison of the one- and two-dose vaccine regimens revealed a significant difference only at 8 weeks, between the two multivalent vaccine groups. Because groups B and D received a booster vaccination at 4 weeks, antibody titers were elevated in almost all horses at 8 weeks. In the monovalent vaccine groups (groups A and B), the median antibody titer at 8 weeks under the two-dose regimen was 5.16 UI/mL—higher than that under the one-dose regimen at 1.83 UI/mL. However, because of the large inter-individual variation in antibody titers this difference was not significant. In the multivalent vaccine groups (groups C and D), the median antibody titer at 8 weeks under the two-dose regimen was 2.65 UI/mL—significantly higher than that under the one-dose regimen at 0.70 UI/mL (P=0.007). This significant difference was present only at 8 weeks and did not persist at 6 months (26 weeks) or 1 year (52 weeks). The median antibody titers 1 year after the initial study vaccination were actually lower with the multivalent vaccine than with the monovalent vaccine because of the differences in antigen amounts and the multivalency itself, as described above: the 1-year titers were 0.83 UI/mL in group A, 1.92 UI/mL in group B, 0.46 UI/mL in group C, and 0.62 UI/mL in group D. However, antibody titers were maintained above the effective titer of 0.01 UI/mL for the entire study period of 1 year in all horses; this maintenance is vital to the effectiveness of the vaccines in preventing tetanus. Our results therefore indicate that two-dose revaccination after a long time (in this case, 2 to 2.5 years), whether with monovalent or multivalent vaccines, does not provide a significant benefit over one-dose revaccination in protecting horses against tetanus for a 1-year period. As we assessed antibody titers for only 1 year after the initial study vaccination, it is not clear in terms of antibody titers whether the commercial tetanus vaccine available in Japan can be used at 2-year intervals, as is practiced in the United Kingdom with a different brand of vaccine [3, 5]. However, our results show that there is little benefit to re-starting with two-dose primary immunization for horses that have not been vaccinated against tetanus in the previous year.

Vaccination is an important veterinary practice to protect horses from various diseases, including tetanus [3]. However, overuse of vaccines should be avoided from animal welfare, economic, time, and labor perspectives, and the vaccination regimen should be based on scientific evidence. From our results, we suggest that a single dose of tetanus vaccine—either the monovalent or the multivalent vaccine commercially available in Japan—is sufficient to maintain beneficial protection for at least 1 year in horses that have not been vaccinated against tetanus in the previous year.

CONFLICT OF INTEREST

The authors have no conflicts of interest to declare.