Clostridial vaccines are effective in preventing serious and typically fatal diseases that result from exposure of domestic ruminants to toxins produced by Clostridium sordellii, Clostridium chauvoei, Clostridium perfringens, and other pathogens of this genus.1–4 Because of their efficacy these vaccines are in widespread use; however, they can cause a marked inflammatory response that causes pain-inducing and unsightly reactions at the site of injection.5–7 Additionally injection-site reactions are a cause of financial loss when persistent lesions must be trimmed from carcasses.8 Moreover, the systemic inflammatory response that follows clostridial vaccination can be associated with decreased feed consumption in vaccinated cattle, which may impact animal growth and productivity5,9 Thus, the development of clostridial vaccine formulations or methods of administration that minimize local and systemic inflammatory responses but still induce an effective immune response could reduce the impact of clostridial vaccination on animal well-being and productivity.
One method of vaccine administration that could minimize injection-site reactions associated with clostridial vaccination of cattle is TD needle-free injection. Vaccines delivered via TD needle-free injection are transferred through the skin by compressed gas, which pushes a liquid or powder vaccine through a tiny orifice in the injection system with great force.10 The force delivered allows the vaccine to penetrate the skin and enter the dermis, subcutaneous tissues, and underlying muscles without the use of a needle. Although TD needle-free injection in humans can lead to relatively larger injection-site reactions after vaccination, compared with results for vaccine administration with a needle,11 TD needle-free delivery of vaccine to cats induces little or no local reaction.12 Moreover, when administered via TD needle-free injection, smaller doses of vaccine can induce immune responses comparable to those induced by larger doses administered with a needle and syringe.13,14 Administration of a smaller volume of vaccine could decrease the size of the reaction in cattle vaccinated with clostridial vaccines. The purpose of the study reported here was to evaluate injection-site reactions and humoral immune responses in cattle vaccinated with a clostridial vaccine administered SC by use of a needle and syringe or via TD needle-free injection.
Materials and Methods
Animals—Sixteen 11- to 12-month-old Herefords (6 heifers and 10 bulls) were allocated into 2 groups (8 cattle/group) by use of a randomization procedure wherein cattle were assigned a random number by a spreadsheet programa; the random numbers were listed in order and the cattle with the 8 smallest numbers were assigned to the TD group, whereas the cattle with the 8 largest numbers were assigned to the SC group. During the study, cattle were housed on pasture. Cattle were fed Bermuda grass–fescue hay and had unlimited access to water. The study was approved by the University of Georgia Institutional Animal Care and Use Committee.
Vaccine administration—On day 0, vaccine was administered SC to 1 group of cattle (6 bulls and 2 heifers) with a needle and syringe, whereas vaccine was administered to the other group of cattle (4 bulls and 4 heifers) via TD needle-free injection. Sex distribution was not equal in the 2 groups because cattle were assigned to blocks on the basis of age a few months before vaccination, with the intent that cattle with residual maternal antibody titers would be distributed equally between the 2 treatments. Cattle were vaccinated by administration of a commercially available multivalent clostridial bacterin-toxoidb labeled to aid in the reduction of disease caused by C sordellii, Clostridium septicum, C chauvoei, Clostridium novyi, and C perfringens types C and D. Cattle in the SC group received a label dose of 2 mL administered SC in the left cervical region cranial to the scapula by use of a needle and syringe. Cattle in the TD group received vaccine administered by use of a needle-free injectorc and a No. 5 nozzle with a pressure of 5,742 Pa/injection. Vaccine administered to cattle via this injector system is dispersed into multiple tissue layers, including the dermis, subcutaneous tissues, and muscular tissues.d Because the maximum dose that can be delivered via the needle-free injector is 0.5 mL, cattle in the TD group were vaccinated with 0.5 mL of the bacterin-toxoid in the left cervical region cranial to the scapula. Vaccines were administered by 2 investigators (PAT and RF); those 2 investigators did not evaluate injection-site reactions. On day 28, cattle in both groups received a booster vaccination via the same route in the right cervical region cranial to the scapula.
Evaluation of injection-site reactions—Injection sites were evaluated by 2 investigators (ARW and DTE) who were unaware of the treatment group assignment of the cattle. The site of the first administration (left cervical region) was evaluated before vaccination on day 0 and on days 4, 7, 14, 21, and 28 after the first vaccination and on days 4, 7, 14, 21, and 28 days after the second vaccination (ie, days 32, 35, 42, 49, and 56). The site of the second administration (right cervical region) was evaluated 4, 7, 14, 21, and 28 days after the second vaccination (ie, days 32, 35, 42, 49, and 56). Sites were evaluated to detect soft or firm swellings, abscesses, or ulcers. The diameter of any swelling was measured by use of a plastic ruler, and the maximum thickness (depth) of any swelling was estimated. If a swelling was irregularly shaped (ie, not round), the largest dimension was recorded as the diameter. Both investigators measured all swellings during the first week of the study to ensure agreement of measurements at subsequent time points.
Evaluation of serum antibody responses after vaccination—Blood samples were collected from cattle on days 0, 7, 10, 14, 21, 28, 32, 35, 42, 49, and 56. Serum antitoxin titers against C perfringens beta toxin, C novyi alpha toxin, and C septicum alpha toxin were determined by use of ELISAs. Recombinant, toxin-derived antigens were used to avoid cross-reactivity with other clostridial products that may have copurified from native preparations. Clostridium chauvoei titers were not measured because a recombinant antigen that would be free of other potentially cross-reacting clostridial products was not available. To generate recombinant antigens, PCR primers were derived from gene sequences obtained from GenBank, and whole genomic bacterial DNA from C perfringens, C novyi, or C septicum was used as a template to amplify the respective toxin gene sequences. Amplification products were digested with appropriate restriction enzymes and ligated into pTrcHis.e Escherichia coli DH5alpha transformed by the resulting plasmids was cultured in Luria-Bertani broth with appropriate antimicrobials, and transcription of the cloned gene was induced by the addition of iso-propyl-β-D-thiogalactoside. Purification by means of metal affinity chromatography was facilitated by adding the hexahistidine N-terminal tag after cloning into pTrcHis. Purity and identity of recombinant toxins were confirmed by means of reaction with specific antisera.
For the ELISAs, 4 plates were coated with 100 μL of antigen diluted to 10 ng/mL in sodium carbonate buffer (pH, 9.6). Plates were incubated overnight at 4°C. Plates were washed twice with PBS solution and 0.3% polysorbate 20 and incubated with 200 μL of 1% bovine serum albumin in PBS solution and 0.3% Tween 20 for 1 hour at 4°C. Plates then were washed in the same manner and incubated with 100 μL of test sera diluted 1:10 through 1:10,240 in PBS solution and 0.3% Tween 20. Plates were washed 9 times, and 100 μL of rabbit anti-bovine IgG (heavy and light chain) peroxidase-conjugated antibodyf (diluted 1:8,000) was added; plates were incubated for 1 hour at 37°C. Plates then were washed 6 times, and 100 μL of O-phenylamine diamine substrate was added; plates were incubated in the dark for 30 minutes at 37°C. The reaction was stopped by adding 50 μL of 3M HCl, and the color was allowed to develop in the dark for an additional 15 minutes at 37°C. Results were measured as the optical density at 490 nm, and the titer of a given serum sample was the highest dilution that resulted in a positive test result (defined as an optical density > 0.4). Samples were tested in duplicate.
Titers of toxin-neutralizing antibody against C sordellii lethal toxin were measured in a cytotoxicity assay, with Clostridium difficile toxin B as a surrogate for C sordellii lethal toxin. Chinese hamster ovary cells were maintained in Iscove modified Dulbecco mediumg with 10% fetal bovine serum and 100 mg of gentamicin/mL. For toxicity assays, cells were trypsinized, washed, and suspended at a concentration of 2 × 105 cells/mL; aliquots (100 μL) were dispensed into wells of a 96-well cell culture plate. After plates were incubated for 4 hours at 37°C in an atmosphere with 5% CO2, 100-μL aliquots of serial dilutions of serum with toxin preparations were added to each well. Cells were incubated for 18 hours, and the titer of each serum sample was the highest dilution that neutralized 100% of the toxin activity, with toxin activity defined as the induction of death of 50% of Chinese hamster ovary cells.
Statistical analysis—Data were analyzed via 1 of 2 data analysis programs.h,i The difference in diameter and thickness of swelling at the administration site between groups was evaluated by use of repeated-measures analysis. Heterogeneous variances across groups were allowed in the model, and observations for the same animal were included in the model via a first-order autoregressive structure, which recognized that measures close together in time are more highly correlated than are measures far apart in time. Fixed effects included in the model were group, day, and the group-by-day interaction.
The geometric mean serum antibody titer against each clostridial agent was compared between groups on each day of sample collection via a mixed-effect model with repeated measures; observations for the same animal were included in the model via a first-order autoregressive structure. Fixed effects included in the model were group, day, and the group-by-day interaction. Titers were logarithmically transformed (log2) to ensure homogeneity of variances among measurements for different days. All tests were 2 sided, and values of P < 0.05 were considered significant.
To determine whether vaccination via either route induced a significant increase in serum antibody titer for each clostridial antigen, the difference between log2-transformed geometric mean titers on day 0 and the highest titer detected after vaccination was compared between groups by use of a t test. To determine whether the second vaccination via either route induced a significant booster effect, the difference between the highest titer (log2-transformed geometric mean) after the first vaccination and the highest titer after the second vaccination was compared between groups by use of a t test. For both comparisons, values of P < 0.05 were considered significant.
Results
Injection-site reactions—After the first vaccination (administered in the left cervical region), all cattle in both groups had a firm swelling at the vaccination site on at least 1 day after vaccination. However, the swellings were smaller in the TD group on all days evaluated, with significantly smaller swellings in the TD cattle on days 4, 14, 21, and 28 (Figure 1). Swellings in the TD group were not as thick as were swellings in the SC group, with lesions in the SC group being significantly thicker on day 35.
Mean ± SEM diameter (A) and thickness (B) of swellings that developed at the site of the first vaccination (left cervical region) in beef cattle vaccinated with 0.5 mL of a multivalent clostridial vaccine via TD needle-free injection (diamonds [n = 8]) or 2.0 mL of the same clostridial vaccine via SC injection (squares [8]) on day 0. A booster vaccination was administered in the right cervical region via the same route on day 28. *Within a day, values differ significantly (P < 0.05) between groups.
Citation: American Journal of Veterinary Research 72, 8; 10.2460/ajvr.72.8.1124
Similarly, swellings that developed in the right cervical region after the booster dose of vaccine was administered on day 28 were significantly smaller in the TD group on all days evaluated (Figure 2). Swellings in the SC group were significantly thicker than those in the TD group on day 32 (4 days after the second vaccination).
Mean ± SEM diameter (A) and thickness (B) of swellings that developed at the site of the second (booster) vaccination (right cervical region) in beef cattle vaccinated with a multivalent clostridial vaccine via TD needle-free injection (diamonds [n = 8]) or SC injection with a needle and syringe (squares [8]) on day 28. *Within a day, values differ significantly (P < 0.05) between groups.
Citation: American Journal of Veterinary Research 72, 8; 10.2460/ajvr.72.8.1124
No abscesses or ulcers were recorded in any cattle. However, cattle in the TD group sometimes had a small amount of crusty serous discharge at the vaccination site on ≥ 1 day of observation. Also, sites of TD administration sometimes developed a small (approx 0.5 to 1 cm in diameter) alopecic area in the center of the vaccination site, which persisted in some cattle for the duration of the study. Subjectively, palpation of vaccination sites in the TD group suggested that the reactions were limited to the dermis; these reactions were localized and could be moved with the skin. In contrast and as would be expected, reactions in the SC group often extended, sometimes extensively, into the subcutaneous region.
C perfringens beta toxin titers—No significant differences were detected in geometric mean C perfringens beta toxin titers between the groups on any day (Figure 3). Vaccination via both routes induced a significant (P < 0.001) increase in antibody titer, compared with the baseline titer; the difference between the log2-transformed geometric mean ± SD baseline titer and the highest titer after vaccination was 3.0 ± 0.76 for the TD group and 4.1 ± 0.90 for the SC group. There was not a significant increase in peak titer after the second dose of vaccine, compared with the peak titer after the first dose of vaccine, in either group, which indicated that neither route induced a booster effect as measured by increased antibody titers following the second vaccination.
Geometric mean ± SEM serum antibody titers against Clostridium perfringens beta toxin (A), Clostridium novyi alpha toxin (B), Clostridium sordellii lethal toxin (C), and Clostridium septicum alpha toxin (D) in beef cattle vaccinated with a multivalent clostridial vaccine via TD needle-free injection (diamonds) or SC injection (squares). Cattle received the first dose of vaccine in the left cervical region via TD needle-free injection or SC injection with a needle and syringe on day 0 and a booster dose of vaccine via the same route in the right cervical region on day 28. Titers were measured by use of ELISAs. Notice that the y-axis scale for panels A and D differs from the y-axis scale for panels B and C. *Within a day, values differ significantly (P < 0.05) between groups.
Citation: American Journal of Veterinary Research 72, 8; 10.2460/ajvr.72.8.1124
C novyi alpha toxin titers—No significant differences were detected in geometric mean C novyi alpha toxin titers between the groups on any day, except on days 10 and 56, when the titers in the TD group were significantly lower than the titers in the SC group (Figure 3). Vaccination via both routes induced a significant increase in antibody titer over the baseline titer; the difference between the log2-transformed geometric mean ± SD baseline titer and the highest titer after vaccination was 1.8 ± 0.68 (P = 0.002) for the TD group and 2.5 ± 1.77 (P = 0.005) for the SC group. There was not a significant increase in peak titer after the second dose of vaccine, compared with the peak titer after the first dose of vaccine, in either group, which indicated that neither route induced a booster effect on serum antibody titer.
C sordellii lethal toxin titers—Geometric mean C sordellii lethal toxin titers were significantly lower in the TD group than in the SC group on days 10, 14, 21, 35, and 42 after the first vaccination (Figure 3). However, vaccination via both routes induced a significant increase in antibody titer over the baseline titer, as indicated by the fact that cattle in both groups were seronegative at baseline and titers increased following the first vaccination. There was a significant (P = 0.018) booster effect in the TD group; the difference between the highest log2-transformed geometric mean ± SD titer after the second vaccination and that after the first vaccination was 1.9 ± 1.73. The difference between the highest titer after the second vaccination and that after the first vaccination did not differ significantly for the SC group.
C septicum alpha toxin titers—No significant differences were detected in the geometric mean C septicum alpha toxin titers between groups on any day. Vaccination via both routes induced a significant increase in antibody titer over the baseline titer; the difference between the log2-transformed geometric mean ± SD baseline titer and the highest titer after vaccination was 2.1 ± 0.83 (P < 0.001) for the TD group and 3.1 ± 1.20 (P = 0.002) for the SC group. There was not a significant increase in peak titer after the second dose of vaccine, compared with the peak titer after the first dose of vaccine, in either group, which indicated that neither route induced a booster effect on serum antibody titer.
Discussion
Yearling cattle vaccinated with a commercially available multivalent clostridial vaccine by means of a TD needle-free injector had smaller reactions at the site of vaccination than did cattle vaccinated SC with the same product. Although the size of the reactions was not significantly different at all observation times (probably attributable, in part, to the large range of sizes of injection-site reactions in the 2 groups on several days), the subjective impression of the evaluators was that the reactions differed in a clinically important way in that the lesions in the TD group appeared limited to the dermis and to involve little if any subdermal tissue. Although postmortem evaluation would be required to confirm this, it appeared that clostridial vaccination via the TD route would be less likely to lead to lesions requiring trimming at harvest, compared with results after vaccination via the SC route. Postmortem histologic evaluation of the injection-site reactions in cattle of the present study would have added useful information but unfortunately was not possible; this would be an important outcome to evaluate in future comparisons of TD versus SC administration of clostridial vaccines.
The route of vaccination likely affected the nature of vaccine site reactions in the cattle in the study reported here, but the difference was also likely affected by the fact that the SC group received 2 mL of vaccine, whereas the TD group received 0.5 mL of vaccine. The TD group was administered a smaller volume of vaccine because 0.5 mL is the maximum dose that can be administered via TD injection by use of the needle-free injector. The SC group received the label-indicated dose of the vaccine to induce an antibody response consistent with the administration of an approved product via an approved route of administration; this provided a meaningful control treatment with which to compare the antibody response induced via the TD route. Another logical control treatment for inclusion in the study would have been a group receiving 0.5 mL of vaccine SC, and that comparison should be made in future studies. Although the difference in volume of vaccine administered to each of the 2 groups could be considered a weakness of this study, the comparison of different volumes of the same product via these 2 routes was clinically relevant because ID or TD administration of vaccine with a smaller vaccine volume induces an immune response comparable to that obtained with a larger vaccine volume administered SC or IM.13,14 This principle was supported by the data in the present study: despite the fact that the TD group received a smaller volume of vaccine, cattle in that group had a significant increase in serum antibody titer for all of the clostridial toxins tested and mean titers after vaccination often did not differ significantly from titers induced by administration of the vaccine at an approved label dose via the SC route.
Although titers after vaccination often did not differ significantly between the 2 groups, an exception was that C sordellii lethal toxin titers were significantly lower in the TD group at most times after the first vaccination and until 21 days after the second vaccination (ie, day 49). The second vaccination administered via the TD route induced a significant increase in the C sordellii lethal toxin titer. The reason for the greater between-group differences in response to the C sordellii vaccine component, compared with responses to the other clostridial toxins tested, is not known. The lack of an anamnestic serum antibody response to C pefringens beta toxin, C novyi alpha toxin, and C septicum alpha toxin in both treatment groups after the second vaccination at day 28 was unexpected; however, an inconsistent anamnestic response to vaccine components following booster vaccination with a multivalent clostridial vaccine in cattle has also been reported by other investigators.7 It may be that the timing of administration of the booster vaccination was not optimal to induce an anamnestic response; however, the impact of the timing of booster vaccination on antibody responses to clostridial vaccines has been investigated,4,15 with investigators generally recommending that booster vaccinations be administered 28 to 42 days after the initial vaccination, which we did in this study. Moreover, administration 28 days after the primary dose was consistent with the label recommendations for the commercial clostridial vaccine. Serum antibody titers against C perfringens beta toxin, C novyi alpha toxin, and C septicum alpha toxin had returned nearly to baseline values by day 56 for most cattle in both treatment groups; similarly, other investigators4,7 have found that serum antibody titers return to baseline values within 2 to 4 months after vaccination with commercially available multivalent clostridial vaccines. Whether an appropriately administered series of primary and booster vaccinations can lead to an anamnestic response to challenge exposure several months after serum antibody titers have returned to baseline values is not known because studies2,16,17 have consistently been conducted to evaluate protection against challenge exposure within 2 months after vaccination and before serum antibody titers have returned to baseline values.
Although titers against C perfringens, C septicum, and, at most time points, C novyi were not significantly different between the 2 groups after vaccination, they were consistently lower in the TD group. This may have been attributable to lower reactivity at the site of vaccination because a relationship between size of the reaction and antibody response in ruminants receiving clostridial vaccines was observed in one study7 but not in another study.6 Serum antibody titers against clostridial toxins are associated with protection against disease.2,16,17 Serum agglutination titers of 1:147 were associated with complete protection against lethal challenge exposure with C septicum in sheep vaccinated with a commercially available bacterin-toxoid, and challenge exposure led to death of all control sheep that had serum agglutination titers of < 1:4.2 Similarly, serum antitoxin titers (measured by means of a mouse protection bioassay) of 1.6 to 3.2 antitoxin U/mL were associated with complete protection of vaccinated sheep exposed to lethal amounts of C novyi, but all control sheep with titers of < 0.4 antitoxin U/mL succumbed to challenge exposure.16 Because cattle in the study reported here were not challenge exposed, it is not known whether the titers measured in cattle vaccinated via TD and SC routes would have been equally protective. However, the fact that the TD route of vaccination induced significant increases in serum antibody titers against all toxins evaluated, with significantly smaller reactions at the site of vaccination than in cattle vaccinated SC, indicated that this route of vaccination merits further study as a method for protecting cattle against clostridial disease and minimizing pain-inducing and potentially costly local reactions.
ABBREVIATION
| TD | Transdermal |
Microsoft Excel, version 2003, Microsoft Corp, Redmond, Wash.
Caliber 7 clostridia vaccine, Boehringer Ingelheim, St Joseph, Mo.
Derma-Vac needle-free device, Bioject Inc, Tualatin, Ore.
Research and Development, Merial, Athens, Ga: Unpublished data, 2002.
Invitrogen, Carlsbad, Calif.
No. 14-12-06, Kirkegaard and Perry Laboratories, Washington, DC.
IMDM, Cambrex, East Rutherford, NJ.
SAS, version 8.2, SAS Institute Inc, Cary, NC.
STATXACT, version 7.0, Cytel Inc, Cambridge, Mass.
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