Clostridial Vaccination Efficacy on Stimulating and Maintaining an Immune Response in Beef Cows and Calves 1,2

Clostridial Vaccination Efficacy on Stimulating and Maintaining an Immune Response in Beef Cows and Calves 1,2 T. R. Troxel*,3, G. L. Burke*, W. T. Wallace*, L. W. Keaton*, S. R. McPeake*, D. Smith, and I. Nicholson *University of Arkansas Cooperative Extension Service, Little Rock, AR 72203 and Boehringer Ingelheim Animal Health, Inc., St. Joseph, MO 64506 ABSTRACT: Experiments were conducted to determine the efficacy in stimulating and maintaining an immune response in the presence of maternal antibodies, compare the extent of the anamnestic responses to revaccination, and compare the maternal antibody response of 2- or 5-mL clostridial vaccination. In Exp. 1, 118 nursing calves were randomly assigned to receive a 2-mL (Alpha-7, A7) or a 5-mL clostridial vaccine (Ultrabac 7; UB7) at 50.4 ± 15.30 ( X ± SD) d of age ( d 0 = date of calving). Calves were revaccinated with the same treatment on d 170. Blood samples were collected from 10 calves of each treatment group on d 50, 170, and 191 to determine antitoxin units for Clostridium perfringens type C (PC) and D (PD) and agglutination titers for Cl. chauvoei (CC). The A7-treated calves tended ( P <.10) to have higher PC units on d 170, an increased rate of change in PD units from d 170 to d 191 ( P <.06), and a tendency ( P <.10) for enhanced CC titers on d 191 compared with UB7-treated calves. In Exp. 2, 109 pregnant cows and 83 pregnant heifers were randomly assigned within a 2 2 2 factorial design. The main effects were dam age (cow or heifer), dam treatment (A7 or UB7), and calf treatment (A7 or UB7). Dams were vaccinated with A7 or UB7 d 124 prepartum (d 124) and d 53 after birth. At d 53.4 ± 12.88, calves were vaccinated with A7 or UB7 (d 53). Calves were revaccinated with the same treatment on d 173. Blood samples were collected from 10 dams per treatment group on d 124, 53, and 173 and from their calves on d 53, 173, and 194. Cows had higher antitoxin levels for PC ( P <.01) and PD ( P <.05) than heifers. The A7-treated dams had higher ( P <.01) PD units on d 53 and d 173 and CC on d 173 than did UB7-treated dams. Calves from A7-treated cows had higher ( P <.03) PD units on d 53 than calves from UB7-treated cows. The A7-treated calves had higher titers for CC ( P <.01) on d 173 than did UB7- treated calves, and this enhanced level seemed to continue to d 194 ( P <.08). In conclusion, titers for clostridial diseases in 50- to 53-d-old calves can be enhanced if dams are vaccinated approximately 4 mo before calving, and 120 d between clostridial vaccinations seems to be too long for adequate protection. Key Words: Clostridium, Vaccines, Beef Cows, Calves J. Anim. Sci. 1997. 75:19 25 Introduction 1 Mention of trade names, proprietary product, or specific equipment does not constitute a guarantee of warranty of the product by the University of Arkansas and does not imply approval to the exclusion of other products that may also be suitable. 2 Appreciation is expressed to Ron Everett and Joe Pattie for the use of their cattle and their assistance. 3 To whom correspondence should be addressed. Received November 3, 1995. Accepted August 28, 1996. Clostridial diseases have been a concern of cattle producers for many years. Because clostridial diseases are often rapidly fatal and usually affect cattle 6 mo to 2 yr of age, most producers view vaccination as cheap insurance. Many clostridial vaccines require revaccination 4 to 6 wk following the initial treatment (Compendium of Beef Products, 1993), but many cowcalf producers fail to revaccinate their calves at that time (personal observation). A previous study of the cow-calf segment revealed that approximately 60% of producers vaccinated calves for clostridial diseases before weaning (NAHMS, 1994). The effects of maternal antibody on active immunizations and the period of time between injection of vaccines that require multiple doses are reasons for immune failure (Schultz, 1994). The objectives of these experiments were to compare the effectiveness of a single injection of a 2-mL vaccine with a single injection of a 5-mL vaccine in stimulating and maintaining an immune response in the presence of maternal antibodies, to compare the extent of the anamnestic response to revaccination of a 19

20 2- and 5-mL vaccine approximately 120 d after the initial vaccination, to compare the maternal antibody response to 2- or 5-mL vaccine, and to evaluate the incidence of injection site lesions due to vaccination. Materials and Methods Experiment 1. Nursing crossbred beef calves ( n = 118, from multiparous beef cows) born in February and March were used. At the time of treatment, the average calf age was 50.4 ± 15.30 d ( X ± SD). All calves were individually identified and randomly assigned (based upon working order) to receive 2-mL clostridial vaccine (Alpha-7, A7) or 5-mL clostridial vaccine (Ultrabac 7, UB7). Alpha-7 (Boehringer Ingelheim) bacterium-toxoid uses an oil adjuvant and is labeled for a single 2-mL injection. Ultrabac 7 is labeled for a 5-mL injection with revaccination in 4 to 6 wk and uses an aluminum hydroxide adjuvant. Calves assigned to UB7 were not revaccinated 4 to 6 wk following d 50. Revaccination was not completed because many cow-calf producers fail to revaccinate their calves according to label directions. This revaccination treatment schedule was conducted under veterinarian supervision. Both products protect beef cattle against Cl. chauvoei (blackleg), Cl. septicum (malignant edema), Cl. novyi (black disease), and Cl. perfringens types C and D (Compendium of Beef Products, 1993). All injections were administered s.c. in the neck region using the tented technique. Sixty calves received A7, and 58 calves received UB7. Day 50 was used to designate the time of the initial clostridial treatment (d 0 = date of calving). All calves were revaccinated s.c. in the neck region with their assigned treatment on d 170. The dams of these calves do not receive annual clostridial vaccinations. They did, however, receive clostridial vaccinations as preweaning calves. Blood was collected via jugular venipuncture from 10 randomly selected calves of each treatment group immediately before each clostridial treatment ( d 50 and 170) and on d 191. The same calves from each treatment group were sampled throughout the experimental period. Blood samples were placed in crushed ice immediately after collection. The serum was harvested and stored at 20 C until assayed. Antitoxin units were determined for Cl. perfringens type C ( PC) and D ( PD) by the antitoxin neutralization test as described by USDA:APHIS:VS (1985 and 1993, respectively), and agglutination titers were determined for Cl. chauvoei ( CC) by the serum agglutination test modified from Claus and Macheak (1972). Standard antitoxin (10 IU/mL and 1 IU/mL for PC and PD, respectively) and toxin for PC and PD was obtained from USDA. The standard toxin was diluted to contain 10 L 0 doses/ml, and another sample was diluted to contain 10 L + doses/ml of antitoxin for both TROXEL ET AL. PC and PD. Ten international units of the PC standard antitoxin was combined with 10 L 0 doses of diluted PC standard toxin, and 10 IU of standard PC antitoxin was combined with 10 L + doses of diluted standard toxin. One international unit of PD standard antitoxin was combined with 10 L 0 doses of PD diluted standard toxin, and 1 IU of PD standard antitoxin was combined with 10 L + doses of PD diluted standard toxin. One milliliter of undiluted test serum was combined with 10 L 0 doses of PC diluted standard toxin. Additionally, 1 ml of the test serum was diluted with 1 ml of diluent, and a 1-mL aliquot of this solution was combined with 10 L 0 doses of PD diluted standard toxin. All toxin-antitoxin mixtures (PC and PD) were neutralized at room temperature for 1 h and held in ice water until mouse injection. Five Swiss white mice, each weighing 16 to 20 g, were used for each PC and PD toxin-antitoxin mixture. A dose of 2 ml (PC or PD mixture) was injected i.v. into each mouse. The test was terminated in 24 h, and deaths were recorded. The test was interpreted as follows: 1) if any mice inoculated with the mixture of 10 IU (PC) or 1 IU (PD) of standard antitoxin and 10 L 0 doses of standard toxin died, the results were inconclusive and the test was repeated, 2) if less than 80% of the mice inoculated with the mixture of 10 IU (PC) or 1 IU (PD) of standard antitoxin and 10 L + doses of standard toxin died, the results were inconclusive and the test was repeated, and 3) if any mice inoculated with the mixture of serum and 10 L 0 doses of standard PC and PD toxin died, the serum was considered to contain less than 10 IU/mL for PC and 1 IU/mL for PD. Clostridium chauvoei agglutination antigen (.4 ml) was added to 6 50 mm tubes in order to test a range of titers (.4,.2,.1, and.05 ml). Unknown serum was added to each tube. A negative control was made by mixing.4 ml of chauvoei agglutination antigen with.4 ml of chauvoei agglutination buffer. Tubes were covered with non-absorbent paper and mixed by inverting eight to 10 times. Tubes were then placed in a 50 C water bath so that the water covered approximately the bottom half of each tube. Tubes were incubated for 1 h. Tubes were removed from the water bath, and agglutination was observed by back lighting the tubes in a darkened room. Any agglutination (clumping) observed was designated by a + (positive) and no agglutination was designated (negative). The end point was the last tube to show agglutination. Titers were calculated as 1/serum volume 2 serum dilution. Experiment 2. One hundred nine pregnant multiparous crossbred cows and 83 pregnant crossbred heifers were used. On approximately d 124 prepartum (d 124), beef females were randomly assigned (based upon working order) to receive A7 or UB7. On d 124, 56 pregnant cows and 39 pregnant heifers received A7, and 53 pregnant cows and 44 pregnant heifers received UB7. All injections were administered

CLOSTRIDIAL VACCINATION EFFICACY 21 s.c. in the neck region using the tented technique. Dams were revaccinated with their assigned treatment on d 53 postpartum. Blood samples were collected from 10 A7-treated cows and heifers and 10 UB7-treated cows and heifers. The samples were handled and analyzed in the same manner as described in Exp. 1. Blood samples were collected via jugular venipuncture immediately prior to each clostridial treatment (d 124 and d 53) and on d 173. On d 53.4 ± 12.88 ( X ± SD) postpartum, the nursing calves were randomly assigned to receive A7 or UB7. Twenty-eight calves that came from A7- treated cows and 25 calves that came from UB7- treated cows received A7. Twenty-eight calves that came from A7-treated cows and 28 calves that came from UB7-treated cows received UB7. Of the 39 heifers that received A7, 20 of their calves received A7 and 19 received UB7. Of the remaining 44 calves that came from UB7-treated heifers, 24 received A7 and 20 received UB7. This resulted in a 2 2 2 randomized factorial design. The main effects were dam age (cows or heifers), dam treatment (A7 or UB7), and calf treatment (A7 or UB7). Calves received clostridial treatment s.c. in the neck region on d 53 (d 0 = date of calving) and on d 173. The calf treatment schedule of Exp. 2 followed the same reasoning and supervision guidelines as in Exp. 1. Blood samples were collected via jugular venipuncture from five calves of each treatment combination on d 53, 173, and 194. Blood sampling and analysis were conducted as described in Exp. 1. Table 1 summarizes the treatment and bleeding schedule for Exp. 1 and 2. All cattle were observed for injection site lesions (knots) 3 wk following the time of the second calf vaccination (d 170 in Exp. 1 and d 173 in Exp. 2). Cattle from both experiments grazed native pasture with trace mineral salt supplementation. Data Analysis. Cows (Exp. 2) and calves (Exp. 1 and 2) served as experimental units. The data from Exp. 1 and Exp. 2 were analyzed as a complete randomized design and as a 2 2 2 randomized factorial design, respectively. The data from both experiments were tested for normality by the Shapiro- Will test (SAS, 1992). The null hypothesis was rejected ( P <.05) for both experiments. Therefore, we concluded that the data were not normally distributed. Data within each time period were ranked, and ANOVA procedures were used to perform the Krushal- Wallis test (SAS, 1994). Ratio between two time periods were determined and ranked for analysis. If data were below detectable levels (1,.1, and 10 for PC, PD, and CC, respectively), the midpoint between the detectable level and 0 was assigned (.5,.05, and 5 for PC, PD, and CC, respectively). Because in Exp. 2 the number of animals assigned to each treatment group was not the same, the general linear model procedure (Type III SS) was used. One calf in the A7 Table 1. Summary of the treatment and bleeding schedules for Experiments 1 and 2 a d 0 = day of calving. b Experiment 1. c Experiment 2. d Alpha-7 or Ultrabac 7. e Collection of blood sample. Experimental period, d Item 124 a 50 b or 53 c 170 b or 173 c 191 b or 194 c Experiment 1 Calves Trt, d B e Trt, B B Experiment 2 Dams Trt, B Trt, B B Calves Trt, B Trt, B B cow-a7 calf sub-treatment group died at birth. Therefore, the cow and calf data from this sub-treatment group were removed from the experiment. All other sub-treatment groups had 5 treated cows and 5 treated calves. Because the data for both experiments were not normally distributed, the variation around each mean value is not reported. Results and Discussion Experiment 1. The mean PC antitoxin unit level was 12.1 on d 50 (probably due to maternal antibodies) and was not different across treatments (A7 or UB7; Table 2). The A7-treated calves had a tendency ( P <.10) to have higher PC serum antitoxin units on d 170 than the UB7-treated calves (1.7 vs 1.2). There also was a tendency ( P <.10) for the change in PC antitoxin unit levels of the UB7-treated calves from d 170 to d 191 to be greater than the change for the A7- treated calves (1.2 to 7.6 and 1.7 to 4.3). This interaction was partially caused by the lower antitoxin unit levels of the UB7-treated calves on d 170; however, the PC antitoxin unit levels on d 191 were not different ( P >.10) across treatment groups. Clostridium perfringens type C causes severe enteritis with diarrhea and dysentery in young lambs, calves, pigs, and foals. Usually calves 7 to 10 d old are affected by PC, but calves up to 10 wk of age may also be affected. In very acute cases, death occurs in a few hours, sometimes without diarrhea being evident (Radostits et al., 1994). Antitoxin unit levels for PD were below detectable levels ( <.1) on d 50, and the mean antitoxin unit across treatment groups on d 170 was still very low (.13). Clostridium perfringens type D or pulpy kidney can cause sudden death in calves between 1 and 4 mo of age. It is a short-term inhabitant that does not usually persist in the soil for more than 1 yr (Radostits et al., 1994). The UB7-treated calves had a greater antitoxin unit increase ( P <.06) from d 170 to d 191 than did the A7-treated calves (.03 to 7.20 and.23 to 6.52).

22 TROXEL ET AL. Table 2. The mean antitoxin units for Clostridium perfringens C and D and agglutination titers for Cl. chauvoei in serum of calves in Experiment 1 Experimental period Cl. perfringens C Cl. perfringens D Cl. chauvoei A7 a UB7 a A7 UB7 A7 UB7 d 50 12.1 12.0 0 b 0 22.0 46.0 d 170 e,f 1.7 c 1.2 d.23.03 34.0 16.5 d 191 4.3 7.6 6.52 7.20 153.5 c 78.5 d a Alpha-7 or Ultrabac 7. b Below detectable levels (.1). c,d Within rows, means not followed by a common superscript letter differ ( P <.10); in the absence of superscripts, means are not different. e,f Means change across sampling period d 170 to d 191 due to treatment effects: e: Cl. perfringens C, P <.10; f: Cl. perfringens D, P <.06. Agglutination titers for CC were similar for treatment groups on d 50 and d 170. There was a tendency ( P <.10) for the A7-treated calves to have higher agglutination titers on d 191 compared with UB7- treated calves (153.5 vs 75.8). Clostridium chauvoei affects growing cattle 6 mo to 2 yr old on good nutritional plans. It is a soil-borne infection that causes severe death losses unless cattle are vaccinated (Radostits et al., 1994). Because calves were not sampled from d 50 to 170, it is not known if antitoxin unit levels (PC and PD) or agglutination titers (CC) increased following the first treatment ( d 50) and then decreased or if maternal antibodies prevented response of the calves immune system. Twenty-one days following revaccination ( d 191), antitoxin unit levels for PC and PD increased compared with d 170 levels, but this was not the case for agglutination titers for CC. Kennedy et al. (1977) reported an enhanced response to the second injection for CC in yearling calves when the time between injections was 4 or 6 wk. This may suggest that waiting 120 d (over 17 wk) may be too long for an enhanced CC response to the second injection. Experiment 2. Dam age had a significant effect on the PC and PD antitoxin unit response to dam treatment (Table 3). Regardless of treatment, cows had higher PC antitoxin units ( P <.01) for all three sampling periods ( d 124, 53, and 173) and higher PD antitoxin units for d 124 and 53 ( P <.05) with a tendency on d 173 ( P <.11) compared with heifers. Cows antitoxin unit level responses for PC were 9.2, 15.3, and 10.0 for d 124, 53, and 173, respectively, compared with the heifers antitoxin unit response levels of 6.0, 8.7, and 4.7 for the same sampling Table 3. The mean antitoxin units for Clostridium perfringens C and D and agglutination titers for Cl. chauvoei in serum of dams in Experiment 2 Experimental period Cows Heifers A7 a UB7 a A7 UB7 Cl. perfringens C b d 124 8.9 9.5 3.9 8.1 d 53 20.0 11.0 8.2 9.1 d 173 13.9 c 6.5 c 3.8 c 5.6 c Cl. perfringens D b d 124 d,e 1.04.48.35.10 d53 e 1.24 f.28 g.53 f.10 g d 173 1.91 f.48 g 1.12 f.10 g Cl. chauvoei d 124 e 61.1 53.5 65.5 54.0 d53 e 301.1 187.5 204.5 171.0 d 173 228.3 57.0 268.0 76.0 a Alpha-7 or Ultrabac 7. b Main effects for dam age differ: Cl. perfringens C, P <.01; Cl. perfringens D, P <.05. c Dam age treatment ( P <.05). d,e Means change across sampling periods due to treatment effects: d: d 124 to d 53, P <.01; e: d 124 to d 173 and d 53 to d 173, P <.01. f,g Within rows, means not followed by a common superscript differ ( P <.01); in the absence of superscripts, means are not different.

CLOSTRIDIAL VACCINATION EFFICACY 23 periods. The cows PD antitoxin unit responses for d 124, 53, and 173 were.71,.70, and 1.10 respectively, whereas the heifers PD antitoxin unit levels were.23,.32, and.61. There were no differences detected with the main effect of age on the CC response ( P >.10). The anamnestic response to PC and PD antitoxin unit due to dam age may be due to cows being exposed to more annual vaccinations (on the average) throughout their lifetime than the heifers. There also were no differences detected ( P >.10) due to dam age in the relative changes in PC and PD antitoxin units or CC titers from one sampling period to another. Even though the A7-treated females had lower PC antitoxin units than UB7-treated females on d 124 (6.3 vs 8.8, P <.01), the relative changes from d 124 to 53 (6.3 to 13.8 and 8.8 to 10.1, P <.01) and from d 124 to d 173 (6.3 to 8.6 and 8.8 to 6.1, P <.01) were greater for the A7-treated females than for the UB7- treated females. There were no treatment effects ( P >.10) on the relative change from d 53 to 173. There was an age treatment interaction on d 173 ( P <.05) for the PC antitoxin units response. The A7-treated cows and heifers had antitoxin unit responses of 13.9 and 3.8 compared with the UB7-treated cows and heifers responses of 6.5 and 5.6. Therefore, to obtain higher antitoxin unit levels based upon this treatment schedule, different clostridial vaccines could be selected based upon dam s age. Additional blood samples were not collected after d 173 to determine how long the enhanced antitoxin unit response was maintained in the UB7-treated heifers. There was no age treatment interaction detected on d 173 for PD or CC ( P >.10). There were significant PD and CC titers responses due to treatment. Clostridium perfringens type D antitoxin units response was higher ( P <.01) for the A7-treated females than for the UB7-treated females on d 53 (.87 vs.19) and d 173 (1.49 vs.29). Unlike the PC levels, there was no difference for PD levels on d 124 by treatment groups. The only sampling period where CC titers were different ( P <.01) due to treatment was d 173. The A7-treated females had a mean CC titer of 249.2 compared with UB7-treated females CC titer of 66.5. The relative change for the dam PD titer response for all three time periods was different due to treatment. The change from d 124 to d 53, from d 124 to d 173, and from d 53 to d 173 was.68 to.87 vs.29 to.19 ( P <.05),.68 to 1.49 vs.29 to.29 ( P <.01), and.87 to 1.49 vs.19 to.29 ( P <.01) for A7- and UB7-treated females. Although there was no difference in the CC titer response from d 124 to 53 ( P >.10) due to A7- or UB7-treatment, there was a difference ( P <.01) from the time period of d 124 to 173 (63.4 to 249.2 vs 53.8 to 66.5) and from d 53 to 173 (241.3 to 249.2 vs 179.3 to 66.5). A possible explanation for the A7-treated females having a greater response for PC, PD, and CC titers may be due to A7 being used in this cow herd for the first time, whereas UB7 had been used in previous years. A second possible explanation for the enhanced response to A7 may be due to the different adjuvants used by A7 and UB7. Alpha-7 uses an oil adjuvant with gentamicin as a preservative, and UB7 uses aluminum hydroxide as an adjuvant. The A7 oil-based adjuvant may cause a slower and longer release of the bacterium-toxoid, resulting in the enhanced immune response. The main effects of dam treatment, calf treatment, or dam s age were not significant for PC as measured by the serum in the suckling calf. Other research reported that PC does produce measurable antitoxin levels in the colostrum of pigs (Djurickovie et al., 1975) and cows (Lozano et al., 1971). One would have suspected a higher level of PC antitoxin units in the suckling calves from the cows because the cows had higher response rate to treatment. The PC antitoxin unit levels on d 50 and 170 for the suckling calves in Exp. 1 were lower ( P <.01) than the suckling calves PC antitoxin unit levels that came from A7-treated cows on d 53 in Exp. 2, suggesting an enhanced response in suckling calf PC titer levels due to A7 mature cow treatment. Unlike in Exp. 1, there were detectable levels of PD antitoxin unit levels in the serum of suckling calves on d 53 (Tables 2 and 4), once again suggesting higher calf PD titers due to dam treatment. Calves of A7- treated dams had higher levels of PD antitoxin units on d 53 than calves of UB7-treated dams (1.62 vs.23, P <.05). Transfer of maternal antibody to lambs via the colostrum has been demonstrated by Oxer et al. (1971). Vaccinating pregnant dams with A7 124 d before calving increased the PD maternal response in the suckling calf compared with UB7 treatment, and therefore vaccinating calves at 50 d of age may not be necessary. Antitoxin unit levels (PD), however, were enhanced following the second injection ( d 191 or d 194) in calves that came from non-vaccinated dams (Exp. 1) over calves from A7- or UB7-treated dams (6.82, 3.37, and 4.12 for non-vaccinated, A7-, and UB7-treated dams, respectively). In addition, A7- treated calves had higher levels of PD antitoxin units on d 173 than UB7-treated calves (.55 vs.02, P <.01). Although no calves died of PD, it is unknown how well the UB7-treated calves with low antitoxin unit levels (.02) would have responded to a PD challenge on d 173. The PD antitoxin unit rate of decline from d 53 to 173 was increased for the UB7-treated calves compared with the A7-treated calves (.95 to.02 and.80 to.55, P <.01), suggesting that, given this treatment schedule, A7 would maintain PD levels in the calf longer than UB7. Dam treatment had a significant effect on the rate of the calves CC titer change from d 53 to 173. The calves from A7-treated dams had increased titer losses from d 53 to 173 compared with UB7-treated dams

24 TROXEL ET AL. Table 4. The mean antitoxin units for Clostridium perfringens C and D and agglutination titers for Cl. chauvoei in serum of calves in Experiment 2 Experimental period Cow treatment Heifer treatment A7 a UB7 a A7 UB7 A7 UB7 A7 UB7 Calf treatment A7 A7 UB7 UB7 A7 A7 UB7 UB7 Cl. perfringens C d53 b 20.0 5.0 27.5 8.0 7.0 13.0 10.0 13.0 d 173 2.6.8 6.5.4.4.2 2.6 2.6 d 194 11.0 8.4 1.5 10.6 4.6 26.2 8.4 13.2 Cl. perfringens D d 53 2.44 c.10 d 3.03 c.10 d.26 c.46 d 1.02 c.26 d d 173.26 f.44 f.05 g 0 e,g.06 f 1.44 f 0 g.02 g d 194 7.00 6.06 4.25.42 1.60 7.00.80 3.00 Cl. chauvoei d53 h 159.0 22.0 73.8 33.0 95.0 85.0 66.0 70.0 d 173 55.0 75.0 2.5 0 e 0 95.0 0 22.0 d 194 57.0 240.0 121.3 44.0 188.0 200.0 53.0 24.0 a Alpha-7 or Ultrabac 7. b Means change across sampling period d 53 to d 173 due to dam age ( P <.05). c,d Within rows, dam treatment means not followed by a common superscript letter differ (P <.05). e Below detectable levels: Cl. perfringens D=1,Cl. chauvoei = 10. f,g Within rows, calf treatment means not followed by a common superscript letter differ (P <.01). h Means change across sampling period d 53 to 173 due to dam ( P <.05) and calf treatment effects ( P <.01). (99.0 to 15.0 vs 52.5 to 48.0, P <.05). Age of dam or dam treatment had no effect ( P >.10) on calf CC serum agglutination titers on d 53. On d 173, however, the A7-treated calves had CC titers of 56.3 compared with titers of 6.3 for the UB7-treated calves ( P <.01). This would suggest that A7 maintained higher CC titers for the 120-d period compared with UB7. There also was a tendency ( P <.08) for the A7-treated calves CC agglutination titers to increase at a faster rate from d 173 to 194 compared with UB7-treated calves (56.3 to 180.3 vs 6.3 to 54.5). The interaction of dam treatment dam age was significant for PC, PD, and CC titers in the serum of suckling calves. Calves of A7-treated cows had higher PC antitoxin units on d 53 than calves of UB7-treated cows (23.3 vs 6.5, P <.01), but there were no differences between calves of A7- or UB7-treated heifers (8.5 vs 13.0). The same titer response ( P <.05) was detected with PD and CC titers for d 53 (2.70,.10,.64, and.36 for PD titers of calves from A7- cows, UB7-cows, A7-heifers, and UB7-heifers, respectively, and 121.1, 27.5, 79.0, and 77.5 for CC titers of calves from A7-cows, UB7-cows, A7-heifers, and UB7- heifers, respectively). There were higher PC antitoxin unit levels for calves that came from A7-treated cows on d 173 than for calves that came from UB7-treated cows (4.33 vs.60), but the reverse was true for calves that came from A7-treated and UB7-treated heifers (.30 vs 1.30), resulting in a dam treatment dam age interaction ( P <.01). The same interaction on d 173 was significant for PD ( P <.04) and CC ( P <.03). There was, however, a difference with these interactions as compared with the d 53 interaction of PC antitoxin units. The calves from A7- or UB7-treated cows for PD or CC were not different (.17 and.22 for PD and 31.7 and 37.5 for CC), but rather the calves antitoxin units from A7- or UB7-treated heifers were different for PD and CC (.03 and.70 for PD and 0.0 and 58.0 for CC). The only interaction that was significant for calf antitoxin unit response on d 194 was for PD ( P <.01). This interaction followed the same pattern, with calf antitoxin units from A7- and UB7-treated cows being 5.8 and 3.2 and calf antitoxin units from A7- and UB7-treated heifers being 1.2 and 5.0. There were dam treatment dam age interactions ( P <.01) for the relative change in calf antitoxin units for PD and CC titers from d 173 to 194. The relative changes in PD antitoxin units of calves from A7- and UB7-treated cows from d 173 to 194 were.17 to 5.78 and.22 to 3.2, whereas the relative changes for calves from A7- or UB7-treated heifers were.03 to.12 and.73 to 5.0. These interactions may provide additional support to anamnestic response detected with treatment and to age of dam. These data may also suggest that to enhance titer response in suckling calves in this reported treatment schedule would be to use A7 to treat pregnant mature cows and UB7 to treat pregnant heifers. Data on the incidence of injection site lesions (knots) were collected from both experiments. There were no differences ( P >.10) between experiments or treatments (A7 or UB7) on the incidence of or the size of injection site lesions. Overall, 12% of the treated

CLOSTRIDIAL VACCINATION EFFICACY 25 animals (calves and dams) had lesions 3 cm or less on d 191 or 194. Vaccinating cows with A7 approximately 124 d before calving provided enhanced maternal antibody protection in the suckling calf for PD as compared with cow UB7 vaccination and no prepartum cow vaccination and CC as compared with no vaccination prepartum. The older dams ( > 3 yr) showed an increased response to clostridial vaccination compared with younger dams ( < 3 yr). When calves were vaccinated for clostridial diseases on d 50 or 53 of age, antitoxin units levels for PC and PD and agglutination titers for CC decreased to low levels by d 170 or 173. Samples were not collected between d 50 or 53 and d 170 or 173 to determine if the vaccinations caused an enhanced immune response in the suckling calf or if maternal antibodies prevented a response of the calves immune system. The immunity of PC, PD, and CC was enhanced by d 21 following the second vaccinations. It has also been shown that in sheep a useful degree of immunity could be obtained after a single injection of a combined clostridial vaccine but a second inoculation to reinforce the early immunity was important (Sterne et al., 1962; Kerry and Craig, 1979). Additional samples past d 191 or 194 were not collected to determine the duration of the d 170 or 173 revaccinated enhanced response. There were no differences in the incidence of injection site lesions between the two vaccines evaluated. Implications These data indicate that titers against clostridial diseases in 50- to 53-d-old calves can be enhanced if their dams are vaccinated approximately 4 mo before calving. Many producers vaccinate suckling beef calves for clostridial diseases with one injection even though many vaccination labels state that two injections should be given. The data implied that vaccinating calves at d 50 of age, and not again until weaning, may not provide adequate protection against clostridial diseases. Also, if pregnant females are vaccinated approximately d 124 before calving, vaccinating calves may not be necessary until the calves are older than d 50 to 53. Some producers have reported calf losses to clostridial diseases between the time of calf vaccination (approximately d 60 of age) and weaning (personal observation). With proper management and vaccination timing, these losses may be prevented. Literature Cited Claus, K. D., and M. E. Macheak. 1972. Preparation of a Clostridium chauvoei antigen and determination of protective immunity by plate agglutination test. Am. J. Vet. Res. 33:1045. Compendium of Beef Products. 1993. North American Compendiums, Port Huron, MI. Djurickovie, S. M., J. E. Dworak, and K. L. Wickham. 1975. Antitoxin titer in colostrum and milk after vaccination of sows with Clostridium perfringens type C toxoid vaccine. Veterinary Medicine Small Animal Clinician (March) pp 283 285. Kennedy, K. K., S. J. Norris, W. H. Beckenhauer, and R. G. White. 1977. Vaccination of cattle and sheep with a combined Clostridium perfringens type C and D toxoid. Am. J. Vet. Res. 38, No. 10. Kerry, J. B., and G. R. Craig. 1979. Field studies in sheep with multicomponent clostridial vaccines. Vet. Rec. 105:551. Lozano, E. A., J. E. Catlin, and W. W. Hawkins. 1971. Effects of Clostridium perfringens beta antitoxin and colostral absorption on incidence of calf scours. Cornell Vet., Vol. LXI: No. 2. National Animal Health Monitoring System. 1994. Beef Cow/Calf Health and Productivity Audit. USDA:APHIS:VS, Fort Collins, CO. Oxer, D. T., D. W. Minty, and C. E. Liefman. 1971. Vaccination trials in sheep with clostridial vaccines with special reference to passively acquired Cl. welchii type D antitoxin in lambs. Aust. Vet. J. 47:134. Radostits, O. M., D. C. Blood, and C. C. Gay. 1994. Veterinary Medicine A Textbook of the Diseases of Cattle, Sheep, Pigs, Goats and Horses (8th ed.). Bailliere Tindall, London, England. SAS. 1992. SAS Procedures Guide (Version 3 Ed.). SAS Inst. Inc., Cary, NC. SAS. 1994. SAS/STAT User s Guide (Version 6 Ed.). SAS Inst. Inc., Cary, NC. Schultz, R. D. 1994. Certain factors to consider when designing a bovine vaccination program. Bovine Proceedings, No. 26. Sterne, M., I. Batty, A. Thompson, and J. M. Robertson. 1962. Immunization of sheep with multi-component clostridial vaccines. Vet. Rec. 74:909. USDA:APHIS:VS. 1985. Supplemental assay method for potency testing products containing Clostridium perfringens type C beta antigen. 9CFR 113.111. USDA:APHIS:VS. 1993. Supplemental assay method for potency testing products containing Clostridium perfringens type D epsilon antigen. 9CFR 113.112.