Vaccination of Turkeys with Clostridium septicum Bacterin-Toxoid: Evaluation of Protection Against Clostridial Dermatitis Author(s): Anil J. Thachil, Brian McComb, Michelle Kromm, and Kakambi V. Nagaraja Source: Avian Diseases, 57(2):214-219. 2013. Published By: American Association of Avian Pathologists DOI: http://dx.doi.org/10.1637/10421-101512-Reg.1 URL: http://www.bioone.org/doi/full/10.1637/10421-101512-Reg.1
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AVIAN DISEASES 57:214–219, 2013
Vaccination of Turkeys with Clostridium septicum Bacterin-Toxoid: Evaluation of Protection Against Clostridial Dermatitis Anil J. Thachil,A Brian McComb,B Michelle Kromm,C and Kakambi V. NagarajaAD A
Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, 1971 Commonwealth Avenue, Saint Paul, MN 55108 B Willmar Poultry Company, Willmar, MN 56201 C Jennie-O Turkey Store, 2505 Willmar Avenue, Willmar, MN, 56201 Received 15 October 2012; Accepted 23 January 2013; Published ahead of print 25 January 2013 SUMMARY. Clostridial dermatitis is an acute disease causing high mortality in turkeys. Both Clostridium septicum and Clostridium perfringens have been isolated from these cases; however, reports from several diagnostic laboratories indicate an increased isolation rate of C. septicum compared with C. perfringens from cases of clostridial dermatitis in recent years. Previous studies suggested C. septicum was more potent than C. perfringens in causing clostridial dermatitis in turkeys. The objective of this study was to develop and evaluate the use of a C. septicum bacterin-toxoid to control clostridial dermatitis in turkeys. A C. septicum bacterin-toxoid was prepared and was initially tested in 6-wk-old commercial turkeys under laboratory conditions for its safety and efficacy. Subsequently, the bacterin-toxoid was evaluated for use in commercial turkey farms with a consistent history of clostridial dermatitis. Birds in the field were vaccinated subcutaneously once at 6 wk of age with C. septicum bacterin-toxoid, and then mortality in both vaccinated and unvaccinated groups was recorded and compared. Blood samples from birds in both groups were examined using ELISA to detect antibody response to the C. septicum toxoid. The C. septicum bacterin-toxoid was found to be safe and to elicit antibodies against the toxoid. In vaccinated commercial turkeys, control of clostridial dermatitis was achieved via antibiotic use and clostridial dermatitis mortality was significantly reduced compared with that of birds in the unvaccinated group. The C. septicum bacterin-toxoid seems to be a valuable tool for the turkey industry to reduce losses due to clostridial dermatitis. RESUMEN. Vacunacio´n de pavos con una bacterina-toxoide de Clostridium septicum: Evaluacio´n de la proteccio´n contra la dermatitis clostridial. La dermatitis clostridial es una enfermedad aguda que causa una alta mortalidad en pavos. Tanto Clostridium septicum como Clostridium perfringens se han aislado de estos casos. Sin embargo en los u´ltimos an˜os, informes de varios laboratorios de diagno´stico indican un aumento de la tasa de aislamiento de C. septicum en comparacio´n con C. perfringens a partir de casos de dermatitis clostridial. Los estudios previos han sugerido que C. septicum era ma´s potente que C. perfringens para causar dermatitis clostridial en pavos. El objetivo de este estudio fue desarrollar y evaluar el uso de una bacterina-toxoide de C septicum para controlar la dermatitis clostridial en los pavos. La bacterina-toxoide contra C. septicum se preparo´ y fue probada inicialmente en pavos comerciales de seis semanas de edad, bajo condiciones de laboratorio, para determinar su seguridad y eficacia. Posteriormente, la bacterina-toxoide se evaluo´ para su uso en granjas de pavos comerciales con un historial de dermatitis clostridial constante. Las aves en el campo fueron vacunadas por vı´a subcuta´nea una vez a las seis semanas de edad con la bacterina-toxoide de C. septicum bacterina y se registro´ y comparo´ la mortalidad en los grupos vacunados y no vacunados. Las muestras de sangre de aves en ambos grupos se examinaron usando una prueba de ELISA para detectar la respuesta de anticuerpos contra el toxoide de C. septicum. Se determino´ que la bacterina-toxoide de C. septicum era segura y que inducı´a anticuerpos contra el toxoide. En pavos comerciales vacunados, el control de la dermatitis clostridial se logro´ mediante el uso de antibio´ticos y se redujo significativamente la mortalidad por dermatitis clostridial en comparacio´n con las aves del grupo no vacunado. La bacterina-toxoide contra C. septicum parece ser una herramienta valiosa para la industria de los pavos para reducir las pe´rdidas debidas a la dermatitis clostridial. Key words: clostridial dermatitis, cellulitis, Clostridium septicum, vaccine, turkey Abbreviations: BHI 5 brain heart infusion; CFU 5 colony-forming unit(s); dpv 5 days postvaccination; HE 5 hemorrhagic enteritis; OD 5 optical density; RAR 5 Research Animal Resources
Clostridial dermatitis and cellulitis is one of the leading causes of economic loss in the turkey industry in the United States (3,4). The severity of these conditions varies depending on the virulence characteristics of the bacteria. Although Clostridium perfringens and Clostridium septicum can cause clostridial dermatitis in turkeys, C. septicum has been found to be more pathogenic than C. perfringens in causing clostridial dermatitis (4). Clostridial dermatitis in turkeys is characterized by inflammation of skin and subcutaneous tissue, resulting in accumulation of frothy sanguinous exudate in the subcutis of breast and tail regions (4,5,14). Mortality, increased condemnation rates, and expensive medication costs for treatment have accounted for the huge economic losses (3,8). D
Corresponding author. E-mail: [email protected]
Clostridium septicum is reported to cause traumatic gas gangrene and clostridial myonecrosis in animals and humans (9,11). The exotoxins produced by C. septicum are responsible for tissue damage and toxemia (16). Clostridium septicum toxins were reported to be more potent than those of C. perfringens in causing clostridial dermatitis in turkeys (4,14). Clostridial vaccines containing toxoid and killed bacteria are very effective in humans and animals against many clostridial infections. Clostridium perfringens infections have been reported to be successfully controlled in suckling piglets by administration of a toxoid vaccine (10). The use of C. septicum toxoid induced protection against spore challenge in sheep (2). Some studies (12,13) have drawn attention to secretory toxins from C. septicum and their role in causing clostridial dermatitis in turkeys.
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Fig. 1. Gross lesions and histopathology of subcutaneous and muscle tissue in clostridial dermatitis due to C. septicum in turkeys. (A) Presence of frothy sanguineous exudate in subcutaneous tissue and underlying muscle necrosis in a bird infected with C. septicum spore culture. Bar 5 1 cm. (B) Fragmented muscle fibers infiltrated with C. septicum in a clostridial dermatitis–affected bird. Section was stained with hematoxylin and eosin. Bar 5 50 mm.
The use of a bivalent C. perfringens and C. septicum bacterintoxoid against clostridial dermatitis was found to reduce loses due to clostridial dermatitis in turkeys (7,15). Because of the increased isolation rate of C. septicum alone in many cases of clostridial dermatitis, studies have started to look into developing a C. septicum bacterin-toxoid alone for effective use in the field for controlling clostridial dermatitis in turkeys. Here, we report the use of a C. septicum bacterin-toxoid against clostridial dermatitis in turkeys. MATERIALS AND METHODS Bacteria. Clostridium septicum isolate (UMNCS 106), originally isolated from breast lesions of clostridial dermatitis in a turkey, was used in this research. This isolate possessed the ability to express high toxin titers and heat-resistant spore counts (14). In brief, C. septicum was first grown in brain heart infusion (BHI) broth at 37 C for 18 hr under anaerobic conditions, and incubation was continued for additional 6 hr at 24 C. The presence of toxin in the culture supernate was ascertained by mouse lethal assay (14), and the culture was then treated with 0.5% formalin to inactivate toxins in it. The complete inactivation was confirmed by subculturing an aliquot of culture supernate in BHI broth anaerobically. Failure of any growth was taken as complete inactivation of bacteria. The inactivation of toxins was confirmed by hemolysis assay (17). This inactivated Clostridium culture contained 4.8 3 107 colony-forming units (CFU)/ml bacteria and 3 g/ml toxoid, which was later used for making a bacterin-toxoid. Preparation of C. septicum bacterin-toxoid. An experimental bacterin-toxoid vaccine was prepared using the inactivated culture of C. septicum mentioned under ‘‘Bacteria’’ section. Dakreol-6VR (Pennsylvania Refining Company, Butler, PA), a vaccine-grade mineral oil, and the emulsifier Arlacel-A (Sigma-Aldrich, St. Louis, MO) were used to complete the preparation of the vaccine. Arlacel-A was used at the rate of 10% (v/v) in the oil component. A 1:1 water/oil emulsion was prepared after slowly mixing the oil component (mineral oil and ArlacelA) in a blender to which the aqueous inactivated culture component was added over a period of 5 min. The final preparation contained 1.5 g
(128 mouse lethal dose50) of toxoid per milliliter of vaccine. The experimental birds were vaccinated at different doses to evaluate the efficacy of the vaccine postchallenge. Laboratory evaluation of C. septicum toxoid in turkey poults. The experimental bacterin-toxoid was first tested for its safety and efficacy in 6-wk-old commercial Nicholas White male turkey poults obtained from a source with no history of clostridial dermatitis. The birds were inoculated subcutaneously with either a 1-ml dose containing 1.5 g of toxoid or with a 2-ml dose containing 3 g of toxoid. In brief, ninety-six 6-wk-old commercial Nicholas White turkey poults obtained from a source with no history of clostridial dermatitis were divided into four groups (groups I–IV) of 24 birds each. Each bird in group I was inoculated with the 1-ml dose and each bird in group II were inoculated subcutaneously with the 2-ml dose of the toxoid preparation at the wing web. The birds in group III were nonvaccinated positive controls for clostridial challenge, and the birds in group IV were treated as nonvaccinated negative controls. On the 14th day postvaccination, half of the birds from groups I and II were given a booster dose at the same dose as mentioned. The blood from all the groups was collected at 0-, 14-, and 28-days post-one vaccination and on 7-days post-booster vaccination for serologic examination. All the birds were monitored twice daily for any adverse effects of the toxoid. Any bird showing severe pain or distress or that was unable to move to feeders and waterers was euthanized in a carbon dioxide chamber. After 28-days post-one vaccination, 12 birds each from groups I and II that received one vaccination and 12 birds from nonvaccinated control group III were challenged with 2 ml of fresh C. septicum culture (4.8 3 107CFU/ml) subcutaneously on the breast region. Similarly, after the 14days post-booster vaccination, the remaining 12 birds from groups I and II and 12 birds from the nonvaccinated control group III were challenged with C. septicum culture. The challenge dose and route with C. septicum culture were optimized in our previous study (14). The sera from all the groups were examined for seroconversion to the vaccine by using C. septicum–specific ELISA as described previously (12,14). All the birds at the end of the study were euthanized in a carbon dioxide chamber. All animal experimental protocols were approved by the Institutional Animal Care and Use Committee, and the procedures were performed
Table 1. Clostridium septicum ELISA serum antibody titer (optical density [OD] values) after vaccination with C. septicum toxoid in vaccinated and nonvaccinated birds (n 5 12).A Post-one vaccination OD values
Vaccine at 1-ml dose Vaccine at 2-ml dose Nonvaccinated A
Post-two vaccination OD values
0 dpv
14 dpv
28 dpv
7 dp2v
0.2459 (0.13)a 0.2691 (0.09)a 0.2708 (0.54)a
0.6574 (0.24)b 0.7645 (0.23)b 0.2182 (0.17)a
0.704 (0.10)b 0.7254 (0.09)b 0.2814 (0.05)a
0.9217 (015) 1.0686 (0.14) 0.2689 (0.05)a
Means (SD) within a row with no common lowercase letter are considered significantly different (P , 0.05).
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in accordance with their requirements. The experimental birds were reared at Research Animal Resources (RAR) isolation facilities at the University of Minnesota, Saint Paul, MN. Field trials with C. septicum toxoid in commercial turkeys. With permission from the Minnesota State Board of Animal Health and University of Minnesota and consent from a leading commercial turkey producer, two field trials were done to test the efficacy of C. septicum bacterin-toxoid. The birds were vaccinated subcutaneously in the neck region by using a vaccine applicator. In the first field trial, vaccinated and nonvaccinated birds were separated by a wire mesh in the middle and subjected to similar management conditions. In the second field trial, vaccinated and nonvaccinated birds were kept together in the same barn to eliminate any bias toward exposure to clostridia from the environment within the barn. Age of birds at vaccination was 6 wk in both field trials. Age at marketing was at the end of 22 wk in field trial I and 19 wk in the field trial II. Field trial I. Eight thousand male Nicholas White turkey poults in a flock of 16,000 in a brooder barn were selected randomly at the age of 6 wk and vaccinated with C. septicum bacterin-toxoid at a 1-ml dose. They were vaccinated subcutaneously in the neck region by using a vaccine applicator. All the poults were previously vaccinated for Newcastle disease at 3 days 3 wk of age and for hemorrhagic enteritis (HE) at 4 wk of age. After vaccination, 4000 birds each from vaccinated and nonvaccinated groups were transferred to two grow-out farms, hereafter referred to as Farm I and Farm II, where there was a consistent history of clostridial dermatitis. The assumption was that all birds were likely to be exposed equally to clostridia found in that barn environment. Being in a commercial setting, penicillin G potassium (Alpharma Inc., Bridgewater, NJ) use in water was in place as a precautionary measure as and when clostridial dermatitis–related mortality rose above 0.1% per day. Daily mortality and packs of penicillin used to control clostridial dermatitis–related mortality were recorded in both vaccinated and nonvaccinated groups from 10 wk of age until 22 wk of age, when the birds are marketed. On the farms, routine monitoring of all the sick, moribund, and dead birds was conducted twice daily, and birds identified as sick or moribund were euthanized. The results were analyzed to detect differences in the occurrence of mortality between vaccinated and nonvaccinated birds. When birds were 10 wk of age, 10 birds each from the vaccinated and nonvaccinated groups from the field-vaccinated trial were transferred to RAR facilities at the University of Minnesota. All the birds were challenged as mentioned above. Clostridial dermatitis development and mortality between the groups were recorded. All the birds left at the end of the study were euthanized in a carbon dioxide chamber. Statistical analysis was performed by SAS software, version 9.2 (SAS Institute, Cary, NC). Mortality data after challenge were analyzed using Fisher exact test. A survival analysis of maximum likelihood estimates using proportional hazards model (PH-REG procedure) was conducted to analyze the mortality rates in vaccinated and nonvaccinated groups. A Pearson chi-square test was used to compare the use of antibiotics between vaccinated and nonvaccinated groups. The serologic data were analyzed using a GLM procedure for repeated measures analysis of variance, where a P value of 0.05 was considered significant. Field trial II. In brief, 4000 poults in a flock of 20,000 male Nicholas White turkey poults in a brooder barn at the age of 6 wk were banded and vaccinated with C. septicum bacterin-toxoid at the 1-ml dose. The vaccine was administered subcutaneously at the neck region by using a vaccine applicator. All birds were vaccinated previously for Newcastle disease at 3 days and 3 wk of age and for HE at 4 wk of age. At 7 wk of age, half of the birds from each of the vaccinated and nonvaccinated groups were transferred to two different grower farms, hereafter referred to as Farm IA and Farm IIA, where there was a consistent history of clostridial dermatitis. The vaccinated and nonvaccinated birds were kept together and subjected to similar management conditions. A therapeutic antibiotic penicillin G potassium use in water was in place as a precautionary measure as and when the mortality due to clostridial dermatitis rose above 0.1% per day. Daily mortality with visible clostridial dermatitis lesions was recorded in both vaccinated and
Table 2. Comparison of clostridial dermatitis–related mortality and antibiotic use postvaccination with C. septicum bacterin-toxoid in field trial I from 10 wk of age to 22 wk of age.A
No. of birds Mortality % Mortality Penicillin useB (packs) Penicillin use (days)
Nonvaccinated group
Vaccinated group
8000 970 12.1 204 68
8000 840a 10.5a 84a 28a
A
Values followed by a lowercase letter indicate P , 0.05. One pack of penicillin contains 1.0 billion international units (601.2 g) of penicillin G potassium. B
nonvaccinated birds until the birds were marketed. The results were compared to detect any observable differences in the occurrence of mortality between vaccinated and nonvaccinated birds. Laboratory challenge of birds with C. septicum postvaccination. Ten birds each from vaccinated and nonvaccinated groups from field trial I were transferred to RAR facilities at University of Minnesota. Blood was collected at 3 wk postvaccination from all the birds and challenged with C. septicum. Challenge inoculum and dose used were the same as mentioned above.
RESULTS
Laboratory evaluation of bacterin-toxoid in turkey poults. No gross adverse effects were noticed in any of the birds at the site of inoculation of experimental C. septicum bacterin-toxoid–inoculated groups. All the birds challenged with C. septicum in nonvaccinated group III developed severe clostridial dermatitis lesions and died within 24 hr of challenge. The lesions that developed in these birds included marked fibrin exudation, frothy serosanguineous fluid in the subcutis, and underlying muscle necrosis (Fig. 1A). Histopathologic examination revealed subcutaneous tissue and muscle fibers that were infiltrated with C. septicum (Fig. 1B). The lesions were similar to the classical clostridial dermatitis lesions observed in birds from the field (3,14) One bird from group I that was given a 1-ml dose of vaccine one time also developed clostridial dermatitis and died within 48 hr of challenge. However, no clostridial dermatitis lesion development or mortality was observed in any of the birds vaccinated twice with either doses or vaccinated once with a 2-ml dose. A significant rise in C. septicum antibody titers via ELISA was noticed in vaccinated groups I and II on 14 and 28 days postvaccination (dpv) compared with nonvaccinated group IV birds (Table 1). Field trials with bacterin-toxoid in commercial turkeys. The mortality due to clostridial dermatitis in vaccinated and nonvaccinated birds from field trial I are shown in Table 2 and Fig. 2. There were significant differences in the mortality (P , 0.005) in the vaccinated group compared with the nonvaccinated groups. The use of experimental C. septicum toxoid reduced clostridial dermatitis– related mortality from 12.1% to 10.5%. This effect accounts for a 14% reduction in clostridial dermatitis–related mortality in vaccinated birds compared with nonvaccinated birds. The hazard ratio was 1.14 for the nonvaccinated group over the vaccinated group of birds. A hazard ratio is the rate at which proportion of events (e.g., mortality) happen, so that the probability of an event happening in a short time interval is the length of time multiplied by the hazard. A Kaplan-Meier survival curve comparing the survival distribution function of nonvaccinated and vaccinated birds is shown in Fig. 1. No significant differences were noted in terms of mortality between the farms (P 5 0.6343).
Clostridium septicum vaccine for clostridial dermatitis in turkeys
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Fig. 2. Mortality after vaccination with C. septicum bacterin-toxoid in field trial I in nonvaccinated (solid line) and vaccinated birds (broken line) from 10 wk of age to 22 wk of age.
There was a reduced use of the antibiotic penicillin because of vaccination (P , 0.001), from 204 packs in nonvaccinated birds to 84 packs in vaccinated birds, accounting for a 59% reduction in the use of antibiotics in vaccinated birds compared with nonvaccinated birds. The number of days required to use penicillin to control clostridial dermatitis also was reduced from 68 days in nonvaccinated group to 28 days in vaccinated group (Fig. 3). In field trial II, C. septicum toxoid significantly reduced clostridial dermatitis mortality from 1.68% in nonvaccinated birds to 0.87% in
vaccinated birds, accounting for an approximately 50% reduction in mortality (Table 3; Fig. 4). Clostridium septicum was isolated from dermatitis lesions from a few cases of vaccinated and nonvaccinated birds, but there was no isolation of C. perfringens in this study. When field birds were challenged, all birds from the vaccinated group resisted challenge and did not develop any signs of clostridial dermatitis lesions or mortality, and they were found to be completely protected. Vaccinated birds had a C. septicum ELISA serum antibody titer of 0.6870 6 0.12, and nonvaccinated birds had a background
Fig. 3. Antibiotic use after vaccination with C. septicum bacterin-toxoid in field trial I in nonvaccinated birds (white bar) and vaccinated birds (black bar) from 10 wk of age to 22 wk of age.
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Table 3. Comparison of clostridial dermatitis–related mortality in C. septicum bacterin-toxoid in field trial II from 7 wk to 19 wk of age.A
No. of birds Clostridial dermatitis mortality % Clostridial dermatitis mortality
Nonvaccinated group
Vaccinated group
16,000 268 1.68
4000 35a 0.87a
A
Values followed by a lowercase letter indicate P , 0.05.
titer of 0.3626 6 0.09. We observed a mortality of 100% (10/10) in 24 hr in nonvaccinated birds after challenge with C. septicum. DISCUSSION
Clostridium septicum alpha-toxoid was reported to be protective in guinea pigs (Cavia porcellus) against experimental challenge with C. septicum spores (1). In broiler chickens, the use of a polyvalent C. perfringens, C. septicum, and Pasteurella bacterin reduced mortality due to gangrenous dermatitis (6). However, the use of these vaccines in controlling clostridial dermatitis in turkeys has not been examined. In this study, we showed that a single administration of C. septicum bacterin-toxoid at 6 wk of age was protective against clostridial dermatitis in commercial turkeys. The use of this vaccine reduced the number of clostridial dermatitis cases, mortality, and antibiotic use in a commercial turkey operation. The reduction of mortality by 14% in field trial I and by 50% in field trail II in vaccinated birds compared with nonvaccinated birds was highly significant, considering that the disease appears in birds at marketing age and any mortality at this stage causes significant economic loss to turkey producers. In our laboratory experiments, higher serum antibody titers and better protection were noted in birds administered the 2-ml vaccine compared with the 1-ml vaccine. However, administration of a volume of .1 ml was not examined under field conditions. The indication of better protection with the 2-ml dose was assumed to be
due to higher antigen concentration in the vaccine. Being a killed vaccine, multiple vaccinations may be required for prolonged protection. The use of C. septicum bacterin-toxoid in this study elicited antibody response against C. septicum in immunized turkeys that was similar to that of a previous report (12). However, there are no reports available regarding the protective efficacy of any C. septicum vaccines against C. septicum challenge causing clostridial dermatitis in turkeys. The results from this study demonstrated protection in C. septicum bacterin-toxoid–vaccinated birds against clostridial dermatitis in turkeys caused by C. septicum. The results from previous vaccine trials with a bivalent vaccine containing both C. perfringens and C. septicum also showed a reduction in clostridial dermatitis cases; however, the results were not comparable (15). During that study, C. perfringens was isolated in higher number of turkey dermatitis cases than C. septicum. Moreover, the amount of C. septicum antigen contained in the vaccine was much lower than the amount used in this study (15). The incidence of C. perfringens– and C. septicum–associated diseases in poultry has increased significantly in the recent years, possibly due to reduced use of antimicrobial growth promoters (18). Clostridial dermatitis is commonly controlled in a preventative manner in endemic farms by incorporation of antimicrobial drugs in the feed or water, but this practice has come under increased criticism or has been objected in many countries. Because clostridial dermatitis cases in turkeys are also rising at an alarming rate, there is a need to investigate alternative biologic approaches for its effective control. We were able to reduce the dose and duration of antibiotic penicillin G potassium use significantly in turkeys where the vaccine was used to control clostridial dermatitis and mortality compared with nonvaccinated controls. It is widely accepted that natural outbreaks of clostridial dermatitis in turkeys are associated with proliferation of pathogens in the poultry environment. Under field conditions, the commercial poults are reared on deep litter systems, and the litter in grow-out
Fig. 4. Kaplan-Meier survival curve showing the survival distribution function after vaccination with C. septicum bacterin-toxoid in field trial II in nonvaccinated birds (broken line) and vaccinated birds (solid line) from 10 wk of age to 22 wk of age.
Clostridium septicum vaccine for clostridial dermatitis in turkeys
barns is not replaced for every flock. Buildup of clostridial load may occur during this time. A greater environmental load of these clostridial spores and their persistence may increase the chance of exposure over time. Under such scenarios, a high dose of clostridial challenge may take place, unlike in laboratory studies. However, additional studies are required to know the effect of clostridial load in the litter on the incidence of clostridial dermatitis cases under field conditions. In this study, with single vaccination in two different farms, the industry used considerably less antibiotics in vaccinated birds. There was a saving on antibiotic cost. When reduction in mortality also is taken into account, the average savings per barn could be substantial. Our results establish that C. septicum bacterin-toxoid could be used effectively to mitigate clostridial dermatitis cases in turkeys in the field. In summary, the experimental C. septicum bacterin-toxoid that we developed offered complete protection against clostridial dermatitis after homologous challenge under experimental conditions. The same vaccine enabled us to provide statistically significant reduction in mortality and use of antibiotics in preventing clostridial dermatitis in commercial turkeys. Serum ELISA results showed significant antibody response against C. septicum in vaccinated birds. Multiple vaccinations as well as use of higher doses of vaccine may offer better protection against clostridial dermatitis. Subcutaneous inoculation of C. septicum cultures consistently produced clostridial dermatitis lesions and mortality in nonvaccinated turkeys, as in our previous studies (13,14). Our results offer promise for the use of C. septicum toxoid to successfully control clostridial dermatitis in commercial turkeys. REFERENCES 1. Amimoto, K., T. Ohgitani, O. Sasaki, E. Oishi, S. Katayama, M. Isogai, and S. Ota. Protective effect of Clostridium septicum alpha-toxoid vaccine against challenge with spores in guinea pigs. J. Vet. Med. Sci. 64:67–69. 2002. 2. Cardella, M. A., and D. R. Kolbe. Potency testing of Clostridium septicum bacterins in sheep and laboratory animals. Dev. Biol. Stan. 32:115–122. 1976. 3. Carr, D., D. Shaw, D. A. Halvorson, B. Rings, and D. Roepke. Excessive mortality in market-age turkeys associated with cellulitis. Avian Dis. 40:736–741. 1996. 4. Clark, S., R. Porter, B. McComb, L. Ron, O. Steve, N. Sheilina, and H. L. Shivaprasad. Clostridial dermatitis and cellulitis: an emerging disease of turkeys. Avian Dis. 54:788–794. 2010. 5. Fenstermacher, R., and B. S. Pomeroy. Clostridium infection in turkeys. Cornell Vet. J. 29:25–28. 1939. 6. Gerdon, D. Effects of a mixed clostridial bacterin on incidence of gangrenous dermatitis. Avian Dis. 17:205–206. 1973.
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7. Nagaraja, K. V., A. J. Thachil, and D. A. Halvorson. Role of Clostridium perfringens and Clostridium septicum in cellulitis in turkeys. In: Proc. 58th Western Poultry Disease Conference, Sacramento, CA. p. 11. 2009. 8. Olkowski, A. A., L. Kumor, D. Johnson, M. Bielby, M. ChirinoTrejo, and H. L. Classen. Cellulitis lesions in commercial turkeys identified during processing. Vet. Rec. 145:228–229. 1999. 9. Smith-Slatas, C. L., M. Bourque, and J. C. Salazar. Clostridium septicum infections in children: a case report and review of the literature. Pediatrics 117:796–805. 2006. 10. Springer, S., and H. J. Selbitz. The control of necrotic enteritis in sucking piglets by means of a Clostridium perfringens toxoid vaccine. FEMS Immunol. Med. Microbiol. 24:333–336. 1999. 11. Susan, E. A. Breast blisters. In: The Merck veterinary manual, 8th ed. Merck and Co., Inc., Whitehouse Station, NJ. pp. 675–676. 2003. 12. Tellez, G., N. R. Pumford, M. J. Morgan, W. Amanda, and B. Hargis. Evidence for Clostridium septicum as a primary cause of cellulitis in commercial turkeys. J. Vet. Diagn. Invest. 21:374–377. 2009. 13. Thachil, A. J., D. A. Halvorson, and K. V. Nagaraja. Cellulitis in turkeys: Clostridium septicum being a primary pathogen. In: Proc. American Association of Avian Pathologists, 145th American Veterinary Medical Association Annual Convention, New Orleans, LA. p. 26. 2008. 14. Thachil, A. J., B. McComb, M. Andersen, D. P. Shaw, D. A. Halvorson, and K. V. Nagaraja. Role of Clostridium perfringens and Clostridium septicum in causing turkey cellulitis. Avian Dis. 54:795–801. 2010. 15. Thachil, A. J., B. McComb, M. E. Michelle, C. Heeder, and K. V. Nagaraja. A bivalent Clostridium perfringens and Clostridium septicum toxoid to control cellulitis in turkeys. J. Appl. Poult. Res. 21:358–366. 2012. 16. Timoney, J. F., J. H. Gillespie, F. Scott, and J. E. Barlough. The genus Clostridium. In: Hagan and Bruner’s microbiology and infectious diseases of domestic animals, 8th ed. Comstock Publishing Associates, London, United Kingdom. pp. 214–240. 1998. 17. Titball, R. W., C. E. Naylor, and A. K. Basak. The Clostridium perfringens alpha-toxin. Anaerobe 5:51–64. 1999. 18. Van Immerseel, F., J. DeBuck, F. Pasmans, G. Huyghebaert, F. Haesebrouck, and R. Ducatelle. Clostridium perfringens in poultry: an emerging threat for animal and public health. Avian Pathol. 33:537–549. 2004.
ACKNOWLEDGMENTS We thank Dr. Binu T. Velayudhan (Diagnostic Virology, Texas A&M Veterinary Medical Diagnostic Laboratory–Amarillo, TX) for valuable help and comments and Dr. Louis Espejo for help with statistical analysis. This research was supported by funding from Minnesota Turkey Research and Promotion Council and Midwest Poultry Consortium.
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AVIAN DISEASES 57:214–219, 2013
Vaccination of Turkeys with Clostridium septicum Bacterin-Toxoid: Evaluation of Protection Against Clostridial Dermatitis Anil J. Thachil,A Brian McComb,B Michelle Kromm,C and Kakambi V. NagarajaAD A
Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, 1971 Commonwealth Avenue, Saint Paul, MN 55108 B Willmar Poultry Company, Willmar, MN 56201 C Jennie-O Turkey Store, 2505 Willmar Avenue, Willmar, MN, 56201 Received 15 October 2012; Accepted 23 January 2013; Published ahead of print 25 January 2013 SUMMARY. Clostridial dermatitis is an acute disease causing high mortality in turkeys. Both Clostridium septicum and Clostridium perfringens have been isolated from these cases; however, reports from several diagnostic laboratories indicate an increased isolation rate of C. septicum compared with C. perfringens from cases of clostridial dermatitis in recent years. Previous studies suggested C. septicum was more potent than C. perfringens in causing clostridial dermatitis in turkeys. The objective of this study was to develop and evaluate the use of a C. septicum bacterin-toxoid to control clostridial dermatitis in turkeys. A C. septicum bacterin-toxoid was prepared and was initially tested in 6-wk-old commercial turkeys under laboratory conditions for its safety and efficacy. Subsequently, the bacterin-toxoid was evaluated for use in commercial turkey farms with a consistent history of clostridial dermatitis. Birds in the field were vaccinated subcutaneously once at 6 wk of age with C. septicum bacterin-toxoid, and then mortality in both vaccinated and unvaccinated groups was recorded and compared. Blood samples from birds in both groups were examined using ELISA to detect antibody response to the C. septicum toxoid. The C. septicum bacterin-toxoid was found to be safe and to elicit antibodies against the toxoid. In vaccinated commercial turkeys, control of clostridial dermatitis was achieved via antibiotic use and clostridial dermatitis mortality was significantly reduced compared with that of birds in the unvaccinated group. The C. septicum bacterin-toxoid seems to be a valuable tool for the turkey industry to reduce losses due to clostridial dermatitis. RESUMEN. Vacunacio´n de pavos con una bacterina-toxoide de Clostridium septicum: Evaluacio´n de la proteccio´n contra la dermatitis clostridial. La dermatitis clostridial es una enfermedad aguda que causa una alta mortalidad en pavos. Tanto Clostridium septicum como Clostridium perfringens se han aislado de estos casos. Sin embargo en los u´ltimos an˜os, informes de varios laboratorios de diagno´stico indican un aumento de la tasa de aislamiento de C. septicum en comparacio´n con C. perfringens a partir de casos de dermatitis clostridial. Los estudios previos han sugerido que C. septicum era ma´s potente que C. perfringens para causar dermatitis clostridial en pavos. El objetivo de este estudio fue desarrollar y evaluar el uso de una bacterina-toxoide de C septicum para controlar la dermatitis clostridial en los pavos. La bacterina-toxoide contra C. septicum se preparo´ y fue probada inicialmente en pavos comerciales de seis semanas de edad, bajo condiciones de laboratorio, para determinar su seguridad y eficacia. Posteriormente, la bacterina-toxoide se evaluo´ para su uso en granjas de pavos comerciales con un historial de dermatitis clostridial constante. Las aves en el campo fueron vacunadas por vı´a subcuta´nea una vez a las seis semanas de edad con la bacterina-toxoide de C. septicum bacterina y se registro´ y comparo´ la mortalidad en los grupos vacunados y no vacunados. Las muestras de sangre de aves en ambos grupos se examinaron usando una prueba de ELISA para detectar la respuesta de anticuerpos contra el toxoide de C. septicum. Se determino´ que la bacterina-toxoide de C. septicum era segura y que inducı´a anticuerpos contra el toxoide. En pavos comerciales vacunados, el control de la dermatitis clostridial se logro´ mediante el uso de antibio´ticos y se redujo significativamente la mortalidad por dermatitis clostridial en comparacio´n con las aves del grupo no vacunado. La bacterina-toxoide contra C. septicum parece ser una herramienta valiosa para la industria de los pavos para reducir las pe´rdidas debidas a la dermatitis clostridial. Key words: clostridial dermatitis, cellulitis, Clostridium septicum, vaccine, turkey Abbreviations: BHI 5 brain heart infusion; CFU 5 colony-forming unit(s); dpv 5 days postvaccination; HE 5 hemorrhagic enteritis; OD 5 optical density; RAR 5 Research Animal Resources
Clostridial dermatitis and cellulitis is one of the leading causes of economic loss in the turkey industry in the United States (3,4). The severity of these conditions varies depending on the virulence characteristics of the bacteria. Although Clostridium perfringens and Clostridium septicum can cause clostridial dermatitis in turkeys, C. septicum has been found to be more pathogenic than C. perfringens in causing clostridial dermatitis (4). Clostridial dermatitis in turkeys is characterized by inflammation of skin and subcutaneous tissue, resulting in accumulation of frothy sanguinous exudate in the subcutis of breast and tail regions (4,5,14). Mortality, increased condemnation rates, and expensive medication costs for treatment have accounted for the huge economic losses (3,8). D
Corresponding author. E-mail: [email protected]
Clostridium septicum is reported to cause traumatic gas gangrene and clostridial myonecrosis in animals and humans (9,11). The exotoxins produced by C. septicum are responsible for tissue damage and toxemia (16). Clostridium septicum toxins were reported to be more potent than those of C. perfringens in causing clostridial dermatitis in turkeys (4,14). Clostridial vaccines containing toxoid and killed bacteria are very effective in humans and animals against many clostridial infections. Clostridium perfringens infections have been reported to be successfully controlled in suckling piglets by administration of a toxoid vaccine (10). The use of C. septicum toxoid induced protection against spore challenge in sheep (2). Some studies (12,13) have drawn attention to secretory toxins from C. septicum and their role in causing clostridial dermatitis in turkeys.
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Fig. 1. Gross lesions and histopathology of subcutaneous and muscle tissue in clostridial dermatitis due to C. septicum in turkeys. (A) Presence of frothy sanguineous exudate in subcutaneous tissue and underlying muscle necrosis in a bird infected with C. septicum spore culture. Bar 5 1 cm. (B) Fragmented muscle fibers infiltrated with C. septicum in a clostridial dermatitis–affected bird. Section was stained with hematoxylin and eosin. Bar 5 50 mm.
The use of a bivalent C. perfringens and C. septicum bacterintoxoid against clostridial dermatitis was found to reduce loses due to clostridial dermatitis in turkeys (7,15). Because of the increased isolation rate of C. septicum alone in many cases of clostridial dermatitis, studies have started to look into developing a C. septicum bacterin-toxoid alone for effective use in the field for controlling clostridial dermatitis in turkeys. Here, we report the use of a C. septicum bacterin-toxoid against clostridial dermatitis in turkeys. MATERIALS AND METHODS Bacteria. Clostridium septicum isolate (UMNCS 106), originally isolated from breast lesions of clostridial dermatitis in a turkey, was used in this research. This isolate possessed the ability to express high toxin titers and heat-resistant spore counts (14). In brief, C. septicum was first grown in brain heart infusion (BHI) broth at 37 C for 18 hr under anaerobic conditions, and incubation was continued for additional 6 hr at 24 C. The presence of toxin in the culture supernate was ascertained by mouse lethal assay (14), and the culture was then treated with 0.5% formalin to inactivate toxins in it. The complete inactivation was confirmed by subculturing an aliquot of culture supernate in BHI broth anaerobically. Failure of any growth was taken as complete inactivation of bacteria. The inactivation of toxins was confirmed by hemolysis assay (17). This inactivated Clostridium culture contained 4.8 3 107 colony-forming units (CFU)/ml bacteria and 3 g/ml toxoid, which was later used for making a bacterin-toxoid. Preparation of C. septicum bacterin-toxoid. An experimental bacterin-toxoid vaccine was prepared using the inactivated culture of C. septicum mentioned under ‘‘Bacteria’’ section. Dakreol-6VR (Pennsylvania Refining Company, Butler, PA), a vaccine-grade mineral oil, and the emulsifier Arlacel-A (Sigma-Aldrich, St. Louis, MO) were used to complete the preparation of the vaccine. Arlacel-A was used at the rate of 10% (v/v) in the oil component. A 1:1 water/oil emulsion was prepared after slowly mixing the oil component (mineral oil and ArlacelA) in a blender to which the aqueous inactivated culture component was added over a period of 5 min. The final preparation contained 1.5 g
(128 mouse lethal dose50) of toxoid per milliliter of vaccine. The experimental birds were vaccinated at different doses to evaluate the efficacy of the vaccine postchallenge. Laboratory evaluation of C. septicum toxoid in turkey poults. The experimental bacterin-toxoid was first tested for its safety and efficacy in 6-wk-old commercial Nicholas White male turkey poults obtained from a source with no history of clostridial dermatitis. The birds were inoculated subcutaneously with either a 1-ml dose containing 1.5 g of toxoid or with a 2-ml dose containing 3 g of toxoid. In brief, ninety-six 6-wk-old commercial Nicholas White turkey poults obtained from a source with no history of clostridial dermatitis were divided into four groups (groups I–IV) of 24 birds each. Each bird in group I was inoculated with the 1-ml dose and each bird in group II were inoculated subcutaneously with the 2-ml dose of the toxoid preparation at the wing web. The birds in group III were nonvaccinated positive controls for clostridial challenge, and the birds in group IV were treated as nonvaccinated negative controls. On the 14th day postvaccination, half of the birds from groups I and II were given a booster dose at the same dose as mentioned. The blood from all the groups was collected at 0-, 14-, and 28-days post-one vaccination and on 7-days post-booster vaccination for serologic examination. All the birds were monitored twice daily for any adverse effects of the toxoid. Any bird showing severe pain or distress or that was unable to move to feeders and waterers was euthanized in a carbon dioxide chamber. After 28-days post-one vaccination, 12 birds each from groups I and II that received one vaccination and 12 birds from nonvaccinated control group III were challenged with 2 ml of fresh C. septicum culture (4.8 3 107CFU/ml) subcutaneously on the breast region. Similarly, after the 14days post-booster vaccination, the remaining 12 birds from groups I and II and 12 birds from the nonvaccinated control group III were challenged with C. septicum culture. The challenge dose and route with C. septicum culture were optimized in our previous study (14). The sera from all the groups were examined for seroconversion to the vaccine by using C. septicum–specific ELISA as described previously (12,14). All the birds at the end of the study were euthanized in a carbon dioxide chamber. All animal experimental protocols were approved by the Institutional Animal Care and Use Committee, and the procedures were performed
Table 1. Clostridium septicum ELISA serum antibody titer (optical density [OD] values) after vaccination with C. septicum toxoid in vaccinated and nonvaccinated birds (n 5 12).A Post-one vaccination OD values
Vaccine at 1-ml dose Vaccine at 2-ml dose Nonvaccinated A
Post-two vaccination OD values
0 dpv
14 dpv
28 dpv
7 dp2v
0.2459 (0.13)a 0.2691 (0.09)a 0.2708 (0.54)a
0.6574 (0.24)b 0.7645 (0.23)b 0.2182 (0.17)a
0.704 (0.10)b 0.7254 (0.09)b 0.2814 (0.05)a
0.9217 (015) 1.0686 (0.14) 0.2689 (0.05)a
Means (SD) within a row with no common lowercase letter are considered significantly different (P , 0.05).
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in accordance with their requirements. The experimental birds were reared at Research Animal Resources (RAR) isolation facilities at the University of Minnesota, Saint Paul, MN. Field trials with C. septicum toxoid in commercial turkeys. With permission from the Minnesota State Board of Animal Health and University of Minnesota and consent from a leading commercial turkey producer, two field trials were done to test the efficacy of C. septicum bacterin-toxoid. The birds were vaccinated subcutaneously in the neck region by using a vaccine applicator. In the first field trial, vaccinated and nonvaccinated birds were separated by a wire mesh in the middle and subjected to similar management conditions. In the second field trial, vaccinated and nonvaccinated birds were kept together in the same barn to eliminate any bias toward exposure to clostridia from the environment within the barn. Age of birds at vaccination was 6 wk in both field trials. Age at marketing was at the end of 22 wk in field trial I and 19 wk in the field trial II. Field trial I. Eight thousand male Nicholas White turkey poults in a flock of 16,000 in a brooder barn were selected randomly at the age of 6 wk and vaccinated with C. septicum bacterin-toxoid at a 1-ml dose. They were vaccinated subcutaneously in the neck region by using a vaccine applicator. All the poults were previously vaccinated for Newcastle disease at 3 days 3 wk of age and for hemorrhagic enteritis (HE) at 4 wk of age. After vaccination, 4000 birds each from vaccinated and nonvaccinated groups were transferred to two grow-out farms, hereafter referred to as Farm I and Farm II, where there was a consistent history of clostridial dermatitis. The assumption was that all birds were likely to be exposed equally to clostridia found in that barn environment. Being in a commercial setting, penicillin G potassium (Alpharma Inc., Bridgewater, NJ) use in water was in place as a precautionary measure as and when clostridial dermatitis–related mortality rose above 0.1% per day. Daily mortality and packs of penicillin used to control clostridial dermatitis–related mortality were recorded in both vaccinated and nonvaccinated groups from 10 wk of age until 22 wk of age, when the birds are marketed. On the farms, routine monitoring of all the sick, moribund, and dead birds was conducted twice daily, and birds identified as sick or moribund were euthanized. The results were analyzed to detect differences in the occurrence of mortality between vaccinated and nonvaccinated birds. When birds were 10 wk of age, 10 birds each from the vaccinated and nonvaccinated groups from the field-vaccinated trial were transferred to RAR facilities at the University of Minnesota. All the birds were challenged as mentioned above. Clostridial dermatitis development and mortality between the groups were recorded. All the birds left at the end of the study were euthanized in a carbon dioxide chamber. Statistical analysis was performed by SAS software, version 9.2 (SAS Institute, Cary, NC). Mortality data after challenge were analyzed using Fisher exact test. A survival analysis of maximum likelihood estimates using proportional hazards model (PH-REG procedure) was conducted to analyze the mortality rates in vaccinated and nonvaccinated groups. A Pearson chi-square test was used to compare the use of antibiotics between vaccinated and nonvaccinated groups. The serologic data were analyzed using a GLM procedure for repeated measures analysis of variance, where a P value of 0.05 was considered significant. Field trial II. In brief, 4000 poults in a flock of 20,000 male Nicholas White turkey poults in a brooder barn at the age of 6 wk were banded and vaccinated with C. septicum bacterin-toxoid at the 1-ml dose. The vaccine was administered subcutaneously at the neck region by using a vaccine applicator. All birds were vaccinated previously for Newcastle disease at 3 days and 3 wk of age and for HE at 4 wk of age. At 7 wk of age, half of the birds from each of the vaccinated and nonvaccinated groups were transferred to two different grower farms, hereafter referred to as Farm IA and Farm IIA, where there was a consistent history of clostridial dermatitis. The vaccinated and nonvaccinated birds were kept together and subjected to similar management conditions. A therapeutic antibiotic penicillin G potassium use in water was in place as a precautionary measure as and when the mortality due to clostridial dermatitis rose above 0.1% per day. Daily mortality with visible clostridial dermatitis lesions was recorded in both vaccinated and
Table 2. Comparison of clostridial dermatitis–related mortality and antibiotic use postvaccination with C. septicum bacterin-toxoid in field trial I from 10 wk of age to 22 wk of age.A
No. of birds Mortality % Mortality Penicillin useB (packs) Penicillin use (days)
Nonvaccinated group
Vaccinated group
8000 970 12.1 204 68
8000 840a 10.5a 84a 28a
A
Values followed by a lowercase letter indicate P , 0.05. One pack of penicillin contains 1.0 billion international units (601.2 g) of penicillin G potassium. B
nonvaccinated birds until the birds were marketed. The results were compared to detect any observable differences in the occurrence of mortality between vaccinated and nonvaccinated birds. Laboratory challenge of birds with C. septicum postvaccination. Ten birds each from vaccinated and nonvaccinated groups from field trial I were transferred to RAR facilities at University of Minnesota. Blood was collected at 3 wk postvaccination from all the birds and challenged with C. septicum. Challenge inoculum and dose used were the same as mentioned above.
RESULTS
Laboratory evaluation of bacterin-toxoid in turkey poults. No gross adverse effects were noticed in any of the birds at the site of inoculation of experimental C. septicum bacterin-toxoid–inoculated groups. All the birds challenged with C. septicum in nonvaccinated group III developed severe clostridial dermatitis lesions and died within 24 hr of challenge. The lesions that developed in these birds included marked fibrin exudation, frothy serosanguineous fluid in the subcutis, and underlying muscle necrosis (Fig. 1A). Histopathologic examination revealed subcutaneous tissue and muscle fibers that were infiltrated with C. septicum (Fig. 1B). The lesions were similar to the classical clostridial dermatitis lesions observed in birds from the field (3,14) One bird from group I that was given a 1-ml dose of vaccine one time also developed clostridial dermatitis and died within 48 hr of challenge. However, no clostridial dermatitis lesion development or mortality was observed in any of the birds vaccinated twice with either doses or vaccinated once with a 2-ml dose. A significant rise in C. septicum antibody titers via ELISA was noticed in vaccinated groups I and II on 14 and 28 days postvaccination (dpv) compared with nonvaccinated group IV birds (Table 1). Field trials with bacterin-toxoid in commercial turkeys. The mortality due to clostridial dermatitis in vaccinated and nonvaccinated birds from field trial I are shown in Table 2 and Fig. 2. There were significant differences in the mortality (P , 0.005) in the vaccinated group compared with the nonvaccinated groups. The use of experimental C. septicum toxoid reduced clostridial dermatitis– related mortality from 12.1% to 10.5%. This effect accounts for a 14% reduction in clostridial dermatitis–related mortality in vaccinated birds compared with nonvaccinated birds. The hazard ratio was 1.14 for the nonvaccinated group over the vaccinated group of birds. A hazard ratio is the rate at which proportion of events (e.g., mortality) happen, so that the probability of an event happening in a short time interval is the length of time multiplied by the hazard. A Kaplan-Meier survival curve comparing the survival distribution function of nonvaccinated and vaccinated birds is shown in Fig. 1. No significant differences were noted in terms of mortality between the farms (P 5 0.6343).
Clostridium septicum vaccine for clostridial dermatitis in turkeys
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Fig. 2. Mortality after vaccination with C. septicum bacterin-toxoid in field trial I in nonvaccinated (solid line) and vaccinated birds (broken line) from 10 wk of age to 22 wk of age.
There was a reduced use of the antibiotic penicillin because of vaccination (P , 0.001), from 204 packs in nonvaccinated birds to 84 packs in vaccinated birds, accounting for a 59% reduction in the use of antibiotics in vaccinated birds compared with nonvaccinated birds. The number of days required to use penicillin to control clostridial dermatitis also was reduced from 68 days in nonvaccinated group to 28 days in vaccinated group (Fig. 3). In field trial II, C. septicum toxoid significantly reduced clostridial dermatitis mortality from 1.68% in nonvaccinated birds to 0.87% in
vaccinated birds, accounting for an approximately 50% reduction in mortality (Table 3; Fig. 4). Clostridium septicum was isolated from dermatitis lesions from a few cases of vaccinated and nonvaccinated birds, but there was no isolation of C. perfringens in this study. When field birds were challenged, all birds from the vaccinated group resisted challenge and did not develop any signs of clostridial dermatitis lesions or mortality, and they were found to be completely protected. Vaccinated birds had a C. septicum ELISA serum antibody titer of 0.6870 6 0.12, and nonvaccinated birds had a background
Fig. 3. Antibiotic use after vaccination with C. septicum bacterin-toxoid in field trial I in nonvaccinated birds (white bar) and vaccinated birds (black bar) from 10 wk of age to 22 wk of age.
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Table 3. Comparison of clostridial dermatitis–related mortality in C. septicum bacterin-toxoid in field trial II from 7 wk to 19 wk of age.A
No. of birds Clostridial dermatitis mortality % Clostridial dermatitis mortality
Nonvaccinated group
Vaccinated group
16,000 268 1.68
4000 35a 0.87a
A
Values followed by a lowercase letter indicate P , 0.05.
titer of 0.3626 6 0.09. We observed a mortality of 100% (10/10) in 24 hr in nonvaccinated birds after challenge with C. septicum. DISCUSSION
Clostridium septicum alpha-toxoid was reported to be protective in guinea pigs (Cavia porcellus) against experimental challenge with C. septicum spores (1). In broiler chickens, the use of a polyvalent C. perfringens, C. septicum, and Pasteurella bacterin reduced mortality due to gangrenous dermatitis (6). However, the use of these vaccines in controlling clostridial dermatitis in turkeys has not been examined. In this study, we showed that a single administration of C. septicum bacterin-toxoid at 6 wk of age was protective against clostridial dermatitis in commercial turkeys. The use of this vaccine reduced the number of clostridial dermatitis cases, mortality, and antibiotic use in a commercial turkey operation. The reduction of mortality by 14% in field trial I and by 50% in field trail II in vaccinated birds compared with nonvaccinated birds was highly significant, considering that the disease appears in birds at marketing age and any mortality at this stage causes significant economic loss to turkey producers. In our laboratory experiments, higher serum antibody titers and better protection were noted in birds administered the 2-ml vaccine compared with the 1-ml vaccine. However, administration of a volume of .1 ml was not examined under field conditions. The indication of better protection with the 2-ml dose was assumed to be
due to higher antigen concentration in the vaccine. Being a killed vaccine, multiple vaccinations may be required for prolonged protection. The use of C. septicum bacterin-toxoid in this study elicited antibody response against C. septicum in immunized turkeys that was similar to that of a previous report (12). However, there are no reports available regarding the protective efficacy of any C. septicum vaccines against C. septicum challenge causing clostridial dermatitis in turkeys. The results from this study demonstrated protection in C. septicum bacterin-toxoid–vaccinated birds against clostridial dermatitis in turkeys caused by C. septicum. The results from previous vaccine trials with a bivalent vaccine containing both C. perfringens and C. septicum also showed a reduction in clostridial dermatitis cases; however, the results were not comparable (15). During that study, C. perfringens was isolated in higher number of turkey dermatitis cases than C. septicum. Moreover, the amount of C. septicum antigen contained in the vaccine was much lower than the amount used in this study (15). The incidence of C. perfringens– and C. septicum–associated diseases in poultry has increased significantly in the recent years, possibly due to reduced use of antimicrobial growth promoters (18). Clostridial dermatitis is commonly controlled in a preventative manner in endemic farms by incorporation of antimicrobial drugs in the feed or water, but this practice has come under increased criticism or has been objected in many countries. Because clostridial dermatitis cases in turkeys are also rising at an alarming rate, there is a need to investigate alternative biologic approaches for its effective control. We were able to reduce the dose and duration of antibiotic penicillin G potassium use significantly in turkeys where the vaccine was used to control clostridial dermatitis and mortality compared with nonvaccinated controls. It is widely accepted that natural outbreaks of clostridial dermatitis in turkeys are associated with proliferation of pathogens in the poultry environment. Under field conditions, the commercial poults are reared on deep litter systems, and the litter in grow-out
Fig. 4. Kaplan-Meier survival curve showing the survival distribution function after vaccination with C. septicum bacterin-toxoid in field trial II in nonvaccinated birds (broken line) and vaccinated birds (solid line) from 10 wk of age to 22 wk of age.
Clostridium septicum vaccine for clostridial dermatitis in turkeys
barns is not replaced for every flock. Buildup of clostridial load may occur during this time. A greater environmental load of these clostridial spores and their persistence may increase the chance of exposure over time. Under such scenarios, a high dose of clostridial challenge may take place, unlike in laboratory studies. However, additional studies are required to know the effect of clostridial load in the litter on the incidence of clostridial dermatitis cases under field conditions. In this study, with single vaccination in two different farms, the industry used considerably less antibiotics in vaccinated birds. There was a saving on antibiotic cost. When reduction in mortality also is taken into account, the average savings per barn could be substantial. Our results establish that C. septicum bacterin-toxoid could be used effectively to mitigate clostridial dermatitis cases in turkeys in the field. In summary, the experimental C. septicum bacterin-toxoid that we developed offered complete protection against clostridial dermatitis after homologous challenge under experimental conditions. The same vaccine enabled us to provide statistically significant reduction in mortality and use of antibiotics in preventing clostridial dermatitis in commercial turkeys. Serum ELISA results showed significant antibody response against C. septicum in vaccinated birds. Multiple vaccinations as well as use of higher doses of vaccine may offer better protection against clostridial dermatitis. Subcutaneous inoculation of C. septicum cultures consistently produced clostridial dermatitis lesions and mortality in nonvaccinated turkeys, as in our previous studies (13,14). Our results offer promise for the use of C. septicum toxoid to successfully control clostridial dermatitis in commercial turkeys. REFERENCES 1. Amimoto, K., T. Ohgitani, O. Sasaki, E. Oishi, S. Katayama, M. Isogai, and S. Ota. Protective effect of Clostridium septicum alpha-toxoid vaccine against challenge with spores in guinea pigs. J. Vet. Med. Sci. 64:67–69. 2002. 2. Cardella, M. A., and D. R. Kolbe. Potency testing of Clostridium septicum bacterins in sheep and laboratory animals. Dev. Biol. Stan. 32:115–122. 1976. 3. Carr, D., D. Shaw, D. A. Halvorson, B. Rings, and D. Roepke. Excessive mortality in market-age turkeys associated with cellulitis. Avian Dis. 40:736–741. 1996. 4. Clark, S., R. Porter, B. McComb, L. Ron, O. Steve, N. Sheilina, and H. L. Shivaprasad. Clostridial dermatitis and cellulitis: an emerging disease of turkeys. Avian Dis. 54:788–794. 2010. 5. Fenstermacher, R., and B. S. Pomeroy. Clostridium infection in turkeys. Cornell Vet. J. 29:25–28. 1939. 6. Gerdon, D. Effects of a mixed clostridial bacterin on incidence of gangrenous dermatitis. Avian Dis. 17:205–206. 1973.
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ACKNOWLEDGMENTS We thank Dr. Binu T. Velayudhan (Diagnostic Virology, Texas A&M Veterinary Medical Diagnostic Laboratory–Amarillo, TX) for valuable help and comments and Dr. Louis Espejo for help with statistical analysis. This research was supported by funding from Minnesota Turkey Research and Promotion Council and Midwest Poultry Consortium.
