Efforts to Transmit and to Enhance the Virulence of an Avirulent Strain of Pasteurella multocida in Turkeys

Transcription

1 Efforts to Transmit and to Enhance the Virulence of an Avirulent Strain of Pasteurella multocida in Turkeys B. W. BlERER AND W. T. DERIETJX South Carolina Agricultural Experiment Station, Clemson University, Clemson, South Carolina (Received for publication November 13, 1972) ABSTRACT Nonvaccinated turkeys were added to groups of turkeys that had been recently vaccinated with the avirulent Clemson University vaccine strain of Pasteurella multocida by the drinking water route. Separate control groups were nonvaccinated. Five weeks after treatment, all groups were challenged with a highly virulent P-1S9 strain. None of the turkeys added to the vaccinated groups developed clinical evidence of fowl cholera disease during the S-weeks exposure period. After challenged with the highly virulent P-159 culture, the total number infected was 95% in the nonvaccinated groups, 12.5% in the turkeys that had received the avirulent vaccine strain, and 85% in the nonvaccinated turkeys added to the vaccinated groups. These results suggest, under the conditions of this experiment, (1) that if the vaccine strain is transmittible by cohabitation, then this transmission is not easily accomplished and (2) that the unvaccinated turkeys remained suscedtible to fowl cholera infection. In a second experiment, the vaccine strain was injected intravenously into turkeys, was isolated from them after death and was then injected into additional turkeys, etc., for a total of 1 turkey passages. Separate groups of turkeys were then exposed by the drinking water route to the 5-passage isolate, to the 1-passage isolate, and to the original vaccine strain. Other groups were not exposed. Two weeks post treatment, all groups were then challenged with a highly virulent P-159 strain of P. multocida. Under the conditions of the second experiment, one of the 2 turkeys exposed to the 1- turkey passage isolate developed an infection of the tibiometatarsal area. None of the turkeys exposed to either the 5-turkey passage isolate or to the vaccine strain developed clinical evidence of fowl cholera disease. After challenge with the highly virulent P-159 strain, the total number infected was 5% in the turkeys that had been exposed to the 1-turkey passage isolate and 1% in those that had been exposed to the 5-turkey passage isolate. None of the turkeys that had received the vaccine strain developed evidence of infection and the infection rate in those not vaccinated was 85%. The results of the second experiment suggest that there was little or no increase in the virulence of the avirulent vaccine strain after either 5-turkey passages or after 1-turkey passages and, also, that the ability of the vaccine strain to immunize did not appear to be seriously impaired, if at all, by these passages. INTRODUCTION BIERER and Derieux (1972) have reported on the successful experimental use of an avirulent Pasteurella multocida vaccine administered to turkeys in the drinking water and McDowell et al. (1972) have reported on their successful efforts to immunize turkeys against fowl cholera with the use of an avirulent Pasteurella multocida mutant. A rather universal objection to the use of vaccines containing either avirulent or attenuated living bacteria stems from the fear that either avirulent or attenuated Published with approval of the director as Technical Contribution No POULTRY SCIENCE 52: , 1973 bacteria will become virulent after an opportunity for animal passage, contaminate the premises with virulent bacteria and, also, infect unvaccinated animals in the immediate vicinity. Experiments were thus conducted to endeavor to increase the virulence of the avirulent Clemson University vaccine strain by animal passage and to determine if this vaccine strain would spread from vaccinated turkeys to unvaccinated pen mates. 151 MATERIALS AND METHODS In the first experiment, 2 8-weeks-old turkeys were placed in each of 4 research Downloaded from at Penn State University (Paterno Lib) on September 18, 216

2 PASTEURELLA MVLTOCIDA 1511 chambers. The chambers were part of a concrete block building that contained 8 separate research units each with a separate entrance into an anteroom. The anteroom contained feed and water facilities for the adjacent chambers and each chamber was 2.7 X 3.4 meters. These units were routinely cleaned and disinfected and the chambers prepared with fresh, clean shavings litter prior to their use. There was no direct air communication between the chambers and there were no other disease experiments in progress on the premises at the time. Twenty turkeys in each of 2 of these chambers were exposed to a P. multocida vaccine in the drinking water and 2 turkeys in each of 2 additional chambers were maintained as unvaccinated groups. The avirulent vaccine was used with the same precautions described in a previous paper (Bierer and Derieux, 1972) and was prepared from the same avirulent Clemson University vaccine strain. Vaccination was accomplished by placing 3 ml. of a 24-hours old brain-heart-infusion broth culture (incubated at 37 degrees C.) to each 4, ml. of drinking water daily, for 4 consecutive days. To encourage prompt consumption of the inoculated water, the drinking water was withheld 2 hours prior to exposure to the vaccine. On each of the second, third, and fourth days, a separate flask of 24- hours old broth culture was used. A tenfold dilution plate count technique (Heddleston, 1972) on dextrose starch agar established that each ml. of water vaccine contained approximately 3,5, viable bacilli when prepared. On the day after water vaccination was completed, 1 turkeys from the same initial source were added to each of the 2 chambers of vaccinated turkeys and to each of the 2 pens of nonvaccinated turkeys. Each of the added turkeys were wing-banded for identification purposes. Five weeks after these turkeys were added, all turkeys in each of the 4 chambers were exposed to a highly virulent P-159 strain of P. multocida. This was accomplished through the drinking water route and the inoculum was prepared and applied in the same way and manner that the use of the vaccine was described in the foregoing. Two weeks after exposure to the virulent P-159 strain the trial was terminated, using the method described by Alls et al. (197). Thus, the number of surviving turkeys that were morbid or unthrifty (including torticollis, enlarged tibiometatarsal area or any other clinical evidence of chronic pasteurellosis) was added to the number that died to arrive at the total number infected figure for the turkeys in each of the 4 chambers. In the second experiment, 5 8-weeksold turkeys were placed in a structure located in an isolated field used for horticultural purposes only. The structure was of frame construction and was completely covered to provide excellent shelter from inclement weather. This provided a dirt floor pen area 7.6 X 7.6 meters. Since construction this facility had only been used as a holding pen for healthy turkeys. In an attempt to establish the minimum lethal dose of the Clemson University vaccine strain when injected i.m., 18 of the turkeys were removed to a separate dirt floor structure that was similar to the holding pen described, but was located in a separate field approximately one-quarter mile from the holding pen. The maximum dose used was 1 ml., undiluted, of a 24- hours old broth culture, which contained approximately 2 million viable bacilli per kilogram of body weight. Since none of these turkeys died by the third day post injection, the i.v. route was inves- Downloaded from at Penn State University (Paterno Lib) on September 18, 216

3 1512 B. W. BlERER AND W. T. DERIEUX tigated. An i.v. dose of approximately 2 million viable bacilli per kilogram of body weight resulted in deaths of each of 3 turkeys within 24 to 48 hours. The dead turkeys were then subjected to a postmortem and bacteriological examination. The liver was the only organ cultured. Colonies on dextrose starch agar were examined using the method described by Bond et al. (197) and an iridescent colony was selected for culture material for the second animal passage. This procedure was repeated until 1 isolates, representing 1 animal passages, had been obtained. To compare the pathogenicity of isolates obtained by turkey passage to the original vaccine strain, 1 8-weeks-old turkeys were placed in each of 8 concrete block chambers described in the foregoing paragraphs. Turkeys in 2 of these chambers were exposed to the Clemson University vaccine strain by the drinking water route. The 1 turkeys in each of 2 additional chambers were similarly exposed to the isolate that was obtained from the fifth turkey passage and the turkeys in 2 additional chambers to the isolate from the tenth passage. The turkeys in the 2 remaining chambers were non-exposed. Two weeks after these treatments were applied, all the turkeys in all of the chambers were exposed by the drinking water route to a highly virulent P-159 strain. The turkeys used in the experiments described in the foregoing paragraphs were from a single commercial flock for each experiment. In each instance, the flock did not have a history of an infectious disease problem and the turkeys selected had not been previously used for any kind of research work. All turkeys in each of the chambers in each experiment were observed at least once daily and mortality and morbidity recorded. When deaths occurred, a representative number of dead turkeys were subjected to a postmortem and bacteriological examination using the same procedures and techniques described in a previous paper (Bierer and Derieux, 1972). P. multocida isolates were subjected to an agar-gel-diffusion test (Heddleston, 1971) that was conducted by W. T. Derieux at the Clemson Livestock Laboratory at Columbia, South Carolina. The highly virulent P-159 culture used for challenge purposes was obtained from Kenneth L. Heddleston, Senior Research Bacteriologist at the National Animal Disease Laboratory at Ames, Iowa. RESULTS AND DISCUSSION In the first experiment, in which normal turkeys were added to groups of turkeys after they had received drinking water vaccine, none of the added turkeys died or developed clinical evidence of fowl cholera infection during a 5 weeks period of exposure. Exposure by cohabitation is routinely used as a method to spread fowl cholera infection from artificially inoculated birds to pen mates that have not been inoculated (Anonymous, 1971). When the highly virulent P-159 isolate of P. multocida was applied to all turkeys of all groups, 38 of 4 turkeys that were neither vaccinated nor exposed to vaccinated turkeys either died or exhibited other evidence of infection during the 2 weeks post exposure period. The turkeys exposed to vaccinated turkeys, as pen mates, experienced a similar mortality with a total of 17 of these 2 becoming infected, compared to only 5 of the 4 turkeys that had received the water vaccine. A summary of the data is given in Table 1. Under the conditions of this experiment, nonvaccinated turkeys did not develop symptoms of fowl cholera infection Downloaded from at Penn State University (Paterno Lib) on September 18, 216

4 PASTEURELLA MULTOCIDA 1513 TABLE 1. Efforts to transmit an avirulent Pasteurella multocida strain from vaccinated turkeys to their nonvaccinated pen mates Chamber Treatment Number of turkeys Total number infected (mortality+morbidity) :** 5 weeks 2 weeks post expost vacci- posure to virulent nation P-159 strain (%) Vaccinated Vaccinated Nonvaccinated Nonvaccinated * These turkeys were added to the turkeys in chambers 1 & 2 the day after vaccination was completed in those facilities. ** The vaccine was administered at 8 weeks of age, through the drinking water route, to the turkeys to be vaccinated in chambers 1 & 2. The virulent P-159 strain was administered via the drinking water to all groups at 13 weeks of age. after a 5 weeks period of exposure to vaccinated pen mates. In addition, these same nonvaccinated turkeys did not exhibit evidence of an immunologic response suggesting that the vaccinated turkeys either did not shed the avirulent P. multocida bacilli after vaccination or did not shed these bacilli in numbers sufficient to incite an immunological response in their nonvaccinated pen mates. That is, nonvaccinated turkeys exposed to vaccinated turkeys did not become vaccinated as a result of this exposure. Presumably, it appears that each member of any given flock must have an opportunity to drink some of this type of vaccine if it is to succeed under field conditions. In the second experiment, in which the avirulent P. multocida strain was compared to the pathogenicity of an isolate obtained after 5 turkey passages and to another isolate obtained after 1 passages, none of the turkeys exposed to either the avirulent vaccine strain or the 5-turkey passage strain developed evidence of fowl cholera infection during the 2 weeks post exposure period. One of the 2 turkeys exposed to the 1-turkey passage isolate developed a swelling in the tibiometatarsal area. This turkey was clinically normal except for this apparent evidence of fowl cholera infection. The bird was killed on the 14th day post exposure and subjected to a bacteriological examination. P. multocida bacilli were isolated from fluid obtained from the swollen tibiometatarsal area. This isolate was serologically indistinguishable from the original avirulent vaccine strain, which has certain distinguishing characteristics that will be described in a succeeding paragraph. Bierer and Derieux (1972), in an earlier paper, reported on overall 4.2% infection rate in 12 turkeys exposed to the same avirulent vaccine strain used in these present experiments. That is, an occasional clinically infected turkey in small groups of vaccinated birds is not an unusual observation. The results of this second experiment suggest that there was little or no increase in the virulence of the Downloaded from at Penn State University (Paterno Lib) on September 18, 216

5 1514 B. W. BlERER AND W. T. DERIEUX TABLE 2. Results of efforts to enhance virulence of an avirulent Pasteurella multocida strain by passage through turkeys Chamber Treatment* Avirulent strain Avirulent strain Five turkey passage strain Five turkey passage strain Ten turkey passage strain Ten turkey passage strain Nonexposed Nonexposed Number of turkeys Total number infected (mortality + morbidity): 2 weeks 2 weeks post expost posute to virulent treatment P-159 strain** 1 /1 /1 2/1 /1 /9 1/1 9/1 8/1 * Treatment was administered at 8 weeks of age, exposure to the various strains being accomplished through the drinking water route. ** The virulent P-159 strain was administered at 1 weeks of age, exposure being accomplished through the drinking water. avirulent vaccine strain after either 5 turkey passages or after 1 turkey passages under the conditions of this experiment (see Table 2). An excellent discussion on the conditions under which pathogenicity or virulence can be increased or decreased is in the text by Merchant and Packer (1967). These authors note that successive passage through mice and egg embryos increased the virulence of certain hemorrhagic septicemia (P. multocida) bacilli. It is rather commonly believed that rapid animal passage of pathogens of low virulence will result in increased virulence and that virulent isolates sometimes become even more virulent. On the other hand, Brucella abortus Strain 19 (the subject of a decade of bitter controversy during the 194's) is an example of a stable gramnegative avirulent bacterial vaccine. An outstanding example of an attenuated bacterial vaccine is the Bacille Calmette- Guerin (BCG) vaccine (Gell and Coombs, 1968) used to immunize the human species against tuberculosis. Merchant and Packer (1967) note that the loss as well as the acquisition of virulence appears to be involved in the dissociation phenomenon. Heddleston et al. (1964) have observed that a highly virulent P. multocida culture dissociated, in vitro, from iridescent colonies to blue colonies and from blue colonies to gray colonies. In vivo, however, the gray colonies reverted back to blue colonies and the blue colonies back to iridescent colonies. These workers further detected a direct relationship of virulence to colony morphology with regard to the particular isolate studied: the iridescent colonies were highly virulent, the blue colonies less virulent and the gray colonies without virulence. The Clemson University avirulent vaccine strain was reported by Heddleston (197) to produce iridescent colonies on dextrose starch agar and that the cells were encapsulated. Recent examinations of this culture at the Clemson Livestock Laboratory (Blalock, 1972) confirm that these characteristics are still present. Since this strain produces iri- Downloaded from at Penn State University (Paterno Lib) on September 18, 216

6 PASTEURELLA MULTOCIDA 1515 descent colonies and iridescent colonies are a form that are not dissociated, it appears that the avirulence of this strain is not associated with the process of dissociation described by Merchant and Packer (1967) and by Heddleston et al. (1964). Presumably, then, there is a likelihood of a greater degree of stability being associated with the Clemson University vaccine strain than would ordinarily be encountered because of the fact that it does not appear to be a dissociated strain. With reference to the isolates from the livers of the turkeys that died following i.v. injection of the vaccine strain, agargel diffusion test results provided evidence that all of the isolates selected for use were serologically indistinguishable from each other. While the vaccine strain has been classified as a P-159 isolate (Heddleston, 197), this culture also possesses P-1662 antigens. This characteristic was useful in distinguishing the vaccine strain from, for instance, the P-159 challenge culture isolates, which possessed P-159 antigens only. Several years ago the senior author of this present paper observed that an initially virulent P. multocida culture that was transferred weekly on triple-sugariron agar slants had lost its virulence. Subsequently, this isolate was used for oral vaccination (Bierer and Eleazer, 1968) with encouraging results. Presumably, this loss of virulence was due to dissociation that resulted in attenuation. After some 2 years of weekly transfer, however, this isolate lost its ability to stimulate a favorable immunogenic response in turkeys vaccinated by the drinking water route. The avirulent vaccine strain used and referred to in this present paper was isolated during March of 197. It has been transferred weekly on bovine-blood-agar plates since that time and appears to have retained its original characteristics up to the present time. Merchant and Packer (1967) note that certain strains of avirulent bacteria can be made fully virulent when grown on culture media containing blood and blood serum. It would appear then that the maintenance of the Clemson University vaccine strain on bovine-blood-agar plates has been a deterrent to the dissociation of this culture. A simple method of guarding against dissociation, of course, is either by lyophilization or by storing the culture in sterile skim milk at 23 C. or lower (Anonymous, 1971; Watko and Heddleston, 1966). The data of the experiments reported in this paper failed to demonstrate that vaccinated pen mates were harmful to the presence of nonvaccinated siblings or the virulence of the Clemson University vaccine strain was effectively enhanced by as many as 1 turkey passages. One must keep in mind, however, that field conditions involving thousands of animals frequently produce different results than the results obtained with the small numbers used under laboratory conditions. Heddleston and Rebers (1972) note that "the stability of avirulent or lowvirulence strains in birds remains in question, which invites caution when such strains are proposed for use under field conditions." There is no doubt, of course, that caution is the only safe approach as a general rule. On the other hand, when one examines a number of fowl cholera outbreaks in turkeys and detects certain premises that have experienced serious fowl cholera problems year after year, and when the owners of these flocks have found that various treatments and prophylactics (Peterson, 1972) are sometimes either ineffective or economically unsound, and when these same individuals are on the verge of quitting turkey production as an unprofitable ven- Downloaded from at Penn State University (Paterno Lib) on September 18, 216

7 1516 B. W. BIERER AND W. T. DERIEUX ture, it appears that situations of this kind have little to lose and a chance of something to gain by the introduction of a relatively stable avirulent or attenuated P. multocida vaccine into the flocks of these premises. REFERENCES Alls, A. A., G. S. Appleton and J. R. Ipson, 197. A bird contact method of challenging turkeys with Pasteurella multocida. Avian Diseases, 14: 175. Anonymous, Methods for Examining Poultry Biologies and for Identifying and Quantifying Avian Pathogens, National Academy of Sciences, Washington, D. C. 2, page 171. Bierer, B. W., and T. H. Eleazer, Continuous use of a live vaccine in the drinking water against fowl cholera infection in turkeys. Poultry Sci. 47: Bierer, B. W., and W. T. Derieux, Immunologic response in turkeys to an avirulent Pasteurella multocida vaccine in the drinking water. Poultry Sci. 51: Blalock, G. H., Personal communication, July 5, 1972, P.O. Box 1771, Clemson Livestock-Poultry Health Department, Columbia, South Carolina Bond, R. E., J. M. Donahue and L. D. Olson, 197. Colony features of Pasteurella multocida and their use in diagnosing fowl cholera in turkeys. Avian Diseases, 14: Gell, P. G. H., and R. R. A. Coombs, Clinical Aspects of Immunology, F. A. Davis Co., Philadelphia, Pa. 191, 2nd Ed., page The Tenaha, Texas branch has been closed and sold. Texas customers will be served from Carthage, Mississippi, and Springdale, Arkansas. The Albertville, Alabama branch will be phased out during 1973, and customers will be served from Blairsville, Georgia, and Carthage. The Ashville, North Carolina branch will be sold, and Western Carolina customers will be served from the Concord, North Carolina branch. The Delmarva branch at Princess Anne has been closed, and delivery will be made to area customers from the Concord branch. This realignment leaves four strategically lo- NEWS AND NOTES {Continued from page 159) Heddleston, K. L., 197. Official laboratory report, April 1, 197. National Animal Disease Laboratory, Ames, Iowa 51. Heddleston, K. L., Reported at the 2th Western Poultry Disease Conference, University of California, Davis, California, March 23-24, Heddleston, K. L., Personal communication, January 24, Heddleston, K. L., and P. A. Rebers, Fowl cholera: cross immunity induced in turkeys with formalin-killed in-vivo-propagated Pasteurella multocida. Avian Diseases, 16: Heddleston, K. L., L. P. Watko and P. A. Rebers, Dissociation of a fowl cholera strain of Pasteurella multocida. Avian Diseases, 8: McDowell, J. R., S. K. Maheswaran, D. S. Boulcy and B. S. Pomeroy, The immunization against fowl cholera using an avirulent Pasteurella multocida mutant. Reported at the 19th Annual AVMA Meeting, New Orleans, Louisiana, July Abstract in Program of 19th Annual Amer. Vet. Med. Ass. Meeting, page 174. Merchant, I. A., and R. A. Packer, Veterinary Bacteriology and Virology, The Iowa State University Press, Ames, Iowa 51, 7th Ed., page 118. Peterson, E. H., Serviceman's Poultry Health Handbook, Better Poultry Health Company, P. O. Box 1144, Fayetteville, Arkansas 7271, 1972 Printing, pages Watko, L. P., and K. L. Heddleston, Survival of shell-frozen, freeze-dried, and agar slant cultures of Pasteurella multocida. Cryobiology, 3: cated broiler breeder branches Springdale, Arkansas; Carthage, Mississippi; Blairsville, Georgia; and Concord, North Carolina. Canadian customers will be served from Bedford, Quebec, and Atwood, Ontario. PFIZER NOTES Pfizer Inc. has been selected as one of the nation's five best-managed companies for 1972 by the Editors of Dun's Review. The five were singled out for excellence in different management categories. Pfizer was selected for its achievements in multinational development. Downloaded from at Penn State University (Paterno Lib) on September 18, 216 {Continued on page 153)