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Exposing Turkeys by Various Routes to an Avirulent and Virulent Strains of Pasteurella multocida
Exposing Turkeys by Various Routes to an Avirulent and Virulent Strains of Pasteurella multocida B . W . BlERER AND W . T . DERIUEX South Carolina Agricultural Experiment Station, Clemson University, Clemson, South Carolina 29631 (Received for publication March 1, 1973)
POULTRY SCIENCE 52: 2290-2298, 1973
INTRODUCTION
MATERIALS AND METHODS
IERER and Derieux (1972) have reported on their successful experimental use of an avirulent Pasteurella multocida vaccine administered to turkeys in the drinking water. McDowell et al. (1972), investigating various routes of administration, have reported on their successful efforts to immunize turkeys against fowl cholera with the use of an avirulent P. multocida mutant in the drinking water and also established that the highest degree of protection resulted from endotracheal vaccination. It was the purpose of the following described experiments to determine whether or not certain routes of vaccination other than water application may have a potential commercial application.
In the first experiment, 10 7-weeks-old turkeys were placed in each of 8 research chambers. The chambers were part of a concrete block building that contained 8 separate research units, each with a separate entrance into an anteroom. There was no direct air communication between the chambers and there were no other disease experiments in progress on the premises at the time. The anteroom contained feed and water facilities for the adjacent chambers, and each chamber was 2.7 x 3.4 meters. The units were routinely cleaned and disinfected and the chambers prepared with fresh, clean shavings litter prior to their use for each experiment. The avirulent P. multocida strain used was the Clemson University vaccine strain described in a previous paper (Bierer and Derieux, 1972, 1973). The basic vaccine was
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ABSTRACT In three separate experiments, an avirulent Pasteurella multocida vaccine was administered to separate groups of turkeys by the following routes: ocular (conjunctival sac), nostril, palatine cleft, oral, drinking water, cloacal, intradermal, feather follicle and wing web. All groups were challenged with a virulent P-1059 P. multocida strain 2 to 5 weeks post vaccination. Under the conditions of these experiments, the results suggest that a favorable immunogenic response of some kind occurred in all groups except those vaccinated by the nostril and cloacal routes. In the latter two groups there was little or no evidence of a favorable response. Turkeys vaccinated by the stick-wing-web method exhibited an excellent immunogenic response, but a large cheesy mass at the site of vaccination would probably result in condemnation of the vaccinated wing areas of some turkeys at the time of slaughter. This method uses little vaccine, is accurate, easy and rapid and, presumably, may have an application in nations where edible food of any kind is a primary objective. In a fourth experiment, the virulent P-1059 challenge strain mentioned above was used as the vaccinating agent and was administered to separate groups of turkeys by the same various routes mentioned in the foregoing. One hundred percent of the turkeys exposed by the feather follicle route were dead within 48 hours and 90% to 100% of the groups exposed by the ocular, palatine cleft, intradermal and stick-wing-web routes were dead by the fourth day. The lowest mortality during a given number of days was in the groups exposed by the nostril and cloacal routes. Under the conditions of the fourth experiment, the results suggest that the eye is a possible route of infection under natural conditions, and that scratches and puncture wounds to the skin and crushed feather follicles, when accompanied by virulent P. multocida containing material, may possibly serve as portals of entry.
PASTEURELLA
MULTOCIDA EXPOSURE
a 24-hours old brain-heart-infusion broth culture which had been incubated at 37 degrees C. Plate counts on dextrose-starch agar, using a method suggested by Heddleston (1972), established that the vaccine contained approximately 1 billion viable bacilli per ml. of vaccine. Treatment applied to the turkeys in chambers 1 through 8 were:
6. Drinking water vaccination was applied as described in a previous paper (Bierer and Derieux, 1972) and consisted of 30 ml. of the basic vaccine (24 hours old brain-heart-infusion broth culture) placed into each 4000 ml. of drinking water, daily for four consecutive days. To encourage prompt consumption, water was withheld for two hours prior to the time the water vaccine was administered. Each ml. of drinking water contained approximately 7,500,000 viable bacilli when freshly prepared. 7. Intradermal vaccination consisted of the injection of vaccine between the layers of the loose skin (wattle) under the lower jaw. A tuberculin syringe fitted with a 25 gauge needle was used for injecting the vaccine. 0.02 ml. of vaccine was injected and this amount contained approximately 20 million viable bacilli. 8. Cloacal vaccination was accomplished with the same type cotton swab used for the swabbed palatine cleft vaccination. The swab containing the vaccine was inserted into the cloaca and firmly rotated for 10 complete rotations. A separate freshly prepared swab was used for each turkey. Fourteen days after applying the treatments described in the foregoing, all turkeys in each of the 8 chambers were exposed to a virulent P-1059 P. multocida strain by the drinking water route. The virulent P-1059 strain was obtained from K. L. Heddleston, Senior Research Microbiologist at the National Animal Disease Laboratory at Ames, Iowa. It was in a vacuum sealed vial in lyophilized form when received. This culture was inoculated into brain-heart-infusion broth, which was then incubated at 37 degrees C. for 24 hours. The resulting culture was lyophilized, the final product being in vacuum sealed glass vials, which were stored at 18 degrees C. When used in connection with an experiment,
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1. Nonvaccinated. 2. Ocular vaccination or 1 drop of the vaccine placed into the conjunctival sac. This drop contained approximately 34 million viable bacilli, and was administered from the nipple portion of a sterile plastic syringe (non leur-lock type) held at a 45 degree angle. Average drop size was 0.034 ml. 3. Nostril vaccination or 1 drop of the vaccine placed in the nostril, and applied in the same manner as the ocular vaccination. 4. Palatine cleft vaccination was accomplished with the use of a sterile cotton swab that had been dipped into the vaccine. The swab was partially inserted through the palatine cleft or choanae area into the nasal cavity and gendy, but firmly, rotated to and fro. Each swab contained approximately 0.25 ml. of vaccine or approximately 250 million viable bacilli. A separate freshly prepared cotton swab was used for each turkey. 5. Oral vaccination was accomplished with the use of a sterile plastic syringe, the needle portion of which was covered with small caliber rubber tubing. The mouth of the subject was held open by the operator with the fingers of the left hand and the delivery end of the syringe placed in the oral cavity of the turkey. One ml. of vaccine was then slowly injected into the oral cavity. The 1 ml. of vaccine contained approximately one billion viable bacilli.
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TABLE 1.—Results of vaccination by various routes with (Experiments ;rim«
3
Number infected 2 weeks post vaccination
Two weeks post exposure
Daily record of deaths: & Treatment* 6 3 4 5 2 1 u Nonvaccinated 0 4 0 0 2 0 1 Nonvaccinated 0 2 2 2 1 0 2 — Ocular 1 0 0 0 1 1 2** 0 Ocular 0 0 1 0 0 0 0 2 Nostril 1 1 0 2 0 0 0 1 Nostril 3 1 0 2 0 0 1 2 Palatine cleft 0 1 0 0 0 0 0 1 Palatine cheft 0 1 0 0 1 0 2 0 Oral 0 0 1 1 0 0 0 0 Oral 1 1 0 0 0 1 2 0 Drinking water 0 0 0 0 0 0 1 0 Drinking water 0 0 0 2 0 0 0 It Intradermal 0 0 1 0 0 0 1 0 Intradermal 0 0 0 0 0 2 0 0 Cloacal 0 1 1 1 0 0 0 1 Cloacal 1 1 0 0 1 2 2 0 Feather follicle 0 0 0 0 0 0 2 2** *Ten turkeys for each separate listing. ** Killed on 14th day post vaccination for examination, prior to the use of the virulent P-1059 strain. tDied on the 14th day post vaccination.
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the lyophilized contents of a vial were sus consisted of 10 turkeys in a portable woodpended in a small amount of brain-heart-infu- floor building with a floor space of 1.2 x sion broth and inoculated on to a blood agar 2.4 meters. This building provided excellent plate. This initial isolate was then incubated shelter and was constructed in such a manner at 37 degrees C. for 24 hours. When used that feed and water could be placed inside in the drinking water, it was prepared and without entering the building. It had been administered in the same manner that the cleaned and disinfected prior to use and drinking water vaccination was administered supplied with a thick layer of fresh, clean to the turkeys in Chamber 6, as described shavings. It was located in the general vicinity in the foregoing, and in a previous paper of the research units already described, but (Bierer and Derieux, 1972). The experiment was in no way in direct communication with was terminated 14 days after exposure to the these units. The turkeys in this building were virulent P. multocida strain. At that time the subjected to a feather follicle vaccination number of turkeys that died in each chamber which consisted of the plucking of four or was added to the number of survivors that five of the large feathers from the upper were morbid to arrive at a total number exterior surface of the thigh and rubbing the infected value for the turkeys in each of the exposed feather follicles with a cotton swab 8 treatment groups. This method of evaluation saturated with vaccine. Feather follicle vacwas first described by Alls et al. (1970) and cination is one of the methods used in fowl has since been used routinely by the authors pox vaccination in chickens and turkeys (Barger et al., 1958). of this present paper. The third experiment utilized the 8 research The second experiment was a repetition of the first experiment with the exception chambers used in the first experiment; 10 of the addition of a ninth treatment that 8-weeks-old turkeys were placed in each of
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MULTOCIDA EXPOSURE
lent P-1059 P. multocida strain was used as the vaccine. All turkeys used were from flocks that did not have a disease problem history and were from a single source for each experiment and had not been previously used for any type of research work. All groups of turkeys were observed at least once daily and data relative to mortality and morbidity were recorded. When deaths occurred, representative numbers from a single group were subjected to a bacteriological examination as described in a previous paper (Bierer and Derieux, 1972). Isolates were identified biochemically and subjected to an agar-gel diffusion test at the Clemson Livestock Laboratory, Columbia, South Carolina. Records of feed consumption and of body weights were not kept during the course of the experiments described in the foregoing. Evaluation was based on mortality and morbidity results. RESULTS AND DISCUSSION Under the conditions of the first and second
an avirulent strain of Pasteurella multodica 1,2) to virulent P-1059 strain:
7 1 0 0 0 1 0 1 0 0 0 0 0 0 0 1 0 0
8 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0
9 0 0 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0
10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
14 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Total number died 7 7 2 1 6 7 2 2 1 3 0 0 2 1 4 5 0
Total number morbid 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0
Total number infected 7 7 4 1 6 7 3 3 1 3 0 1 2 1 4 5 2
Average number infected
<%) 70 25 65 30 20 5 15 45 20
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these chambers. The turkeys in Chambers 1 through 4 were nonvaccinated and the turkeys in Chambers 5 through 8 were subjected to a stick-wing-web vaccination with the Clemson University vaccine strain. This was accomplished with the use of the double needle applicator that is supplied for use with commercial fowl pox vaccine. The vaccine was applied at a rather central location through the wing-web area. The two needles contained a maximum of 0.004 ml. of vaccine which contained approximately 4 million viable bacilli. Five weeks post vaccination, all eight groups of turkeys were challenged with the same virulent P-1059 strain and in the same manner that this strain was used in the first experiment. This third experiment was also terminated 14 days from the time the challenge was initiated in the same manner that termination was accomplished in the first and second experiments. The fourth experiment was essentially a repetition of the second experiment, except that a 24-hours-old broth culture of the viru-
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by the feather follicle method of vaccination, the wing-web method of vaccination was investigated by the treatments applied in the third experiment. The results of this experiment were quite impressive since all of the turkeys survived challenge with the virulent strain and all of the survivors were clinically healthy. A summary of the data is given in Table 2. One of 40 turkeys died after wingweb vaccination: on postmortem examination, a solidified lung was observed from which P. multocida bacilli were isolated. The agar-gel-diffusion test established that the isolate possessed both P-1059 and P-1662 antigens which characterized the Clemson University vaccine strain that was administered to these turkeys (Bierer and Derieux, 1973). The wing-web vaccination sites were examined, in each of the 40 vaccinated turkeys, 24 hours post vaccination and a small cheesy swollen area was observed. A small reaction of this type was considered to be most desirable. By the second and third days post vaccination, however, the cheesy swollen areas surrounding the sites of vaccination had increased considerably in size to eventually produce a large, irregularly shaped cheesy mass sometimes approximately 50 mm. in diameter. By the fifth week post vaccination, a number of the enlargements had ruptured and the rather dry cheesy mass had been expelled leaving a gaping break in the skin on the lower side of the wing-web area. During this entire five weeks post vaccination period all of the turkeys, except the one that died, appeared to be clinically normal and not distressed by the wing-web lesion described in the foregoing. The wing-web or stick method of vaccination has been used for some years as the method of choice to vaccinate chickens and turkeys against fowl pox disease. Barger et al. (1958) and Hall (1956) note that the stick method is "fast, accurate, and economical of vaccine." These individuals also called attention to the fact that the two needle
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experiments, the results suggest that some kind of a favorable immunogenic response occurred following the various vaccination routes with the exception of the nostril and cloacal vaccination treatments. In these latter two groups there was little or no evidence of a favorable immunogenic response. In the first experiment, 2 of the turkeys that had received the ocular vaccination became morbid by the ninth day after vaccination. These 2 turkeys were killed on the fourteenth day post vaccination. Lesions of fowl cholera were not detected on postmortem examination and cultures of the internal organs were nonproductive. Nevertheless, the 2 turkeys were included under the total number infected for this group. In the second experiment, one of the turkeys that had received the vaccine in the drinking water died on the fourteenth day after vaccination; a solidified lung was observed on postmortem examination, from which P. multocida bacilli were isolated. The feather follicle method of vaccination resulted in a cheesy skin lesion at the site of vaccination that increased in size to approximately 25 mm. in diameter. Two of the 10 turkeys in this group developed an enlargement of the tibiometatarsal area. The affected legs were not the same legs to which the feather follicle vaccination had been applied. Fourteen days post vaccination, these two turkeys were killed and subjected to a bacteriological examination. Each of the swollen areas contained clear fluid and cultures failed to establish that a bacterial infection was present. Perhaps these two turkeys were injured at the time of vaccination. Whatever the cause may have been, the two turkeys were included in the total number infected for this group as tabulated in Table 1. All eight of the remaining turkeys in this group survived challenge with the virulent P-1059 strain. A summary of the data for experiments 1 and 2 is given in Table 1. In view of these seemingly favorable results
PASTEURELLA MULTOCIDA EXPOSURE
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inoculation used results in four cutaneous inoculations, two on each side of the wingweb. In evaluating vaccination results one must think in terms of possible world-wide application. What may not be useful to the commercial turkey industry of the United States because of the rules and regulations of poultry inspection could be an acceptable procedure by nations where edible food of any kind is a primary objective. The results of the third experiment suggest that the stick method or wing-web method of vaccination may be an effective method that can be easily and rapidly applied at low cost. The nature of the lesion that develops at the site of vaccination suggests that a portion of the wing of some of the turkeys vaccinated by this method would be removed at the time of inspection when slaughtered. This would result in increased slaughter time and a down grading of the trimmed turkeys. In the nations of the world where edible food of any kind is a primary objective, the removal of a portion of the wing at the time of slaughter would make little or no difference. It was the purpose of the fourth experiment to determine whether or not, under the conditions of this trial, a highly virulent strain of P. multocida could be applied at one or more of the locations described with a resulting
favorable immunogenic response. While the outcome was more or less anticipated, the results were considered to be sufficiently interesting to be included in this paper (Table 3). Under the conditions of this experiment all 10 of the turkeys exposed by the feather follicle route were dead within 48 hours. It would appear that crushed feather follicles could occur during the treading activities of larger turkeys. If the feather follicles were crushed in the presence of material containing virulent P. multocida bacilli, then a fatal septicemic fowl cholera infection could possibly occur under these conditions. By the end of the fourth day 90% to 100% of the turkeys died in the groups exposed by ocular, palatine cleft, intradermal and stickwing-web routes. These results suggest that scratches and puncture wounds, in the presence of material containing virulent P. multocida bacilli, are possible avenues to a fatal septicemic fowl cholera infection under natural conditions. The methods of exposure resulting in the least mortality, under the conditions of this experiment and during a given number of days, were the nostril and the cloacal applications. These same two methods were also the least effective in inciting a favorable immunogenic response when the Clemson University vaccine strain was used as the vaccinating agent and as
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TABLE 2.—Results of vaccination with avirulent Pasteurella multocida by the stick-web-wing method (Experiment 3) Two weeks post exposure to virulent P-1059 strain: Number Average infected number Total Total 5 weeks Total infected number number post vaccnumber infected morbid ination died Treatment* (%) 5 1 4 Nonvaccinated 3 0 3 Nonvaccinated 55 7 0 7 Nonvaccinated 7 0 7 Nonvaccinated 0 0 0 Vaccinated 0 j** 1 0 0 Vaccinated 0 3 0 0 Vaccinated 0 0 0 0 Vaccinated 0 *Ten turkeys for each separate listing. **Died on 7th day after vaccination.
Treatment* 1 2 3 4 Ocular 6 5 4 0 Nostril 0 0 0 1 Palatine cleft 0 7 0 2 Oral 0 1 5 1 Drinking water 0 0 3 1 Intradermal 0 5 3 1 Cloacal 0 2 0 0 Feather follicle 1 9 — — Stick-wing-web 0 7 2 i Nonvaccinated 0 0 0 0 *Ten turkeys for each separate listing.
6 0
7 0
8 6
9 0
1 0 1 0 1 0 1 — — 0 0 0 0 0 1 0 1 0 0 i _ _ _ _ _ 1 0 1 0 0 0 — — — — — _ _ _ _ _ _ 0 0 0 0 0 0
1 0 2 2
5 0
Daily record of deaths
Two weeks post vaccination: Total number 10 11 14 died 0 0 0 9 2 1 8 — — — 10 0 0 9 0 0 8 _ _ 10 1 0 5 — — — 10 _ _ 10 0 0 0
TABLE 3.—Results of vaccination by various routes with a virulent strain of Pas (Experiment 4)
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MULTOCIDA EXPOSURE
chronic fowl cholera in turkeys that is often characterized by torticollis, have reported that the outstanding lesion was the caseous exudate in the air spaces of the spongy portions of the calvarium. In view of this earlier work by Olson et al. (1966), Donahue and Olson (1971) developed a method of exposing turkeys to fowl cholera by inoculating P. multocida bacilli into the spongy portion of the calvarium via the eustachian orifice and were able to produce uniform and consistent infections. The eustachian orifice, of course, is a pharyngeal structure distinctly separate from the palatine cleft. The nasal opening of the nasolacrimal duct, referred to.in discussing ocular exposure, is not in close association with the eustachian orifice but it is, however, in close proximity with the sphenoid bone, which was one of the portions of the calvarium incriminated by Olson et al. (1966) with reference to gross lesions of chronic fowl cholera. REFERENCES Alls, A. A., G. S. Appleton and J. R. Ipson, 1970. A bird contact method of challenging turkeys with Pasteurella multocida. Avian Diseases, 14: 172-178. Barger, E. H., L. E. Card and B. S. Pomeroy, 1958. Diseases and Parasites of Poultry, Lea and Febiger, Philadelphia, Penna. 19100, Fifth Ed., page 170. Bierer, B. W., and W. T. Derieux, 1972. Immunologic response of turkeys to an avirulent Pasteurella multocida vaccine in the drinking water. Poultry Sci. 51: 408-416. Bierer, B. W., and W. T. Derieux, 1973. Efforts to transmit and to enhance the virulence of an avirulent strain of Pasteurella multocida in turkeys. Poultry Sci. 52: 1510-1516. Donahue, J. M., and L. D. Olson, 1971. Research technique—inoculation of Pasteurella multocida into the palatine air spaces as an exposure method for fowl cholera in turkeys. Avian Diseases, 15: 158162. Dorsey, T. A., 1963. Studies on fowl cholera. II. The correlation between biochemic classification and the serologic and immunologic nature of avian Pasteurella multocida strains. Avian Diseases, 7: 399. Hall, W. J., 1956. Fowlpox. In U.S. Department of Agriculture Yearbook, pages 464-466. Heddleston, K. L., 1962. Studies on pasteurella. V.
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already described in a preceding paragraph. The results of the fourth experiment also suggest that ocular conjunctival sac exposure, with a given number of virulent P. multocida bacilli, would be more apt to induce acute fowl cholera and sudden deaths than exposure with the same number of bacilli by the nostril route. Ocular conjunctival sac exposure is the route of choice to infect cattle with virulent Brucella abortus isolates (Hendricks and Ray, 1970) under experimental conditions. This route was selected because it consistently gave uniform results (Hendricks, 1973). Ocular exposure, as described under material and methods in this present paper, consisted of one drop of the vaccine placed in the conjunctival sac. Presumably, the inoculum is carried by way of the nasolacrimal duct to the nasal palatine cleft area since a local inflammatory reaction was not observed. If this line of reasoning is correct, then ocular conjunctival sac exposure should be an excellent approach to nasal palatine cleft exposure, without the injury or trauma to the mucous membrane that must sometimes occur when a cotton swab is applied directly to the nasal palatine cleft area. Swabbing the palatine cleft with virulent P. multocida bacilli has been used routinely for some years (Heddleston, 1962; Dorsey, 1963; Heddleston et al., 1970; Alls et al., 1970; and Donahue and Olson, 1971). Dorsey (1963) noted that this method more closely simulated natural exposure to the disease. There appear to be variations in the manner in which the palatine cleft area is inoculated when swabbed. Donahue and Olson (1971) note that a cotton swab was "wiped across the roof of the mouth." When the turkeys referred to in this present report were inoculated, the cotton swab was partially inserted through the cleft area or choanae (McLeod et al, 1964) into the nasal cavity area and, presumably, Heddleston and Rebers (1972) use a similar technique. Olson et al. (1966), studying the type of
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adjuvant Brucella abortus vaccines. Proceedings of 74th Annual Meeting, U.S. Animal Health Association 1970, page 152. McDowell, J. R., S. K. Maheswaran, D. S. Bouley and B. S. Pomeroy, 1972. The immunization against fowl cholera using an avirulent Pasteurella multocida mutant. Reported at the 109th Annual A.V.M.A. Meeting, New Orleans, Louisiana, July 18-20. Abstract in Program of 109th Annual A.V.M.A. Meeting, page 174. McLeod, W. M., D. M. Trotter and J. W. Lumb, 1964. Avian Anatomy, Burgess Publishing Co., Minneapolis, Minnesota 55400, pages 103-104. Olson, L. D., E. L. McCune and B. L. Moseley, 1966. Gross and histopathological description of the cranial form of chronic fowl cholera in turkeys. Avian Diseases, 10: 518-529.
NEWS AND NOTES (Continued from page 2289) DIAMOND AUTOMATION NOTES J. Stephens has been appointed Product Manager for Keenco cage layer systems. The line, manufactured by Diamond International Corporation, Diamond Automation Division, Farmington, Michigan, consists of Keenco's contour " A " Line two-tier system, Triple " A " Line three-tier system, and single tier multicage system. He will be responsible also for associated products used with Keenco systems but manufactured by other companies. Stephens has had 23 years of experience in marketing and product management of agricultural products. BIG DUTCHMAN NOTES Gary L. Voss has been appointed Sales Manager, Bernard Brownstein, Western Sales Mannager, and Harry W. Cronise, Southeastern Sales Manager for the Big Dutchman Staalkat egg processing equipment line. Prior to his recent appointment, Voss was Big Dutchman Cage Systems Coordinator. Prior to joining Big Dutchman, Brownstein served in various management positions with Kimber Farms, and most recently was Broiler Production Manager for Zacky Farms in southern California. Cronise has had more than 30 years of experience in the shell egg processing field. A new Ecology Division has been established at Big Dutchman, Zeeland, Michigan. John F. Bergdoll, who has been Big Dutchman New Products Manager,
has been appointed General Manager of the new Division. He has had more than 20 years of experience in poultry, animal health, and feed management. HY-LINE NOTES Dennis Dixon has been appointed Marketing Coordinator for Hy-Line International, Des Moines, Iowa. He will be on the staff of Dr. G. Waters, Director of Marketing, and will help expedite the firm's marketing policies and aid in maintaining communications with Hy-Line distributors throughout the world. He has served Hy-Line in a number of capacities. Most recently he was Sales Director for Latin America, Wallace International, St. Petersburg, Florida. OHIO NOTES J. H. Florea, former Editorial Director of Poultry Tribune, received the "Distinguished Alumni Award" from the Department of Poultry Science, Ohio State University, at a special ceremony on April 10. He graduated from Ohio State in 1928, and retired from the Watt Publishing Company on June 30, 1969, after 40 years of service. WASHINGTON NOTES Dr. Paul C. Harrison, formerly Associate Professor, Department of Animal Sciences, Washington State University, Pullman, is not on the staff of the Depart-
(Continued on page 2305)
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Two immunogenic types of Pasteurella multocida associated with fowl cholera. Avian Diseases, 6: 315-321. Heddleston, K. L, J. E. Gallagher and P. A. Rebers, 1970. Fowl cholera: immune response in turkeys. Avian Diseases, 14: 626-635. Heddleston, K. L., 1972. Personal communication, January 24, 1972. Heddleston, K. L., and P. A. Rebers, 1972. Fowl cholera: cross immunity induced in turkeys with formalin-killed in-vivo-propagated Pasteurella multocida. Avian Diseases, 16: 583. Hendricks, J. B., 1973. Personal communication, January 2, 1973, Clemson Livestock Laboratory, P. O. Box 1771, Columbia, S.C. 29202. Hendricks, J. B., and W. C. Ray, 1970. Studies on cattle vaccinated with live strain 19 and/or 45/20
POULTRY SCIENCE 52: 2290-2298, 1973
INTRODUCTION
MATERIALS AND METHODS
IERER and Derieux (1972) have reported on their successful experimental use of an avirulent Pasteurella multocida vaccine administered to turkeys in the drinking water. McDowell et al. (1972), investigating various routes of administration, have reported on their successful efforts to immunize turkeys against fowl cholera with the use of an avirulent P. multocida mutant in the drinking water and also established that the highest degree of protection resulted from endotracheal vaccination. It was the purpose of the following described experiments to determine whether or not certain routes of vaccination other than water application may have a potential commercial application.
In the first experiment, 10 7-weeks-old turkeys were placed in each of 8 research chambers. The chambers were part of a concrete block building that contained 8 separate research units, each with a separate entrance into an anteroom. There was no direct air communication between the chambers and there were no other disease experiments in progress on the premises at the time. The anteroom contained feed and water facilities for the adjacent chambers, and each chamber was 2.7 x 3.4 meters. The units were routinely cleaned and disinfected and the chambers prepared with fresh, clean shavings litter prior to their use for each experiment. The avirulent P. multocida strain used was the Clemson University vaccine strain described in a previous paper (Bierer and Derieux, 1972, 1973). The basic vaccine was
B
Published with approval of the director as Technical Contribution No. 1081
2290
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ABSTRACT In three separate experiments, an avirulent Pasteurella multocida vaccine was administered to separate groups of turkeys by the following routes: ocular (conjunctival sac), nostril, palatine cleft, oral, drinking water, cloacal, intradermal, feather follicle and wing web. All groups were challenged with a virulent P-1059 P. multocida strain 2 to 5 weeks post vaccination. Under the conditions of these experiments, the results suggest that a favorable immunogenic response of some kind occurred in all groups except those vaccinated by the nostril and cloacal routes. In the latter two groups there was little or no evidence of a favorable response. Turkeys vaccinated by the stick-wing-web method exhibited an excellent immunogenic response, but a large cheesy mass at the site of vaccination would probably result in condemnation of the vaccinated wing areas of some turkeys at the time of slaughter. This method uses little vaccine, is accurate, easy and rapid and, presumably, may have an application in nations where edible food of any kind is a primary objective. In a fourth experiment, the virulent P-1059 challenge strain mentioned above was used as the vaccinating agent and was administered to separate groups of turkeys by the same various routes mentioned in the foregoing. One hundred percent of the turkeys exposed by the feather follicle route were dead within 48 hours and 90% to 100% of the groups exposed by the ocular, palatine cleft, intradermal and stick-wing-web routes were dead by the fourth day. The lowest mortality during a given number of days was in the groups exposed by the nostril and cloacal routes. Under the conditions of the fourth experiment, the results suggest that the eye is a possible route of infection under natural conditions, and that scratches and puncture wounds to the skin and crushed feather follicles, when accompanied by virulent P. multocida containing material, may possibly serve as portals of entry.
PASTEURELLA
MULTOCIDA EXPOSURE
a 24-hours old brain-heart-infusion broth culture which had been incubated at 37 degrees C. Plate counts on dextrose-starch agar, using a method suggested by Heddleston (1972), established that the vaccine contained approximately 1 billion viable bacilli per ml. of vaccine. Treatment applied to the turkeys in chambers 1 through 8 were:
6. Drinking water vaccination was applied as described in a previous paper (Bierer and Derieux, 1972) and consisted of 30 ml. of the basic vaccine (24 hours old brain-heart-infusion broth culture) placed into each 4000 ml. of drinking water, daily for four consecutive days. To encourage prompt consumption, water was withheld for two hours prior to the time the water vaccine was administered. Each ml. of drinking water contained approximately 7,500,000 viable bacilli when freshly prepared. 7. Intradermal vaccination consisted of the injection of vaccine between the layers of the loose skin (wattle) under the lower jaw. A tuberculin syringe fitted with a 25 gauge needle was used for injecting the vaccine. 0.02 ml. of vaccine was injected and this amount contained approximately 20 million viable bacilli. 8. Cloacal vaccination was accomplished with the same type cotton swab used for the swabbed palatine cleft vaccination. The swab containing the vaccine was inserted into the cloaca and firmly rotated for 10 complete rotations. A separate freshly prepared swab was used for each turkey. Fourteen days after applying the treatments described in the foregoing, all turkeys in each of the 8 chambers were exposed to a virulent P-1059 P. multocida strain by the drinking water route. The virulent P-1059 strain was obtained from K. L. Heddleston, Senior Research Microbiologist at the National Animal Disease Laboratory at Ames, Iowa. It was in a vacuum sealed vial in lyophilized form when received. This culture was inoculated into brain-heart-infusion broth, which was then incubated at 37 degrees C. for 24 hours. The resulting culture was lyophilized, the final product being in vacuum sealed glass vials, which were stored at 18 degrees C. When used in connection with an experiment,
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1. Nonvaccinated. 2. Ocular vaccination or 1 drop of the vaccine placed into the conjunctival sac. This drop contained approximately 34 million viable bacilli, and was administered from the nipple portion of a sterile plastic syringe (non leur-lock type) held at a 45 degree angle. Average drop size was 0.034 ml. 3. Nostril vaccination or 1 drop of the vaccine placed in the nostril, and applied in the same manner as the ocular vaccination. 4. Palatine cleft vaccination was accomplished with the use of a sterile cotton swab that had been dipped into the vaccine. The swab was partially inserted through the palatine cleft or choanae area into the nasal cavity and gendy, but firmly, rotated to and fro. Each swab contained approximately 0.25 ml. of vaccine or approximately 250 million viable bacilli. A separate freshly prepared cotton swab was used for each turkey. 5. Oral vaccination was accomplished with the use of a sterile plastic syringe, the needle portion of which was covered with small caliber rubber tubing. The mouth of the subject was held open by the operator with the fingers of the left hand and the delivery end of the syringe placed in the oral cavity of the turkey. One ml. of vaccine was then slowly injected into the oral cavity. The 1 ml. of vaccine contained approximately one billion viable bacilli.
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B. W. BIERER AND W. T. DERIEUX
TABLE 1.—Results of vaccination by various routes with (Experiments ;rim«
3
Number infected 2 weeks post vaccination
Two weeks post exposure
Daily record of deaths: & Treatment* 6 3 4 5 2 1 u Nonvaccinated 0 4 0 0 2 0 1 Nonvaccinated 0 2 2 2 1 0 2 — Ocular 1 0 0 0 1 1 2** 0 Ocular 0 0 1 0 0 0 0 2 Nostril 1 1 0 2 0 0 0 1 Nostril 3 1 0 2 0 0 1 2 Palatine cleft 0 1 0 0 0 0 0 1 Palatine cheft 0 1 0 0 1 0 2 0 Oral 0 0 1 1 0 0 0 0 Oral 1 1 0 0 0 1 2 0 Drinking water 0 0 0 0 0 0 1 0 Drinking water 0 0 0 2 0 0 0 It Intradermal 0 0 1 0 0 0 1 0 Intradermal 0 0 0 0 0 2 0 0 Cloacal 0 1 1 1 0 0 0 1 Cloacal 1 1 0 0 1 2 2 0 Feather follicle 0 0 0 0 0 0 2 2** *Ten turkeys for each separate listing. ** Killed on 14th day post vaccination for examination, prior to the use of the virulent P-1059 strain. tDied on the 14th day post vaccination.
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the lyophilized contents of a vial were sus consisted of 10 turkeys in a portable woodpended in a small amount of brain-heart-infu- floor building with a floor space of 1.2 x sion broth and inoculated on to a blood agar 2.4 meters. This building provided excellent plate. This initial isolate was then incubated shelter and was constructed in such a manner at 37 degrees C. for 24 hours. When used that feed and water could be placed inside in the drinking water, it was prepared and without entering the building. It had been administered in the same manner that the cleaned and disinfected prior to use and drinking water vaccination was administered supplied with a thick layer of fresh, clean to the turkeys in Chamber 6, as described shavings. It was located in the general vicinity in the foregoing, and in a previous paper of the research units already described, but (Bierer and Derieux, 1972). The experiment was in no way in direct communication with was terminated 14 days after exposure to the these units. The turkeys in this building were virulent P. multocida strain. At that time the subjected to a feather follicle vaccination number of turkeys that died in each chamber which consisted of the plucking of four or was added to the number of survivors that five of the large feathers from the upper were morbid to arrive at a total number exterior surface of the thigh and rubbing the infected value for the turkeys in each of the exposed feather follicles with a cotton swab 8 treatment groups. This method of evaluation saturated with vaccine. Feather follicle vacwas first described by Alls et al. (1970) and cination is one of the methods used in fowl has since been used routinely by the authors pox vaccination in chickens and turkeys (Barger et al., 1958). of this present paper. The third experiment utilized the 8 research The second experiment was a repetition of the first experiment with the exception chambers used in the first experiment; 10 of the addition of a ninth treatment that 8-weeks-old turkeys were placed in each of
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lent P-1059 P. multocida strain was used as the vaccine. All turkeys used were from flocks that did not have a disease problem history and were from a single source for each experiment and had not been previously used for any type of research work. All groups of turkeys were observed at least once daily and data relative to mortality and morbidity were recorded. When deaths occurred, representative numbers from a single group were subjected to a bacteriological examination as described in a previous paper (Bierer and Derieux, 1972). Isolates were identified biochemically and subjected to an agar-gel diffusion test at the Clemson Livestock Laboratory, Columbia, South Carolina. Records of feed consumption and of body weights were not kept during the course of the experiments described in the foregoing. Evaluation was based on mortality and morbidity results. RESULTS AND DISCUSSION Under the conditions of the first and second
an avirulent strain of Pasteurella multodica 1,2) to virulent P-1059 strain:
7 1 0 0 0 1 0 1 0 0 0 0 0 0 0 1 0 0
8 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0
9 0 0 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0
10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
14 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Total number died 7 7 2 1 6 7 2 2 1 3 0 0 2 1 4 5 0
Total number morbid 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0
Total number infected 7 7 4 1 6 7 3 3 1 3 0 1 2 1 4 5 2
Average number infected
<%) 70 25 65 30 20 5 15 45 20
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these chambers. The turkeys in Chambers 1 through 4 were nonvaccinated and the turkeys in Chambers 5 through 8 were subjected to a stick-wing-web vaccination with the Clemson University vaccine strain. This was accomplished with the use of the double needle applicator that is supplied for use with commercial fowl pox vaccine. The vaccine was applied at a rather central location through the wing-web area. The two needles contained a maximum of 0.004 ml. of vaccine which contained approximately 4 million viable bacilli. Five weeks post vaccination, all eight groups of turkeys were challenged with the same virulent P-1059 strain and in the same manner that this strain was used in the first experiment. This third experiment was also terminated 14 days from the time the challenge was initiated in the same manner that termination was accomplished in the first and second experiments. The fourth experiment was essentially a repetition of the second experiment, except that a 24-hours-old broth culture of the viru-
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B. W. BIERERANDW. T. DERIEUX
by the feather follicle method of vaccination, the wing-web method of vaccination was investigated by the treatments applied in the third experiment. The results of this experiment were quite impressive since all of the turkeys survived challenge with the virulent strain and all of the survivors were clinically healthy. A summary of the data is given in Table 2. One of 40 turkeys died after wingweb vaccination: on postmortem examination, a solidified lung was observed from which P. multocida bacilli were isolated. The agar-gel-diffusion test established that the isolate possessed both P-1059 and P-1662 antigens which characterized the Clemson University vaccine strain that was administered to these turkeys (Bierer and Derieux, 1973). The wing-web vaccination sites were examined, in each of the 40 vaccinated turkeys, 24 hours post vaccination and a small cheesy swollen area was observed. A small reaction of this type was considered to be most desirable. By the second and third days post vaccination, however, the cheesy swollen areas surrounding the sites of vaccination had increased considerably in size to eventually produce a large, irregularly shaped cheesy mass sometimes approximately 50 mm. in diameter. By the fifth week post vaccination, a number of the enlargements had ruptured and the rather dry cheesy mass had been expelled leaving a gaping break in the skin on the lower side of the wing-web area. During this entire five weeks post vaccination period all of the turkeys, except the one that died, appeared to be clinically normal and not distressed by the wing-web lesion described in the foregoing. The wing-web or stick method of vaccination has been used for some years as the method of choice to vaccinate chickens and turkeys against fowl pox disease. Barger et al. (1958) and Hall (1956) note that the stick method is "fast, accurate, and economical of vaccine." These individuals also called attention to the fact that the two needle
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experiments, the results suggest that some kind of a favorable immunogenic response occurred following the various vaccination routes with the exception of the nostril and cloacal vaccination treatments. In these latter two groups there was little or no evidence of a favorable immunogenic response. In the first experiment, 2 of the turkeys that had received the ocular vaccination became morbid by the ninth day after vaccination. These 2 turkeys were killed on the fourteenth day post vaccination. Lesions of fowl cholera were not detected on postmortem examination and cultures of the internal organs were nonproductive. Nevertheless, the 2 turkeys were included under the total number infected for this group. In the second experiment, one of the turkeys that had received the vaccine in the drinking water died on the fourteenth day after vaccination; a solidified lung was observed on postmortem examination, from which P. multocida bacilli were isolated. The feather follicle method of vaccination resulted in a cheesy skin lesion at the site of vaccination that increased in size to approximately 25 mm. in diameter. Two of the 10 turkeys in this group developed an enlargement of the tibiometatarsal area. The affected legs were not the same legs to which the feather follicle vaccination had been applied. Fourteen days post vaccination, these two turkeys were killed and subjected to a bacteriological examination. Each of the swollen areas contained clear fluid and cultures failed to establish that a bacterial infection was present. Perhaps these two turkeys were injured at the time of vaccination. Whatever the cause may have been, the two turkeys were included in the total number infected for this group as tabulated in Table 1. All eight of the remaining turkeys in this group survived challenge with the virulent P-1059 strain. A summary of the data for experiments 1 and 2 is given in Table 1. In view of these seemingly favorable results
PASTEURELLA MULTOCIDA EXPOSURE
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inoculation used results in four cutaneous inoculations, two on each side of the wingweb. In evaluating vaccination results one must think in terms of possible world-wide application. What may not be useful to the commercial turkey industry of the United States because of the rules and regulations of poultry inspection could be an acceptable procedure by nations where edible food of any kind is a primary objective. The results of the third experiment suggest that the stick method or wing-web method of vaccination may be an effective method that can be easily and rapidly applied at low cost. The nature of the lesion that develops at the site of vaccination suggests that a portion of the wing of some of the turkeys vaccinated by this method would be removed at the time of inspection when slaughtered. This would result in increased slaughter time and a down grading of the trimmed turkeys. In the nations of the world where edible food of any kind is a primary objective, the removal of a portion of the wing at the time of slaughter would make little or no difference. It was the purpose of the fourth experiment to determine whether or not, under the conditions of this trial, a highly virulent strain of P. multocida could be applied at one or more of the locations described with a resulting
favorable immunogenic response. While the outcome was more or less anticipated, the results were considered to be sufficiently interesting to be included in this paper (Table 3). Under the conditions of this experiment all 10 of the turkeys exposed by the feather follicle route were dead within 48 hours. It would appear that crushed feather follicles could occur during the treading activities of larger turkeys. If the feather follicles were crushed in the presence of material containing virulent P. multocida bacilli, then a fatal septicemic fowl cholera infection could possibly occur under these conditions. By the end of the fourth day 90% to 100% of the turkeys died in the groups exposed by ocular, palatine cleft, intradermal and stickwing-web routes. These results suggest that scratches and puncture wounds, in the presence of material containing virulent P. multocida bacilli, are possible avenues to a fatal septicemic fowl cholera infection under natural conditions. The methods of exposure resulting in the least mortality, under the conditions of this experiment and during a given number of days, were the nostril and the cloacal applications. These same two methods were also the least effective in inciting a favorable immunogenic response when the Clemson University vaccine strain was used as the vaccinating agent and as
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TABLE 2.—Results of vaccination with avirulent Pasteurella multocida by the stick-web-wing method (Experiment 3) Two weeks post exposure to virulent P-1059 strain: Number Average infected number Total Total 5 weeks Total infected number number post vaccnumber infected morbid ination died Treatment* (%) 5 1 4 Nonvaccinated 3 0 3 Nonvaccinated 55 7 0 7 Nonvaccinated 7 0 7 Nonvaccinated 0 0 0 Vaccinated 0 j** 1 0 0 Vaccinated 0 3 0 0 Vaccinated 0 0 0 0 Vaccinated 0 *Ten turkeys for each separate listing. **Died on 7th day after vaccination.
Treatment* 1 2 3 4 Ocular 6 5 4 0 Nostril 0 0 0 1 Palatine cleft 0 7 0 2 Oral 0 1 5 1 Drinking water 0 0 3 1 Intradermal 0 5 3 1 Cloacal 0 2 0 0 Feather follicle 1 9 — — Stick-wing-web 0 7 2 i Nonvaccinated 0 0 0 0 *Ten turkeys for each separate listing.
6 0
7 0
8 6
9 0
1 0 1 0 1 0 1 — — 0 0 0 0 0 1 0 1 0 0 i _ _ _ _ _ 1 0 1 0 0 0 — — — — — _ _ _ _ _ _ 0 0 0 0 0 0
1 0 2 2
5 0
Daily record of deaths
Two weeks post vaccination: Total number 10 11 14 died 0 0 0 9 2 1 8 — — — 10 0 0 9 0 0 8 _ _ 10 1 0 5 — — — 10 _ _ 10 0 0 0
TABLE 3.—Results of vaccination by various routes with a virulent strain of Pas (Experiment 4)
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chronic fowl cholera in turkeys that is often characterized by torticollis, have reported that the outstanding lesion was the caseous exudate in the air spaces of the spongy portions of the calvarium. In view of this earlier work by Olson et al. (1966), Donahue and Olson (1971) developed a method of exposing turkeys to fowl cholera by inoculating P. multocida bacilli into the spongy portion of the calvarium via the eustachian orifice and were able to produce uniform and consistent infections. The eustachian orifice, of course, is a pharyngeal structure distinctly separate from the palatine cleft. The nasal opening of the nasolacrimal duct, referred to.in discussing ocular exposure, is not in close association with the eustachian orifice but it is, however, in close proximity with the sphenoid bone, which was one of the portions of the calvarium incriminated by Olson et al. (1966) with reference to gross lesions of chronic fowl cholera. REFERENCES Alls, A. A., G. S. Appleton and J. R. Ipson, 1970. A bird contact method of challenging turkeys with Pasteurella multocida. Avian Diseases, 14: 172-178. Barger, E. H., L. E. Card and B. S. Pomeroy, 1958. Diseases and Parasites of Poultry, Lea and Febiger, Philadelphia, Penna. 19100, Fifth Ed., page 170. Bierer, B. W., and W. T. Derieux, 1972. Immunologic response of turkeys to an avirulent Pasteurella multocida vaccine in the drinking water. Poultry Sci. 51: 408-416. Bierer, B. W., and W. T. Derieux, 1973. Efforts to transmit and to enhance the virulence of an avirulent strain of Pasteurella multocida in turkeys. Poultry Sci. 52: 1510-1516. Donahue, J. M., and L. D. Olson, 1971. Research technique—inoculation of Pasteurella multocida into the palatine air spaces as an exposure method for fowl cholera in turkeys. Avian Diseases, 15: 158162. Dorsey, T. A., 1963. Studies on fowl cholera. II. The correlation between biochemic classification and the serologic and immunologic nature of avian Pasteurella multocida strains. Avian Diseases, 7: 399. Hall, W. J., 1956. Fowlpox. In U.S. Department of Agriculture Yearbook, pages 464-466. Heddleston, K. L., 1962. Studies on pasteurella. V.
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already described in a preceding paragraph. The results of the fourth experiment also suggest that ocular conjunctival sac exposure, with a given number of virulent P. multocida bacilli, would be more apt to induce acute fowl cholera and sudden deaths than exposure with the same number of bacilli by the nostril route. Ocular conjunctival sac exposure is the route of choice to infect cattle with virulent Brucella abortus isolates (Hendricks and Ray, 1970) under experimental conditions. This route was selected because it consistently gave uniform results (Hendricks, 1973). Ocular exposure, as described under material and methods in this present paper, consisted of one drop of the vaccine placed in the conjunctival sac. Presumably, the inoculum is carried by way of the nasolacrimal duct to the nasal palatine cleft area since a local inflammatory reaction was not observed. If this line of reasoning is correct, then ocular conjunctival sac exposure should be an excellent approach to nasal palatine cleft exposure, without the injury or trauma to the mucous membrane that must sometimes occur when a cotton swab is applied directly to the nasal palatine cleft area. Swabbing the palatine cleft with virulent P. multocida bacilli has been used routinely for some years (Heddleston, 1962; Dorsey, 1963; Heddleston et al., 1970; Alls et al., 1970; and Donahue and Olson, 1971). Dorsey (1963) noted that this method more closely simulated natural exposure to the disease. There appear to be variations in the manner in which the palatine cleft area is inoculated when swabbed. Donahue and Olson (1971) note that a cotton swab was "wiped across the roof of the mouth." When the turkeys referred to in this present report were inoculated, the cotton swab was partially inserted through the cleft area or choanae (McLeod et al, 1964) into the nasal cavity area and, presumably, Heddleston and Rebers (1972) use a similar technique. Olson et al. (1966), studying the type of
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adjuvant Brucella abortus vaccines. Proceedings of 74th Annual Meeting, U.S. Animal Health Association 1970, page 152. McDowell, J. R., S. K. Maheswaran, D. S. Bouley and B. S. Pomeroy, 1972. The immunization against fowl cholera using an avirulent Pasteurella multocida mutant. Reported at the 109th Annual A.V.M.A. Meeting, New Orleans, Louisiana, July 18-20. Abstract in Program of 109th Annual A.V.M.A. Meeting, page 174. McLeod, W. M., D. M. Trotter and J. W. Lumb, 1964. Avian Anatomy, Burgess Publishing Co., Minneapolis, Minnesota 55400, pages 103-104. Olson, L. D., E. L. McCune and B. L. Moseley, 1966. Gross and histopathological description of the cranial form of chronic fowl cholera in turkeys. Avian Diseases, 10: 518-529.
NEWS AND NOTES (Continued from page 2289) DIAMOND AUTOMATION NOTES J. Stephens has been appointed Product Manager for Keenco cage layer systems. The line, manufactured by Diamond International Corporation, Diamond Automation Division, Farmington, Michigan, consists of Keenco's contour " A " Line two-tier system, Triple " A " Line three-tier system, and single tier multicage system. He will be responsible also for associated products used with Keenco systems but manufactured by other companies. Stephens has had 23 years of experience in marketing and product management of agricultural products. BIG DUTCHMAN NOTES Gary L. Voss has been appointed Sales Manager, Bernard Brownstein, Western Sales Mannager, and Harry W. Cronise, Southeastern Sales Manager for the Big Dutchman Staalkat egg processing equipment line. Prior to his recent appointment, Voss was Big Dutchman Cage Systems Coordinator. Prior to joining Big Dutchman, Brownstein served in various management positions with Kimber Farms, and most recently was Broiler Production Manager for Zacky Farms in southern California. Cronise has had more than 30 years of experience in the shell egg processing field. A new Ecology Division has been established at Big Dutchman, Zeeland, Michigan. John F. Bergdoll, who has been Big Dutchman New Products Manager,
has been appointed General Manager of the new Division. He has had more than 20 years of experience in poultry, animal health, and feed management. HY-LINE NOTES Dennis Dixon has been appointed Marketing Coordinator for Hy-Line International, Des Moines, Iowa. He will be on the staff of Dr. G. Waters, Director of Marketing, and will help expedite the firm's marketing policies and aid in maintaining communications with Hy-Line distributors throughout the world. He has served Hy-Line in a number of capacities. Most recently he was Sales Director for Latin America, Wallace International, St. Petersburg, Florida. OHIO NOTES J. H. Florea, former Editorial Director of Poultry Tribune, received the "Distinguished Alumni Award" from the Department of Poultry Science, Ohio State University, at a special ceremony on April 10. He graduated from Ohio State in 1928, and retired from the Watt Publishing Company on June 30, 1969, after 40 years of service. WASHINGTON NOTES Dr. Paul C. Harrison, formerly Associate Professor, Department of Animal Sciences, Washington State University, Pullman, is not on the staff of the Depart-
(Continued on page 2305)
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Two immunogenic types of Pasteurella multocida associated with fowl cholera. Avian Diseases, 6: 315-321. Heddleston, K. L, J. E. Gallagher and P. A. Rebers, 1970. Fowl cholera: immune response in turkeys. Avian Diseases, 14: 626-635. Heddleston, K. L., 1972. Personal communication, January 24, 1972. Heddleston, K. L., and P. A. Rebers, 1972. Fowl cholera: cross immunity induced in turkeys with formalin-killed in-vivo-propagated Pasteurella multocida. Avian Diseases, 16: 583. Hendricks, J. B., 1973. Personal communication, January 2, 1973, Clemson Livestock Laboratory, P. O. Box 1771, Columbia, S.C. 29202. Hendricks, J. B., and W. C. Ray, 1970. Studies on cattle vaccinated with live strain 19 and/or 45/20