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Animal Models for the Study of Whooping Cough and the Testing of Vaccine Materials

ANIMAL MODELS FOR THE STUDY OF WHOOPING COUGH

Bordetella pertussis does not naturally cause disease in animals. Nevertheless, experiments in animals have made important contributions to the present, although incomplete, understanding of pertussis. Mice, rats, rabbits, dogs, ferrets, and primates have been used. The respiratory colonization of mice by B. pertussis mimics that of humans, but mice do not cough, and so the infection is not spread from mouse to mouse (Pittman et al., 1980). Among experimental animals, only primates have been found to develop a paroxysmal cough and mucus production; they do transmit the infection from one animal to another (Weiss and Hewlett, 1986). However, adult primates can become resistant to pertussis, so that newborn animals are needed for use in experiments, and this is impractical. Rats are very hard to infect with B. pertussis, and rabbits carry the organism for months without showing signs of disease (Ashworth et al., 1982; Weiss and Hewlett, 1986).

Most of the information about pertussis gained from animal models has come from the study of mice. Three sites of infection have been used: intraperitoneal, respiratory, and intracerebral. Mice can rapidly kill B. pertussis when the organism is injected intraperitoneally. But, given enough bacteria by this route, they will die of apparent toxemia in 1 to 3 days (Pittman, 1970; Proom, 1947). These responses do not represent a model of whooping cough. Unlike the situation in humans, virulence by the intraperitoneal route in mice is inversely related to intracerebral virulence (Pittman, 1970),



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Page 333 C Animal Models for the Study of Whooping Cough and the Testing of Vaccine Materials ANIMAL MODELS FOR THE STUDY OF WHOOPING COUGH Bordetella pertussis does not naturally cause disease in animals. Nevertheless, experiments in animals have made important contributions to the present, although incomplete, understanding of pertussis. Mice, rats, rabbits, dogs, ferrets, and primates have been used. The respiratory colonization of mice by B. pertussis mimics that of humans, but mice do not cough, and so the infection is not spread from mouse to mouse (Pittman et al., 1980). Among experimental animals, only primates have been found to develop a paroxysmal cough and mucus production; they do transmit the infection from one animal to another (Weiss and Hewlett, 1986). However, adult primates can become resistant to pertussis, so that newborn animals are needed for use in experiments, and this is impractical. Rats are very hard to infect with B. pertussis, and rabbits carry the organism for months without showing signs of disease (Ashworth et al., 1982; Weiss and Hewlett, 1986). Most of the information about pertussis gained from animal models has come from the study of mice. Three sites of infection have been used: intraperitoneal, respiratory, and intracerebral. Mice can rapidly kill B. pertussis when the organism is injected intraperitoneally. But, given enough bacteria by this route, they will die of apparent toxemia in 1 to 3 days (Pittman, 1970; Proom, 1947). These responses do not represent a model of whooping cough. Unlike the situation in humans, virulence by the intraperitoneal route in mice is inversely related to intracerebral virulence (Pittman, 1970),

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Page 334 an observation that illustrates the difficulties of using animal models to represent human disease. Older mice are relatively resistant to respiratory infection; infant or suckling mice have reproducible symptoms and mortality from pertussis pneumonia, and the disease resembles the disease in humans (Pittman et al., 1980; Sato and Sato, 1988; Sato et al., 1981). Infection induced by intranasal inoculation (Pittman et al., 1980) has been reported to be less reproducible than that induced by aerosol inhalation (Sato and Sato, 1988). The strain of mice used can affect the results (Pittman et al., 1980).  Survivors of a sublethal dose of organisms can develop a chronic infection that lasts for weeks or months (Dolby et al., 1961; Sato et al., 1981; Weiss et al., 1984). Using intranasal inoculation of infant mice, Weiss and colleagues (1983, 1984) showed that mutant strains of B. pertussis lacking pertussis toxin (PT) or extracytoplasmic adenylate cyclase were much less virulent than the wild-type (naturally occurring) organism. A mutant deficient in filamentous hemagglutinin was nearly as virulent as the wild-type strain. The results obtained with these carefully engineered strains raise a question about the contribution of filamentous hemagglutinin to virulence. Such a contribution had been suggested by data from other models. These and other considerations warrant reservations about the general applicability of the results obtained with this or the other models to the disease in humans. Mice infected intracerebrally have been the most widely used animal model for pertussis. To achieve this model, anesthetized mice are injected with various numbers of organisms, in some cases after immunization with bacteria or bacterial products (usually given intraperitoneally). Only one strain of B. pertussis, strain 18-323, works well in the model, which raises further questions regarding the applicability of this model to the natural disease in humans. In fact, analysis of isoenzyme patterns suggests that this bacterial strain is genetically more closely related to Bordetella bronchiseptica than it is to other strains of B. pertussis (Musser et al., 1986). In mice, the bacteria attach to the ciliated cells of the ependymal lining of the ventricles (Berenbaum et al., 1960), which simulates attachment to the respiratory cilia in humans with whooping cough. However, this infection within the skull otherwise deviates rather markedly from the presentation of the disease in humans. Despite these obvious differences from the infection in humans, protection in this model has correlated with vaccine efficacy in humans (Medical Research Council, 1959; Standfast, 1958). STANDARDIZED ANIMAL TESTS OF VACCINE MATERIALS The intracerebral mouse protection test (Kendrick et al., 1947, 1949) has served importantly in the progress in vaccine development that has been made to date. The test uses a standardized strain of bacteria (strain 18-323)

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Page 335 stored in liquid nitrogen (Cameron, 1988), standardized mice (strain HSFS/ N) (Manclark et al., 1976), a freeze-dried reference vaccine (Armitage and Perry, 1957), and an interval between immunization and injection of 14 to 17 days (Cameron, 1988). The intranasal mouse protection test has been improved by use of a standardized system for delivery of bacteria by aerosol (Sato and Sato, 1988). This test has been used for the study of the role in pathogenesis of bacterial adherence proteins, for example, the 69-kilodalton outer membrane protein (Shahin et al., 1990). The toxicities of vaccines have been studied by the mouse weight gain test. This test depends on the observation that intraperitoneal injection of vaccine into young mice leads to a weight loss within hours, followed by total recovery of weight within the next 7 days (Cameron, 1988).  The causes of toxicity (manifested as poor weight gain) in the test are not well understood; the test is not very sensitive to endotoxin (Cameron, 1977). Results of the test have been shown to vary with the adjuvant or absorbent used with the vaccine, mouse strain, diet, size of cage, ambient temperature, and duration of exposure to light (Cameron, 1988). These vagaries further illustrate the difficulty of generalizing to humans the results obtained from studies in animals. A sensitive assay for the particularly important toxin PT and for anti-PT has been developed by using Chinese hamster ovary (CHO) cells (Gillenius et al., 1985; Hewlett et al., 1983). In the presence of PT, CHO cells undergo a characteristic clumping, which can be blocked with antibody to PT. The test can detect PT at levels one-fiftieth those of the next most sensitive assay (Cameron, 1988). In summary, B. pertussis is a complex organism, multiple factors having been proposed as possible contributors to its virulence. Their role in whooping cough has not been clearly established. Without better understanding of the organism and the human disease, it cannot be concluded with confidence that data from animal models relate to findings in humans.  REFERENCES Armitage P, Perry WLM. 1957. British standard for pertussis vaccine: its use in routine control of commercial vaccines. British Medical Journal 2:501-505. Ashworth LAE, Irons LI, Dowsett AB. 1982. Antigenic relationship between serotype-specific agglutinogen and fimbriae of Bordetella pertussis. Infection and Immunity 37:1278-1281. Berenbaum MC, Ungar J, Stevens WK. 1960. Intracranial infection of mice with Bordetella pertussis. Journal of General Microbiology 22:313-322. Cameron J. 1977. Pertussis vaccine: mouse-weight-gain (toxicity) test. Developments in Biological Standardization 34:213-215. Cameron J. 1988. Evaluation of control testing of pertussis vaccines. In: Wardlaw AC, Parton R, eds. Pathogenesis and Immunity in Pertussis. New York: John Wiley & Sons.

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Page 336 Dolby JM, Thow DCW, and Standfast AFB. 1961. The intranasal infection of mice with Bordetella pertussis. Journal of Hygiene 59:191-216. Gillenius P, Jaatmaa E, Askelof P, Granstrom M, Tiru M. 1985. The standardization of an assay for pertussis toxin and antitoxin in microplate culture of Chinese hamster ovary cells. Journal of Biological Standardization 13:61-66. Hewlett EL, Sauer KT, Myers GA, Cowell JL, Guerrant RL. 1983. Induction of a novel morphological response in Chinese hamster ovary cells by pertussis toxin. Infection and Immunity 40:1198-1203. Kendrick PL, Eldering G, Dixon MK, Misner J. 1947. Mouse protection tests in the study of pertussis vaccine. American Journal of Public Health 37:803-810. Kendrick PL, Updyke EL, Eldering G. 1949. Comparison of pertussis cultures by mouse protection and virulence tests. American Journal of Public Health 39:179-184. Manclark CR, Hansen CT, Treadwell PE, Pittman M. 1976. Selective breeding to establish a standard mouse for pertussis vaccine bioassay. 2. Bioresponses of mice susceptible and resistant to sensitisation by pertussis vaccine HSF. Journal of Biological Standardardization 3:353-363. Medical Research Council. 1959. Vaccination against whooping cough: the final report. British Medical Journal 1:994-1000. Musser JM, Hewlett EL, Peppler MS, Selander RK. 1986. Genetic diversity and relationships in populations of Bordetella spp. Journal of Bacteriology 166:230-237. Pittman M. 1970. Bordetella pertussis—bacterial and host factors in the pathogenesis and prevention of whooping cough. In: Mudd S, ed. Infectious Agents and Host Reactions. Philadelphia: W.B. Saunders Co. Pittman M, Furman BL, Wardlaw AC. 1980. Bordetella pertussis respiratory tract infection in the mouse: pathophysiological response. Journal of Infectious Diseases 142:56-66. Proom H. 1947. The immunological aspects of experimental Haemophilus pertussis infection. Journal of Pathology and Bacteriology 59:165-180. Sato Y, Sato H. 1988. Animal models of pertussis. In: Wardlaw AC, Parton R, eds. Pathogenesis and Immunity in Pertussis. New York: John Wiley & Sons. Sato Y, Izumiya K, Sato H, Cowell JL, Manclark CR. 1981. Role of antibody to leukocytosis-promoting factor hemagglutinin and to filamentous hemagglutinin in immunity to pertussis. Infection and Immunity 31:1223-1231. Shahin RD, Brennan MJ, Li ZM, Meade BD, Manclark CR. 1990. Characterization of the protective capacity and immunogenicity of the 69-kD outer membrane protein of Bordetella pertussis. Journal of Experimental Medicine 171:63-73. Standfast AFB. 1958. The comparison between field trials and mouse protection tests against intranasal and intracerebral challenges with Bordetella pertussis. Immunology 2:135-143. Weiss AA, Hewlett EL. 1986. Virulence factors of Bordetella pertussis. Annual Review of Microbiology 40:661-686. Weiss AA, Hewlett EL, Myers GA, Falkow S. 1983. Tn5-induced mutations affecting virulence factors of Bordetella pertussis. Infection and Immunity 42:33-41. Weiss AA, Hewlett EL, Myers GA, Falkow S. 1984. Pertussis toxin and extracytoplasmic adenylate cyclase as virulence factors in Bordetella pertussis. Journal of Infectious Diseases 150:219-222.