The chick embryo neutralization test in the assay of meningococcal antibody*

Bull. Org. mond Sante 11969, 40, 235-240 The Chick Embryo Neutralization Test in the Assay of Meningococcal Antibody* 1. Infection of the Embryo with Neisseria meningitidis K. UEDA,1 B. B. DIENA 2 & L. GREENBERG 3 Present methods of controlling meningococcal cerebrospinal meningitis have failed to contain the disease. This has led to the search for effective vaccines and to the development ofmethods for assaying the potency of these vaccines, as well as for measuring the immune response of the individual. The feasibility of using the sero-protection test in embryonated eggs for the assay of meningococcal antibody has been investigated. The paper describes detailed investigations of the infectious process established by injecting Neisseria meningi- tidis by various challenge routes. Embryos, even 10-12 days old, showed high susceptibility to meningococci injected via the yolksac or chorioallantoic vein, and lesser susceptibility when injected via the intra-allantoic and chorioallantoic routes. The deaths ofintravenously inoculated embryos coincided with the multiplication of the organisms and ensuing septi- caemia. A study of the infection in embryos which had died, or were killed, 18-24 hours after inoculation showed a specific localization of the organisms in the brain. Epidemics of meningococcal cerebrospinal menin- gitis are still of considerable importance in some parts of the world (Lapeyssonnie, 1963); elsewhere, sporadic cases occur in special-risk groups, despite the general effectiveness of chemotherapy (Feldman, 1966; Vedros, Hunter & Rust, 1966). Present methods of control have to date failed to contain the disease and this has led to the search for an effective vaccine and to the development of methods to assay the potency of vaccines and to measure the immune response of the individual. The adoption of a sero- protection test in embryonated eggs for the assay of meningococcal antibody was one of the recommen- dations made at a meeting convened by the World Health Organization in 1965 to discuss research on Neisseria. The feasibility of using this technique has been investigated in our laboratory and this report outlines the first part of the study and deals with the infection of the chick embryo with N. meningitidis. * From the Biologics Control Laboratories, Laboratory of Hygiene, Department of National Health and Welfare, Ottawa, Canada. I Post-doctorate Fellow. 2 Head of Reseach Section. 3Chief, Biologics Control Laboratories. MATERIALS AND METHODS Chick embryo Fertile eggs were incubated in a Brower 4 egg incubator at a temperature of 38.5°C-39.5°C, with adequate humidity and air circulation. Strain of Neisseria N. meningitidis strain 1027 (group A) was used (see Greenberg & Cooper, 1965). The strain was maintained by daily transfer on nutrient blood agar plates (5% sheep red blood cells), incubated at 36°C-38°C in a carbon dioxide incubator.5 Diluent for bacterial suspensions Sorensen's phosphate-buffered saline, pH 7.2, containing I % medium 199 and 0.1 % heat-inactivat- ed absorbed guinea-pig serum, was the diluent. The serum component was prepared by pooling normal guinea-pig sera and absorbing the pool overnight in the cold with heat-killed (at 65°C for 1 hour) N. meningitidis (100 mg wet weight meningococci to ' Manufactured by the Brower Manufacturing Co., Quincy, Illinois, USA. I Manufactured by Hotpack, Waterloo, Ontario, Canada. 2293 - 235- K. UEDA, B. B. DIENA & L. GREENBERG I ml of serum). The absorbed pooled serum was then inactivated at 56°C for 30 minutes and kept frozen. Inoculation and observation of embryos Intra-yolksac (IYS), intra-allantoic (IAC), chorio- allantoic membrane (CAM) and intravenous (IV) inoculations were made according to the meth- ods of Goodpasture & Buddingh (1948) with the following modifications: IAC inoculation was made via the air-sac through a slit in the shell, and CAM inoculation was performed by making an artificial air-sac through a slit on the side of the shell. All shell openings were sealed with cellulose tape. The eggs were candled daily for 1 week and ob- served for viability. Viable counts of organs and embryonic fluids Allantoic fluid, blood, amniotic fluid and yolk were withdrawn at appropriate intervals (see below) by syringe, and 10-fold dilutions were made with the diluent and cultured on blood-agar medium. Pieces of organs (left lobe of the liver, a longitudinal half of the brain, including the mid-brain and cerebellum, one side of the mesonephros and of the lung, small pieces of the chorioallantoic membrane and yolksac) were weighed and homogenized in glass tissue-grind- ers (100 mg of tissue per 1 ml of diluent). Appro- priate dilutions of these homogenates were plated for counts on the same media. RESULTS Age susceptibility of the chick embryo to N. meningi- tidis following inoculation by different routes Results of several experiments are listed together in Table 1. Chick embryos 5 and 8 days old were very susceptible by all the routes tested. Ten-day-old embryos showed high susceptibility when inoculated by either the IYS or IV routes but lower susceptibi- lity to IAC and none at all to CAM injections. Twelve-day-old embryos were susceptible only to IV inoculations in the doses used. In order to study the optimal conditions for the establishment of meningococcal infectiop, the fol- lowing inoculation routes were studied in greater detail because they provided the best relationship between the infecting dose of meningococci and the death of the embryos: IYS route in 10-day-old embryos, and the IV route in the 12-day-old em- bryos. TABLE 1 AGE SUSCEPTIBILITY OF EMBRYOS TO N. MENINGITIDIS INFECTION Age No. of micro- Route of injection a of embryo organisms (days) in dose IYS IAC CAM IV , |~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 5 8 10 12 14 1.4x 103 102 10' 100 (diluent only) 1.2x 10' 106 10' 104 10' 102 10' 100 2.5x 107 106 105 104 10' 102 101 100 1.3x 107 100 10 104 10' 102 1.6x 10 107 10' 10' 5/5 5/5 5/5 1/5 0/5 6/6 6/6 4/6 6/6 1/6 5/5 5/6 4/6 4/6 3/6 1/6 1/6 5/6 2/6 3/6 2/6 4/6 3/6 4/6 2/6 0/6 4/6 4/6 2/6 2/6 1/6 1/6 2/6 0/6 2/6 0/10 0j8 0/10 0/10 0/10 6/6 5/6 5/6 4/6 5/6 4/6 2/6 0110 0/8 0/10 0/10 1/10 0/10 0/8 0/10 0/10 1/10 8/8 7/8 7/8 4/8 4/8 2/6 8/10 0. 6/10 8/10 0 4 7/10 0/6 2/10 5/10 b.e4 2/10 2/104 2/10 0/6 a Results expressed as dead/tested. 236 ,~~ ~ ,~ CHICK EMBRYO NEUTRALIZATION TEST FOR ASSAY OF MENINGOCOCCAL ANTIBODY. I TABLE 2 ISOLATION OF N. MENINGITIDIS FROM ORGANS OF CHICK EMBRYOS AT VARIOUS TIMES AFTER IYS INOCULATION a Egg No. 2 3 4 5 6 7 8 9 10 11 12 Killed (K) or died (D) K K K D D D K K KK\ Recovery of organisms b from: Blood c Not done Not done Not done K K K Liver c Brain c ±J ++ +++ I~ ++ +++ +++ ++ ++ Yolk c +++ a Dose: 1.8x 104 organisms per egg. Age of embryo: 10 days. b Keyto symbols: - = no growth; 4- = afew colonies; + = lessthan 50 colonies; ++ = isolated colonies, more than 50; +++ = confluent colonies. c Blood (0.1 ml of the blood sample from the chorioallantoic vein) was inoculated on a plate. Liver and brain: a cut surface was stamped on a plate. Yolk: a loopful was streaked on a plate. Intra-yolksac infection Ten-day-old embryos inoculated via the IYS gen- erally died in 1-5 days. An attempt was made to recover N. meningitidis from the blood, liver, brain and yolksac of these embryos in order to determine whether the tissues had been invaded and whether multiplication of the organisms had occurred. The results shown in Table 2 reveal that numerous meningococci can be recovered from the organs of dead or killed embryos 24-28 hours after the injection of 1.8 x 104 organisms. Infection by the intravenous route With an inoculum of 130 000-200 000 N. meningiti- dis, 12-day-old embryos began to die 6-8 hours after being inoculated. Mortality was 60% after 12 hours and 90% within 24 hours (Table 3). Here again, to determine whether multiplication of the organisms occurred before death, viable counts were made from the blood, liver, brain and allantoic fluid. Fig. I shows that multiplication occurred 2 hours after injection of the organisms, with peak incidence of mortality and bacterial multiplication by the TABLE 3 TIME OF DEATH OF INFECTED EMBRYOS AFTER CHALLENGE No. Hours after inoculation:N. meningitidis of(inoculum size) eggs o 3 5 7 9 11 13 15 17121 25 (1.3x105/egg) 40 3 1 3 11 7 12 3 (2.0x10'/egg) 48 _22 6 4 7 6 2 1 Time after inoculation 1 hour 24 hours 48 hours Experi- ment No. II 237 K. UEDA, B. B. DIENA & L. GREENBERG FIG. 1 GROWTH OF N. MENINGITIDIS IN CHICK EMBRYO ORGANS, AND TIME OF DEATH AFTER CHALLENGE 0 route of inoculation: IV inoculum size :1.3 x 105/egg ,n fl 2 4 6 HOURS 0 2 7 HOURS AFTER INJECTION 7th hour. With one exception, the blood more bacteria than any other fluid or org Distribution of N. meningitidis in organs nously inoculated embryos To assess more precisely the outcome c tions, embryos inoculated with 2.0 x 10 and surviving until the 12th hour, were and viable counts were made (Fig. 2). degree of congestion was noted in the si and subcranial meninges; the counts ind the blood, liver and mesonephros conta organisms than other fluids or organs. which survived more than 24 hours o showed conspicuous haemorrhages in the meninges and mid-brain ventricles; the li spleen were enlarged. Some embryos killed and autopsied after surviving for 18 hours contained more organisms in the brain than in the liver and mesonephros, which is the reverse of the finding at the 12th-hour stage of infection (Fig. 3). DISCUSSION Infection of embryos with N. meningitidis by vari- ous inoculation routes was investigated to establish the method best suited to the development of an assay for meningococcus vaccines. Embryos 5-8 dayslo 12 old were killed by small challenge doses, regardless of the route of administration. However, these youn- ger embryos may have acted simply as a nutrient medium, which would therefore considerably limit the usefulness of this type of test. Increasing the age of the embryo led to a more specific infection. Ten-day-old embryos are very susceptible to meningococci injected into the yolksac, as has been shown by Schoenback (1948), and the infection so produced has some characteristics in common with the disease in man. Multiplication of bacteria in the embryo was suggested by our ex- periments because of the presence of numerous organisms in the liver and brain, in spite of the low level of bacteraemia. However, it is probable that the organisms may multiply in the yolk before they reach the tissue of the developing egg. If such is the case, the use of this infection route for an effective passive protection test would be ruled out. The IV infection route was next studied, and meningococcal infection was successfully estab- lished. Buddingh & Polk (1939) found that IV injection of N. meningitidis kills almost all the em- bryos within 24 hours, but they were unable to demonstrate the presence of organisms in the blood contained in the 18-24-hour period following such injection. On an tested. the basis of these results, they suggested that the meningococci do not survive for any length of time of intrave- in vivo and that death is presumably caused by toxic products released by disintegrating organisms. Al- f IV infec- though in our experiments the embryos also died 5 bacteria, within 24 hours, multiplication of the organisms autopsied occurred before death. This is a point of consider- A slight able interest for the establishment of a sero-protec- pleen, liver tive test in the chick embryo because it shows that an icated that actual infection had in fact been established, even mined more though the immediate cause of death might be Embryos attributed to toxic substances released by the menin- ccasionally gococci. subcranial Blood from embryos inoculated intravenously and ver and the killed within 12 hours contained more organisms 1- E -c 0 "oC' 20 o- o0 0 C) 10 0C a01aU0 a) 0. 01 E 0 0 a) ._ 0 0 a, 0 * x 238 8 FIG. 2 OF N. MENINGITIDIS IN THE CHICK EMBRYO 12 HOURS AFTER CHALLENGE route of inoculotion: IV inoculum size : 2.0 X 105/egg I ABCDEFGHI J A. blood F. B. liver G. C. brain H. D. kidney 1. E. lung J. ABCDEFGH I J C A M yolk sac allontoic fluid amniotic fluid yolk FIG. 3 OF N. MENINGITIDIS IN THE CHICK EMBRYO 18 HOURS AFTER CHALLENGE route of inoculation : IV inoculum size: I.0 X 105/egg ABCDEFdH1 J F. G. H. I. J. C A M yolk sac allantoic fluid omniotic fluid yolk DISTRIBUTION 01- to C, E 0 0 b. S C. 60 8- C *_ 0 5 8 7_ 6 4 3 ABCDEFGHI J DISTRIBUTION 7h 0 Uw 0' E 0 0 I- by 0 .o D0 C.) C-) 0 61- 51- 4 3L A. blood B. liver7 C. brain D. kidney E. lung S _ 8r 240 K. UEDA. B. B. DIENA & L. GREENBERG than any other organs or fluids. This infection is similar to the " septicaemia " described by Buddingh & Polk (1938) in chick embryos infected by the CAM route with a larger challenge than that used in our experiments. The distribution of the organisms revealed that the liver and the mesophrenos harbour more organisms than either the brain or lung. This may be explained by preferential trapping of phago- cytized N. meningitidis by liver and mesonephros. When embryos were autopsied 18 hours after infection by the IV route, meningococci were found to be localized in specific areas, namely the cranial sinuses, lung, brain and meninges, and haemor- rhages were seen in the meninges and mid-brain ventricles. The selective localization of the organ- isms in the brain at the later stage of infection is interesting in relation to the pathogenesis of menin- gococcal cerebrospinal meningitis. In conclusion, the intravenous inoculation of 12- day-old embryos is considered to be the best method to establish N. meningitidis infection for a sero- protection test. ACKNOWLEDGEMENTS Thanks are due to Dr E. T. Bynoe, Director, Laboratory of Hygiene, Department of National Health and Welfare, Ottawa, Canada, and to Mr J. L. Byrne, Assistant Chief, Biologics Control Laboratories, Labora- tory of Hygiene, Department of National Health and Welfare, Ottawa, Canada, for their suggestions in the preparation of this report. RESUME tPREUVE DE NEUTRALISATION SUR EMBRYON DE POULET POUR LA MESURE DES ANTICORPS MENINGOCOCCIQUES: 1. INFECTION DE L'EMBRYON PAR NEISSERIA MENINGITIDIS En deux articles, les auteurs exposent leurs recherches en vue de definir un test de seroprotection permettant d'evaluer l'activite des vaccins antimeningococciques et la reponse immunitaire postvaccinale. Ils decrivent ici la technique utilisee pour realiser l'infection par N. meningi- tidis chez l'embryon de poulet. Les embryons ages de 10 a 12 jours sont tres receptifs a l'infection par le meningocoque, que l'inoculation ait lieu dans le sac vitellin ou par voie intraveineuse. Dans le premier cas, la mort survient en general apres 1 a 5 jours; 24 a 48 heures apres l'inoculation, de nombreux meningo- coques peuvent etre isoles des organes de l'embryon. Si N. meningitidis est injecte par voie intraveineuse, le taux de mortalite des embryons est de 60% apres 12 heures et atteint 90% apres 24 heures. La mort survient apres une phase precoce de multiplication des micro-organismes. L'examen des embryons sacrifies a la 21 e heure decele des meningocoques dans les divers organes et liquides orga- niques, avec une concentration maximale dans le sang. Si l'examen necropsique est effectue 18 heures apres l'inoculation, on constate des lesions cerebrales selectives avec presence de meningocoques. L'inoculation de N. meningitidis par voie intraveineuse a des embryons de poulet ages de 10 a 12 jours semble s'affir- mer comme la meilleure m6thode pour obtenir une infec- tion se pretant a 1'execution d'un test de seroprotection. REFERENCES Buddingh, G. J. & Polk, A. D. (1939) J. exp. Med., 70, 485-497 Feldman, H. A. (1966) Ann. intern. Med., 65, 1148 Goodpasture, E. W. & Buddingh, G. J. (1948) Chick- embryo technics. In: Rivers, T. M., ed., Viral and rickettsial infection of man, Philadelphia, Lippincott, pp. 97-113 Greenberg, L. & Cooper, M. Y. (1965) Bull. Wid Hlth Org., 33, 21-26 Lapeyssonnie, L. (1963) Bull. Wid Hlth Org., 28, Suppl. Schoenback, E. B. (1948) The meningococci. In: Dubos, R. J., ed., Bacterial and mycotic infection of man, Philadelphia, Lippincott, p. 508 Vedros, N. A., Hunter, D. H. & Rust, J. H. (1966) Milit. Med., 131, 1413-1417