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

Bull. Org. mond. Sante} 1969, 40, 241-244Bull. Wid Hith Org. The Chick Embryo Neutralization Test in the Assay of Meningococcal Antibody* 2. Response of the Embryo to Meningococcal Endotoxin and to Infection K. UEDA,1 B. B. DIENA,2 H. SATO 3 & L. GREENBERG 4 A previous report described the infection of the chick embryo with Neisseria meningi- tidis. Although death of embryos was preceded by multiplication of the organisms, the possibility exists that death is caused by the endotoxin contained in the inoculum or released by the bacteria lysing in vivo. A second important condition for the establishment of a satisfactory sero-protection test is that the embryo should act as a host and not merely as a growth medium for N. meningitidis. Results of tests with cortisone-treated embryos show that the pathogenic effect of live meningococcal challenge is mainly due to infection elicited by the meningococci and not to toxaemia. Also, the rate of clearance of meningococci from the blood of the embryo, increased by the addition of meningococcal immune serum, suggests that at this stage of embryonic development (12 days) the phagocytes are active in combating meningococcal infection. In a previous report (Ueda, Diena & Greenberg, 1969), a method for the challenge of chick embryos with Neisseria meningitidis was described. Although we have found that death of the embryo is preceded by multiplication of the organisms, the possibility exists that death is actually caused by endotoxin contained in the inoculum or released from bacteria lysing in vivo. A second important condition for the establishment of a satisfactory sero-protection tech- nique is that the chick embryo should act as a host and not merely as a growth medium for N. meningi- tidis. If the presence of a phagocytic function of the embryo can be confirmed, it would indicate that the chick embryo is actively reacting against the chal- lenge. This paper deals with these 2 points, namely, the differentiation between the endotoxic activity of meningococci and their infectivity, and the clearance of the organisms from the bloodstream. * From the Biologics Control Laboratories, Laboratory of Hygiene, Department of National Health and Welfare, Ottawa, Canada. Post-doctorate Fellow. 'Head of Research Section. 3Bacteriologist. 4 Chief, Biologics Control Laboratories. MATERIALS AND METHODS Chick embryo challenge Chick embryos, the strain of Neisseria used in these experiments, the diluent and the intravenous method of challenge are described in Ueda, Diena & Greenberg (1969). Heat-killed meningococci A 24-hour growth of meningococci on blood-agar plates was harvested with saline, and a suspension was prepared with 20 mg wet weight per ml. The bacteria were heated at 1000C for 1 hour. Cortisone treatment Hydrocortisone sodium succinate (Solu-Cortef, Upjohn) at a concentration of 4 mg/ml was used. Equal volumes of bacterial suspensions-live or killed-and cortisone were mixed, and 0.1 ml of each mixture was injected intravenously into the embryos. Controls received either cortisone alone or bacterial suspension. Eggs were candled daily for 3 days to test for viability. 2294 -241- 6 K. UEDA AND OTHERS Immune serum White rabbits weighing about 2.5 kg were immu- nized with live meningococci, strain 1027, grown in NCDM (Kenny et al., 1967) for 6 hours (viable counts averaged 2.7 x 108/ml). The immunization schedule consisted of 9 injections (0.5, 0.75, 1.0 ml, weekly for 3 weeks). The 0.5-ml dose was given intraperitoneally, and the other doses intravenously. The rabbits were bled 28 days after the first injection, the serum was inactivated at 560C for 30 minutes and kept in a frozen state. Viable count of infected embryo blood In the first clearance experiment, embryos inocu- lated intravenously with meningococci were bled from the chorioallantoic vein at appropriate inter- vals and 0.2 ml of blood was taken for viable counts. In the second clearance experiment (opson- ization studies), equal volumes ofnormal or meningo- coccus-immune rabbit sera (diluted 1: 50) and bac- terial suspensions at the desired concentrations were mixed and injected and the embryos were bled from the chorioallantoic vein at 10 minutes, and 1, 2 and 4 hours and counts were made on blood-agar plates. For the in vitro incubation test, the injection of serum-bacteria mixtures was made as for opsonization studies. However, in this latter test, embryos were bled immediately after injection, 0.4 ml of blood was EFFECT OF CORTISONE ON TABLE 1 EFFECT OF CORTISONE a ON THE TOXICITY OF HEAT-KILLED N.MENWNGITIDIS FOR THE CHICK EMBRYO Dose of killed With Without organisms b cortisone c cortisone c 1 2/8 919 0.3 3/8 6/9 0.1 1/9 3/10 0.03 1/8 2/9 Contro 3/10 a 0.2 mg cortisone per embryo. b mg of wet weight per embryo. c Results (expressed as dead/tested) were read 3 days after injection. withdrawn and 0.1 ml of blood was used for the viable count; the remainder (0.3 ml) was incubated at 37°C on a shaker for 3 hours. After incubation, viable counts were made. RESULTS Toxicity of meningococci for the chick embryo It is known that cortisone decreases the resistance of the host to various bacterial infections while, on TABLE 2 THE INFECTIVITY OF N.MENINGITIDIS IN THE EMBRYO Dose (viable counts of Dead/ With cortisone b Dead/ Without cortisone borganisms/ Itested a tested a embryo) 1.2 x 108 10/10 (1,1,1,1,2,2,2,2,2,3) 8/8 (1,1,1,1,1,1,1,1) 1.2 x 107 10/10 (1,1,1,1,2,2,2,2,3,3) 8/8 (1,1,1,1,1,1,1,1) 1.2 x 106 9/10 (1,1,1,2,2,2,2,2,3,S) 6/8 (1,1,1,2,2,3,S,S) 1.2 x 105 7/10 (1,1,2,2,2,2,3,S,S,S) 6/8 (1,1,1,1,,2,S,S) 1.2 x 104 8/10 (2,2,2,2,2,2,3,3,S,S) 4/8 (1,1,1,1,S,S,S,S) 1.2 x 103 6/10 (2,2,2,2,2,2,S,S,S,S) 2/8 (1,1,S,S,S,S,S,S) 1.2 x 102 4/10 (2,2,3,3,S,S,S,S,S,S) 1/8 (1,S,S,S,S,S,S,S) 1.2 x 101 2/10 (1,3,S,S,S,S,S,S,S,S) 1/8 (2,S,S,S,S,S,S,S) 1.2 x 10° 1/10 (2,S,S,S,S,S,S,S,S,S) 2/8 (1,2,S,S,S,S,S,S) Cortisone only | 1/8 (3,S,S,S,S,S,S.S) Within 3 days after challenge. b Numerals in parentheses indicate the times of death (days) after injection; " S" indicates survival at 3rd day of infection. 242 CHICK EMBRYO NEUTRALIZATION TEST FOR ASSAY OF MENINGOCOCCAL ANTIBODY. 2 the other hand, it provides protection from the lethal activity of endotoxin (Smith & Thomas, 1956). This dual role of cortisone was utilized to study the challenge of the chick embryo with killed and living meningococci. Table 1 shows that cortisone protected against the lethal activity of killed meningococci in spite of the toxicity of cortisone itself for the chick embryos. The data in Table 2 show that cortisone did not protect against challenge by living meningo- cocci but merely delayed the deaths of embryos inoculated with relatively large numbers of living organisms (challenge dose: 107_107) as compared with the control groups. These results indicate that in our meningococcal challenge test, the infection brought about by multiplication of the organisms is the main cause of death, and that the endotoxic effect of the living bacterial inoculum may be discounted, pro- vided a suitable injection dose is used. Clearance of organisms from the bloodstream If phagocytes located along the blood vessels of the embryo are functional at 10-12 days of age, it would be expected that direct inoculation of organ- isms into the bloodstream would be cleared by these cells, provided the inoculum was not so large as to overcome the phagocytic activity of these cells, and that the clearance rate would be enhanced by the addition of immune serum as an opsonin source. The results are shown in the accompanying figure; a slight decrease in viable N. meningitidis was found in the blood counts taken at varying intervals after challenge, and the clearance rate became much more pronounced when immune serum was used to opso- nize N. meningitidis. To determine whether this decrease in the counts was due to the phagocytic or to the bactericidal activity of the embryo blood, the bactericidal activity of the blood was tested. Blood samples were withdrawn shortly after challenge with N. meningitidis mixed with either normal or immune rabbit serum and incubated for 3 hours at 37°C. During this incubation period, the number of organ- isms increased (Table 3) indicating that embryo blood in itself had no bactericidal activity and that the increased clearance of bacteria from the blood of the embryo was indeed due to phagocytic activity. DISCUSSION The endotoxin from Gram-negative bacteria, in- cluding meningococci, are known to be toxic in the chick embryo. This toxicity, described by Smith & THE EFFECT OF NORMAL OR IMMUNE SERUM ON THE CLEARANCE OF N. MENINGITIDIS FROM THE CHICK EMBRYO BLOODSTREAM 6 Inocu lu m size: 9.7 x 105 /egg E 5 0. a) 4 D) 0 __. 3 cn -6. c o 2 a) Z i . 1/6 42 HOURS AFTER CHALLENGE TABLE 3 INCUBATION OF BLOOD OF EMBRYOS INFECTED WITH N. MENINGITIDIS IN VITRO a Group______ E Viable counts (in 0.1 ml of blood)Group Eg Before incubation 3 h after incubation 1i 6.6 x10 2.2 x 103 With normal 2 7.4 x102 5.2 x 10' serum 3 9.3x102. 5.2 x10' 4 7.8x102 1.9x103 5 2.7 x102 8.7 x 102 With antiserum 6 6.1 x 102 1.5 x 103 7 7.0 x 102 5.Ox 10' a Inoculum - 0.5x 104 organisms per embryo. Thomas (1956), Finkelstein (1964) and Hook, Lut- trell & Wagner (1961), among others, causes lesions similar to those observed when living meningococci are injected (Ueda, Diena & Greenberg, 1969; Bud- 243 244 K. UEDA AND OTHERS dingh & Polk, 1939) either intravenously or into the chorioallantoic membrane. Endotoxin is known to produce cerebral lesions (Hook, Luttrell & Wagner, 1961) similar to those found after challenge with living meningococci in the 12-day-old embryos. However, this toxic effect decreases with the age of the embryo. In the establishment of a sero-protection test for the assay of meningococcal antibody, the death of the chick embryo should be the result of a meningo- coccal infection, similar to that found in humans, rather than to a toxaemia, even though it may be difficult to separate the effect of the toxin from that of the infection itself. Results of our experiments indicate that cortisone, which enhances bacterial infections but protects against toxins (Smith & Thomas, 1956), had nb protective activity and only delayed the onset of death in the chick embryos which were inoculated intravenously with- high chal- lenge doses of meningococci. Therefore, death of the embryos is mainly due to infection rather than toxaemia. It is well established that the reticulo-endothelial system of the chick embryo is functional at an early stage of embryonic development, and that the rate of clearance of organisms from the blood varies with the bacterial species involved (Karthigasu & Jenkin, 1963). Our results with the 12-day-old embryos show a slight decrease in the number of viable meningococci found in the blood at varying intervals after challenge. Although it is possible that this clearance effect may be due to a mechanical trap- ping, unrelated to phagocytosis, our data, showing an enhanced clearance by the addition of immune serum, suggest that phagocytes of 12-day-old em- bryos are functional against meningococci. RtSUME EPREUVE DE NEUTRALISATION SUR EMBRYON DE POULET POUR LA MESURE DES ANTICORPS MENINGOCOCCIQUES: 2. REACTION DE L'EMBRYON A L'ENDOTOXINE MENINGOCOCCIQUE ET A L'INFECTION Apr6s avoir decrit prec6demment une technique d'in- fection de 1'embryon de poulet par Neisseria meningitidis, les auteurs etudient le mecanisme de I'action l6tale du germe et les moyens de defense mis en jeu par 1'embryon. Lorsqu'on injecte par voie intraveineuse a des em- bryons de poulet un m6lange d'une suspension de meningocoques vivants ou tues et de cortisone, on voit que la cortisone protege 1'embryon contre I'action des menin- gocoques tues; par contre, si l'inoculum Contient une dose relativement elev6e de germes vivants, 1' cortisone a simplement pour effet de retarder la mort-de 1'embryon. L'action l6tale semrble donc resulter de la prdlif6ration de N. meningitidis eti 1'effet de l'endotoxine ces bact6ries vivantes peut etre neglige si l'on utilise un inoculum renfermant un nombre suffisant de germes. Le rythme de disparition de N. meningitidis vivant du sang de 1'embryon est accelere si l'on ajoute a l'inoculum de l'immunserum antimeningococcique. On peut en conclure que chez l'embryon de 12 jours le systeme r6ticulo-endoth6lial est actif et que l'elimination de l'agent infectieux est la consequence d'une activite phagocytaire. Ces resultats montrent que 1'embryon de poulet r6agit activement A la presence de N. meningitidis et il semble possible d'utiliser ce modele d'infection experimen- tale en vue de la mise au point d'un test de sero- protection. REFERENCES Buddingh, G. J. & Polk, A. D. (1939) J. exp. Med., 70, 485-497 Finkelstein, R. A. (1964) Proc. Soc. exp. Biol. (N.Y.), 115, 702-707 Hook, E. W., Luttrell, C. N. & Wagner, R. R. (1961) Bull. Johns Hopk. Hosp., 108, 21-35 Karthigasu, K. & Jenkin, C. R. (1963) Immunology, 6, 255-263 Kenny, C. P., Ashton, F. E., Diena, B. B. & Greenberg, L. (1967) Bull. Wld Hlth Org., 37, 569-573 Smith, R. T. & Thomas, L. (1956) J. exp. Med., 104, 217-231 Ueda, K., Diena, B. B. & Greenberg, L. (1969) Bull. Wld Hlth Org., 40, 235-240