Evaluation of the Chick as a Test Animal in the Assayof Diphtheria Toxoids

SARA E. BRANHAM, MARION W. GRABOWSKI, AND DONALD B. RIGGS

Division of Biologics Standards, National Institutes of Health, Public Health Service, U. S. Department of Health,Education, and Welfare, Bethesda, Maryland

Received for publication February 14, 1957

In a previous report (Branham and Wormald, 1954),the authors have described the titration of therapeuticdiphtheria antitoxins in 7- to 8-day old chicks. Theseantitoxins had a titer of several thousand units per ml.Reference was made to an adaptation of this techniqueto the determination of smaller amounts of antitoxin inthe sera of guinea pigs vaccinated with precipitatedtoxoids. An effective means of titrating small amounts ofantitoxin is especially desirable because of the wide-spread use of toxoids for active immunization of childrenand the trend towards vaccination of adults. Therecommended guinea pig test (National Institutes ofHealth, 1947) is sometimes inconvenient to perform in-asmuch as suitable guinea pigs are expensive and attimes are difficult to obtain. Different strains of guineapigs vary in susceptibility to diphtheria toxin; sex too isa factor of considerable importance (Kolb et al., 1955),and variation among individual pigs also occurs. Thereliability of chicks in the studies already describedsuggested further investigation of their adaptabil-ity to the evaluation of prophylactic diphtheria tox-oids.

MATERIALS AND METHODS

The animals, materials, and methods employed werethe- same as previously described (Branham andWormald, 1954).The White Rock chicks used throughout these studies

were obtained when 1 day old from a reliable hatchery;on the eighth day, they were divided into groups andinjected intraperitoneally. Such chicks were usually 45to 80 g in weight and were very uniform in suscepti-bility to diphtheria toxin. Chicks of this breed in thisweight range were designated as "standard" chicks, anda group of 10 as a standard group.The same lot of toxin that was used in the previous

study, No. 4617-4618, was employed throughout thesestudies for testing. The NIH Standard DiphtheriaAntitoxin, Lot No. A, containing 6 units per ml, wasused as a basis for comparison in determining all anti-toxic values in titrations. Mixtures of toxin and seradiluted with 0.85 per cent NaCl solution were incu-bated for 1 hr at 37 C, and then placed in an icebox at4 C overnight before injection intraperitoneally intochicks.

The term LDm is used to indicate that amount oftoxin which killed one-half of a group of standardchicks in 24 hr. The LDm of toxin 4617-4618 in chickswas so nearly the same as the MLD in guinea pigs thatthe latter was used in a study on the use of the chick instandardizing Schick toxins (Kolb et al., 1955). Like-wise the "test dose" of this toxin for chicks was re-markably similar to the L+ used with guinea pigs. The"test dose" may be defined as that amount which, in thepresence of 1 unit of antitoxin, killed one-half of thechicks in a standard group in 24 hr. The validity of thechoice of a 24-hr end point in work with chicks wasshown by Branham and Wormald (1954). The time ofdeath of 50 per cent of the chicks is designated Tm, ormedian time.

RESULTS

Alum precipitated toxoids. Diphtheria toxoids thatare in use for human immunization are either plain orwith a mineral adjuvant. The preliminary experimentwith toxoids (Branham and Wormald, 1954) was donewith alum precipitated preparations (AP); hence, itwas with this form of toxoid that a more detailed studywas now begun.The official method of the Division of Biologics

Standards for titrating alum precipitated toxoids re-quires that groups of guinea pigs weighing 450 to 500g to be injected subcutaneously with /1 of the humanimmunizing dose, bled afterA4 weeks, and a pool madeof equals amounts of serum from each animal. Thesepools are titrated in guinea pigs for antitoxin content.Minimum requirements for diphtheria toxoid demand atleast 2 units per ml of antitoxin in the sera tested. In thepresent study, the same procedure was followed in thepreparation of guinea pig serum pools, but largergroups of guinea pigs were immunized (20 with eachtoxoid) in order to have sufficient serum for intensivestudy. These sera were titrated in chicks.

In every test, a control group of chicks received 1unit of Standard Antitoxin plus the test dose of toxin.Results obtained with this control group were the basisfor determining the value of the serum under investi-gation. The end point was the Tm or the median timein hr when 50 per cent of the chicks had died, and wasapproximately 24 hr for this control group of chicks.

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TABLE 1. Sample titration of 2 pools of sera from guinea pigs vaccinated with alum precipitated (AP)toxoids; test done at 1-unit level

Serum Pool from Units Tested Serum Dilution 0.5 Ml Hr t D Units at MmGuinea Pigs Given for Per Chick aHr to Death of Individual Chicks Tm Neardt

ml units/ml.

Toxoid B (3 dil)t 7.0 135 (0.143) 22 22 24 24 27* 27 29 29 35 St 27 7+8.0 X (0.125) 15 19 19 19. 19 22 22 22 22 24 199.0 1/i (0.111) 15 15 15 15 16 15 19 19 19 19 15

Toxoid C (3 dii) 2.5 1!4.25 (0.4) 22 35 35 39 39 43 47 S S S 393.0 -.5 (0.33) 22 22 22 24 24 24 24 27 27 S 24 33.5 1/75 (0.28) 9 19 19 19 19 19 22 22 24 S 19

NIH Standard 6.0 (0.166) 22 22 22 24 24 27 35 35 35 35 24 6

Toxin dose = 0.5 mI Of Y5 4 dilution per chick (0.0197 mi/chick).* Tm = median death time in hr.t Dilutions based on results from preliminary tests.t S = survived.

Determination of the test dose has been previouslydescribed by Branham and Wormald (1954).' Witheach serum pool, preliminary tests were usually done for3 and 5 units with small groups of five or six chicks andthe results, as compared with the control group, sug-gested the range of dilutions to use for more accuratedeterminations with the larger groups of ten.Most of the titrations were done first at a 0.1-unit

level and later checked at 1 unit. By using this plan,use of the serum was increased 10-fold. One-tenth of theregular amounts of serum was used, including the con-trol; at the 0.1-unit level the NIH Standard Antitoxinwas used in dilution 1 to 30 instead of 1 to 3. The toxindose necessarily had to be adjusted experimentally forthis 0.1-unit level; it was found to be 0.5 ml of 1 to 200dilution at the beginning of the study, becoming 1 to170 before the work was completed. A sample titrationat the 1-unit level is shown in table 1.Seven alum precipitated (AP) toxoids were studied

as described. The serum pools were adequate for re-peated testing. Such tests were done in chicks at 1-unitand at 0.1-unit levels and by the official guinea pig testat 1 unit.

Table 2 presents the results obtained with theseseven alum precipitated toxoids as tested by themethods cited. Results obtained at a 1-unit level weresimilar in chicks and guinea pigs; in only one instancewere the values not identical. Values found at the 0.1-unit level tended to be slightly less than those found at 1unit. On the whole, there was excellent correlationamong all of these titrations.

Direct immunization of chicks. Titration in chicks of

1 At the beginning of these experiments, it was 0.5 ml of a1 to 26 dilution of the toxin, 4617-4618, or 0.0192 ml, for tests atthe 1-unit level. Because toxin very slowly decreases in po-tency, it was necessary to adjust this test dose at intervals;from 1 to 26 it had increased to 1 to 24.5 by the end of thesestudies.

TABLE 2. Unit values of pools of sera from guinea pigs vaccinatedwith AP toxoids

Units of AntitoxinToxoid Given Guinea pigs test Chick test

1 Unit 1 Unit 0.1 Unit

B 7 7 6C 3 3 2.5D 2 2.5 2E 6 6 6F 3 3 2.5G 5 5 4H 7 7 6

pooled serum from vaccinated guinea pigs gave reliableresults. Nevertheless it was thought desirable to tryimmunizing the chicks directly to test the possibility ofeliminating the use of guinea pigs altogether. Two ex-periments were performed:

(1) One hundred 8-day-old chicks were used for thispurpose. Seventy-six were given M of the total humanimmunizing dose (0.5 ml in this case) of AP toxoid J,and 24 were kept as controls. Injections were made sub-cutaneously into the loose tissue at the back of theneck.

(2) Six other chicks 2 to 3 weeks old were similarlyvaccinated.The plan for study of these was as follows:In the first experiment, a group of vaccinated chicks

and unvaccinated controls was challenged at each of thefollowing intervals after injection: 2, 3, 4, and 6 weeks.Survivors were bled and the sera tested separately in8-day chicks for antitoxin.

In the second experiment, the six older chicks weregiven a series of immunizing injections, and sera takenfrom them at intervals were tested in chicks for anti-toxin content.Only five of the first group showed measurable anti-

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toxin, one showing 1 unit per ml following the challenge3 weeks after vaccination, two showing 1 and 1.5 unitsper ml after the 6 weeks' challenge, and the remainingtwo less than 1 unit. Of the six older chicks given sev-

eral immunizing injections, two survived for 3 monthsand showed 2 and 3 units per ml respectively. This poor

antitoxin response is a confirmation of a similar obser-vation made by Mauss and Frobisher (1943). Chickswere not found suitable for antitoxin production.Plain toxoids. The official guinea pig test for anti-

genicity of plain toxoids is different from that for alumprecipitated preparations. According to the procedurerecommended in the minimum requirements for plaintoxoids (National Institutes of Health, 1947), smallerguinea pigs (270 to 320 g) are given not more than 16

of the total human immunizing dose subcutaneously,and after 6 weeks are challenged with 10 MLD of toxinin a volume of 2 ml. The adoption of this procedure,merely substituting chicks for guinea pigs, would be un-

satisfactory as, in addition to giving poor antitoxinresponse, chicks grow very rapidly, becoming fullgrown during this 6-week period. They eat tremen-dously, are noisy and untidy, and require much space.

When guinea pigs, vaccinated with only 16 of the totalhuman dose, as in the official test, were bled instead ofbeing challenged, and the pooled sera tested in 8-daychicks, as described for the alum precipitated toxoids, a

very low antitoxin content, often well below the re-

quired 2 units per ml, was found. Obviously changes inprocedures were necessary if the pooled sera from vac-

cinated guinea pigs were to be titrated for antitoxin inchicks. Studies were made to determine the effect ofamount of inoculum, number and spacing of injections,and time of bleeding upon the response of guinea pigs tovaccination with plain toxoids. Groups of pigs weighing250 to 350 g were given subcutaneously:

1 injection of / the total human immunizing dose(THD),

3 injections of 16 THD, weekly intervals, and2 injections of 14 THD, some with 1 week and others

with 2 weeks between the doses.Groups were bled at 3 weeks and 4 weeks after the

first injection and the serum pools from each group

titrated in chicks for antitoxin. Results are summarizedin table 3. Obviously one injection of 2 THD of plaintoxoid was inadequate. Usually, highest antitoxintiters were obtained when two injections of 14 THDwere given 2 weeks apart, and the pigs bled a week afterthe second injection. Good results were also obtainedwith three injections of 16 THD a week apart withbleeding a week after the last injection. In most cases,

a 3-week period after first injection (1 week after lastinjection) for bleeding gave better results than a

4-week period. The schedule finally adopted was that oftwo injections of 14 the THD given subcutaneously to

250 to 350 g guinea pigs, 2 weeks apart, with bleedingfrom the heart 3 weeks after the first injection.

Seven plain toxoids were studied in this way. Sched-ules of injections and bleedings and the antitoxin con-tent of the serum pools found by testing in chicks atboth the 1-unit and the 0.1-unit levels are shown intable 3.

Certain of these serum pools were checked for anti-toxin content with the official guinea pig technique atthe 1-unit level. These results are also shown in table 3.Very good correlation is obtained in these various

TABLE 3. Titration in chicks of sera obtained by vaccinatingguinea pigs with plain toxoids by various schedules

Method Units of AntitoxinNo.

Toxoid Guinea Weeks to Chick test Gui-(Anti- Group Pigs No' rc rqecBedn apggen) Vacci- of inFac FeunyBleig psnated jecn tion of ofI Iti- THD* injection1 0. 1tions ~~~~~unit unit unit

K a 5 1 ½3 - 1b 5 3 16 weekly 3 11 9c 15 3 IJ weekly 4 6 5 6

6 2 % 2 weeks 3 11 10apart

k 6 2 1/4 2 weeks 4 6 5apa rt

L a 5 1 ½ 3 1b 5 3 /f weekly 3 11 9c 15 3 /% weekly 4 7 6j 6 2 }/4 2 weeks 3 9 7 9

apartk 6 2 4 2 weeks 4 4 4

apart

M a 5 1 3 3 1b 5 3 M weekly 3 10 8c 15 3 1/ weekly 4 6 6 6

N d 5 3 M weekly 3 5 4 5e 5 3 M weekly 4 3 3f 5 2 1/4 weekly 3 7 6g 5 2 Y4 weekly 4 - 2

O d 5 3 /J weekly 3 8 7 7e 5 3 M weekly 4 5 5f 5 2 '4 weekly 3 6 5g 5 2 1J weekly 4 3 3

P d 5 3 } weekly 3 8 7e 5 3 M weekly 4 3 2f 5 2 4 weekly 3 6 5 6g 5 2 4 weekly 4 4 3

Q h 6 3 1/ weekly 3 8 6i 6 3 1M weekly 4 9 8

6 '2 2 weeks 3 13 11apart

k 6 2 Y4 2 weeks 4 5 4 4apart

* THD = Total human immunizing dose.

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1957] CHICKS FOR ASSAY OF DIPHTHERIA TOXOIDS 289

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tests, especially between those done at the 1-unit levelin guinea pigs and chicks. Here again, as with the alumprecipitated toxoids, somewhat lower unitage is foundat the 0.1-unit per ml level than at 1.0 unit per ml.

Relationship between doses of antitoxin and toxin atTm = 24 hr at various levels. In previous studies with a

commercial antitoxin of high potency prepared inhorses, it was shown that when tests were performed atseveral different unit levels the end points, obtainedwhen the amount of toxin and antitoxin represented bythe Tm were plotted on log log paper, fell along a

straight line. This straight line was parallel with thatobtained with the NIH Standard Antitoxin which was

tested similarly. It was observed that comparableresults could be obtained at any point in the region oflinearity; hence, these findings were taken as addi-tional evidence that a comparison between these sera

could be made at any dosage level, and gave addedweight to a belief in the validity of the test (Branhamand Wormald, 1954).

Could sera of low potency, such as was obtained byvaccinating guinea pigs with toxoids, give similarquantitative results? Four serum pools, prepared byimmunizing guinea pigs with toxoids, two plain and twoalum precipitated, were studied in an attempt to answerthis question. Two series of experiments were doneabout a year apart, using the same materials. In theearlier part of the study, groups of chicks were givenserum-toxin mixtures representing several antitoxinunit levels, ranging from 0.1 to 1.0 unit, and higher, inone case up to 4 units. With so many antitoxin levelsonly one or two serum pools could be included in one

test. Each was repeated a number of times. Always a

group receiving the NIH Standard Antitoxin at the 1unit level was included. In the later experiments allserum pools were included in each test, using fewer doselevels and a smaller number of chicks in each group.

This latter series was done to compare these sera with

all experimental conditions equal. The amounts ofserum and toxin which gave end points represented bya Tm of approximately 24 hr were plotted on log logpaper. These points are shown for the serum poolsrepresenting these four toxoids in the graphs shown infigure 1. The upper part of the graph represents thefirst series in which many points were taken with eachserum pool. Many different tests are included. In thesecond series, all serum pools were tested simultaneouslythree times at three levels. There is no significant de-parture from linearity in either series of experiments. Ithas been pointed out previously that end points ob-tained below the 0.1-unit level tend to deviate from a

straight line (Branham and Wormald, 1954).Are the straight lines obtained with these low titered

antitoxin pools parallel to each other and to that shownby the NIH Standard Antitoxin? To answer thisquestion the slopes of these lines were calculated usingthe method of least squares. The slope of a line indi-cates the rise in ml in toxin required per unit of ml in-crease in antitoxin. The slopes and intercepts calcu-lated for each experiment, as well as their appropriateaverages and their standard errors, are shown in table 4for four serum pools and the NIH Standard Antitoxin.The various slopes do not differ significantly from eachother.

DIscUSSIONIn seeking an accurate means of evaluating diphtheria

toxoids more convenient and less expensive than thecommonly performed test in guinea pigs, the authorshave found the 7- to 8-day old chick very useful. Asimple substitution of chicks for guinea pigs throughoutthe guinea pig test as usually performed is, however,not the complete solution of the problem. Chicks do notform antitoxin well; hence, it has been found inadvisableto vaccinate them with toxoids. A more successful pro-

cedure is to vaccinate guinea pigs with the toxoids and

TABLE 4. Slopes and intercepts of linear relationship of log dose toxin for Tm = 24 to log dose antitoxin

Serum Pools from Guinea Pigs Immunized with Toxoids Indicated Below:

AP toxoid F, Plain toxoid L, Plain toxoid M, AP toxoid H, Standard antitoxin A, Standard antitoxin A,5 tests 4 tests 4 tests 5 tests 4 early tests 4 later tests

S I* n S I n S I n S I n S I n S I n

0.846 -1.016 3 0.855 -0.984 3 0.886 -0.976 3 1.010 -0.827 3 0.805 -1.090 3 0.912 -0.992 30.804 -1.042 2 0.793 -1.029 3 0.852 -1.016 3 0.908 -0.895 3 0.871 -1.029 3 0.859 -1.017 30.874 -0.970 3 0.861 -0.984 7 0.907 -0.955 5 1.190 -0.567 2 0.868 -1.033 4 0.954 -0.891 20.841 -1.013 4 0.866 -0.961 4 0.826 -1.051 5 0.814 -0.954 2 0.898 -1.023 3 0.900 -0.955 30.851 -1.026 6 0.857 -1.021 3 0.972 -0.857 4

Average ..... 0.844-1.014 0.846-0.988 0.861-1.008 0.966-0.864 0.861-1.044 0.889-0.993

Standard erroroftheavg.. +10.038 +0. 052 -+0.0384±0.054 +0.032 +0. 044 +0. 0404+0.048 40.026+40.034 40.0301-0.036* S = slopes; I = intercepts; n = number of points determining line.Note: In computing means and standard errors, the factor of differing values of n and differing amounts of antitoxin have been

considered.

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to use chicks for the titration of the antitoxic serumobtained from them.With alum precipitated toxoids it was a simple

matter to follow the usual procedure of giving guineapigs one injection of ½ of the total human dose (THD)of toxoid, and bleeding them after 4 weeks. With plaintoxoids one injection of ½ THD did not produce aresponse comparable with that following ½ THD ofalum precipitated toxoid. Two injections of Y4 THD aweek apart were finally decided upon for use in thisstudy, with bleeding at the end of the third week. It isrecognized that two injections are not altogether de-sirable, since a second response is thereby invoked.Subsequent work should be done to clear up thiscomplication.The end point chosen in these titrations is the Tm, or

median time of death in hours in each group of chicksas compared with that of the NIH Standard Antitoxinwhich is always included in the test. The Tm in theStandard approximates 24 hr; this Tm is the basis forcalculating the unit value of the antitoxin under test.There are two factors which tend to affect the

precision of the results obtained:(1) The number of dead chicks was recorded every

2 hr so that the exact time of death could vary withinthis span, and

(2) The Tm obtained with the test serum sometimeswas an hour or more greater or less than that of theStandard Antitoxin.

Excellent correlation is obtained between the unitvalues obtained thus in chicks and those found in theofficial guinea pig method at the 1-unit level. Tests inguinea pigs at the 0.1-unit level were not performed.Skin test titrations in guinea pigs were also done, butare not shown here. Such skin tests consistently gave alower value than was found in either chick or guineapig.When chick titrations on some of the serum pools

included in this study were performed at various unitlevels (that is, 1.0, 0.5, 0.25, and 0.1 unit and so forth),and the amounts of serum and toxin used at the Tm'splotted on log log paper, the points were found to lieapproximately along straight lines. The lines represent-ing these sera were parallel to each other and to that ofthe NIH Standard Antitoxin. The slopes of these lines

and of their standard errors show a high degree ofuniformity. Serum representing toxoid B was the onlyone which showed any appreciable variation in theslopes determined in five tests. Toxoid B was the firststudied and was employed in working out the tech-nique subsequently used throughout later studies.

It is worth noting that this linear relationship can beutilized in determining appropriate levels of toxin andantitoxin for use in repeated assays. Tests with any ofthese sera could be performed at any level along thestraight line. Below the 0.1 unit level the Tm's havebeen found to deviate from a straight line and to. swingto the left (Branham and Wormald, 1954).

ACKNOWLEDGMENTSThe authors wish to thank Mr. Jacob Lieberman and

Dr. Max Halperin for their help and advice with themathematical and statistical phases of this work.

SUMMARYA method is described for the assay of diphtheria

toxoids in which guinea pigs are vaccinated with thetoxoid, bled after appropriate intervals, and the pooledserum titrated for antitoxin in 7- to 8-day chicks. Suchtitrations may be performed at any level in linearitydown to 0.1 unit. The values obtained give good cor-relation with those found in the official guinea pigtest. The chick affords an accurate, rapid, and inex-pensive means of measuring small amounts, as well aslarger quantities, of diphtheria antitoxin, and is wellsuited for use in the evaluation of toxoids, both alumprecipitated and plain.

REFERENCESBRANHAM, SARA E. AND WORMALD, MARION F. 1954 The use

of the chick in titration of diptheria antitoxin. J. Im-munol., 72, 478-484.

KOLB, R. W., BRANHAM, SARA E., AND RIGGS, D. B. 1955Comparison of the guinea pig and chick in evaluation ofdiphtheria toxin for the Schick test. Appl. Microbiol., 3,242-247.

MAUSS, EVELYN A. AND FROBISHER, M., JR. 1943 The anti-genicity of diphtheria toxoid in chicks, mice, and rats.J. Hygiene, 37, 234-238.

National Institutes of Health 1947 Minimum requirements:diphtheria toxoid, 4th Revision, National Institutes ofHealth, Bethesda, Md.

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