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Titration of Antigen
Hemolysin Sheep's titre NaCl (dil. 1 : 2) Anti- (dil. 1: 50) corpuscles 0.002 0.85% titre 0.03 gen
Titre of the antigen is 0.05 cc. In the complement fixation test we employ 4 units of antigen. As the binding power of the above antigen with complement is exhibited only in amounts of antigen greater than 0.5 cc, there is no danger from complement absorption with the required amount of the antigen.
In the above titration 5 times the titre of immune serum is used, namely 0.01 cc; one and a half units of complement and 5 units of hemolysin are employed.
The Complement Fixation Test Measured amounts of the sera under test are placed into tubes containing 1.5 cc of saline solution. The diluted sera are inactivated by heating in a water bath at 56-58°C. for 20-30 minutes. It should be made a rule to inactivate all sera, as traces of complement may, otherwise, remain even after several days of holding in the ice box.
Complement and antigen of known strength are then added, as is shown in the following tables, and the tubes incubated at 37°C. in a water bath for one hour in order to allow complete binding of complement in the presence of positive sera. Hemolysin and sheep's corpuscles* are then added, and after thorough mixing of the contents the tubes are returned to the water bath for a period of 50-60 minutes.
A known negative and a known positive serum should be set up as controls in every fixation test, in the same manner as the sera on which tests are sought.
* Hemolysin and corpuscles may be combined in the proper proportion, before being added to the serum-complement-antigen mixture, thus neces. sitating only one pipetting operation instead of two
The Fixation Test
cells Tube used sol. Titre Titre
Incu- Complete hemolysis 0.1 0.2 bation
bated 3 0.05
for i hr.
50-60 0.02 at 37° C.
at 37° C. (Cow is a non-reactor, according to test) (2) Known Negative Serum 1 0.2 1:5 0.045 none Incu0.25
Incu- Complete hemolysis 2 0.1 0.2 bation
bated 3 0.05
for 1 hr.
50-60 4 0.02
at 37° C.
at 37° C. (3) Known Positive Serum 1 0.2 1..5 0.045 none Incu0.25
Complete hemolysis 2 0.1 0.2 bation
bated No 3 0.05
for i hr.
50-60 4 0.02
at 37° C.
minutes 5 none
at 37° C. Complete hemolysis 6
The method employed here allows adequate checks on all reagents used.
In all tests (4 tubes) tube No. I should be hemolyzed, since antigen is absent and hence the complement cannot be deviated. If laking does not take place an error in technique has occurred or an anti-complementary substance is interfering. Few cows' sera have been anti-complementary, however.
If the given animal is a reactor, tubes 2, 3 and 4 will show no hemolysis. Negative sera do not fix or deviate complement, and therefore all four tubes should be completely hemolyzed when such sera are employed.
In the table for known positive serum eight tubes are set up. Tube No. I serves as control in each individual serum test. Tubes 5, 6, 7 and 8 are additional checks. If 5 and 6 fail to hemolyze the antigen has become anti-complementary and should be discarded. Numbers 7 and 8 are useful chiefly as further checks on accuracy in manipulation.
DISCUSSION OF METHODS The Agglutination Method. Serological pipettes are employed for the transfer of serum from the blood sample to the agglutination tubes. This is necessitated, both in abortion and in white
diarrhea diagnosis, by the large numbers of daily tests. For the same reason, no account is taken of the difference in the total volume of fluid in tubes i and 2, and in the corresponding slightly unequal dilution of serum in the two tubes.
Results of routine tests are recorded as "+", "?", and "O", largely as a matter of convenience. Furthermore, this method of reporting is desirable as a matter of accuracy, and of justice to the owner. Animals should never be pronounced positive unless they are unquestionably so, particularly in herds in which there are no other positive animals.
Experience has shown that the time and method of incubation employed here are highly desirable. Though final readings are not made before the end of 3 days, the sharpness of the reactions is full compensation for the time allowed. Incubation at temperatures above 37°C. would probably considerably shorten the period, but few laboratories are supplied with high temperature incubators and furthermore reactions would as a rule not be so sharp.
The Complement Fixation Test. Here again the serum is transferred directly from the blood tube to the different dilution tubes, with serological pipettes, and no provision is made to have the same volume of total fluid in all of the tubes. As it is well known that, within reasonable limits, the serum reactions depend on amount of agents in solution, and not on their dilution, the saving of time effected by this method is fully justified. Experience over quite a number of years has shown the results to be consistent.
All along, the routine fixation results have been recorded in terms of hemolysis, rather than fixation. The symbols "+", "?", and “O” are used to designate the individual tube reactions. It may appear to many persons more logical to report the results in terms of fixation, and of course this may be done. It is true that to the lay mind our system may be somewhat confusing, as complete hemolysis in the four tubes is recorded as "O" in each, though the fixation test is negative, and on the same page positive agglutination is indicated by “+”. For our own permanent record, however, the method now used appears to us the more desirable of the two. THE DIAGNOSTIC VALUE OF THE AGGLUTINATION AND
COMPLEMENT FIXATION TESTS These tests have been employed for the past ten years as a basic part of the investigation of infectious abortion at the Storrs Experiment Station. During this period at least ten thousand bovine blood samples have been subjected to the tests.
No method of disease diagnosis has yet been devised which is absolutely free from criticism. Even the most recent improvements in the methods of conducting the agglutination test for typhoid fever in man are still far from perfect. The writers believe
that the agglutination and complement fixation tests for infectious abortion constitute an indispensable part of an intelligent study of the abortion problem and of any effective system of eradication.
Evidence of the value of such tests may be sought in the following ways:
(1) By establishing a definite relation between the serological reactions and the calving incidence.
(2) By observing close agreements between the results of the two tests.
(3) By testing the same animals again and again and noting agreements between the data obtained at different times.
I. RELATION BETWEEN THE SERUM REACTIONS AND THE LENGTH
OF THE GESTATION PERIODS It is a well-known fact that many cows which are infected with Bact. abortus carry their calf to full term. Therefore, positive reactions are not necessarily indications of premature calving. This is not due to any defects in the tests, but to the ability of some cows to retain the fetus to full time, in spite of Bang bacillus infection.
On the other hand, if the tests are of real merit very few nonreacting heifers and cows should abort, providing of course that no other bacteria or agencies are operating to cause premature calving. In the work of the Storrs Station the results have been such throughout the extended investigation as to preclude other organisms, except perhaps in a few isolated instances.
The numbers of reacting and non-reacting cows which calved normally and those which aborted are summarized in the following tables :
It will be seen at once that the figures in Table I are of no significance except that they show that normal calving animals may and often are reactors to both the agglutination and fixation tests. While in a few instances the positive animals constitute at least 45 per cent. of the given groups, and in two cases 66.6%, in most of the groups the negative heifers and cows greatly preponderate. It should be stated that in each of the herds recorded here infectious abortion had been thoroughly established, and that a large proportion of the animals were reactors to the serological tests.
Table II brings out a strong correlation between abortions and positive serological reactions, and demonstrates the specificity and diagnostic value of the blood tests.
In herd A in the years 1916 and 1917, the reactors constituted only 25% and 33 1/3 % respectively of the aborting animals. No explanation can be offered for this except that other organisms may have played some important part in the bringing about of the abortions. In these two years the numbers of animals tested were so small as not to affect the total calving and reacting records materially. In the 1917 figures are included a premature fetus which was decidedly deformed.
TABLE I. Giving Blood Reactions of Normal Calving Animals.
Note: The differences in the proportions of reacting, and non-reacting animals noted from year to year are not due to changes in individual animals, but to removal of old and addition of new animals.
TABLE II. Blood Reactions of Aborting Animals (1914-1923).