II. Irregularities Observed in the Isoagglutination Reaction of the Blood of Bovines
Authors: Little Ralph B. 
Published: December 2022

Discussion and Summary
In the foregoing pages there have been submitted brief notes of representative cases in which the agglutinating properties of the blood of cattle have fluctuated.
In man, after the appearance of the isoagglutinins or agglutinogens in the blood of an individual, the grouping once established generally holds until advanced age is reached. In recent publications both Doan (2) and Snyder (3) mention certain changes in agglutination which had been observed by some investigators after the administration of drugs, prolonged ether anesthesia and roentgen-ray irradiation. Other investigators found no change in the blood grouping under similar conditions.
The appearance of isoagglutinins in the blood of some cattle and the attenuation or depletion of isoagglutinins in the blood of others occurred without any definable reason. In the following pages some of the fluctuating conditions, both internal and external or environmental, to which the cows were subjected are briefly given.
With one cow, no. 18, the repeated intravenous injections of a heated culture of B. coli may have affected the composition of the blood during the immunization period. It is very doubtful whether tuberculosis was responsible, for there was a marked gain in weight with improvement in health soon after this animal was under our care. The disease was not generalized but confined to the mediastinal lymph nodes.
The relation of pregnancy to agglutination reactions of the blood was not consistent. In certain cases the appearance of isoagglutinins (4 individuals) was first demonstrated shortly after conception. On the other hand isoagglutinins appeared in the blood of cattle (4 cows) which were not pregnant. With cow no. 32 after a five months gestation period the serum still failed to agglutinate, although the agglutination had been complete when the serum was first examined. The most potent sera of Group I examined immediately before and after parturition showed no change in the titer. Pregnancy so far has not shown any relation to the aggutinability of the corpuscles of Group II.
The effect of mastitis and infectious abortion on the agglutination reactions is in doubt. Both infections were about equally distributed in each group.
All but two of the cows were in milk throughout the entire period of examination. The lactating animals, with the exception of no. 18, were handled alike; a few cows were milked three times a day. It was arranged so that each barn of fifty cows were exercised twice a week in a paddock. The grain ration was of the same composition for all. During the summer months fresh cut alfalfa from the field was artificially dried and ground up for feeding in place of last year's ground alfalfa hay. All the cows were fed ensilage. This year it happened that the supply of silage was exhausted late in July and fresh silage was fed from then on. Harper and Byron (4) in 1922, working with human bloods, found an increased strength in the agglutinability of Group III cells when a diet rich in greens was taken. The cells of Groups I and II were not affected. It is of interest to note that with a number of bloods the appearance of agglutinins was first detected late in the summer after the cows had been fed fresh alfalfa hay and ensilage.
It may be of some significance to mention that with a number of cattle the first examinations were made shortly after the animals had calved. At this time the cows had not advanced to their maximum milk yield for the current lactation period. In later examinations when the depletion of the isoagglutinins in the blood was first detected the cows were either in or approaching the full milk yield. After a period of six to eight months there occurred a gradual diminution in milk secretion and with some cattle the isoagglutinins were again present in the blood about this time.
With Group I the potency of the serum was generally stable after one or two examinations. If the fresh sera completely clumped the agglutinable corpuscles without exception when examined with control sera the agglutination was generally maintained. A serum which failed on the first or second re-examination to agglutinate all the corpuscles of Group II, was then placed in Subgroup B of Group I. The fluctuation in the agglutinophilic properties of the serum was apparent on the first examination, or there might follow a gradual decline in the avidity of the agglutinins until the diminution had reached a low level. At this point any appreciable agglutination was only determined by a microscopic examination of the dilution.
The question arises whether these irregular agglutination reactions might be attributable in part to a fluctuating condition of the corpuscles from one examination to another. In the preparation of the cell suspensions minute quantities of serum were carried over with the clot to the normal saline. However, some sera would occasionally clump suspensions of washed cells, whereas cell suspensions prepared from the blood clot was either negative or partially agglutinated. It also happened that the concentration of the cells varied on different examinations, for at times the cells were not easily dispersed from the clots. If variation in the cell suspension did influence the agglutination reaction this fact would soon be apparent in the character of the agglutination shown by the control sera. If the control sera on the whole completely clumped all the agglutinable corpuscles into a firm mass, it was evident that sufficient corpuscles had been supplied. Nevertheless the cell suspensions of some cattle were regularly more agglutinable than were the cells of other cattle. The fluctuation of the agglutinating properties of these sera may then be referred to a temporary depletion of the isoagglutinins rather than an insufficient quantity of serum. In a recent experiment (1) it was shown that with Group I serum a complete agglutination occurred with a dilution of 0.1 and 0.05 cc.
It is evident from protocols that between Group III and Subgroup IIIa there exists a close similarity in the agglutinating properties of the serum. In Group III the serum was not agglutinative, however; cases were found in which isoagglutinins normally appeared in the blood. In Subgroup IIIa the sera irregularly and only weakly agglutinated, nevertheless in this group definite agglutination with some sera has been shown. The serum of Group IV will occasionally agglutinate the corpuscles of Group I. The avidity of these sera for certain corpuscles of Group III and Subgroup IIIa has been shown. This agglutination, then, may indicate the bloods in which isoagglutinins may later appear.