THE AGGLUTINATION TEST IN UNDULANT FEVER DUE TO BRUCELLA ABORTUS. A PRELIMINARY NOTE ON THE VALUE OF THE "ABORTOSCOPE."

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1 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE. Vol. XXI. No THE AGGLUTINATION TEST IN UNDULANT FEVER DUE TO BRUCELLA ABORTUS. A PRELIMINARY NOTE ON THE VALUE OF THE "ABORTOSCOPE." BY G. R. ROSS, M.B,, Ch.B., Ph.D., D.P.H. Rhodesian Research Fellow, London School of Hygiene ~.~ Tropical Med~%ine. There can be no dispute that the most valuable diagnostic sign in undulant fever is the presence of agglutinins in the serum of the infected. Since, however, it has recently been claimed that, in addition to Brucella melitensis, Br. abortus may be of etiological importance in the disease, it is pertinent to enquire how far the reliability of the agglutination test, as usually performed with a suspension of Br. melitensis, is affected should the pathogenicity of Br. abortus to man be established.

2 58 THE AGGLUTINATION TEST IN UNDULANT FEVER DUE TO BRUCELLA ABORTUSo MICROSCOPIC AGGLUTINATION; Investigations into the serology of the two organisms have produced results which demonstrate their close relationship. BURNET (1925), B~GUET (1926), FAVlLLI (1926), and ORCUTT (1926) have failed to distinguish between the two. On the. other hand, EVANS (1918), FEUSIER and MEYER (1920) and KHALEO (1921), insist on serological distinction between Br. abortus and Br. melitensis. Such differentiation, however, is possible only by the absorption-of-agglutinin test and, when simple agglutination is employed, both agglutinate equally with either Br. melitensis or Br. abortus immune serum. It is not in the province of this present communication to diseuss which of the above statements is correct, but it can be pointed out that both agree in this important fact that immune serum prepared against either of the organisms will agglutinate both to titre. Thus, from theoretical grounds, the conclusion can be drawn that there can be no objection to the use of a suspension of Br. melitensis as the test suspension in the diagnosis of a case of undulant fever caused by Br. abortus or vice versa. A further and perhaps more important conclusion that can be drawn is that, because the serum of a case of undulant fever shows agglutinins for Br.abortus, it cannotbe assumed that Br. abortus is the infecting organism. The fact that the agglutinin response to either Br. melitensis or Br. abortus infection is not specific, as far as simple agglutination is concerned, has been lost sight of and has lead to considerable confusion in the literature. The first conclusion, namely, that suspensions of both organisms, provided that each is active in agglutination, can be employed in the agglutination test, has been tested in Southern Rhodesia. For some time it has been the custom in this laboratory to test the sera of suspected cases of undulant fever against Br. melitensis and The test as performed in the routine examination is a microscopic one, dilutions of 1 in 50 and 1 in 400 being employed. During 1926, thirty-four tests gave positive results. Of these, twenty-two were done with a suspension of Br. abortus which was afterwards discarded. In this first series only five cases gave agglutination with both suspensions, ten gave agglutination with Br. melitensis only, while seven cases gave a doubtful reaction with Br. abortus only. These results were reviewed and it was decided to employ fresh strains in the test, one being a strain of Br. melitensis from the Lister Institute, and the second a strain of Br. abortus isolated from a bovine in South Africa. In the remaining tests, twelve in number, these strains were employed. In eight, both organisms were equally agglutinated, while in the remaining four Br. abortus only was agglutinated. Four of the eight cases which gave agglutination with both, and one in which Br. abortus only agglutinated, were treated in hospital, and h~emoculture resulted in the isolation of an organism serologically identical with The serological data establishing this will be published separately.

3 G. R. ROSS. 59 MACROSCOPIC AGGLUTINATION. The results of microscopic agglutination, then, undoubtedly show that when suitable suspensions are employed both organisms, in the majority of cases, are agglutinated by the sera of cases of undulant fever due to Br. abortus infection. At the same time, however, one case in which infection was proved by hmmoculture failed to agglutinate Br. melitensis. This failure is probably due more to the inherent difficulties of the microscopic method of agglutination than to an error in the theory that both should agglutinate equally. The minute quantity of serum generally available may result in unequal distribution between the two suspensions ; the difficulties of observing agglutination with such small microorganisms allow for considerable error in the personal element, and, finally, the possibility of evaporation in the drop under observation in an inadequately protected slide must always be considered. In every circumstance the macroscopic method must be preferred, This, of course, means that additional serum must be available and that venipuncture is generally necessary to obtain the requisite amount of blood. In order, however, to test the value of the macroscopic method in showing whether agglutination of both suspensions resulted from the presence of sera from undulant fever patients, cases, in the majority of which the causal Organism had been isolated and which had been tested by the microscopic method, were examined. The serum was tested in 0'25 c.cm. amounts, in dilutions of 1 in 25 up to 1 in 3200 in arithmetical progression, against an equal amount of 4 T.M. (4,000,000,000) suspension of Br. mditensis and Br. abortus. Incubation was at 37 C. for eighteen hours, although results conld generally be read after four hours' incubation. The result can best be expressed in tabular form. Case No.! Macroscopic. i,titre positivetitre positive against I against Br: melitensis.! Br. melitensis. Microscopic. Br. aborlus. Organism isolated. 1 in in in 50 1 in in in 4:00 I in in 50 1 in in (+) ~ (2) + Br. para-abort'as. Test not performed. Test not perfmmed. In this table (-}-)signifies doubtful agglutination, + agglutination, 4- marked agglutinaion. These cases, with the exception of No. 6, all exhibited typical clinical sywptoms of undulant fever at the time of the test. Case 6 was apyrexial, but had been in hospital about a year previously with typical symptoms, and at that time an

4 60 THE AGGLUTINATION TEST IN UNDULANT FEVER DUE TO BRUCELLA ABORTUS. organism distinct from both Br. melitensis and Br. abortus had been isolated. It resembled Br. paramelitensis, but as it was serologically identical with an organism isolated from a bovine it is provisionally termed Br. para-abortus. The Table shows that in the cases tested, by the macroscopic method, Br. melitensis and Br. abortus were in every case agglutinated to an equal degree by the sera of declared cases of the disease. By the microscopic method, on the other hand, while in the majority of cases both organisms agglutinate, that agglutination varies in degree, and in one case the result disagrees with the result of the macroscopic method. In order to test the hypothesis further, three guinea-pigs were inoculated, one with a Br. melitensis strain, one with Br. abortus, and the last with a strain isolated from a human case. In every instance agglutination of both Br. melitensis and Br. abortus occurred to an equal degree. The same was found when rabbits were immunised against Br. melitensis and Br. abortus by means of dead cultures. Here, again, the titre was the same for the homologue and the heterologue. The conclusion that can be drawn, both theoretically and practically, is that either Br. melitensis or Br. abortus can be expected to give a reliable result, in case of undulant fever, whether due to Br. melitensis or Br. abortus infection, and that the macroscopic method is the method of choice. It was necessary to establish this fact in the present instance, in which an endeavour was made to simplify the methods of agglutination available to the general practitioner. It is true in most places, and particularly so in Southern Rhodesia, that in the case of practitioners removed from a laboratory, considerable time must elapse before the result of an agglutination test can be available. This delay detracts from the value of the test, since contamination and deterioration of the serum may occur, particularly in a hot climate, which may lead to equivocal results. THE ABORTOSCOPE. In an effort to obviate this, the attention of the present writer was drawn to the " abortoscope " devised by BEVAN for the diagnosis of contagious abortion in cattle. It was described by BEVAN (1925) and has been found in controlled experiments to give rapid and reliable results in the diagnosis of that condition. The instrument consists of a tube containing a specially standardised suspension of Br. abortus, ciosed by a cork to which is attached a double loop of thin wire coated with sterile paraffin to prevent rusting. On the back of the tube is pasted a label marked " Infected " which cannot be read through the suspension. Before use the suspension is thoroughly shaken up. The animal is pricked in the ear and one full loopful of blood is obtained. The loop is then returned to the tube, which is again thoroughly shaken to mix the blood with the suspension,

5 G, R. ROSS, 61 and the whole apparatus is allowed to stand in an upright position at room temperature. In those cases which are infected, the suspension and blood cells settle to the bottom of the tube leaving a clear, supernatant fluid through which the word " Infected " can easily be read. In those cases which give negative reactions, the cells settle down but the suspension remains opaque. The proof of a positive reaction is the ability to read the word " Infected " through the contents of the tube, after eighteen hours at room temperature. It having been established that a suspension of Br. abortus could give reliable results in the diagnosis of cases of undulant fever, the abortoscope was tested on a series of cases of undulant fever in which infection had been proved by the isolation of the organism. Tests were also made on patients suffering from other diseases in order to control the specificity of the results. In three cases Of undulant fever in the pyrexial period, tests repeated at weekly intervals invariably gave positive results. In two of these cases, tested during an apyrexia intervening between two pyrexial periods, the sai;ae was the case. In all these cases agglutination could easily be made out in from fifteen to thirty minutes, and at the end of eighteen hours deposition of suspension and cells was complete, the supernatant fluid being water-clear, the suspension being clumped at the bottom of the tube with the red cells on top. In one undoubted case of the disease, while the word " Infected " could easily be read through the tube, agglutination was not absolutely complete, and in this instance the cells deposited first with the suspension on top. In one case, which had been apyrexial for one month and in which the agglutinin titre was 1 in 50, a negative result was obtained, as was also the finding in a patient who had been apyrexial for over a year and in whom the titre was 1 in 25. The specificity of the test was established by the fact that two cases of typhoid fever, two of amcebie dysentery, three of malaria, two of tuberculosis, one of bilharzia, one of pseudo-typhus, and ten normals gave negative results. In guinea-pigs infected with recent human strains of Br. abortus a positive result was obtained, as well as in rabbits immunised with killed cultures of Br. melitensis and Control rabbits gave negative reaction. This communication is merely a preliminary report, but it indicates that the method may be of the greatest value to practitioners in enabling them to diagnose their cases independent of the laboratory. It requires a drop only of blood from the finger, is done at room temperature, and the result is available, in many cases, almost at once, and in all at the end of eighteen hours. It is, of course, merely an improvement of the agglutination test and cannot be expected to give results which the agglutination test cannot give, as it is dependent upon the agglutinin contents of the patient's serum. Its simplicity is appealing, and it gives hopes of being a reliable and rapid method of diagnosis, not only in undulant fever, but, by employing different suspensions, in any organismat disease which gives a positive agglutination reaction.

6 69~ THE AGGLUTINATION TEST IN UNDULANT FEVER DUE TO BRUCELLA ABORTUS. SUMMARY. Experimental evidence is submitted that both Br. melitensis and Br. abortus can be employed as the test suspension in the diagnostic agglutination test for undulant fever due to Attention is drawn to the " abortoscope " as a promising method of performing the agglutination test in a simple, rapid, and reliable manner. REFERENCES. B~GUET, M. (1926). Les caraet~res diff6rentiels des souches dans le genre Brucella et les essais de classification. Summarised in Trop. Dis. Bull. xxiii, 731. BEVAN, L. E.W. (1925). The Abortoseope. A simple apparatus for the detection of infectious abortion of cattle. Vet. ffl., lxxxi, (1O), 476. BURNET, Et. (1925). Apergu des acquisitions rdcentes sur la fi6vre m~diterrandenne. Summarised in Trop. Dis. Bull., xxii, 938. EVANS, ALICE C. (1918). Further studies on Bacterium abortus and related bacteria. II. A comparison of Bacterium abortus with Bacterium bronchisepticus and with the organism which causes Malta Fever. ffl. lnfec. Dis., xx, (6), FaVILLX, GIOVANNI. (1926). Ricerehe e considerazioni critiche sulla febbre mediterranea e sul' aborto epizootico. Summarised in Trop. Dis. Bull. xxiii, 725. FEUSmR, M. L. and MEYER, K. F. (1920). Principles in serological grouping of Br. abortus and Br. melit.ensis. Correlation between absorption and agglutinin tests. Studies on the genus Brucella. nov. gen. ffl. Infec. Dis., xxvii, (3), 185. KHALED, E. (1921). A comparative study of bovine abortion and undulant fever from the bacteriological point of view. ffl. Hyg., xx, (4), 819. OBCUTT, MARION L. (1926). Agglutination affinities of the abortus-melitensis group of bacteria with special reference to two human strains. ~l. Expr. Med., xliii, (2), 225.