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45 THE RELATION BETWEEN SPECIFIC AND NON-SPECIFIC AGGLUTINATION IN THE BRUCELLA GROUP. BY S. R. PANDIT AND G. S. WILSON. (From the London School of Hygiene and Tropical Medicine.) FOE an understanding of the epidemiology of undulant fever it is desirable that the relations between the different members of the Brucella group should be made clear. The present paper records the results of a preliminary examina- tion, by specific and non-specific agglutination tests, of a number of strains isolated from various sources. A more detailed study by means of specific agglutination and absorption tests is now in progress, the results of which it is hoped to publish at a later date. TECHNIQUE. Altogether 117 strains have been examined. According to their source of origin and other information available at the commencement of the investi- gation they were preliminarily grouped as follows: Br. abortus, porcine type ... 12 strains ,, bovine type ... 47 ,, Br. melitensis ... ... ... 47 ,, Br. paramelitensis ... ... 11 ,, The strains were derived from various countries including England, France, Denmark, Italy, Malta, Tunis, Algiers, Palestine, India, South Africa, the United States, and Peru. The country of origin of some strains was unknown. The following tests were employed: (a) Thermo-agglutination. A 48-hour growth on an agar slope was suspended in normal saline, and standardised to an opacity corresponding to about 8000 million B. coli per c.c. One cubic centimetre was transferred to a glass tube, 13 x 0-8 cm., which was then immersed in a bath of water kept approximately at 95° C.; the level of the water was just above that of the suspension in the tube. The tube was examined after 5, 10, 15, 30, 45, 60 and 120 minutes. The occurrence of definite agglutination visible to the naked eye against an illuminated dark background was recorded as a positive result. The following notation was used: Agglutination within 5 minutes +++ in from 5 to 10 minutes ... ++ 10 to 120 minutes ... + available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0022172400017812 Downloaded from https://www.cambridge.org/core. IP address: 65.21.229.84, on 10 Jan 2022 at 06:30:11, subject to the Cambridge Core terms of use,
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45

THE RELATION BETWEEN SPECIFIC ANDNON-SPECIFIC AGGLUTINATION IN THE

BRUCELLA GROUP.

BY S. R. PANDIT AND G. S. WILSON.

(From the London School of Hygiene and Tropical Medicine.)

FOE an understanding of the epidemiology of undulant fever it is desirablethat the relations between the different members of the Brucella group shouldbe made clear. The present paper records the results of a preliminary examina-tion, by specific and non-specific agglutination tests, of a number of strainsisolated from various sources. A more detailed study by means of specificagglutination and absorption tests is now in progress, the results of which itis hoped to publish at a later date.

TECHNIQUE.

Altogether 117 strains have been examined. According to their source oforigin and other information available at the commencement of the investi-gation they were preliminarily grouped as follows:

Br. abortus, porcine type ... 12 strains,, bovine type ... 47 ,,

Br. melitensis ... ... ... 47 ,,Br. paramelitensis ... ... 11 ,,

The strains were derived from various countries including England, France,Denmark, Italy, Malta, Tunis, Algiers, Palestine, India, South Africa, theUnited States, and Peru. The country of origin of some strains was unknown.

The following tests were employed:

(a) Thermo-agglutination.

A 48-hour growth on an agar slope was suspended in normal saline, andstandardised to an opacity corresponding to about 8000 million B. coli per c.c.One cubic centimetre was transferred to a glass tube, 13 x 0-8 cm., which wasthen immersed in a bath of water kept approximately at 95° C.; the level ofthe water was just above that of the suspension in the tube. The tube wasexamined after 5, 10, 15, 30, 45, 60 and 120 minutes. The occurrence ofdefinite agglutination visible to the naked eye against an illuminated darkbackground was recorded as a positive result. The following notation was used:

Agglutination within 5 minutes + + +„ in from 5 to 10 minutes ... + +„ „ 10 to 120 minutes ... +

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46 The Brucella Group

(6) Salt agglutination.

A 48-hour growth on an agar slope was suspended in distilled water, heatedto 60° C. for 1 hour in a water bath, and standardised to an opacity of 1000million B. coli per c.c. Equal quantities of the suspension and of varyingstrengths of saline were mixed in Dreyer's tubes, so that the final concen-trations of sodium chloride were 1, 2-5, and 5 per cent. The tubes wereincubated in a water bath at 55° C, and the results read after 24 hours. Thefollowing notation was used:

No agglutination ... ... ... ... —Agglutination in 5 per cent, saline ... +

„ „ 2-5 per cent, saline ... + +„ „ 1-0 per cent, saline ... + + +

(c) Acid agglutination.

The same suspension was used as that for salt agglutination. Equalquantities of the heated suspension and of Beniasch's (1912) lactic acid andsodium lactate solutions were mixed in Dreyer's tubes, which were thenincubated for 2 hours in a 37° C. water bath. Colorimetric estimations of theH-ion concentration of the nine different solutions employed gave resultssimilar to those theoretically anticipated (Table I). The tube containing the

Table I. pH. values of Beniasch's acid solutions.Tube 1 2 3 4 5 6 7

Estimated pH 4-77 4-46 415 3-85 3-55 3-26 2-96Observed pR 4-7 4-4 4-1 3-8 3-6 3-2 2-9

most alkaline mixture in which agglutination occurred was recorded, andthe following notation was used:

No agglutination, or agglutination in tubes 8 and 9 only —Agglutination in tube 7 +

„ tube 6 + +„ „ tube 5 or below + + +

(d) Agglutination with specific sera.

Two sera were used obtained from rabbits inoculated with a bovineabortus strain, K 25, of human origin from Denmark, and a paramelitensisstrain, Pm 2062, of caprine origin from Tunis. K 25 serum had a titre of1/5120, Pm 2062 of 1/640. A 48-hour agar growth of the organism to be testedwas washed off in 0-5 per cent, formolised saline, heated to 60° C. for 1 hour,and standardised to an opacity of 1000 million B. coli per c.c. Equal quantitiesof the suspension and of varying dilutions of the serum in 0-85 per cent, salinewere mixed, and incubated in a 55° C. water bath for 24 hours. The tubeswere then examined with a magnifying glass (x 4) against an illuminated

82-662-6

92-362*2

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S. R. PANDIT AND G. S. WILSON 47dark background, and the last tube was recorded in which definite agglutina-tion was visible. For purposes of tabulation a positive result was taken asthat in which agglutination occurred in a dilution equivalent to a quarter ofthe titre of the serum or more. With K 25 serum therefore agglutination indilutions of 1/1280-1/5120, with Pm 2062 serum agglutination in dilutions of1/160-1/640 was recorded as positive.

(e) Agglutination with absorbed specific sera.

K 25 serum was diluted 1/80 with 0-5 per cent, formolised saline, mixedwith an equal quantity of a heat-killed suspension of paramelitensis organismsstandardised to 3000 million B. coli per c.c, and incubated for 2 hours at37° C. The mixture was then centrifuged, and the supernatant fluid withdrawn.Pm 2062 serum was similarly absorbed with K 25 organisms. These absorbedsera were tested in the same way as the unabsorbed sera. Agglutination witheither serum up to one-quarter of the original titre, 1/1280-1/5120 with K 25serum and 1/160-1/640 with Pm 2062 serum, was recorded as a positive result.

The results obtained with the different groups of organisms are recordedin Tables II-V; Table VI is an abbreviated synoptic table comprising allstrains. It will be noted that the primary classification has been based on thethermo-agglutination test.

Table II. Porcine abortus strains.

Thermo-aggluti-nation

++ +

+ + +Totals

No.of

strains11001

12

No. agglutinatingwith salt

Nil11001

12

+ +00000

+ + +00000

No. agglutinatingwith acid

Nil00000

+20002

+ +50005

+ + +40015

Absorbed sera

K25only11000

11

Pm2062only

00011

Both00000

Unabsorbed sera

K 25only

70007

Pm2062only

00011

Both40004

+ +Totals

112

47

Table III. Bovine abortus strains.

Thermo- No.aggluti- of ,nation strains Nil

- 43 43

No. agglutinatingwith salt

112

47

No. agglutinatingwith acid

+ + + + + Nil10000

10

+22

100

23

Absorbed sera

Pm-, K 25 2062

Unabsorbed sera

+ + + + + only only Both only only Both43110

45

00011

43110

45

++ +

+ + +Totals

948

47

Table IV. Melitensis strains.

Thermo- No.aggluti- of ,nation strains Nil

26 26

No. agglutinatingwith salt

924

41

No. agglutinatingwith acid

Absorbed sera

+ + + + + + Nil0 0 0 18

Unabsorbed sera

^ PmK25 2062

+ + + + + + only only Both Neither only only Both

Pm, K 25 2062

l l

100

19

4427

17

15200

17

0128

11

7310

11

17200

19

1127

11

8621

17

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48 The Brucella Group

Table V. Paramelitensis strains.

Thermo-aggluti-nation

_

+ ++ + +Totals

No.of

strains100

3011

No

Nil10023

n4-in«i4-inr»

with salt

+00033

+ + +00044

+ +00011

TVrtli (J.

Nil10001

lif~i n atn

with acid

+00000

+ +00033

+ + \00077

Absorbed sera

K 25- only

10001

Pm2062only

00099

Both00011

Unabsorbed sera1

K 25only

00000

Pm2062only

00077

Both10034

Table VI. Abbreviated synoptic table comprising all strains.

Percentage agglutinating

Thermo-agglutination

_+ and + +

+ + +

No. ofstrains

811521

f

With salt0

1357

With acid+ + +

144781

With K 25absorbed

only8627

0

WithPm 2062absorbed

only0

2090

With both K 25and Pm 2062absorbed sera

or with neither145410

The following observations may be made:1. With a few exceptions the porcine and bovine abortus strains are non-

thermo-agglutinable; on the other hand nearly half the melitensis, and all butone of the paramelitensis strains react positively to the thermo-agglutinationtest. It will be noted, however, that whereas many of the melitensis strainsfall into the one and two plus group, all the thermo-agglutinable paramelitensisstrains fall into the three plus group. The thermo-agglutination test, it maybe remarked, depends on the presence of salt in the suspension, and is thereforeessentially a saline auto-agglutination test. If the organisms are washed threetimes in distilled water, and finally suspended in this medium, they remainstable, even after being subjected to heat at 95° C. for 2 hours and allowed tostand overnight at room temperature.

2. None of the porcine or bovine abortus strains is agglutinated by saltat 55° C, but six of the melitensis and all but three of the paramelitensisstrains show some degree of agglutination. It is apparent that the salt test at55° C, even with a strength of 5 per cent, sodium chloride, is a considerablyless delicate method of detecting differences in the stability of suspensionsthan the thermo-agglutination test at 95° C. in the presence of normal saline.Thus, taking all groups together, only fourteen out of the total 117 strains areagglutinated by salt at 55° C, while thirty-six respond positively to the thermo-agglutination test.

3. There is a general correlation between the thermo-agglutination test andthe acid agglutination test. The notation employed in recording the results ofthe latter test was of course quite arbitrary. Nearly all strains show somedegree of agglutination in the most acid tubes, but few, with the exception ofthose that are thermo-agglutinable, react in tubes lower than the fifth. FromTable VI it will be seen that of the non-thermo-agglutinable strains only

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S. R. PANDIT AND G. S. WILSON 49

14 per cent, show agglutination in the fifth tube; of the one and two plusthermo-agglutinable strains 47 per cent., and of the three plus thermo-agglutinable strains as many as 81 per cent, react in this tube.

4. Of the non-thermo-agglutinable strains 86 per cent, are agglutinated byan abortus serum absorbed with paramelitensis, but not by a paramelitensisserum absorbed with abortus; on the other hand, 90 per cent, of the stronglythermo-agglutinable strains are agglutinated by an absorbed paramelitensis,but not by an absorbed abortus serum; of the one and two plus thermo-agglutinable strains some agglutinate with an absorbed abortus, and some withan absorbed paramelitensis serum, while about half of them agglutinate withboth sera. With unabsorbed sera very much the same results are obtained,but the distinctions tend to be less sharp. That is to say, rather fewer strainsagglutinate with either the one or the other serum, while more agglutinatewith both sera.

DISCUSSION.

The term "paramelitensis" was introduced by Negre and Raynaud(1912 a, b) to denote a strain of melitensis that was sharply distinguished bydirect agglutination from the usual type. Burnet (1925), in studying twenty-eight strains of melitensis from human patients and from goats in Tunis,found that by agglutination and absorption of agglutinins they fell into twodistinct types, which he termed Type I and Type II. The two types weredistinguished in other ways. For example, Type I strains had a greateragglutinogenic power when injected into rabbits than Type II strains. Type IIstrains proved to be thermo-agglutinable, while Type I were not. Moreover,Type II strains were frequently agglutinated by normal human serum to atitre of 1/100 or 1/150, whereas Type I strains were rarely agglutinated bynormal serum. It may safely be deduced that Burnet's melitensis Type IIcorresponds to the paramelitensis of Negre and Raynaud. Of recent yearsseveral workers (for references see De Antoni, 1929) have found that certainBrucella strains are susceptible to agglutination by non-specific agencies, suchas heat, acids, formol, peptone, mercuric chloride, alcohol, and aniline dyes,and there has been a considerable measure of agreement that susceptibility toheat is a characteristic of the para strains. The reactions of individual strains,however, are not absolutely constant, and it has been found possible bycertain experimental procedures, such as repeated sub-culture in broth con-taining specific antiserum, to render a strain thermo-agglutinable (Favilli,1926; Burnet, 1928; Frendzel and Szymanowski, 1930). Hadley (1927) putforward the suggestion, which was endorsed by Ross (1927 b), that the normaltype of melitensis represents the smooth form, whereas the para type isessentially the rough form.

This suggestion receives confirmation from the results recorded in thepresent paper. Careful examination of the tables seems to indicate that thenormal smooth form of Brucella is non-thermo-agglutinable, is not agglutinated

Journ of Hyg. xxxn 4

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50 The Brucella Groupby salt, and reacts to about titre with a specific serum prepared against asimilar smooth strain, but not by a serum prepared against a para strain.On the other hand, strains, no matter what label they bear, which are readilyagglutinated by heat, which are often agglutinated by salt, and which reactwith a para serum but not with a serum prepared against a non-thermo-agglutinable strain, are to be regarded as rough strains.

If this interpretation is correct it appears that about 3 per cent, of thestrains labelled porcine and bovine abortus may be regarded as rough, whileanother 5 per cent, present characteristics intermediate between those ofsmooth and rough. To use the terminology of Sangiorgi (1927) and of Ross(1927 b), the rough strains of abortus may conveniently be referred to as"para-abortus." On the other hand, it will be observed that 21 per cent, of themelitensis strains examined are definitely rough, while 26 per cent, are ofintermediate type. Of the eleven strains labelled paramelitensis, seven aredefinitely rough, three are intermediate in type though approaching the roughmore closely than the smooth form, while one strain is almost entirely smooth.

These results suggest that melitensis strains have a greater tendency tobecome rough than strains of abortus. How far this is true it is impossible tosay. With other groups of bacteria it is known that prolonged cultivation inthe laboratory favours the smooth-rough transformation; and it may be thatin the collection of strains examined the melitensis type were on the whole ofless recent origin than the abortus strains. The higher proportion of roughstrains amongst the melitensis group may therefore be merely accidental,resulting from their longer period of cultivation outside the body, and may notindicate that melitensis strains are on the whole less stable than abortus strains.This possibility is difficult to exclude, because the data available are insufficientto afford an exact estimate of the average age of the strains. It does not,however, seem probable, because with the recently isolated strains, aboutwhich full information was available, there was a far greater tendency amongstthe melitensis group to become rough than amongst the abortus group. Onlyone of the abortus strains that had been isolated within the last five yearsshowed a slight degree of roughness, while twelve of the corresponding melitensisstrains were partially or completely rough. It is moreover the opinion ofFavilli (1927) that abortus strains change far less readily to the para type thanmelitensis strains.

In order to ascertain how readily the smooth-rough transformation couldbe brought about, an experiment was performed in which three strains ofporcine abortus, three of bovine abortus, and three of melitensis, all of com-pletely smooth type, were submitted to serial cultivation in heart extractbroth, the cultures being kept continuously in the 37° C. incubator, and sub-cultured into fresh medium every five days.

From time to time they were examined by the thermo-agglutination, acidagglutination, and absorbed serum tests. The results are given in Table VII;in order to economise space, many of the intermediate tests have been omitted.

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S. R. PANDIT AND G. S. WILSON 51

It will be noticed that two out of the three porcine strains became slightlythermo-agglutinable by the fifth passage, that all three of the melitensis strainsdeveloped some degree of thermo-agglutinability, while none of the bovinestrains did so. After fourteen sub-cultures one strain of porcine type remainedabsolutely smooth, while two had become almost completely rough; the threemelitensis strains had likewise become rough; of the bovine strains two hadbecome partially rough, while one remained smooth. The experiment wascontinued till twenty-four successive sub-cultures had been made, but nofurther change occurred; that is one of the porcine and one of the bovine strainsstill remained fully smooth. These results indicate that the passage of all threetypes from the smooth to the rough form may be brought about by simplecultivation in broth. The number of strains tested was too small to allow anydefinite conclusion to be made as to the ease with which this transformationcan be accomplished with the different types, but there is at least a suggestionthat the melitensis strains are rather less stable than the abortus strains.

Table VII. Results of serial passage at 5-day intervals in broth.At start 5th sub-culture 14th sub-culture

Pm Pm PmThermo Acid K 25 2062 Thermo Acid K 25 2062 Thermo Acid K 25 2062

Porcine 1 — + + — +Porcine 2 — + + — —Porcine 404 — + + + — +Melitensis L I — — + — + +Melitensis h 3 - - + - + +Melitensis Y 2 0 + - +Bovine Oxford — + + — —Bovine A m 1 — — + _ _ _ - ) _ _ _ _ - f - _Bovine R C V 4 2 - + + - - + + - + + + + + +

Finally, a few remarks may be made on the value of the different testsemployed in this work in distinguishing between smooth and rough strains.It has already been pointed out how much more delicate the thermo-agglu-tination test is than the salt agglutination test as ordinarily carried out at55° C. with varying concentrations of salt.

The acid agglutination test presents certain features of interest. Accordingto Ross (1927 a) its main virtue is to separate off melitensis and abortus onthe one hand from paramelitensis and para-abortus on the other. This viewreceives only a limited support from the experimental work recorded in thispaper. It is true that the majority of paramelitensis strains, though by nomeans all, are strongly agglutinated by acid, and it is equally true that themajority of non-thermo-agglutinable abortus and melitensis strains are not;but examination of the tables shows that there are a number of abortus andmelitensis strains which are strongly agglutinated by acid, yet which are non-thermo-agglutinable and are not agglutinated by a paramelitensis serum.A strain, therefore, which has none of the other properties of a para strain,may yet be highly susceptible to agglutination by acids. This conclusion seemsto accord with the results obtained by Favilli (1926), who found that thoughthe thermo-agglutination and peptone agglutination tests ran parallel, the

4-2

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52 The Brucella Group

lactic acid agglutination test had a wider range, affecting certain strains thatdid not respond to the other two tests.

Incidentally it may be noted that the majority of the non-thermo-agglutinable strains which agglutinate with acid belong to the porcine andbovine abortus types. This observation is remarkable in view of the resultsrecorded by Vercellana and Zanzucchi (1926) and Zanzucchi and Vercellana(1926). Using lactic acid, they found that strains of abortus were very muchless susceptible than strains of melitensis. They went so far as to make thestatement that a strain which is agglutinated by lactic acid in dilutions above1/300 is certainly melitensis, while one which is not agglutinated at all, orwhich is agglutinated only in low dilutions, is certainly abortus. It is notimprobable that many of the melitensis strains with which they worked wererough; if they had confined their observations purely to smooth strains, theirresults might have been different. Attempts have been made to confirm theconclusions of these workers by Graziosi (1926), Favilli (1926), Cerruti (1926,1927), and Vidal (1928), but without success. All these authors agree that theacid agglutination test is unreliable for differentiating between strains ofabortus and melitensis.

With regard to specific agglutination by smooth and rough sera, the resultsshow a high correlation with those obtained by the thermo-agglutination test.With the abortus and paramelitensis strains the correlation is almost perfect,but with the melitensis strains certain discrepancies are apparent. Forexample, from Table IV it will be seen that only seventeen of the twenty-sixnon-thermo-agglutinable strains are agglutinated to one-quarter titre or overby a smooth unabsorbed serum; of the remainder one is agglutinated only bya rough serum, while eight strains are agglutinated by both sera. Such aresult indicates that a proportion of non-thermo-agglutinable melitensis strainscontain both smooth and rough antigens. These strains are presumably com-mencing to become rough. If this interpretation is correct, agglutination bya rough serum may be regarded as an even more delicate test of detectingslight degrees of roughness than the thermo-agglutination test.

SUMMARY AND CONCLUSIONS.

1. Altogether 117 strains of Brucella, belonging to different types andisolated from different parts of the world, have been examined by the thermo-agglutination, salt agglutination, acid agglutination, and specific serumagglutination tests.

2. The results obtained by the thermo-agglutination and the serum agglu-tination tests are in close agreement; there is a fairly high degree of correlationbetween these tests and the acid agglutination test, and a rather lowercorrelation with the salt agglutination test.

3. Generally speaking, a strain which is highly thermo-agglutinable isfrequently agglutinated by salt, is usually agglutinated strongly by acid, andreacts to a paramelitensis, but not to an abortus serum.

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S. R. PANDIT AND G. S. WILSON 53

4. A strain which is moderately thermo-agglutinable is seldom agglutinatedby salt, is frequently agglutinated by acid, and reacts either with an abortusor a paramelitensis serum, or with both sera.

5. A strain which is not thermo-agglutinable is not agglutinated by salt,seldom reacts markedly to acid agglutination, and is generally agglutinatedby an abortus, but not by a paramelitensis serum.

6. There remain, however, a certain number of strains, particularly of theporcine and bovine abortus types which, though non-thermo-agglutinable,inagglutinable by salt, and reacting only with an abortus serum, yet showsome degree of acid agglutination.

7. Of the twelve porcine strains examined only one strain was stronglythermo-agglutinable; of the forty-seven bovine strains only two were stronglythermo-agglutinable, a further two showing a milder degree of thermo-agglutinability; of the forty-seven melitensis strains eight were strongly, andthirteen were moderately thermo-agglutinable; while of the eleven para-melitensis strains ten were strongly thermo-agglutinable.

8. These results are taken to indicate, in accordance with the suggestionmade by certain previous workers, that those strains which are non-thermo-agglutinable, are not agglutinated by salt, and are agglutinated by an abortusbut not by a paramelitensis serum, represent the smooth form, while thosestrains which are strongly thermo-agglutinable, are frequently agglutinatedby salt, and are agglutinated by a paramelitensis but not by an abortus serum,represent the rough form.

9. If this interpretation is correct it will be noticed that the great majorityof the porcine and bovine strains examined were of the smooth type, thatnearly half the melitensis strains were partially or completely rough, while allbut one of the paramelitensis strains were rough.

10. Whether melitensis strains have a greater tendency than abortusstrains to undergo the smooth to rough transformation it is difficult to saywith certainty, but the reports in the literature and the observations in thepresent paper render this probable.

11. By serial passage through broth at 5-day intervals, it is possible totransform smooth strains of all three types into the rough form. This trans-formation appears to occur more readily and to proceed further in a given timewith melitensis than with abortus strains; but since only three strains of eachtype were examined, the results may have been determined as much by chanceselection of strains as by any greater inherent tendency of the strains of themelitensis type to undergo variation.

12. It is clear that none of the tests employed suffices to differentiateindividual strains of abortus and melitensis. The thermo-agglutination testand the agglutination test with specific smooth and rough sera do, however,enable a differentiation to be made between smooth and rough strains ofall types.

13. In the present paper no attempt has been made to distinguish abortus

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54 The Brucella Group

and melitensis strains by specific agglutination and absorption tests. Thegeneral failure of workers hitherto to obtain any clear-cut serological distinctionbetween these types may possibly be due to the fact that many of the strainswith which they worked were either partially or completely rough. Since therough antigen seems to be more or less alike in strains of all types, it is clearthat its presence would tend to obscure any difference that might exist betweenthe smooth antigens of the different types. If such a difference does exist itis probable that it will be elicited only by a comparison of absolutely smoothstrains.

REFERENCES.BENIASCH, M. (1912). Zeitschr.f. Immunitdtsf. 12, 268.BUBNET, E. (1925). Arch. Inst. Pasteur Tunis, 14, 247.

(1928). Ibid. 17, 128.CEBBTJTI, C. (1926). Qiorn. di Batter, e Immunol. 1, 422.

(1927). Pathologica, 19, 216.D E ANTONI, V. (1929). Bol. Istituto Sieroterap. Milanese, 8, 651.FAVUXI, G. (1926). Lo Sperimentale, 80, 396.- (1927). Bol. Istituto Sieroterap. Milanese,^, 341.

FEENDZEL, J. and SZYMANOWSKI, Z. (1930). Zentralbl.f. Bakt. 117, 240.GRAZIOSI, A. (1926). Nuova Veterinarian, 306.HADLEY, P. (1927). J. Infect. Dis.40, 1.NEGKE, L. and RAYNAUD, M. (1912 a). C.R. Soc. Biol. 72, 791.

(1912 6). Ibid.72, 1052.Ross, G. R. (1927 a). J. Hygiene, 26, 279.

(1927 6). Ibid. 26, 403.SAKGIOBQI, G. (1927). Pathologica, 19, 3.VERCELLANA, G. and ZANZTJCCHI, A. (1926). Ibid. 18, 247.VIDAL, J. (1928). G.B. Soc. Biol.99, 1279.ZANZTTCCHI, A. and VERCEI*LANA, G. (1926). Pathologica, 18, 395.

(MS. received for publication 14. v. 1931.—Ed.)

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