Proposed standardization of the agglutination—adsorption test for leptospira

Bull. Org. mond. Sante 1971, 44, 795-810 Bull. Wld Hlth Org. Proposed Standardization of the Agglutination-Adsorption Test for Leptospira B. BABUDIERI1 The systematics of the Leptospira is based on the agglutination test and on the cross-agglutination-adsorption test. To obtain reproducible and comparable results these tests must be carried out by standard techniques, but the accuracy of the tests is in any case limited since immune sera prepared from animals do not always react uniformly. Moreover, the micro-organisms used in the tests have specific requirements and do not always develop properly in synthetic media. The various factors that influence the results of agglutination-adsorption tests were investigated and evaluated. Taking the results of these studies into account, a standardized method for agglutination-adsorption tests is proposed and described. The present systematics of the Leptospira is founded on the definition of the serotype that has been established and delimited on the basis of cross-agglutination-adsorption tests. It can be assumed that this reaction will remain valid in the near future, at least for systematic purposes that are intended to have a practical application. Thus there is a need to standardize the techniques for carrying out this reaction since the use of different techniques in various laboratories has often led to variable results, especially from the quantitative point of view. The first suggestions for a technique to be followed in carrying out the agglutination-adsorption reac- tion were formulated by Wolff & Broom (1954), and they were accepted, at least partially, by the Joint WHO/FAO Expert Committee on Zoonoses (1959). The report of this Committee stated, con- ventionally, that a residual agglutinating titre for the adsorbed serum with respect to the homologous strain, corresponding at least to 10% of the original titre-namely, the titre preceding the adsorption- must be considered significant. The Committee also suggested that the immune sera employed in the test should have an agglutinating titre for the homologous strain of at least 1: 3 000 and recom- mended that the adsorption should be carried out with an " adequate quantity " of leptospiral suspen- sion. However, the meaning of the term " adequate 1 Head, Microbiology Laboratory, Istituto Superiore di Sanita, Rome, Italy. quantity" has never been specified very clearly. Wolff & Broom (1954) implied that the residual post-adsorption titre for the adsorbing strain should not exceed 1% of the preadsorption titre if the amount is to be considered adequate and the results of the test are to be significant (unless the titre of the homologous strain is reduced below the accepted 10% limit by an " inadequate quantity " of antigen). Wolff & Broom (1954) employed suspensions of live leptospires, but other workers have preferred killed ones. The relation of immune serum to lepto- spires, the time of the contact, the temperature at which adsorption takes place, and the methods for the agglutination test subsequent to adsorption had not been defined at that time and they differed remarkably from one research worker to another. It is necessary to determine, on the basis of experi- mental studies, which factors may influence the results of adsorption tests and which factors have no practical importance. Thus, a series of experimental studies, taking into consideration the various factors involved in the reaction, is first described before a proposal is made for a standardized agglu- tination-adsorption test. IMMUNE SERUM The immune serum employed in these tests is usually obtained from rabbits. The species of ani- mal used to provide immune serum is not without importance; in fact, the antibody response may also 2692 -795- 6 B. BABUDIERI vary qualitatively from species to species. Studies with immune sera prepared from various mammals and from birds have shown, however, that these differences are not very frequent and, mostly, not very great. One of the differences worth noting is that immune serum prepared from rabbits inocu- lated with the serotypepomona does not contain any significant quantity of antibodies for leptospires of the Icterohaemorrhagiae serogroup. On the con- trary, the human anti-pomona serum agglutinates the leptospires of this serotype at a titre not much lower than that for the homologous type. Immunization of rabbits is usually achieved by intravenous injections of a culture of well-developed leptospires. The leptospires can be inoculated live or killed (in general, by the use of formol). Lepto- spires should not be killed by heating because the heat-labile antigen present in some strains would be destroyed. According to reports in the literature and to the tests that have been made (Addamiano, 1957) there is no appreciable difference in the anti- body response to the two types of antigen. Never- theless, it seems preferable to employ live leptospires, which mostly give a more intense antibody response. It must also be borne in mind that these leptospires, even the virulent ones, are not capable, except in few cases, of causing a fatal infection in the animal. The inoculation can be made with either the whole culture or with a suspension of leptospires, previously washed by centrifugation, in saline solu- tion. From a theoretical point of view, the latter method appears preferable; it has, in fact, been shown by Addamiano (1957) and Castellani (1969) that in a leptospire culture medium soluble antigens, less strictly specific than those present in the bodies of the leptospires, are present in limited quantities. It is obvious, however, that these antigens originate from the lysis of the spirochaetal bodies and that even if they are not inoculated with the culture medium, they will be freed when the leptospires dissolve in the rabbit's tissues. The technique of immunizing a rabbit may be varied without causing any difference in the quali- tative character of the antibody response. It is generally suggested that the second injection should be made 7 days after the first one, and the third after a further 3 days. After 3 more days a bleeding test is made and the agglutinin titre of the serum is checked. If the titre is already high, the animal may be bled; otherwise, the process of immunization should be continued. Before the immunization of the rabbit is begun, it is advisable to make a blood test to check the possibility that antibodies, both for the strain to be employed for immunization and for other strains to be used in the adsorption tests of the immune serum, may already be present in the serum The immune serum, after being separated from the coagulum by centrifugation, should be filtered if its sterility is in doubt. It should then be poured into vials or into hermetically closed containers and preserved at a low temperature (preferably at -60°C) or lyophilized. The agglutinating titre of the immune serum against the homologous strain is theoretically of no great importance but for practical reasons it is advisable that it should attain a value of at least 1:10 000. Thus, the titre will still be high enough after the dilutions following adsorption to permit a satisfactory determination of the residual value. This factor is particularly important whenever, as sometimes happens, the immune serum gives a very much lower titre with the strain employed for adsorption than it does with the homologous strain. It should be pointed out also that immune sera at very low titres (1:1.000 or less) often contain a proportionally much larger quantity of paraspecific antibodies than that in an immune serum at a higher titre (Addamiano, 1957). Consequently, sera whose titres are not very high should be rejected. From studies made by Addamiano (1957) and Alexander (unpublished data) it appears that with leptospires a phenomenon frequently observed in immune sera-namely, reactivity against other micro-organisms-does not occur or, more precisely, that in sera at very high titres there is a relatively high proportion of paraspecific and aspecific anti- bodies. Another problem is whether the immune serum should be prepared from a single rabbit or from a number of animals. The quantitative response may vary markedly from one animal to another but the qualitative response of strains having a certain anti- genic affinity with the strain employed for the immu- nization does not usually show any significant variation from animal to animal. The ratios between titres for the immunizing strain and those for the strains belonging to related serotypes are generally constant but in isolated cases may vary within certain limits (1:10). For example, a 1:10 variation has occurred in 1 rabbit out of 10 immunized with the serotype patoc, and only with regard to the strain Sao Paulo. Therefore, the use of a single immune serum should be sufficient in most cases, but variable results may occur if there are more antibodies for the 796 STANDARDIZATION OF THE AGGLUTINATION-ADSORPTION TEST FOR LEPTOSPIRA particular leptospire strain in the serum sample before being collected to immunize the animal. However, whenever possible, especially if the titre of the serum after adsorption is close to the limit of significance (10%), it is advisable to repeat the adsorption with an immune serum prepared from a second rabbit. This method is statistically more valid than that using a pool of two or more immune sera for adsorption. The test, when repeated on more than one rabbit, is more likely to indicate any errors made in the course of immunization or during the subsequent check of the immune serum than to reveal qualitative differences in the antibody res- ponse, though generally there are none. Some recent findings lead us to suspect that different lines of rabbits might give qualitatively different immunological results with the same anti- gen. This possibility has been proved for other species of animal (the agglutination of strain Patoc 1 by some cattle sera positive for pathogenic leptospires but not by other sera with the same antibody pattern, for example) but is only suspected in rabbits. The use of pooled immune sera from several rabbits cannot avoid this inconvenience because all the rabbits from the same laboratory will probably belong to the same breeding line and will therefore have the same immunological reactivity. Only the repetition of the tests in different laboratories could provide greater reliability in this respect. LEPTOSPIRES Culture medium It has generally been stated that the composition of the medium in which leptospires are developing influences neither their antigenic constitution nor their agglutinability (Borg-Petersen & Fragraeus, 1949). As far as agglutinability is concerned, this opinion can no longer be supported. Castelli (1965) has observed that certain strains belonging to the poi serotype cultivated in some batches of Korthof's medium for several passages show a noticeable increase in agglutinability by antileptospiral anti- bodies, even if the latter are not strictly specific. In some cases, these strains have been agglutinated at a titre of 1: 10 000 or more, while the same strains cultivated in another batch of the same medium were not agglutinated at all, or only at a much lower titre (1: 100). Similar behaviour has been observed also in other strains of leptospires belonging to the serotypes bratislava and grippotyphosa and in some water leptospires. In addition, Mazzonelli (unpublished data) has observed that some leptospiral strains (Pomona, Poi, M 20, Patoc 1) cultivated for at least 5 passages in a medium containing from 0.05% to 0.25 % of potassium chloride are agglutinated by the homologous immune sera at a titre 5-7 times that reached by the same strains cultivated in the same medium containing sodium chloride instead of potassium chloride. Kujumgiev (1964) has reported that the addition of capsaicin to the culture medium strongly increases the agglutinability of many leptospiral strains. Besides these differences of a quantitative nature, others have been observed that qualitatively affect the antigenic structure of leptospires, especially if synthetic culture media are used. Thus, Stalheim (unpublished data) observed that in some lepto- spiral strains cultivated in Ellinghausen's synthetic medium (Ellinghausen & MacCullough, 1965), some antigens are missing and he believed this to result from the absence of some unidentified growth fac- tors from the medium, the presence of which is required for the synthesis of the antigens. The quantitative differences of agglutinability men- tioned above have little importance in the technique for the adsorption of antibodies, but the same cannot be said for the qualitative differences, the occurrence of which, in relation to the use of different media, even synthetic ones, cannot be denied. Consequently, the employment of a standard medium in the adsorption tests for the antibodies appears to be indispensable. In theory, it would be preferable to choose a synthetic medium with a constant composition. Unfortunately, for the rea- sons just mentioned, and also because some lepto- spiral strains grow in the synthetic media at present available only with great difficulty, it is necessary, at least for the time being, to avoid the use of syn- thetic media. Live or killed antigen Wolff & Broom (1954) suggested the use of live leptospires for adsorption; other authors have pre- ferred to use dead leptospires, generally killed with formol. The employment of killed leptospires has two advantages. One advantage is that the risk of infection is avoided, the other is that there is no contamination of the concentrated suspension of leptospires employed for the adsorption. Such con- tamination readily occurs when the cultures are centrifuged and when the sediments are collected, especially if the adsorption is not carried out imme- 797 B. BABUDIERI diately. Even a limited contamination of the lepto- spiral suspension may lead to the subsequent develop- ment of the contaminating organism during the adsorption when the suspension is placed in contact with the immune serum. The problem that must be taken into considera- tion, however, is whether the formol added to the leptospiral suspension affects the antigenic structure so that killed leptospires give results that differ from those obtained with live leptospires. In order to check this possibility the following tests were made. Two samples of anti-Ballico serum (serotype aus- tralis) with an agglutinating titre of 1: 5000 were adsorbed with equal quantities of a concentrated suspension of the strain Esposito (serotype bratis- lava). In one test the leptospires were live, in the other they had been killed with 0.05% formol. The adsorbed sera were then tested with cultures of the strains Ballico and Esposito, as well as with other strains mostly belonging to the serogroup Australis -namely, strains Gastaldi, Jalna, 1-23, Hebdoma- dis C, Saigon, Munchen C 90, Jel Bratislava, and Fudge. No significant quantitative or qualitative difference were observed in the agglutination titres obtained with the sera adsorbed with either live or with killed leptospires. Similar results were obtained by repeating these tests with an anti-Ictero No. 1 serum adsorbed with the strain RGA, and with an anti-Arborea serum adsorbed with the strain Mus 29 (serotype ballum), as well as with immune sera against several strains belonging to the biflexa complex. Addamiano (1957) studied 18 rabbits immunized with the serotype autumnalis, employing a live anti- gen for one group and a formolized antigen for the other, and found that the average homologous titre of the first group was 1: 34 500 and that of the second group was 1:33 600. That is, no significant difference was observed between the two groups. Kmety & Sulzer (1970) also carried out 52 adsorp- tion tests with equal quantities of live and formo- lized leptospires and residual titres were determined for the homologous strain. In 19 tests, the titres were the same in all sera treated with the two types of antigen; in 2 tests the titres were higher when the serum was adsorbed with formolized leptospires; in 32 tests the titres were lower by 1, 2, or even, in two tests, by 3 steps when the serum was adsorbed with formolized leptospires. Kmety suggested that the formol might have a harmful effect on the immune serum employed for the agglutination test, but this hypothesis has not been confirmed experimentally. Kmety did not specify the concentration of formol used in the tests, but it was probably higher than that used by the present author (0.05°/), which seems to have no harmful effect on the serum anti- bodies. One more phenomenon should be considered. White & Simpson (1965) observed by electron- microscopy that formol treatment causes irregular breakages of the leptospiral membranes. These investigations have been repeated and extended by Castellani (1969), who showed that as a consequence of these breakages small quantities of antigens from deeper parts of the leptospires flow into the sur- rounding liquid. However, this effect of formol treatment should not influence the results of the adsorption tests, especially if a suspension of live leptospires is used later to test the adsorbed serum. In fact, the agglutination-adsorption test, and the systematics of the Leptospira generally, is based on the pattern of surface antigens and not of the genus- specific deeper antigens. In the study of 18 rabbits made by Addamiano (1957) no significant difference was observed in the titre of paraspecific agglutinations with immune sera prepared with live or formolized leptospires. Consequently, it seems proper, and even advisable, to use a killed antigen in the adsorption tests. A very low concentration of formol (0.05 %) should be employed to kill the leptospiral culture so that dena- turation of the anti-leptospiral antibodies is, as far as possible, avoided. Quantity of antigen As already mentioned, the Joint WHO/FAO Expert Committee on Zoonoses (1959) recommended that the adsorption should be carried out with an " adequate amount " of leptospiral suspension; this amount is now almost generally interpreted as being the quantity capable of adsorbing about 99% of the antibodies for the adsorbing strain from the serum. For this reason, also, it is advisable that the immune serum should have a titre of at least 1: 10 000 in order to facilitate the determination of 1 % of the original titre. In theory, the optimum result would be obtained with the use of an amount of antigen that would bind the least trace of the corresponding antibodies. This result can be achieved by employing a small excess of antigen but it is necessary to make certain that no disadvantage is attached to this method, e.g., some other antibodies might also be adsorbed. It was therefore considered important that the 798 STANDARDIZATION OF THE AGGLUTINATION-ADSORPTION TEST FOR LEPTOSPIRA specificity of the adsorption should be maintained within certain limits. In order to investigate this point, the following tests were carried out: 1 litre of a culture of the strain Pavia 1 (serotype bataviae) was concentrated by centrifugation and the sedi- ment was suspended in saline to a total volume of 5 ml. A 1-ml quantity of this suspension contained over 1 x 1010 leptospires. To a series of test-tubes, each containing 0.1 ml of immune anti-canicola serum (strain Hond Utrecht IV), 0.5 ml, 1.5 ml, and 3 ml of formblized leptospiral suspension were added, and formolized saline was then added to bring the volume of liquid in each test-tube to 3.1 ml. It should be pointed out here that the sero- types canicola and bataviae have no antigenic affinity. The titre of the control immune serum diluted 1: 31 with saline was 1: 3 200 for the strain Hond Utrecht IV and 1:1 600 for the strain Patane. The latter strain also belongs to the serogroup Canicola (serotype broomi). After adsorption with 0.5 ml of leptospiral suspension, the titre of the serum was found to be unchanged for both Hond Utrecht IV and Patane. After adsorption with 1.5 ml of lepto- spiral suspension the titre was still the same but in the highest dilution the rate of agglutination was lower. In the third test, i.e., adsorption with 3 ml of suspension, the titre fell to 1: 800 for both strains. In another test, an immune anti-Ictero No. 1 serum (with a titre of 1: 32 000) was adsorbed with different amounts of live leptospires of the serotype bataviae (strain Pavia 1). Each dose of antigen was suspended in the same volume of saline, i.e., 0.15 ml of immune serum + 1.5 ml of leptospiral suspension. There was no antigenic affinity between strain Ictero No. 1 and serotype bataviae. Seven adsorption tests have been performed with, respectively, 1 x 109, 2 x 109, 4 x 109, 8 x 109, 16 x 109, 32 x 109, and 64 x 109 leptospires. The agglutination titre of the immune serum remained unchanged in the series up to the adsorption with 32 x 1O0 leptospires and dropped to 50% of the original value in the adsorption test with 64 x109 leptospires. The agglutination titres for many other members of the Icterohaemorrhagiae serogroup fell to 50% after adsorption with 64 x 109 leptospires. In further tests, an anti-Hond Utrecht IV serum was adsorbed with 1 ml and 7 ml of the concentrated suspension of a strain of saprophytic leptospires (strain AM 3). The serum titre remained unchanged in titration against strains Hond Utrecht IV and Patane. These tests demonstrate that very large numbers of leptospires brought into contact with an immune serum for a leptospire serotype with an entirely different antigenic pattern may, in some cases, adsorb, non-specifically, some of the anti- bodies in the serum. The rate of adsorption is not constant, but varies with the strain of leptospire. Variation may also occur when the adsorbed serum is tested either with homologous or with related strains. In the first test the loss was 75% for strain Hond Utrecht IV and 50% for strain Patane. Some other tests have been performed in condi- tions closer to those likely to occur during a normal adsorption test, especially with strains belonging to the same serogroup. An immune anti-lora serum (strain Gastaldi) was adsorbed with serial doses of strain Esposito; these strains both belong to the same serogroup but not to the same serotype. In a test series, 3x108, 7x108, 1.3x109, 2.7x10', 5.3 x 109, 1 x 1010, and 2.1 x 1010 leptospires were added to 0.1-ml quantities of serum. The mixtures were then diluted with formolized saline to a stan- dard volume. The control serum at the same dilu- tion agglutinated strain Gastaldi at a titre of 1: 3 200 and strain Esposito at a titre of 1: 400. After adsorption, strain Esposito was agglutinated only in the test-tube containing 3 x 10 leptospires and not in any of the others, even at a titre of 1: 10, whereas the titre for strain Gastaldi remained practically unchanged. This means that the leptospires had completely adsorbed the antibodies corresponding to the antigens that are common to strains Esposito and Gastaldi. Although strain Esposito adsorbed relative- ly little of the antibodies specific for strain Gastaldi the titre for the latter strain fell to 1:800 in the tube containing 2.7 x 109 leptospires and remained at this level in the rest of the series. Two other similar tests were carried out, two anti-B Sh (serotype semaranga) immune sera being adsorbed with serial doses of leptospires of strain Patoc 1 (serotype patoc). The two strains belong to the serogroup Semaranga. The sera were tested also with other strains belonging to this serogroup. The first test was performed with formol-killed leptospires, doses of 1.4x 109, 2.7 x 109, 5.5 x 109, 1.1 x 1010, 2.25 x 1010, 4.5 x I00°, and 9 x 1010 being added to test-tubes containing 0.1 ml of immune serum. The various amounts of antigen were suspended in the same volume of saline. The titre of the serum adsorbed with 1.1 x 1010 leptospires, which in the control test-tube was 1:1 900 for strain Patoc 1, fell to a value of less than 1 :10. For 799 B. BABUDIERI _ Fig. 1. Agglutination titres of anti-Munchen C90 serum adsorbed with strain Ballico. - Ballico 1/23 Munchen C90 Gastaldi - - Esposito Jalna strain B Sh, the titre was reduced from 1:2 500 to 1: 240. In the test-tubes containing greater numbers of leptospires the titres fell to 1: 160, 1:120, and 1: 30. Live leptospires were used in the second test and the doses ofantigen were 2.7 x 109, 5.4 x 109, 1.1 x 1010, 2.2 x 1010, 4.4 x 1010, and 8.8 x 1010 leptospires, in all cases suspended in the same amount of saline. The results were similar to those obtained in the first test. The serum titre, which was originally 1:1 600 for strain Patoc 1, fell below 1:10 after adsorption with 1.1 x 1010 leptospires. In this test also, the serum adsorbed in the test tubes containing higher doses of antigen, lost a further fraction of its original titre against the other members of the Semaranga serogoup. The results of the tests described here are shown graphically in Fig 1-4. Titres given on the figures are for diluted sera. The experiments demonstrate that when the dose of leptospires is progressively increased above the minimal required to adsorb all antibodies specific for the strain employed in the test, the titre of residual antibodies for the homo- Hebdomadis C --- Saigon Jel Bratislava 0 Fudge logous strain continues to fall. This effect is some- times so marked that the taxonomic relationships between the two strains under investigation are completely obscured. Fig. 2 and 3 show that by increasing the dose of leptospires above the minimum required for total adsorption of the respective antibodies, the serum titre against the homologous strain falls still lower; a result already seen in previous tests. In some cases, as shown in Fig 2, this reduction may have a parti- cular significance from a systematic point of view. In fact, when anti-Patoc 1 antibodies were eliminated the level of anti-B Sh antibodies was still 10% of the original value, whereas the level dropped to 1 % when the adsorbing dose of strain Patoc 1 was in excess. All these tests confirm that very high doses of leptospires may in some, but not all, instances adsorb non-specifically heterologous antibodies brought into contact with them. From Fig. 1-4 it can also be seen that the adsorption of agglutinins corresponding to the different leptospiral strains 800 STANDARDIZATION OF THE AGGLUTINATION-ADSORPTION TEST FOR LEPTOSPIRA Fig. 2. Agglutination titres of anti-Gastaldi serum adsorbed with strain Esposito. Ballico -.-.- 1/23 Munchen C90 &--- Hebdomadis C Gastaldi _ _ Saigon - - Esposito JeS Bratislava .. ....Jalna 0 Fudge 1:2~ 1:1 1; I, 90 WHO 10187 5.5 11 No. of I eptospi res X 10-9 Fig. 3. Agglutination titres of anti-B Sh serum adsorbed with formolized strain Patoc 1. Patoc 1 - .- Madida -_-___- B Sh 060*6*06 Sao Paulo - Lublin Veldrat Semarang 173 801 B. BABUDIERI Fig. 4. Agglutination titres of anti-B Sh serum adsorbed with live strain Patoc 1. Patoc 1 Sao Paulo BSh Veldrat Samerang 173 M. Valerio does not follow a parallel pattern. Consequently, if adsorption is stopped at different levels, a difference of ratio between the various strains is observed that can sometimes be quite considerable. It can therefore be inferred that an " adequate quantity" of leptospires for adsorption is that quantity that does not adsorb heterologous anti- bodies to any significant extent. In practice, this means a quantity that does not give a residual value above approximately 1 % of the pre-adsorption titre of the absorbing strain. This value has no signifi- cance for taxonomic purposes but it proves beyond doubt that the dose employed was not excessive. If this principle were adopted, it would be con- venient to establish the serum titre for adsorption tests at 1: 10 000. If the serum has a titre as low as 1: 3 000, it becomes difficult to estimate 1 % of the original titre, i.e., a titre of 1: 3. ADSORPTION TECHNIQUE Adsorption in one or more steps In order to determine whether there were any differences in the final results when equal quantities of leptospires were added to serum all at once or in three steps, the following adsorption tests were carried out. Anti-Patane serum was adsorbed with strain Bianchi 1 (serotype icterohaemorrhagiae), anti-Hond Utrecht IV serum with strain Patane (serotype broomi), and anti-Bianchi 1 serum with strain Hond Utrecht IV (serotype canicola). Two series of test tubes were prepared, each containing 0.1 ml of one serum. To the first test tube in each series 0.8 ml of leptospiral suspension was added, and 0.2 ml to the second. The test tubes were kept at 37°C. After 24 hours 0.3 ml of suspension was added to the second test tube of each series, and after 48 hours a further 0.3 ml was added. The titres of sera that had been adsorbed in three steps were then deter- mined, taking into consideration the various dilu- tions. The titres of sera adsorbed in a single step were determined at the end of the tests. The results are shown in Table 1. # Two more series of tests were carried out in the same way; in the first series, anti-Munchen C 90 serum was adsorbed with strain Ballico; in the second series, anti-Ballico serum was adsorbed with strain Esposito. The results are given in Table 2. 802 STANDARDIZATION OF THE AGGLUTINATION-ADSORPTION TEST FOR LEPTOSPIRA Table 1. Results of adsorption tests to determine whether titres are affected by mixing leptospires and serum all at one time or in several steps Titre Patane UeHond Bianchi Anti-Patane serum adsorbed with strain Bianchi 1 Control serum 1: 400 1: 400 1 :50 Adsorbed with 0.8 ml of suspension 1:400 1 :400 <1:10 Adsorbed with 0.2 ml of suspension 1:400 1:400 <1:10 Adsorbed with 0.2 + 0.3 ml of suspension 1 :400 1:400 <11:10 Adsorbed with 0.2 + 0.3 + 0.3 ml of suspension 1:300 1:400 <1:10 Anti-Hond Utrecht IV serum adsorbed with strain Patane Control serum Adsorbed with 0.8 ml of suspension Adsorbed with 0.2 ml of suspension Adsorbed with 0.2 + 0.3 ml of suspension Adsorbed with 0.2 + 0.3 + 0.3 ml of suspension 1:400 <1 :10 <1:10 <1:10 <1:10 1:400 1:200 1:200 1:100 1:100 1:10 1<1:10 Anti-Bianchi 1 serum adsorbed with strain Hond Utrecht IV Control serum Adsorbed with 0.8 ml of suspension Adsorbed with 0.2 ml of suspension Adsorbed with 0.2 + 0.3 ml of suspension Adsorbed with 0.2 + 0.3 + 0.3 ml of suspension <1:10 <1:10 <1 :;10 <1:10 <1 :10 1:10 <1:10 <1:10 <1:10 <1:10 1:800 These results indicate that the addition of the same amount of leptospires to the serum in two or three steps gives results that are not very different from those obtained when the leptospires are added in a single step. The multiple-step method may give a slightly higher adsorption of antibodies, but not so much higher that titres are significantly different from those obtained by the other method. Consquently, multiple-step adsorption may be adopted for per- forming the adsorption test with the optimum quantity of leptospires, that is, the dose that pro- duces a residual titre of approximately 1 %. Characteristics ofthe mixture ofserum and leptospires Quick and slow mixing. Tests were made to see whether different results were obtained when sera and leptospires were mixed quickly and slowly (drop by drop). Anti-Munchen C 90 serum was adsorbed with strain Ballico and tested with 10 strains of the Australis serogroup. No differences were noted. However, Kmety & Sulzer (1970) reported that drop-by-drop addition of immune serum to the antigen permits better adsorption of the antibodies, especially if the serum is added in two or three steps at 10-minute intervals (Danysz's phenomenon). Persistence ofresidual antibodies in frozen mixtures of serum and leptospires. Residual titres of two adsorbed sera (anti-Munchen C 90 adsorbed with strain Ballico and anti-Ballico adsorbed with strain Esposito) were tested directly after adsorption and after storage at - 20°C for 3-4 months. No difference in titre was observed. Temperature and reaction times. Some workers keep the mixture of immune serum and leptospires at room temperature, others maintain the mixture at 37°C. Although there do not appear to be any appreciable differences in the results, it seems pre- ferable for constant and reproducible results to use a standard temperature. As far as the reaction time is concerned, this can be considered complete after 1-2 hours incubation at 37°C, but the mixture can be kept at this temperature overnight without any change in the results. Determination of the optimal quantity of leptospires to be employed in the adsorption test From a practical point of view it would be very convenient if one could determine beforehand the dose of leptospires required to reduce the titre of a serum to approximately 1% of its original value. Since the adsorption test is a surface reaction, the factors to be considered for the determination of the required dose should be the total surface area of the leptospires employed in the test as well as the anti- body titre of the serum. However, considering the particular morphology of leptospires, if the total surface area were replaced by the total length of the leptospires the inaccurancy would be very small. Consequently, the calculation could be made by counting the leptospires and determining their average length, according to the procedure described by Borg-Petersen & Fagraeus (1949) in a paper on the standardization of the agglutination test. To determine the average length of a leptospire is, however, a rather complicated operation. If the 803 B. BABUDIERl Table 2. Results of adsorption tests to determine whether agglutination titres are affected by mixing leptospires and serum all at one time or in several steps Agglutination titres for the following strains: Esposito Gastaldi Jalna Erin.ea Hebd.C.b Ballico Saigon Munch.c J.B.d Fudge Anti-Munchen C 90 adsorbed with strain Ballico Control serum 1:800 1:400 1:800 1:800 1:800 1:100 1:100 1:1 600 1:1 600 1:100 Adsorbed in 1 step 1:800 1:50 1:200 1:400 1:200 <1: 10 <1:10 1:800 1:1 600 <1: 10 Adsorbed in 2 steps 1:800 1:100 1:200 1:400 1:200 <1: 10 <1:10 1:800 1:800 <1:10 Anti-Ballico serum adsorbed with strain Esposito Control serum 1:100 1:1 600 1:400 1:100 1:800 1:700 1:800 1:400 1:300 1:800 Adsorbedinlstrep <1:10 1:1600 1:400 <1:10 1:700 1:400 1:800 1:400 <1:10 1:400 Adsorbed in 2 steps |<1:10 1:1 600 1:400 <1:10 1:700 1:400 1:700 1:300 <1:10 1:400 a Erinaceus europeus. b Hebdomadis C. same culture that has been used to determine the titre of the serum to be adsorbed is used for the adsorption itself, variations in length should affect both reactions equally, and in that case it would be necessary to consider only the number of leptospires, and, of course, the serum titre. Another method that could be used is the nephelo- metric measurement of the leptospiral suspension. This type of determination, which permits a deter- mination of the leptospiral mass but not the surface area, gives reliable results only when there are no saline precipitates or other impurities in the sus- pension that could interfere with the reading. In order to find out whether a relationship exists between the serum titre and the number of lepto- spires required to reduce the serum titre to approxi- mately 1% of the original value, a series of tests was carried out with several sera and strains. In these tests, the serum titres are those of undiluted serum, the adsorption of which has been performed by adding 0.9 ml of leptospiral suspension to 0.1 ml of serum. The results are shown in the following tabulation: Serum titre of the absorbing strain No. of leptospires required to adsorb 99%/ of the antibodies 1: 100000 3.2x 101 1: 19 000 7 x 109 1: 10000 3x109 1: 4 000 1 x 10 A rather constant ratio between the serum titre and the number of leptospires required to saturate the serum emerges clearly from there results, and the number may be determined by applying the following formula: N = (3.1 0.5) x T x 105, T being the titre of the serum. The value obtained in this way should be considered as an indication only. The volumetric ratio between the serum and leptospires The volumetric ratio between an immune serum and a leptospiral suspension may differ according to the techniques employed; in any case, these differ- ences cannot influence the result of a test. Some workers use a 1: 1 ratio but the present author pre- fers to add 0.1 ml of serum to 0.9 ml of leptospiral suspension. This method is considered preferable for the following reasons. The amount of serum needed is very small and if its titre is not less than 1 : 10 000 the determinations of the residual titre can very easily be made. The leptospires, after being separated by centrifugation, can easily be resuspended in saline to a total volume of 0.9 ml for addition to the serum, and the density of the suspension, considering the limited volume of the serum that is to be adsorbed, is not too high. If the serum to be adsorbed has a titre below 1: 10 000, a larger volume can be added to the leptospiral suspension; for instance, 0.2 or 0.3 ml may be added instead of 0.1 ml. This method offers the practical advantage of employing a limited quantity of immune serum and of permitting a suspension of centrifuged leptospires to be suspended in a convenient volume of formo- lized physiological saline. c Munchen C90. d Je2 Bratislava. 804 STANDARDIZATION OF THE AGGLUTINATION-ADSORPTION TEST FOR LEPTOSPIRA AGGLUTINATION AFTER ADSORPTION After the adsorption is complete, the mixture of immune serum and leptospires is centrifuged at a low speed and the supernatant liquid is collected. The adsorbed serum is diluted in an arithmetic series of dilutions and, after a check has been made that the titre is not higher than 1 % of the pre- adsorption titre for the adsorbing strain, agglutina- tion is carried out, using the standard technique, the starting serum dilution being taken into conside- ration. It is advisable that when the mixture of immune serum and leptospires is prepared for adsorption, another mixture should be prepared with the same proportions of immune serum and physiological saline, possibly with the addition of formol but without leptospires. This diluted serum should be kept under the same conditions and for the same length of time as the mixture of serum and lepto- spires, and should be used for control agglutination tests simultaneously with titrations of the adsorbed serum. An arithmetic series of serum dilutions gives more precise results than a geometric series. The latter can be considered to give valid results only it the residual titre of the adsorbed serum is 0, or very nearly 100%. Kmety (unpublished data, 1965) proposed that, as a more exact definition of the 10% limit, this limit should be considered to have been reached when the titre of the serum adsorbed by the homo- logous strain is at least 5 steps higher than the resi- dual titre of the serum for the adsorbing strain. For example, if an immune serum agglutinates the homo- logous strain at a titre of 1: 10 000 and the strain employed for the adsorption at a titre of 1: 5 000, and if after the adsorption the latter strain is still agglutinated at a titre of 1: 50, the adsorbing strain should be considered as belonging to a different serotype only if the adsorbed serum will agglutinate the homologous strain at a titre of at least 1:1 600 (i.e. 5 steps). This value does not differ much from 1: 1 000, the value corresponding to 10% of the pre- adsorption titre. However, if, as may happen, the homologous strain is agglutinated at a titre less than that of a heterologous strain, the agreement between the two methods of calculation no longer exists. If, for example, the homologous strain is agglutinated at a titre of 1: 10 000 and the heterologous strain at 1: 20 000, the 10% fraction of the titre for the homologous strain always remains 1: 1 000; the " 5 steps " criterion, starting from the value of 1: 200, corresponding to 1% of the pre-adsorption titre, leads to a titre of no less than 1: 6 400. This result is very different from the previous one. It may also happen that three different lines of the same strain are agglutinated by the same immune serum at different titres; in this case, the application of the " 5 steps " criterion gives inconsistent results. An example of this effect may be seen in results obtained by adsorbing with strain RGA an anti- Ictero No. 1 serum prepared at Bratislava with the heated Paris line of this strain. The serum was tested before and after adsorption with three lines of the strain Ictero No. 1, each being different for agglutinability. The results are shown in the follow- ing tabulation. Strain Pre-adsorbtion titre RGA 1: 10000 Icterohaemorrhagiae: Rome 1 : 20 000 Paris 1: 40 000 Antwerp 1: 10 000 Post-adsorbtion titre 1: 50 1: 1000 1: 2000 1: 500 It must be pointed out that with the three lines of the strain Ictero No. 1 the post-adsorption titres represent the same proportion (5 %) of the pre- adsorption titres. Since the 10% level has not been reached in any of the titrations, it can be concluded that the strains RGA and Ictero No. 1 may belong to the same serotype. If the " 5 steps" criterion had been adopted, implying, for significance, a titre of 1:1 600, this level would have been exceeded by the Paris line but not by the others and it would have appeared that only the Paris line of strain Ictero No. 1 belonged to a new serotype; this result would obviously be absurd. Consequently, when it is intended to maintain the conventional significant limit at 10%, it seems to be necessary to avoid using the criterion of 5 steps. In order to avoid excessive intervals between titres, it is advisable that the sera employed for post-adsorption-agglutination tests should be diluted by simple redoubling and not in a geometric series. It is not necessary to discuss the factors that can influence the results of adsorption tests since Borg- Petersen & Fragraeus (1949) have already very accurately investigated them. The factors are essentially the mass of the antigen (for practical purposes the total number of leptospires multiplied by their average length) and the age of the culture. According to Borg-Petersen & Fagraeus (op. cit.), 805 B. BABUDIERI Table 3. Effect of aging on titre of leptospiral cultures Age Titres of Patane strain against: Titres of Hond Utrecht IV strain against: Titres of Bianchi 1 strain against: of Anti-HondAn____ _ _ -_ _ __ _ __ _ _ __ _ _ __ _ __ _ _ __ _ _ culture Anti- Patane Anti-Hond Anti- Anti-Patane Anti-Hond Anti- Bianchi Anti-Patane iAti- Hond Anti- Bianchi(days) serum Utrecht IV Bianchi serum Utrecht IV serum serum Utrecht IV serum __ _ _ _ ~~~~~serum serum _ _ serum _ _ __ serum _ _ 69 1:50 000 1:2000 c 1 :50000 1 :20000 1 :100 - - - 62 1:50000 '1:2000 1 :50000 c-1:20000 1 :100 1:10000 1:2000 55 1:50000 -1:2000 1:50000 -'1:20000 c 1:200 1:10000 1: 2000 48 1:50 000 1:2 000 1:50 000 1:20 000 Y1:200 1:10 000 1:2 000 41 1:50000 1:5000 -1:50000 - 1:20000 1:200 -1:10000 1:2000 35 1:50 000 1:5 000 1:50 000 1:10 000 1:200 1: 10 000 '1:2 000 1: 50 1:100 000 30 '1:50 000 1:5 000 1:50 000 1:10 000 1:200 -1:10 000 1: 2 000 28 1:50 000 1:5 000 1:50 000 1:10 000 1:200 1:10 000 '1:2 000 20 1:50 000 1:5 000 1:50000 -1:20 000 1:200 1:10 000 -1:2000 10 '1:100000 -'1:10 000 1:100 000 1:20 000 1:200 1-1:10 000 1:2 000 7 1:100 000 1:10 000 1:200 000 Y1:50 000 1:500 1:10 000 1:2 000 3 1:100000 1 1:20000 1:200 000 1:50 000 1:500 1:10 000 1:2 000 1 1 :200 000 the culture medium seems to have no influence on the results, but this point will be considered again later. Usually, very rich cultures yield a lower titre than poorer ones. According to Borg-Petersen & Fagraeus (op. cit.) a rich culture diluted 1 :16 usually gives a titre 4 times as high as the same culture undiluted. However, the titre is often only doubled although it may sometimes be 8 times that of the undiluted culture. Ages of cultures Old cultures generally yield lower titres than do young ones (Borg-Petersen & Fagraeus, 1949, tested cultures up to 21 days), for equal numbers and sizes of leptospires. If this factor is not taken into consideration, the fall in the number of leptospires that is normally observed in aging cultures results in a certain degree of compensation and the fall in titre by comparison with titres of young cultures is less evident than might he expected, and is often not apparent. Some experiments were made to determine what effect the age of a culture has on the titre, and the results confirmed, on the whole, the work of Borg- Peterson & Fagraeus (1949). Three immune sera-namely, anti-Patane (titre = 1: 100 000), anti-Hond Utrecht IV (titre = 1: 50 000), and anti-Bianchi (titre = 1: 20 000)-were tested against their homologous leptospiral strains. The latter were seeded at given intervals into a series of four test tubes prepared with medium from the same batch and the cultures were incubated at 30°C for 69 days after the first seeding, and the titres of the three sera were determined on the same day with cultures of four different ages. The results are shown in Table 3. In another investigation anti-Ballico serum (titre = 1: 8000) was tested with cultures of strains belonging to group Australis 10 and 27 days old. The results are given in Table 4. It can be seen from Tables 3 and 4 that aging reduces the sensitivity of cultures to immune sera. However, this decline in sensitivity is neither con- stant nor equal in all strains, nor does it invariably occur in reactions with all sera. The behaviour of strain Hond Utrecht IV is typical in this respect; anti-Hond Utrecht IV serum, which was tested with this strain, showed a reduction in sensitivity of over 60% between a culture 3 days old and one 69 days old, while the titre of anti-Patane serum fell by 75 % for the same strain. On the other hand, anti-Bianchi 1 serum loses little sensitivity in tests with old cultures, while anti-Patane serum under the same conditions loses from 50% to 90%. From a practical point of view, however, cultures 7-10 days old cause no obvious reduction in the 806 STANDARDIZATION OF THE AGGLUTINATION-ADSORPTION TEST FOR LEPTOSPIRA Table 4. Effect of aging on titres of various leptospiral strains against anti-Ballico serum Strain Titres of 1 0-day- Titres of 1 0-day-old cultures old cultures Ballico 1:8 000 -1:4 000 Esposito 1:1 000 1:1 000 Gastaldi 1:16 000 1:4 000 Jalna 1:4 000 -1:4000 Erinaceus europeus 1 :1 000 1 :1 000 Hebdomadis C 1 :8 000 1 :8 000 Saigon 1:8 000 1: 2 000 Munchen C90 1:4 000 c'1:4 000 Jet Bratislava 1 :4 000 1:4 000 Fudge 1:8 000 1:2 000 serum titre. The maximum age limit of 5 days estab- lished by the Leptospira Commision of the US Department of Health, Education, and W'elfare for cultures to be employed in agglutination tests seems, therefore, unnecessarily strict. The rate of reduction in number of leptospires certainly has some influence on the fall in serum titre when tests are made with old cultures but this effect is not of great importance, as the following investigation showed. A 2-day-old culture of strain Hond Utrecht IV contained 6.8 x 107 leptospires and a 55-day-old culture 2.8 x 107 leptospires. When tested with anti-Patane serum, the first culture gave a titre of 1:20 000, the second culture a titre of 1: 2 000. When the first culture was diluted 1: 2 with physio- logical saline, i.e., when its leptospiral content was reduced to about that of the older culture, the titre was 1:10000 after 11/2 hours and 1:20000 after 24 hours. Therefore, dilution had no practical effect on the titre and the lower titre of the older culture must be attributed to the age factor. These variations in titre due to different numbers of leptospires in a culture or to the different ages of cultures have a considerable significance for the reproducibility of a given reaction but they are much less important in agglutinations with adsorbed sera, provided some precautions are taken. In fact, the values obtained in these tests have only a comparative value, applying to the original values and to those of other sera that are examined. Care should always be taken to use for every strain leptospires of the same age and, if possible, the same numbers. It is particularly important to use the same culture to perform simultaneously tests with both unad- sorbed control serum and with adsorbed serum, so that variations in the age and richness of cultures should influence the various tests equally. If it is considered desirable to use a constant number of leptospires in agglutination tests, the concentration recommended is 1 x 107 organisms, which is not an especially high concentration and gives good results. It is easier to dilute a rich culture to this level than to raise the concentration of a poorer culture. The doses of 175-225 million leptospires/ml recommended by Galton et al. (1962), or 100 mil- lion/ml suggested by a WHO Expert Group on Current Problems in Leptospirosis Research (1967) are certainly much too high. These recommendations are valid if the leptospires in a culture are of normal length; small deviations from the average length have little influence on the titre. If, however, the leptospires are abnormally long or short, this factor must be taken into con- sideration. The culture medium According to Borg-Petersen & Fagraeus (1949), the culture medium should not noticeably affect the agglutinability of the leptospires, but this view cannot be considered valid in all cases. Observations suggest that the culture medium can influence the results of agglutination tests for leptospires. The importance of this effect must not be over- looked because it is much more pronounced with human immune sera than with rabbit immune sera that are usually employed in adsorption tests, and also because tests are evaluated comparatively, the titre at the start of a test being compared with the residual titre after adsorption. If the two titrations are made with the same leptospiral culture, higher or lower levels of agglutinability of the organisms should not, in theory, affect the evaluation. However, it seems inadvisable to use atypically agglutinable cultures in agglutination tests. Considerable differ- ences in agglutination titres have been reported from various WHO/FAO leptospirosis reference labora- tories for the same reference immune sera and the suspicion arises that the different culture media used in the various laboratories may be responsible for the discrepancies. Antigenic differences observed in the same strain in different laboratories (M 20 in Rome and Brisbane, and Ictero No. 1 in Rome and Bratislava, for example) could be a consequence of 807 808 B. BABUDIERI selection of mutants by some component or com- ponents of the culture media. Some recent studies have indicated that leptospires cultivated in synthetic media-e.g., Ellinghausen's medium-may lose some of their capacity to synthesize antigens. This is not a case of mutation but is a phenomenon occurring at the phenotypic level since the lack of certain growth factors in the medium can hinder the synthesis of particular anti- gens in the organism. In fact, the missing antigens are produced as soon as the strain is transplanted into a non-synthetic medium. This observation has two consequences. It leads on the one hand to an exclusion of synthetic media from consideration as standardized culture media for leptospires required for serological purposes, this type of medium being the only one really suitable for standardization. On the other hand, the suspicion is reinforced that even in non-synthetic culture media the absence or defi- ciency of particular growth factors may influence the antigenic pattern of the leptospires. The use of the following non-synthetic culture medium is therefore recommended. Peptone (Witte) 0.8 g NaCl 0.4 g NaHCO3 0.02 g KCl 0.04 g KH2PO4 0.18 g Na2HPO4 . 2H2O 0.96 g Nicotinic acid 0.001 g Vitamin B12 0.001 g Rabbit serum 80 ml Distilled water to a total volume of 1000 ml Some workers prefer to employ suspensions of killed leptospires for agglutination tests but the results of tests are much more obvious if suspensions of living leptospires are used. Differences in the results of agglutination tests arise from various factors and in all the tests described here marked individual particularities have been observed in the various strains of leptospires em- ployed. Observations that apply to a given strain or serum cannot always be assumed to apply to another strain or serum, especially from a quantitative point of view. This limitation has to be borne in mind when small differences that are revealed by the technique, but which cannot always be confirmed, are evaluated for systematic purposes. It should not be forgotten that these differences often are within the limits of probable error for the technique. When the results of tests are very clear and significant, some minor technical details may be neglected, but if the results are close to the limits of significance, all the factors that could influence the results must be taken into consideration and the tests should be repeated carefully. This is, in fact, the only way to determine on which side of an arbitrary line drawn between significance and non-significance a given result falls. STANDARDIZATION OF THE AGGLUTINATION-ADSORPTION TEST It is proposed that the standard technique aggluti- nation-adsorption tests should be as follows. At least two sera obtained from two different rabbits should be employed for the adsorption test and the results of tests should, in order to be con- sidered valid, show the same rates for pre- and post- adsorption titres. The titre of a serum tested against the homologous strain should if possible be at least 1: 10 000, and the titre must be determined with the same leptospiral culture that was employed for the adsorption. The leptospiral culture used for adsorption in Korthof's medium should be killed with 0.05% formol and concentrated by centrifugation at 6000-10 000 rev/min for 1/2 hour. The pellet should be suspended in formolized saline buffered to a pH of 7-7.2 to a total volume of 0.9 ml. The suspen- sion should contain the optimum number of lepto- spires required to absorb about 99% of the homolo- gous antibodies. This number, if the leptospires are of average length, may be calculated from the formula: N = (3.1 4 0.5) T x 105. Counts should preferably be performed in a Hawksley' germ-counting chamber or by nephelo- metric determination. To 0.1 ml of serum in a small test tube, 0.9 ml of leptospiral suspension is added. A control test tube contains 0.1 ml of serum and 0.9 ml of formolized buffered saline. Both test tubes are closed with a rubber stopper and kept at a temperature of 370C overnight. The following day, the residual anti- bodies are determined. For this purpose, the cultures should be well grown, should show no sign of spontaneous agglutination, and should be of the same age but not older than 7-10 days. The standard medium should be used and the leptospires should be tested for normal agglutinability. Preferably, the number of leptospires should be 1 x 107/ml but 1 Obtainable from Hawksley & Sons, Ltd, 12 Peter Road, Lancing, Sussex, England. STANDARDIZATION OF THE AGGLUTINATION-ADSORPTION TEST FOR LEPTOSPIRA small variations on either side of this number are acceptable. The test must be made with living leptospires. The test tube containing the mixture of serum and leptospires is centrifuged briefly and the super- natant fluid collected. Successive scalar dilutions of the adsorbed and control sera are prepared with saline in test tubes or on procelain plates. The first dilution should be 1:2 if low titres are expected or 1: 10 if high titres are expected. Subsequent dilutions are made by doubling the previous dilution. Agglutination is carried out by pouring into test tubes or on to plates 0.1 ml of each dilution of serum, starting from the undiluted one, and 0.9 ml of leptospiral culture. Thus, starting from a dilution of 1: 10, a dilution is reached that corresponds to the titre of the control serum, diluted 1: 10. The test tubes are kept at a temperature of 370C for 11/2 hours and the results are observed in a dark field by the usual technique, the values being recorded as percentages, as suggested by Wolff & Broom (1954). The principles for evaluating the significance of the readings are those suggested by the Joint WHO/FAQ Expert Committee on Zoonoses (1959). CRITERIA FOR USING THE AGGLUTINATION-ADSORPTION TEST FOR SYSTEMATIC PURPOSES According to the system of classification adopted by the International Committee on Bacteriological Nomenclature: Leptospira Subcommittee, a refe- rence strain, which is usually the first strain to be isolated, is designated for each serotype. The characterization of the serotype is based on the reference strain. A serotype may include a number of strains, which often differ from each other in some minor antigenic pattern. In systematic studies, when attempts are made to identify the serotype to which a given strain should be ascribed, comparisons must be made between that strain and the reference strain of the serotype, and never with any other strains belonging to the same serotype. Since small antigenic differences between the reference and other strains may pro- duce quantitative differences in the agglutination absorption test, readings that are close to the 10% limit might be sufficient to indicate a different systematic position for a strain to be tested, depend- ing on whether it is compared with the reference strain or with one of the other strains of the same serotype. For example, in Kmety's (1960) descrip- tion of the antigenic factors of leptospires belonging to the group Australis, the agglutination-adsorption test between strains Ballico and Lora does not allow, for quantitative reasons, the classification of the latter strain as a new serotype. If, however, the strain Ballico were to be compared with the strain Monte, which is very close to the strain Lora-even if not completely identical with it-the results might suggest that strain Monte should be made a serotype. A similar situation arises when the strain Ballico is compared with some other leptospiral strains that are antigenically very close to the strain Lora-for example, strain And 11 and also some others that have been isolated from the kidney of hedgehogs. In this case, it seems reasonable to establish a new sero- type and to choose as the reference strain one that in cross-adsorption tests gives readings beyond the 10% limit. Another problem that should be taken into consideration is the existence of some leptospiral strains that, according the results of cross-absorp- tion tests, could be ascribed to one or other of two different serotypes. In this event, the strain under investigation should probably be attributed to the serotype with which it shows the greater antigenic affinity. For instance, the strain Lora could, ac- cording to cross-absorption tests, be ascribed to the serotype australis, the titre of the residual anti- bodies being a little lower than 10%, but strain Lora displays a higher antigenic affinity to strain Monte, the residual antibody titre being approxima- tely 0. Strain Lora should therefore properly be ascribed to the same serotype as strain Monte, i.e., to serotype lora. A similar situation arises with several strains isolated by Babudieri & Ghysels from Belgian hedgehogs. All these strains are similar to strain Ballico and could be included in the serotype australis; they are, however, more closely related to, and almost identical with, strain And 11 of the serotype lora. In such cases epidemiological and geo- graphical factors should be taken into consideration. For example, the strains isolated in Italy and Bel- gium from hedgehogs and man all display higher affinities for the strain And 11 of the serotype lora of Belgian origin, than for the reference strain of the serotype australis whose origin is Australia. 809 810 B. BABUDIERI ACKNOWLEDGEMENTS I am indebted to Dr A. Alexander, Dr C. Borg-Petersen and Professor J. W. Wolff for their helpful comments and suggestions during the preparation of this article. RtSUMt SUGGESTIONS EN VUE D'UNE NORMALISATION DE L'EPREUVE D'AGGLUTINATION-ADSORPTION DANS LA LEPTOSPIROSE On a etudi6 experimentalement les divers facteurs intervenant dans 1'epreuve d'agglutination-adsorption, utilisee pour le typage des leptospires, et evalue leur importance respective en vue d'une standardisation 6ventuelle de la methode. Pour preparer l'immunserum, il faut inoculer a plu- sieurs lapins, par voie intraveineuse, des leptospires vivants et melanger les serums obtenus, le titre de la pr6paration devant atteindre 1:10 000 au moins. La presence, dans les milieux de culture, de certains consti- tuants peut modifier le pouvoir d'agglutination des leptospires. II est donc pref6rable de les cultiver sur des milieux standards, notamment pour la pratique des tests d'agglutination. On ne dispose cependant d'aucun milieu completement synthetique permettant la croissance de toutes les souches. Pour les 6preuves d'adsorption, on utilisera des lep- tospires vivants ou, mieux encore, des leptospires tues par le formol a faible concentration (0,05 %). L'epreuve doit etre realisee avec une quantite d'antigene capable d'adsorber 99% environ des anticorps contenus dans l'immunserum. L'emploi de doses plus fortes peut etre la cause d'une adsorption non specifique. Des essais ont ete faits afin de preciser la technique de l'epreuve et les conditions optimales de temp6rature, de duree et de rapport volumetrique entre l'immunserum et la sus- pension de leptospires; l'importance pratique des modi- fications apportees 'a ces differents facteurs a e evaluee. Quant 'a I'agglutination, elle doit etre realisee a l'aide de leptospires vivants. Il importe en particulier de con- tr6ler l'age de la culture et le nombre de leptospires qu'elle contient. L'auteur discute ces resultats experimentaux et sou- ligne la necessite de conserver comme critere de la distinction entre serotypes un titre residuel apres adsorp- tion de 10%. Il deconseille la methode o en cinq temps ) qui peut etre a l'origine d'erreurs. Il propose enfin, et decrit, une methode standard utilisable pour l'identifi- cation des serotypes de leptospires. REFERENCES Addamiano, L. (1957) R. C. Ist. sup. Saniti, 20, 482 Babudieri, B. & Ghysels, G. (1966) Ann. Inst. Pasteur, 110, 627-634 Borg Petersen, C. & Fagraeus, A. (1949) Acta Path. microbiol. scand., 26, 555-567 Castellani, P. M. (1969) Azione della formaldeide sulla membrana delle leptospire. In: Proceedings of the 15th Italian Congress on Microbiology, Saint-Vincent Castelli, M. (1965) Ann. Ist. sup. Sanitti, 1, 129-163 Ellinghausen, H. C. & McCullough, W. G. (1965) Amer. J. vet. Res., 26, 45 Galton, M. M. et al. (1962) Leptospirosis: epidemiology, clinical manifestations in man and animals, and methods in laboratory diagnosis, Atlanta, Communicable Disease Center Joint WHO/FAO Expert Committee on Zoonoses (1959) Wid Hlth Org. techn. Rep. Ser., No. 169 Kmety, E. (1960) [An attempt to subdivide the Leptospira australis A serogroup]. In: [Leptospirae and leptospi- rosis in man and animals], Warsaw, Wydawnictwo Polskiej Akademii Nauk, pp. 27-39 Kmety, E., Galton, M. & Sulzer, C. R. (1970) Bull. Wld Hlth Org., 42, 733-738 Kujumgiev, I. (1964) Izv. mikrobiol. Inst., Sof., 16, 16 White, F. & Simpson, C. F. (1965) J. infect. Dis., 115, 123 WHO Expert Group on Current Problems in lepto- spirosis Research (1967) Wld Hlth Org. techn. Rep. Ser., No. 380 Wolff, W. & Broom, J.C. (1954) Docum. Med. geogr. trop. (Amst.), 6, 78-95