REVALUATION OF DIFFERENTIATION FEATURES IN THE GROUP PASTEURELLA

Mniz 0.: Revaluation of Differentiation Features in the Group Pasteurella Actinobacillus. Acta vet. Brno, 52, 1983: 77 82. The objective was to examine decisive criteria on the basis of which to differentiate in the group Haemophilus Pasteurella Actinobacillus, the criteria being the ones reported by Mannheim et a!. (1980). From the results can be concluded: 1) In agreement with the above authors while applying their methods we failed in attempting to differentiate 26 collection (CCM) strains of pasteurellae and actinobacilli, whether with respect to the genera or to individual species. 2) Satisfactory results in these and 688 field strains were obtained when the diagnostic features were used in the following order: the growth ability on MacConkey's agar (BBL 11386), the formation of urease and indole, as well as the type of haemolysis and the fermentation of trehalose for actinobacilli in addition. Actinobacilli, pasteurellae. The Pasteurella and Actinobacillus genera developed from initial type species, namely P. multocida and A. lignieresii, as a consequence of different clinic and pathology of pasteurellosis and actinobacillosis. The alignment of further species was then more or less the result of subjective consideration which, in addition, occurred at the time when more refined biochemical tests were missing. Conceivably, their differentiation is not possible when based on the Bergey's Manual (Buchanan and Gibbons 1974) now available, so that the question of reassessing both these genera has come to the fore. Initiation of this paper was due to an article by Mannheim et a!. (1980) in which the authors attempted to reclassify the group Haemophilus Pasteurella Actinobacillus while using indole reaction, sucrose fermentation, phosphatase formation, xylose fermentation and urease formation. Materials and Methods In view of the fact that the strains CCM of pasteurellae and actinobacilli had not been tested for phosphatase before, an attempt was by us to retest them using 3 5 strains from each species (making a total of 26 strains) together with their examination for p-galactosidase, acetoin formation and ornithine decarboxylase. Moreover, the results in 688 field strains were included, which is evident from the following list: P. gallinarum: CCM 5977, 6061, 6062 and further 40 strains from domestic fowl, P. multocida: CCM 5419, 5420, 5902 and further 194 strains from man (29), cattle (20), sheep (2), goat (2), pig (47), dog (3), cat (4), rabbit (49), goose (1), domestic fowl (22), hare (2), mink (12) and nutria (1), P. pneumotropica: CCM 5775, 5777, 5778 and further 118 strains from white rat (26) and white mouse (92), P. ureae: CCM 5774, 5779, 5781 and further 71 strains from man (12), guinea-pig (19), white rat (8) and white mouse (32),


Materials and Methods
In view of the fact that the strains CCM of pasteurellae and actinobacilli had not been tested for phosphatase before, an attempt was by us to retest them using 3 -5 strains from each species (making a total of 26 strains) together with their examination for p-galactosidase, acetoin formation and ornithine decarboxylase.Moreover, the results in 688 field strains were included, which is evident from the following list: P. gallinarum: CCM 5977, 6061, 6062 and further 40 strains from domestic fowl, P. multocida: CCM 5419, 5420, 5902 and further 194 strains from man (29), cattle (20) Indole formation was examined in 1 % tryptone water.The 5-day culture was shaken with 0,5 ml of Kovacs's reagent (1928) and after 1 min.the colour of the emulsion raised to the surface was evaluated.
The testing for phosphatase followed the method by Barber and Kuper (1951).This technique makes use of phenolphthalein diphosphate agar and the 18-hr culture is exposed to ammonia vapour.In positive cases the culture becomes reddish due to the action of liberated phenolphthalein.
Urease activity of strains was proved using the liquid modification of Christensen's medium (1946); to prepare this, the more nutritive proteose peptone was applied.The heavily inoculated test-tubes were placed in the thermostat and the results read in the course of five consecutive days.
The ONPG (.B-galactosidase) test was performed according to Lowe (1962); with respect to fastidious bacteria, tryptose peptone and greater inoculum were used.
Hydrogen sulphide production was estimated in nutrient broth enriched with 0.01 % of cystine.
Incubation of test-tubes and observation of inserted lead acetate paper covered a period of 7 days.
Unified MR -VP medium with proteose peptone according to Abd-el-Malek and Gibson (1948) was used for detection of acetoin.Incubation at 37°C lasted for 5 days.VP test was carried out using alpha naphthol and KOH (Barrit 1936).

Results
From Tab. 1 (modified) will be evident that Mannheim et al. failed in their attempt to separate haemophili, pasteurellae and actinobacilli by means of the above tests, which resulted in their scepticism concerning homogeneity of these genera in taxonomic respects.
Using the same criteria, it must be admitted that we were not succesful eithepas may be evident from Tab. 2. In addition, we were able to confirm the fact that not only phosphatase (and this in particular), but also ~-galactosidase were enzymes the nature of which was too common here to be utilized for a more distinct bacterial differentiation.

Diagnostic features
---, ----, ---Indole On the other hand, the experience obtained by us has suggested that after transferring Pasteurella haemolytica into the genus Actinobacillus (Mraz 1969;Pohl 1981), not only the studied genera are distinguishable but also individual species within the respective genus can be determined.For a classification of this kind, the growth ability on MacConkey's agar BioQuest (Md.z 1975) can be employed as a starting point; while within the genera the formation of urease and indole is applicable, the type of haemolysis and the fermentation of trehalose for actinobacilli in addition, will serve the purpose (Tabs.3 and 4).

Diagnostic features
------------~- ------------------------------- It is to be admitted that the study conducted with groups each conslstmg of 3-5 collection strains meant a contribution, but it could not cover the whole range of potential variability.For this reason the results were documented also by 12 to 60 multiplies of field strains that had been examined in this laboratory during the past ten years.The aerogenic isolates (8 strains) from nasopharynx of healthy guinea-pigs were not included, but in characteristics they corresponded to H 2 S positive biovar of P. ureae.
As for the methods, the growth ability on MacConkey's agar seemed to be a most delicate feature, although only the selection of the convenient medium was the matter.However, the mention of the BBL agar is intended to point out mainly the fact that such a medium can be prepared, and that its selectivity is at a high level already.Neither any special difficulties occurred with the setting up of a classification scheme.Basing on the experience with different carbohydrates the hydrolysis of urea is taken as ranking first, for it yields quick and unambiguous results.
In conclusion, we want to add that the work done so far by Mannheim and his collaborators has been appreciated.With this paper we only wish to contribute to the solution of a new family in statu nascendi.

Table 2
Application of criteria from Table1in CCM strains of pasteurellae and actinobacilli