AEROMONADS IN SLAUGHTERED CIDCKENS : THEIR SPECIES AND PATHOGENIC FACTORS

Cwikova Olga, Alena Hovorkova, O. Mrtz, Iva Steinhauserova and Z. Matyas: Aeromonads in SlaUghtered Chickens: Their Species and Pathogenic Factors. Acta vet. Brno, 62, 1993: 95-102. Washings from 155 eviscerated chicken carcasses coming from 16 agricultural co-operatives were examined at 14-day intervals during one yeat. The isolation attempts yielded 91 aeromonad strains which were further specified, tested for cytotoxicity and examined for pathogenicity for the white mouse. Aeromonads were found in 13 (81 %) agricultural co-operatives, being detected in 58 (41.4%) out of 140 chickens. Examination of the washings detected 36 (39.6%) A. sobria strains, 21 (23.1)% A. trota strains, 17 (18.7%) A. hydrophila strains, 9 (9.9%) A. caviae strains and 8 (8.8) A. jandaei strains. On the respective farms 2 to 3 species were generally found in cOmbination. Of the 91 strains 89 (97.8%) grew in S-phase, 85 (93.4%) haemolysed blood agar and 67 (73.6%) produced cytotoxic effect on tissue culture of MDBK cells. Agreement between all these characteristics was found in 63 (69.2%) strains. In bioassays the pathogenicity agreed with the results of S-phase -growth in -17 (77.3%) out of 22 strains, with the results of beta-haemolysis in 16 (72.7%) out of 22 strains and with those of cytotoxicity in9 (69.2%) out of 13 strains. Agreement between alI these thr~e characteristics was found in 9 (52.9%) out of 17 virulent strains. As pathogenic were classified 3 out of 4 A. caviae strains, 5 out of 8 A. hydrophila strains, 6 out of 8 A. sobria strains (the remaining two strains grew in R-phase) and all A. trota strains examined. Aeromonads, slaUghter~d chickens, food hygiene Only motile aeromonads and then particularly A. hydrophila, A. sobria-and A. caviae, associated'. with gastroenteritis, were described as important from the viewpoint of food hygiene (Janda et al. 1984). Recently, however, ithas become applfrei:J.fthat some other species, naml'ly A.jandaei (Carnahan et al. 1991b), A. veronii (Hickman Brenner et al. 1987) and possibly alsoA. trota.(Carna,han et al. 1991c) may also be entl;l'Opathogenic. Data on thc;se species are fewerin number and none at all have been recorded in this country. The only published data on food hygiene-related aeromonads in this country so far have been the findings of.i4. hydrophila in faeces (Pauc:kova and Fukalova 1986) and the information contained in two reports (Kamenik 1990, Aldova and Schindler 1991). The present study on aeromona<ls in slaughtered chickens was therefore. carried out to draw attention to this question. in our countrY. Relevant published data along this line provide information only on the species A. hydrophila; these microorganisms were isolated from pOUltry purchased from retail dealers (palumbo et a1. 1989) and from refrigerated raw (Nagel et a1. 1960) or cooked pOUltry (Toule and Murphy1978). Th¢ pnly study approaching our objective ,is#Ult of Barnhart et al. (1989) who reported the recovery of. A. hydrophila from broiler carcasses. Our study is concerned in addition withthe other aeronionad species and their pathogenic factors.

The only published data on food hygiene-related aeromonads in this country so far have been the findings of.i4.hydrophila in faeces (Pauc:kova and Fukalova 1986) and the information contained in two reports (Kamenik 1990, Aldova andSchindler 1991).The present study on aeromona<ls in slaughtered chickens was therefore.carried out to draw attention to this question.in our countrY.
Relevant published data along this line provide information only on the species A. hydrophila; these microorganisms were isolated from pOUltry purchased from retail dealers (palumbo et a1. 1989) and from refrigerated raw (Nagel et a1. 1960) or cooked pOUltry (Toule and Murphy-1978).Th¢ pnly study approaching our objective ,is#Ult of Barnhart et al. (1989) who reported the recovery of.A. hydrophila from broiler carcasses.Our study is concerned in addition withthe other aeronionad species and their pathogenic factors.

Materials and Methods
Washings from 155 slaughtered chickens coming from 16 agricultural co-operatives were -examined at 14-day intervals during one year.The eviscerated carcasses were put into polyethylene bags containing 100 ml 0.1% peptone water and thoroughly shaken.From each washing three ,dilutions in saline, namely 1 : 10, 1 : 100 and 1 : 1,000, were prepared from which 0.1 ml aliquots -were inoculated into two dishes of blood agar and Endo agar.
Mer 24-hour incubation at 37° C suspect colonies were transferred to semisolid medium and -only facultatively a~erobic motile strains were subjected to further examination.Their identifica-.tion was carried out with two LACHEMA enterotests supplemented with the following tests: .growth in broth without NaCl, nitrate reduction, oxidase reaction and gas production in glucose, ;all of them conducted at 30 "C.The strains were classified essentially according to the classm-•cation scheme of Carnahan et aI. (1991a) where only the test for resistance to anti\liotics was .moreor less left out (Table 1).
The characteristics of pathogenicity under study included in addition to beta-haemolysin .(Caselitz and Krebs 1962) growth phase in 0.5% glucose broth (Namdari and Bottone 1988) and the test for cytotoxicity (Chanter et aI. 1986).In this test the monolayer of embryonic •bovine kidney (MDBK) cells was overlaid with nutrient agar and inoculated with the strain tested.In positive cases the cYtotoxic effect was seen after as few as 18 hours of incubation at 37°C in -the form of variously wide zone of disintegrating monolayer (Fig. 1 and 2).
For evaluation of these tests reportedlYl'elated directly to enteropathogenicity (Cumberbatch -et al. 1979, Burke et a!. 1982) 24 strains were used in bioassays on mice (N amdari and Bottone 1988).Pairs of mice were inoculated i. p. with doses of 10' microbe cells suspended in 0.5 ml 'saline; where the result was dubious the bioassay was repeated.Each strain that killed at least .50% of the experimental animals was regarded as pathogenic.
frolll and mates

trota
From stool of humans From abaceases.wounds and pleural liquid of humans •

Discussion
The use of blood and Endo agar for the isolation attempts was dictated by non-existence of a special medium on which all aeromonad species would grow.However, thanks to a very rare occurrence of protei in the diluted washings no difficulties arose and by transferring the suspect colonies from Endo agar possible non-haemolytic strains could also be isolated.
The diagnostic tests were chosen so as to permit differentiation of the strains from related vibria and plesiomonads.As to species characteristics some discordance was recorded more or less only with lysine decarboxylation in the A. jandaei -A.sobria group, whereas in A. hydrophila subspecies where this characteristic is of paramount importance its operation proved satisfactory.A finding of considerable value is the fact that the species A. trota and A. jan.daei were also detected.
No difficulties were encountered in testing for cytotoxicity and the choice of MDBK cells for tissue culture proved adequate.The agreement of this test with the pathogenicity for white mice reached only 69.2% but was only slightly lower  Such conformity was not confirmed by our results~ is not apparent from those lieported by Palumbo et al. (19~5) and was rejected by Morgan et al. (1985).
; For evaluation of possible human health risks posed by an aeromonad strain i!n food it is therefore necessary either to assess its enterotoxin production (a process Ilrighly demanding as yet) or to identify its species.A. hydrophila~ A. sobria and $. caviae in which this toxin was demonstrated (Watson et al. 1985)

Fig. L
Fig. L Negative result of testing for cytotoxicity.Coherent monolayer of MDBK cells.Aeromonas hydrophiia, strain No.7.

Table 2
Some pathopnic lactora and re.ult.01 biological experiment._mJc:eC~?to-I Died/ I I Phase I ~= I c~mo-I Died! than the results recorded for beta-haemolysis (72.7%) and S-phase growth (77 .3%).•The question thus arises as to the conformity between these factors of virulence and enteropathOgenicity of the strains as has been repeatedly claimed in the relevant ijterature(Cumberbatch etal.~Burke et al.~ Namdari and Bottone). I,