Ankara Üııi,'. Vet. Fak. Derg.
43: 267-269, 1996
ISOLATION
OF MOTILE AEROMONAS
SPECIES
FROM CHICKEN
FA ECES
MEHMET AKAN' K,SERDAR DİKER"
Tavuk Dışkılarından Hareketli Aeromonas Tüı-1erinin İzolasyonu Özet: Hareketli Aeromonas türleri. 21 kümesten alman 254 ishalli ve
254 normal dlŞkı örneğinde direkt ve zenginleştirme metodlan ile araştmldı.
Direkt metotta 15 (%2.9), zenginleştirme metodunda ise 89 (%17. 5) örnekte
hareketli Aeromonas türleri saptandı. Hareketli Aeromonaslar, incelenen 254
ishalli örneğin 48'inden (%18.8) ve 254 normal dlŞkı örneğinin 41 'inden
(%16.1) izole edildi. Bu izolatların 53'ü (%59.6) A.hydrophila. 14'ü (%15.7)
A.sohria ve 22 'si (%24.7) A.caviae olarak identifiye edildi. A.hydrophila hem
ishalli hem de normal örneklerde daha yüksek oranda izole edildi.
Anahtar kelimeler: Hareketli Aeromonas türleri. izolasyon. tavuk,
Summary: Motile Aeromonas speeies from 21 dillerent poullJy f/oeks
were investigated in 254 diarrhoeic and 254 apparently normaljaeces samples
hy direct plating and enriehment method,. Aerolnonas spp. were detected in 15
(29%) samples hy the direct plating method and jound in 89 (17.5%) samples
hy enrichment method Motile aeromonads were isolated/rom 48 (18.8%) of
254 diarrhoeic jaeees and 41 (16.1%) ol 254 apparently normal jaeeal
samp/es. tested. Among these isolates, 53 (59.6%). 14 (/5.7%) and 22 (24.7%)
were identıjied as A.hydrophila. A.sohria and A.caviae. respeetively
A.hydrophila was more prevalent ei/her in diarrhoeic or normal chıekens.
Key words: Motıle Aerolnonas species. isolation, ehicken. jaeces Introduction
Bacteria of the motile Aeromonas group (Ahvdrophila, Asobria, Aeaviac) oecur widely in fresh, estuarine waters, chlorinated drinking ,vater, and bottled water (12,23) and are reeognized as pathogens of fısh, amphibians and reptiles (1,14) Motilc Aeromonas speeies have become İncreasingly implicated as the eausative agents of diarrhoea, wound infeetions and septieemia in humans (7,8,1 0,22) They have alsa been recovered from the faeeal material of pigs and eattle, and found to be Calilinon contaminants in foods of animal angın (2,6,9,11,17). The studies related \-vith the motile aeromonads of poultry are so limited. Isolation of motile aeroınonads from the faeces of turkey, pet and aviary birds has been reported in a few oeeasion( 16,2i,24).dditional1y,
pathologic conditions in birds due to these orgal1isms have been defıned only in a few reports (4,18) Reeently, Ahydrophila has been isolated from an outbreak of diarrhoea in a tlock and this agent has been implicated as a cause of infectious enteritis in poultry (3).
The aiın of this studv ,vas to determine the role of motile Aeormona-s spp. in naturaııy oeeuring diarrhoeal diseases of chickens.
Material and Methods
Animals: Six week old ehickens showing signs of watery, mucoid and bloody diarrhoea \vere deterınined in 2 i tloeks Duplicate rcctal samples were col1eetcd from 254 diarrhoeie chickens. As non-diarrhoeic controls, duplieate reetal samples wcre alsa obtained from 254 healthy chickens of same •• Dr .. A.Ü.Veteriner Fakültesi, Mikrobiyoloji Anabilim Dalı. Dışkapı, Ankara
26X
flocks in equal numbers. Healthy chickens were observed along three days for a subsequent diarrhoeal condition. Chickens had not been received antimicrobial agents as feed additİves or theurepatics along 2 weeks prior to sampling.
Isolation: All rectal contents were taken into sterile containers and examined within two hours of sampling. For the isolation of motile aeromonads, direct and emichrnent methods \-yere used. ]n direct platİng, faecal samples \-vere streaked on blood-ampicillin agar (BAA) containing 5'10 sheep blood and iO mg/l ampicillin, using sterile swabs. ]n emichment procedure, ig of faecal sample was inoculated into iO ml of alkaline peptone water (APW, pH 84) and incubated at 28 oC for 24 h. APW was further diluted (I: iO) with phosphate buffered saline and samples were plated on BAA with an inoculating loop. All plates were incubated at 28 oC for 24 h.
Identification: Hemolvtİc colonies from BAA were examined for motility and Gram' s reaction and were transferred to nutrient agar slants. After an overnight incubation at 28 oC, a few drops of a 1% solutİon of N,N-dimethyl-p-phenylenediamine monohydro-chloride were added to the growth to detennine the oxidase activity All Gram-negative, oxidase-positive and motik organisms were screened with the foııowing tests: oxidation/fermentation of glucose (O/F), sensitivity to 2,4-diamino-6,1-diisopropyl-pteridine (vibriostatic agent, 0/129), fermentation of mannitol and salicin, utilisation of arabinose, gas production from glucose, HzS production from cysteine hydrochloride and aesculin hydrolysis. Aeromonas spp. were differentiated according to criteria described by Popoff (19).
Statistical analysis: The significance of difference between groups was tested by chi-square analysis.
Results and Discussion
Motile aeromonads were isolated from 48 (188%) of 254 diarrhoeic and 4 i (16. i%) of 254 apparently normal chickens (Table. I). Difference between carriage rates of two groups ,vas not significant (p>0.05). Motile aeromonads were found in all 21 flocks investigated The isolation rate ranged from 12.3 to 24.6 % from flock to flock. Additionaıı)', Aeromonas carriage rate was not significant in any of the flocks (p>005). Any specific clinical sign was not detected in Aeromonas harbouring animals; all three types of diarrhoea (watery, mucoid and
\lEHMET AKAN - K. SERDAR DIKER
bloody) were present. The findings of this study suggested that motik aeromonads were not the cause of diarrhoea of chickens investİgated. TIüs finding is in contrast to that reported by Efuntoye (3) who isolated A.hvdrophila from
56.0% of diarrhoeic and 154% of
healthy chicken faeces and suggested that specifically A.hydrophila was dosely assocciated with the outbreaks of diarrhoea in the poultry. Since the findings of Efuntoye (3) have been obtained from onlv one flock and an experimental infection has not been performed, the suggestion of researcher is not convincing. Table 1. Motile Aeromona.s spccies in diarrhcİc and normal f,ıeccs. Tahlo i Normal ve ishalli dışkılarda harcketlt Acromonas türleri.
Type of ]'io. of A.hvdrophila A.sobria A.caviae
sanıples isolates (%) (%) (%)
(%)
diarrhoeİc 4X(I KX) 27(56.3) 9( 18.7) 12(25.0)
normal 41(16.1) 26(63.4) 5(122) 10(244)
total 89(17.5) 53(59.6) 14(15.7) 22(24.7)
On the other hand, when the results of present study was evaluated by means of a single species, A.hydrophila, the difference between group s was not significant. Some workers (9,24) have reported the low incidence of motile aeromonads in poultry faeces. lindal ct al(9) reported that Aeromonas spp. wcre isolated from 2 of 10 poultry faeces. Stern et al.(24) found Aeromonas spp. from 3 of 2i turkey faeces. These workers, however have not indicated the clinical condition of animals. A further comparison with other studies was not possİbk as a detaikd study on the isolation of motile aeromonads speci~s in normal and diarrhoeic faeces has not been done before.
When the isolation methods were compared, a significant difference was found (p<O.O]). Motile aeromonads were detected in 15 (2.9%) of 508 samples by direct plating method and 89 (ı 7.5%) by emichment method (Table.2). All direct-plating samplcs were also positive in emichment method. These results have showed that emichrnent step is necessary for the primary isolation of motile aeromonads from faeces. The similar results reported in some studies (6,13). Gray ve Stickler (6) reported that an emichment technigue with APW from feaces increased the total number of isolates by 77.i% for isolatİon of A.hvdrophila Majeed et ai. (13) who detected in motile aeromonads 11% of faccal samplcs by emiclunent methods wcre unable to isoiate an)' motile aeromonads by direct plating methods.
ISOLATION OF !vfOT İLE AEROMONAS SPECIES FROM eBlcKEN FAECES 269
Tablo 2. Method~ of isolation: Comparison in numbers (%) of positive cultures.
Tablo 2. İzolasyon metotları: Pozitif kültürlerin (%)
karşıl<ıştırılm'L~ı. .
:\0.of saıııples positive saıııples positive s3ıııples exaıııined 011direct platiııg aller eıırichmeııt
(isolatioıı %) (isolatioıı %)
50S 15 (2.9) 89 (17.5)
Aıı of the 89 strains sclectcd for identification werc found to be motilc Aeromonas spp. These strains comprised 48 from diarrhoeic and 4 i from normal faecal samples. Aıı strains werc motilc and oxidase-positive. Based on their reactions ın aesculin hydrolysis, L-arabinose utilisation, fermentation of salisin, production of gas from glucose and HeS from cvsteine. the strains were idcntified as A.hvdrophiİa, A.s~bria and A.caviae (Table 3). Of these 89 isolates, 53 (59.6%) wcrc identified as A.hydrophila, 14 (15 7%) as A.sobria and 22 (24.7%) as A.caviac. it was elearly demonstrated that A.hvdrophila predominated in both diarrhoeic and normal chicken faeces. it has also becn generaııy accepted by other researchers that A.hydrophila ıs the most common motile Aeromonas specıes ın cither cnvironmental samples or animal hosts (4,5,6,15,20).
Table 3. Differential characteristics ofmotile Aeromona~ speeies Tablo 3 Hareketli Aeromonas \Or/erinin ayırıcı özellikleri.
Ch.ıracteristics A.hydrnphila A.sulıria A.cayinc
Ca!.,!ase + + ,
Oxid.sc + + +
Motilitv + T .1
Resist.nt lo 01129 • + + i
Oxidation- fermenlation Fermentative :cmıentatİvc :crmcntative;::
Fc'WıWMfı~Y'uf ,. ,. + Aesculin hvdrolvsis T . -Arabinose utilization , T Fe;::ınıent~tion of salicin
-
+ CiasflOm elucasc " + -iı,S from cvsteİnc + ,..2-4 diamino 6-7 diisoprophyl pteridine
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