Pathogenicity of Aeromonas hydrophila in chickens By Mahmoud, A. M

Egypt. J. Comp. Path. & Clinic. Path. Vol. 21 No. 3 (September) 2008; 88 - 110

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Pathogenicity of Aeromonas hydrophila in chickens By

Mahmoud, A. M.* and Tanios, A. I.** *Department of Pathology, Faculty of Veterinary Medicine, Cairo University.

**Serology Unit, Animal Health Research Institute, Dokki, Giza .

SUMMARY

S eventeen isolates of Aeromonas hydrophila were isolated from 250 commercial broiler chicks with an incidence of 6.8 %. Most A. hy-

drophila isolates (88.24 %) were positive for exotoxin assay and congo red binding test, while 52.94 % were positive for crystal violet binding activity. Most strains of A. hydrophila were sensitive to chlorampheni-col, ciprofloxacin and norfloxacin followed by gentamicin and neomycin while nalidixic acid, tetracycline, streptomycin and trimethoprim sul-phamethoxazole had moderate effect. On the other hand, all A. hydro-phila strains were resistant to amoxicillin, cephalothin, erythromycin and penicillin G. Two experiments were done. In the first experiment, chicks of one day old were infected with 1.5 X 109 organisms via subcutaneous and yolk sac. The infected chicks dead within 24 hr. A. hydrophila were isolated from most organs. The lesions observed included congestion in the internal organs and few cases showed hepatic and muscular pete-chiae. The ultrastructural study of this group showed presence of the bacilli inside the hepatocytes and macrophages with marked cellular changes. In the second experiment an attempt was made to determine a correlation between level of exposure and mortality. It was found that the mortality rate was relatively high (52.5 %) after injection of a high dose (3.5 X 107 ) of the organisms while it was (35 %) in the low dose (1.5 X 109 ). A. hydrophila was isolated from most examined organs. In the group treated with the low dose, marked degenerative and necrobi-otic changes were observed in both hepatic and splenic tissue beside to muscular lesions manifested by hemorrhage, degeneration, oedema and myositis. In the group injected with the high dose, the lesions were more severe and characterized by diffuse areas of necrosis in hepatic tissue, thrombus formation in the blood vessels together with large number of bacterial colonies and bacilli in the hepatic tissue. Marked muscular ne-crosis and myophagia were also noticed. The ultrastructural study for this group showed heterocells and hepatocytes contain bacilli. In other cases, the bacilli were present in the phagosomes of phagocytic cells in the splenic tissue. Cytopathological lysis was common evidence in the examined cells.


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Referred byReferred by Prof. Dr. Sohir Sokkar Professor of Pathology, Fac. Vet. Med., Cairo

University Prof. Dr. Magda M. Ali Professor of Poultry Diseases, Fac. Vet. Med.,

Moshtohor, Banha University

INTRODUCTION

A eromonas hydrophila (A. hy-drophila) and other motile

aeromonads (A. sobria and A. caviae) are receiving increasing attention as a human pathogens, especially as causative agents of gastroenteritis, wound infections and septicaemia (Janda and Duf-fey, 1988 and Marki et al., 2003). The microorganism occurs widely in nature, especially in water sup-ply and variety of retail-level food (Palumbo and Buchanan, 1988).

The prevalence of A. hydro-phila in avian species is indicated by studies that documented 10 iso-lations from 45 raptors (Needham et al., 1979) and 20 isolations from 15 species of 200, free-living, and companion birds (Shane et al. 1984). The organism is regarded as pathogenic as it was recovered as the only bacterial isolate from dead canaries, a toucan (Panigrahy et al., 1981), young poults (Gerlach and Bitzer, 1971) and cause en-teritis in birds (Aguirre et al., 1992). Akan et al. (1998) examined faecal and carcass samples of 351 chickens from 15 different flocks in Turkey. All of the 15 flocks were positive for motile aeromo-

nads. A. hydrophila was the pre-dominant species in both faeces and carcass samples. A. hydrophila was isolated from different ages of dead or sacrificed chickens, ducks and turkeys with percentages of 15, 22.5 and 20% respectively by Amal (2007) in Upper Egypt. Sarimehmetoglu and Kuplulu (2001) reported that broiler carcass and carcass parts have been con-taminated to important level with motile Aeromonas species and it has been risk for public health. Furthermore, Glunder (2002) iso-lated A. hydrophila in nearly 3500 wild and pet birds provide statisti-cally significant evidence that the composition of the intestinal flora may depend on dietary habits. The infection was found in 1.9% of the carnivorous and herbivorous spe-cies, in 7.1% of the omnivorous and in 12.4% of the carnivorous and insectivorous birds.

The broad spectrum of infec-tion with A. hydrophila is paral-leled by a range of virulence fac-tors including adhesions, cytotox-ins, haemolysin, and various en-zymes. However, most strains of A. hydrophila produce enterotox-ins, regardless of the source (Morgan and Wood, 1988).The presence of several genes encoding


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for putative virulence factors and phenotypic activities that may play an important role in A. hydrophila infection (Escarpulli et al., 2003). The pathogenicity of A. hydrophila to 5 and 17 day-old embryonated chicken eggs revealed mortality and hatchability rates ranged be-tween (36.7 % and 30 %) and (63.3 % and 70 %) respectively (Youssif and Wafaa, 2003).

From pathological point of

view, A. hydrophila is considered as one of the most important aquatic pathogens and usually in-fects different fish species causing severe pathological lesions includ-ing degenerative changes in he-patic and renal tissues with necro-sis in severe infections together with ulcerative dermatitis (Cipri-ano et al., 1984).

Although tissue reaction against A. hydrophila infection was studied in details at different aquatic species (Wang and Xu, 1985; Viola, 1991 and Yambot and Inglis, 1994), the available literatures did not cover the patho-genicity of this microorganism in chickens either in tissue or ultra-structural level.

Due to public health signifi-cance of A. hydrophila and little information regarding incidence, pathogenicity and ultrastructural cellular changes of A. hydrophila in chickens. The present work was undertaken to study the presence

of A. hydrophila in chickens, the biochemical reactions of the iso-lates and the ability of the isolated strains to exotoxin assays, Congo red (CR) binding test, Crystal vio-let (CV) binding and antibiogram. Experimental infection of broiler chicks with the isolated A. hydro-phila was done to study the post-mortem, histopathological and ul-trastructural changes in this avian species in case of A. hydrophila infection.

MATERIAL AND METHODS Samples:

C loacal or faecal swabs were taken from 250 commercial

broilers chickens apparently healthy prior to slaughter in differ-ent localities in Cairo, Egypt. Chickens originated from different flocks. Commercial sterile swabs incorporating a 1-ml ampoule of modified Stuarts bacterial transport medium were used to sample clo-acal material or faeces. Samples were placed on ice, transferred to the laboratory, and examined within 3 hours of collection. Isolation and identification of A. hydrophila : Each swab was transferred into 10 ml of sterile alkaline pep-tone water (APW, pH 8.4) and in-cubated for 18 hours for enrich-ment. Samples in APW were then plated on MacConkey agar me-dium and blood-ampicillin agar (BAA) containing 5 % sheep blood and 10 mg ampicillin per liter. All


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plates were incubated at 28°C for 24 h (Gray, 1984).

Colonies appear large, flat-tened and non-lactose fermenting with a defined zone of beta-haemolysis were selected and puri-fied on the nutrient agar plates. Films from pure colonies were stained by Gram’s stain and exam-ined microscopically. The identifi-cation of the isolates was carried by determining their morphologi-cal, cultural and biochemical char-acteristics according to criteria of Popoff (1984).

Exotoxin assay : The heat-labile enterotoxin of A. hydrophila was detected in cell-free supernatant fluids by the suck-ling mouse assay (SMA). The fluid accumulation due to the action of enterotoxin is considered signifi-cant if there is an intestinal weight/body weight ratio greater than 0.03 (Burke et al., 1981).

Congo red (CR) binding test: All Aeromonas isolates were

tested for its growth status on Congo red medium. The reaction is best seen after 24 hours of incu-bation at 25°C and then left for ad-ditional 4 days. Congo red positive (CR+) was indicated by the devel-opment of bright or orange red colonies (Gray, 1984).

Crystal violet (CV) binding test: All Aeromonas isolates were

inoculated on trypticase soya agar

and incubated 24 hours at 28°C, then flooded with a solution of 0.5 mg of crystal violet per ml, then the dye was removed after 2 min-utes of contact (Panigua et al., 1990).

Antibiogram: All A. hydrophila strains ob-

tained in this study were tested for antibiogram using the disk diffu-sion technique as described by Finegold and Martin (1982). The following antimicrobial disks were used: amoxycillin 20 mg, cepha-lothin 30 mg, chloramphenicol 30 mg, ciprofloxacin 5 mg, erythro-mycin 15 mg, gentamicin 10 mg, nalidixic acid 30 mg, neomycin 30 mg, norfloxacin 10 mg, penicillin G 10 IU, streptomycin 10 mg, tet-racycline 30 mg, trimethoprim/sulphamethoxazole 1.25/23.75 mg.

Experimental infection : The isolates having the mor-

phological, cultural and biochemi-cal character of A. hydrophila, were subjected for pathogenicity test according to method described by Shane and Gifford (1984) .

Experimental birds : A total of one hundred and eighty White leghorn unsexed un-vaccinated one day old chicks were obtained from commercial hatcheries. Water and commercial unmedicated balanced ration were given ad libitum. Twenty chicks were slaughtered and subjected for laboratory examination to be sure


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that they were free from A. hydro-phila infection.

Preparation of inoculum : A. hydrophila isolates were grown in Brain heart infusion (BHI) broth for 18 hrs at 30°C. Pellets that were obtained by cen-trifugation of the BHI cultures at 2000 rpm for 15 min were resus-pended in 3 ml of saline.

Pathogenicity of A. hydrophila : In first experiment, two groups of 20 two-days-old broiler chicks were inoculated, one subcu-taneously and the other via the yolk sac, with 0.1-ml suspension of 1.5 X 109 organisms in saline per chick. Twenty uninoculated chicks served as controls.

In second experiment, two groups of 40 five-days-old chicks, were inoculated via the yolk sac with suspensions containing 1.5 X 109 or 3.5 X 107 organisms per chick, one dose per group. A third group of 20 chicks, which received a 0.1-ml injection of sterile saline per chick, served as controls. All chicks were kept for four weeks. All birds were examined daily for clinical abnormalities; autopsies were performed on chicks in mori-bund stage and subjected to bacte-rial, histopathological and electron microscope examination.

Preparation of samples for pathological examination:

Collected chickens in mori-bund stage from all groups were

subjected to post-mortem examina-tion and any lesion seen was re-corded. Samples from the muscles, liver and spleen were taken, fixed in 10 % neutral buffered formalin, dehydrated in alcohol, cleared in xylol and embedded in paraffin. 4 m thick sections were prepared and stained with Hematoxylene and Eosin (Carleton, 1976). Preparation of samples for elec-tronmicroscopy:

The specimen from hepatic tissue of chicken group of 5 days old injected with high dose were fixed in 5 % cold cacodylate buffer gluteraldehyde (4°C 0.1 N, pH 7.2). Fixation was done by immer-sion of small pieces of such organ (1 mm x 1 mm) in gluteraldehyde. The tissues were fixed for 2 hours, washed by cacodylate buffer and then stored in the fixative solution at 4°C (Carleton, 1976). Further processing was carried out in the electron microscope unit of Assiut University – Egypt.

Transmission electron micros-copy:

The tissue specimens were then washed in cacodylate buffer (pH 7.2) 3 - 4 times for 20 minutes each and then fixed in 1 % osmium tetraoxide for 2 hours. After that, they were washed in cacodylate buffer four times 20 minutes each. Ascending grades of alcohol (30, 50, 70, 90 & 100%) for dehydra-tion of samples were used. After


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dehydration, the samples were im-mersed in propylene oxide for 30 – 60 minutes then in mixture of pro-pylene oxide and Epon mixture (1:1) for 30 – 60 minutes and fi-nally in pure Epon mixture for 30 – 60 minutes. The samples were embedded in the poly-ethylene capsules containing the embedding mixture (Epon mixture and hard-ner). Tissue blocks were polymer-ized in an oven for 2 – 3 days at 60°C, by using LKB ultra-microtomes, semi-thin sections in thickness of 1 micron thickness were prepared. The sections were stained by toluedine blue method and ex-amined by light microscope. For transmission electron microscope examination, ultra-thin sections were prepared by the same ultra-microtome in thickness of 500 A° then contrasted by uranyl acetate and lead citrate then examined by electron microscope JEM 10 c x 11, and photographed.

RESULTS

S eventeen isolates of A. hydro-phila were isolated from 250

commercial broiler chickens with an incidence of 6.8 % (Table, 1).

Virulence attributes of iso-lated Aeromonas species from ex-amined commercial birds were il-lustrated in Table (2). Most A. hy-drophila isolates (88.24 %) were positive for exotoxin assay and congo red binding test, while 52.94 % were positive for crystal violet binding activity.

Most strains of A. hydrophila were sensitive to chloramphenicol, ciprofloxacin and norfloxacin fol-lowed by gentamicin and neomy-cin while nalidixic acid, tetracy-cline, streptomycin and trimetho-prim sulphamethoxazole had mod-erate effect. On the other hand, all A. hydrophila strains were resistant to amoxicillin, cephalothin, eryth-romycin and penicillin G (Table 3).

All chicks infected with 1.5 X 109 organisms via subcutaneous and yolk sac in the first experiment were dead within 24 hrs. Generally chicks died acutely without show-ing premonitory signs. No specific lesions were observed on postmor-tem examination, although gener-alized venous congestion was evi-dent. A. hydrophila was reisolated from most organs (Table, 4).

In the second experiment, an attempt was made to determine a correlation between level of expo-sure and mortality. It was found that the mortality rate was rela-tively high (52.5 %) than in lower dose (35 %). A. hydrophila was isolated from most organs exam-ined (Table 5). Infected chickens showed depression, ruffled feath-ers. The lesions observed included congestion in the most of the inter-nal organs, few cases of hepatic petechiae and severely congested and unabsorbed yolk sac. No isola-tion or lesions were noted in con-trol.


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Table (1): Incidence of A. hydrophila from faeces of examined commer-cial birds.

No. of examined birds Positive Negative

No. % No. %

250 17 6.8 233 93.2

Table (2): Virulence characteristics of A. hydrophila isolated from com-mercial birds.

No. of tested A. hydrophila

Exotoxin assay Congo red

(CR) binding Crystal violet (CV) binding

+ve % +ve % +ve %

17 15 88.24 15 88.24 9 52.94

Table (3): Antimicrobial susceptibility of 17 A. hydrophila isolated from commercial birds.

Antimicrobial agents

Susceptibility Susceptible Intermediate Resistance No. % No. % No. %

Amoxycillin 0 0 0 0 17 100

Cephalothin 0 0 0 0 17 100

Chloramphenicol 16 94.12 0 0 1 5.88

Ciprofloxacin 15 88.24 1 5.88 1 5.88

Erythromycin 0 0 0 0 17 100

Gentamicin 13 64.71 3 17.65 1 5.88

Nalidixic acid 11 68.57 1 5.88 5 29.41

Neomycin 12 71.43 2 11.76 2 11.76

Norfloxacin 15 88.24 0 0 2 11.76

Penicillin G 0 0 0 0 17 100

Streptomycin 8 47.06 2 11.76 7 41.18

Tetracycline 9 52.94 1 5.88 7 41.18

Trimethoprim / Sulphamethoxazole

9 52.94 1 5.88 7 41.18


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Table (4): Mortality associated with A. hydrophila infection in 2-day-old chicks.

Mortality day A. hyrophila isolation from organs Route of

infection Dose Number

1 2 3 4

Cumulative

total (%) Yolk sac Heart Liver Spleen Cloaca

Subcutaneous

Yolk sac

Control

1.5 X 109

1.5 X 109

0

20

20

20

20*

20

0

0

0

0

0

0

0

0

0

0

100

100

0

20/20**

20/20

0/20

20/20

20/20

0/20

20/20

20/20

0/20

20/20

20/20

0/20

17/20

16/20

0/20

Table (5): Mortality associated with 2 dosages of A. hydrophila administered via the yolk sac to 5-day-old chicks.

Mortality day A. hyrophila isolation from organs Dose/

organism Number

1 2 3 4 5 6

Cumulative

total (%) Yolk sac Heart Liver Spleen Cloaca

1.5 X 109

3.5 X 107

0 (control)

40

40

20

11*

7

0

6

4

0

0

1

0

2

1

0

1

1

0

1

0

0

52.5

35

0

21/21**

14/14

0/20

21/21

14/14

0/20

21/21

13/14

0/20

20/21

13/14

0/20

18/21

12/21

0/20

* Number dead. ** Isolation of A. hydrophila / specimens examined of dead chicks.


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Pathological results:

In the experiment one (infected group of 2 days old chicks), the histopathological lesions were se-vere and characterized by diffuse necrosis in the all parenchymatous organs. In the liver, the hepatic tis-sue showed loss of cellular details and massive necrosis of the blood vessel's wall in portal area (Fig. 1). The spleen showed diffuse necro-sis where the tissue replaced by homgenous structurless necrotic areas (Fig. 2). In muscles, promi-nent oedema and myositis were common lesions. Oedematous fluid was accumulated between the muscle fibers in epimyceum and perimyceium together with mono-nuclear cells infiltration indicating myositis (Fig. 3). The electron mi-croscope examination revealed presence of large number of bacte-rial bacilli in the cytoplasm of he-patic cells (Fig. 4). Evidence of nuclear destruction was noticed (Fig. 5). Mitochondrial swelling and phagosomes containing bacilli were also observed (Fig. 6). The observed bacilli in all examined cases in this group were not sur-rounded by any zone of lysis.

In the experiment two (infe-cted group of 5 days old chicks), injected with high dose, the patho-logical lesions were prominent, he-patic tissue showed congestion of the portal and sinusoidal blood

vessels with focal areas of hepato-cellular necrosis (Fig. 7) after 1 day post infection.Various stages of necrobiotic changes were prominent in the examined hepato-cytes including karyopyknosis chromatolysis and karyolysis with evidence of cytoplasmic lysis. Kupffer cells of such specimens were markedly activated (Fig. 8). In spleen, prominent depletion of the lymphoid tissue was a common picture after four days post infec-tion (Fig. 9). The lesions in muscu-lar tissue at this time were charac-teristic where inter muscular oe-dema was noticed together with myolysis, myophagia and the char-acteristic bacterial bacilli were ob-served between muscle fibers (Fig. 10). At five and six days post in-fection, the lesions were closely similat to those at four days.

In the experiment two (infec-ted group of 5 days old chicks), injected with low dose, the histo-pathological lesions were nearly similar to the previous group but in mild extent. Blood vessels conges-tion of hepatic tissue was a com-mon picture while the hepatocytes showed prominent degenerative changes including granular and vacuolar degeneration. Some nu-clei of hepatocytes appeared shrunken with prominent chroma-tolysis (Fig. 11). In spleen, necro-biotic changes was observed in the hemopoietic cells together with


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heterophiles infiltration (Fig. 12). The lesions in the muscular tissue of this group was very clear. Prominent vascular and cellular inflammatory changes were ob-served. Muscular oedema, hemor-rhage, congestion and inflamma-tory cells infiltration mainly heter-ophils were demonstrated (Fig. 13). Zenker's necrosis, myolysis and myophagia and heterophils in-filtration was a common picture in this group (Fig. 14).

On the ultrastructural level,

the hepatocytes of experimentally infected chicks in this group showed mitochondrial swelling and phagosomes containing bacilli (Fig. 15). The bacilli were also no-ticed inside the macrophage (Fig. 16). Cytopathological lysis of the hepatic cells was noticed with the presence of the organism (Fig. 17). The characteristic heterophils con-taining granules was also noticed closely related to hepatocytes con-taining bacilli (Fig. 18). Cytoplas-mic degeneration of the hepato-cytes was noticed in the cells con-tained bacilli of the microorganism (Fig. 19). In the infected cells with bacilli, a zone of cytoplasmic lysis was usually noticed surrounding the microorganism (Fig. 20).


98

Fig. (1): Liver of chicken in experiment one showed loss of cellular details and massive necrosis of the blood vessel's wall (arrows). H&E stain (X 200). Fig. (2): Spleen of chicken in experiment one showed diffuse necrosis (arrow). H&E stain (X 200). Fig. (3): Muscles of chickens in experiment one showed accumulation of Oede-matous fluid between the muscle fibers in epimyceum and perimyceium to-gether with mononuclear cells infiltration (arrows). H&E stain (X 200). Fig. (4): Trnasmission electronmicrograph of hepatocyte of chicken in experi-ment one showed large number of bacilli in the cytoplasm without zone of lysis (arrow), (X 10000). Fig. (5): Trnasmission electronmicrograph of hepatocyte of chicken in experi-ment one showed nuclear destruction (arrow), (X 10000). Fig. (6): Trnasmission electronmicrograph of hepatocyte of chicken in experi-ment one showed mitochondrial swelling and phagosomes containing bacilli


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Fig. (7): Liver of chicken in experiment two showed congestion of the portal and sinusoidal blood vessels with focal areas of hepatocel-lular necrosis (arrows). H&E stain (X 200).

Fig. (8): Liver of chicken in experiment two showed various stages of ne-crobiotic changes. Notice: karyopyknosis, chromatolysis and karyolysis (arrows). H&E stain (X 400).

Fig. (9): Spleen of chicken in experiment two showed prominent deple-tion in the white pulp of lymphoid tissue. H&E stain (X 200).

Fig. (10): Muscle of chicken in experiment two showed muscular oe-dema, myolysis. Notice the bacterial bacilli (arrows). H&E stain (X 400).


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Fig. (11): Liver of chickens in experiment two showed granular and vacuolar degeneration.. Notice: nuclei of hepatocytes appeared shrunken with prominent chromatolysis (arrow). H&E stain (X 400).

Fig. (12): Spleen of chicken in experiment two showed necrobiotic changes in hemopoietic cells together with heterophiles infiltra-tion (arrow). H&E stain (X 400).

Fig. (13): Muscle of chicken in experiment two showed muscular oe-dema, congestion, hemorrhage and Inflammatory cells infiltra-tion. H&E stain (X 200).

Fig. (14): Muscle of chicken in experiment two showed Zenker's necrosis, myolysis and heterophils infiltration (arrow). H&E stain (X 400).


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Fig. (15): Transmission electronmicrograph of hepatocyte of chicken in experi-ment two showed mitochondrial swelling and phagosomes containing bacilli (arrow). (X 8000). Fig. (16): Transmission electronmicrograph of macrophage of chicken in ex-periment two. Notice: The bacilli inside the phagosome (arrow). (X 10000). Fig. (17): Transmission electronmicrograph of hepatocyte of chicken in experi-ment two showed cytopathological lysis of the cell (arrow). (X 10000). Fig. (18): Transmission electronmicrograph of hepatocyte of chicken in experi-ment two showed the characteristic heterophils containing granules closely re-lated to hepatocytes containing bacilli (arrow). (X 10000). Fig. (19): Transmission electronmicrograph of hepatocyte of chicken in experi-ment two showed cytoplasmic degeneration of the hepatocytes.the cells con-taine bacilli of the microorganism (arrow). (X 8000) . Fig. (20): Transmission electronmicrograph of hepatocyte of chicken in experi-ment two showed zone of cytoplasmic lysis surrounding the microorganism (arrow). (X 67000).


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DISCUSSION

I ncreased awareness of Aeromo-nas species in animals and hu-

man has stimulated interest about possible existence and distribution among chickens in Egypt. The Aeromonas organism appears to be wide spread in nature, epidemiol-ogical studies have shown that it is present in water, fruits and vegeta-bles (Martins et al., 2002 and Dumontet et al., 2003 ). At the same time Aeromonas species has been implicated in several out-breaks of food and water illness (Martins et al., 2002 and Bor-chardt et al., 2003) .

In the present study, 17 iso-lates of A. hydrophila were iso-lated from 250 commercial broiler chickens with an incidence of 6.8 % (Table 1). The faecal load of A. hydrophila appeared to be rela-tively low in examined chickens. The low incidence of A. hydro-phila in examined chickens be-cause samples were taken from ap-parently healthy birds prior to slaughter. This assumption is con-firmed with Jindal et al. (1993) and Akan et al. (1998) who re-ported a similar low incidence of A. hydrophila in chickens. Stern et al. (1987) found a low incidence of Aeromonas spp. in livestock (pig, beef, sheep & turkey) faeces and suggested that many of food strains may not be of faecal origin. On the other hand, Amal (2007)

isolated 45 isolates from 300 chickens (15%) of different ages in Upper Egypt. Also our result was disagreed with Sarimehmetoglu and Kuplulu (2001) whom iso-lated Aeromonas spp. from 116 (82.9 %) of total 140 broiler car-casses and carcasses parts pur-chased at different supermarkets in Ankara.

Although the prevalence of motile aeromonads in poultry fae-ces was found to be low, it was very high in carcass samples. These data suggested that during the slaughtering process motile aeromonads which originated from the intestinal contents of chickens spread to the carcasses via water used during processing (Akan et al., 1998).

Virulence attributes of iso-lated Aeromonas species from ex-amined commercial birds were il-lustrated in Table (2). Most A. hy-drophila isolates (88.24 %) were positive for exotoxin. In this as-pect, Krovacek et al. (1989) noted that the enterotoxin is highly pro-duced by A. hydrophila and is re-sponsible for the long term of diar-rhoeal diseases. Higher results were obtained by Burke et al. (1982) who noted that 93 % of A. hydrophila strains were enteropa-thogenic. Also, Turnbull et al. (1984) found 94.59 % strains of Aeromonas species isolated from different sources were enterotoxi-


103

genic. On the other hand, lower enterotoxin production was re-ported by Singh and Sanyel (1992) found 56 % of Aeromonas species have the ability to produce enterotoxin.

In the present study, most A. hydrophila isolates (88.24 %) were positive for Congo red binding test (Table 2). Panigua et al. (1990) and Mona (2003) reported that all motile Aeromonas take up Congo red dye with different degree of colour from pale orange to deep red. There is no biochemical un-derstanding of Congo red binding mechanism by bacteria but it is suggested to be associated with presence of B-Dgylycan in the bacterial cell wall (Vinal, 1988).

In the present study, 52.94% A. hydrophila isolates were posi-tive for crystal violet binding ac-tivity (Table 2). The obtained re-sults agree with Panigua et al. (1990). Moreover Mona (2003) found the ability of isolated motile A. hydrophila species to bind Crystal violet were 64.7%.

In vitro determination of an-timicrobial sensitivity test for local isolates of A. hydrophila is impor-tant in order to guide the choice of the most appreciated drugs re-quired to produce a therapeutic ef-fect. The obtained results recorded in Table (3) revealed that, most strains of A. hydrophila were sen-sitive to chloramphenicol, cipro-

floxacin and norfloxacin followed by gentamicin and neomycin while nalidixic acid, tetracycline, strepto-mycin and trimethoprim sul-phamethoxazole had moderate ef-fect. On the other hand, all A. hy-drophila strains were resistant to amoxicillin, cephalothin, erythro-mycin and penicillin G (Table 3). These results nearly similar to that reported by Rudin (1970) who found that most strains of A. hy-drophila were highly sensitive to chloramphenicol, tetracycline, neomycin and streptomycin. Gado (1988) reported that gentamicin and chloramphenicol were the most antibiotics used against A. hydrophila. Amal (2007) also re-ported that gentamicin was the most effective drug (100%) while neomycin was moderately sensi-tive (80%). Kampfer et al. (1999) recorded that the most A. hydro-phila isolates were highly suscepti-ble to quinolones as ciprofloxacin and chloramphenicol.

In this study, all chicks in-fected with 1.5 X 109 organisms via subcutaneous and yolk sac in the first experiment were dead within 24 h (Table 4). No specific lesions were observed on postmor-tem examination, although gener-alized venous congestion was evi-dent. A. hydrophila was isolated from most organs. In this aspect, Shane and Gifford (1984) show-ed susceptibility of poults and chicks to exposure to A. hydro-


104

phila. They reported that death oc-curred with 24 h in both 2-day-old chicks and 4-day-old poults, irre-spective of the route of administra-tion, indicating the virulence of the organism at the level of exposure.

In the second experiment, an attempt was made to determine a correlation between level of expo-sure and mortality. It was found that the mortality rate in birds in-jected with high dose was rela-tively high (52.5 %) than those in-fected with low dose (35 %). A. hydrophila was isolated from most organs examined (Table 5).The clinical signs observed were in agreement with those reported by El- Kashab (2001) and Amal (2007). The lesions observed in-cluded few cases of hepatic pete-chiae, venous congestion and un-absorbed and severely congested yolk sac. No isolation or lesions were noted in the control group. These results are in agreement with that reported by Shane and Gifford (1984) and Amal (2007).

In this study, it was clear that A. hydrophila infection in young chicks induced severe septicemic pathological lesions in hepatic, muscular and particularely he-mopoietic tissues and so the micro-organism is considered to be a cause for mortality problem in some chicken farms. In this con-cern, Shane and Gifford, 1984 and Shane et al., 1984 paid atten-

tion to prevalence and pathogenic-ity of A. hydrophila to different avian species including chicks, ducks and turkey poults. Water born infection could be the main source for infection in the farms.

In this study, the detected le-sions could be attributed to the dif-ferent virulence factors induced by the microorganism. A. hydrophila pathogenecity is the expression for exotoxin and endotoxin (lipop-olysachaide) production (Janda 1991).

In the ultrastructure study, the presence of bacteria within macrophages in hepatic tissue sur-rounded by hallow zone was agree with Easa et al. (1989). The zone of lysis considered as indicator for the ability of the microorganism to produce proteolytic enzymes (Mahmoud, 1996).The absence of lysis zone in experiment one may be time dependent because all chicks in this group died after 24 hours post infection.

The information given by the achieved results revealed that Aeromonas species (primarily a pathogen of aquatic biota) existed in the examined chickens and the microorganism induced pathologi-cal lesions in the internal organs. The effect of toxin products was detected bacteriologically and on ultrastructural level. Therefore, from our study, it was clear that Aeromonas species could be a


105

cause of serious disease problems in chicken farms and may play a significant role in the epidemiol-ogy of motile Aeromonas species infections. Finally, resolution of pathogenic mechanisms awaits fur-ther studies of Aeromonas species virulence markers.

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Pathogenicity of Aeromonas hydrophila in chickens By Mahmoud, A. M

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