VIRULENCE OF ISOLATED PSEUDOMONAS AERUGINOSA INFECTING DUCKLING AND ANTIBIOTIC RESISTANCE WITH AN EXPERIMENTAL TREATMENT TRIAL

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VIRULENCE OF ISOLATED PSEUDOMONAS AERUGINOSA INFECTING DUCKLING AND ANTIBIOTIC RESISTANCE WITH AN EXPERIMENTAL TREATMENT TRIAL

MOSTAFA S. ABDOU¹; ATEF A.SALIM² AND EL DAKROURY, M.F.³   

¹BacteriobiologyUnit, Animal Health Research Institute, Provisional Lab Kafer El-Sheikh,

 Agricultural Research Center (ARC), Egypt.

²Poultery Diseases Unit, Animal Health Research Institute, Provisional Lab Kafer El-Sheikh,

Agricultural Research Center (ARC), Egypt.

³Pharmacology Dep., Faculty of Vet Medicine MatrouhUniv., Egypt

Received:26 March 2021;     Accepted:27April 2021

Corresponding author:Mostafa S. Abdou

E-mail address:dr.mostafaabdou@yahoo.com

Present address:Bacteriobiology Unit, Animal Health Research Institute, Provisional Lab Kafer El-Sheikh, Agricultural Research Center (ARC), Egypt.

INTRODUCTION

Pseudomonas aeruginosaan opportunistic pathogen, decrease host defenses causeing infection of yolk sac andomphalitisresulting in deaths of young ducks and hatched ducks. Mortality begins from hatching and continue for 10-14 days or more, and the infection with P.aeruginosa is responsible for mortality and clinical signs including respiratory signs and septicaemia (Bapatet al. 1985). P. aeruginosa can infect all tissues in poultry (Khattabet al., 2015). P. aeruginosa causes problems in ducks as septicemia, respiratory manifestation, lameness, conjunctivitis and diarrhea.

(Saifet al., 2008)Stated that the clinical signs include lameness; incoordination; swelling of head, and hock joint or footpads; diarrhea, and septicemia. (Barnes, 2003)mention that The affected ducks showed greenish watery diarrhea and paralytic signs with lameness in some birds and necropsy revealed echymotic and petechial haemorrhages on heart and liver, respectively (Qureshi, S.D.et al., 2010 and Hamza, M. Eidet al., 2019) isolated P.aeruginosa from trachea, heart, lung, liver and spleen of the affected ducksand it is gram-negative, aerobic gammaproteo-bacteria that can cause disease in animals and humans. P. aeruginosa is one of bacteria responsible for drug-resistant nosocomial infections. (Mena and Gerba, 2009(.

P. aeruginosais important in the etiology of many infectious diseases seen in humans (Silbyet al., 2011).P. aeruginosa is one of the environmental associated diseases and serious diseases in poultry farms which has multifarious virulence genes and plays a major role in poultry outbreaks.P. aeruginosa produces cheesy deposits on air sacs, dyspnea and congestion of internal organs, pericarditis and peri-hepatitis (Kheir El din and Awaad1986). The  development  of  antibiotic  resistance  due  to    low  permeability  of  outer  membrane thus potentially compromisesthe  effective  therapeutic  use  of  antimicrobialagents (Davies  and  Davies,  2010(.Pseudomonas species demonstrated a high resistance to monotherapy of penicillins, tetracyclines, cephalosporins, fluoroquinolones, and macrolides. Only combination of drugs asTicarcillin + Clavulanic acid, Cefoperazone + Sulbactum,Cefotaxime + Sulbactum,Piperacillin + Tazobactum,Ceftriaxome + Sulbactum according to (Javiyaet al., 2008).Emergence and spread ofresistance in bacteria may be due to mutationalevents introduced during bacteria replication and vertical transmission of genetic variantsthrough generations in a particular bacteriastrain.(Walker et al., 2002)Reported that P. aeruginosa is non-spore forming rod, motile and gram-negative. This organism is resistant to many antimicrobials and can invade hatched ducks. Virulent strains of this bacterium can cause diarrhea, dehydration, dyspnea, septicemia, and death. P.aeruginosa produce hemolysis on blood agar due to activity of phospholipase C which is potent toxins can damage cells including red blood cells in vivo, resulting in hemorrhage, edema,and tissue necrosis.

P.aeruginosa has proteolytic enzymes degrade lipoproteins in the yolk, this permits its proliferation, enhance other bacteria to multiply (Garcia and Isenberg, 2007).

PCR is simple highly sensitivemore rapid, than cell culture assay in detection ofbacteria. So it could be recommended as screening method in laboratory) Schmidt et al., 1995. (

MATERIALS AND METHODS

I. Samples Collectionaccording to (Middleton et al., 2005):A total of 120 samples were collected from diseased ducklings showing profuse diarrhea, respiratory manifestations, yellowish nasal secretion, ruffled feather and conjunctivitisand samples included liver, heart, yolk sac,cloacal swab and tracheal swabs were collected frommany different  farms inKafrelsheikh governorate for isolation and identification of P.aeruginosa. All samples were handled and collected aseptically to prevent cross contamination using sterile sampling materials and transmitted immediately in ice box to be examined in the laboratory.

II. Isolation and Biochemical identification of p. aeruginosa:

Samples inoculated in nutrient broth and incubated at 37 °c for 24 hours, then sub cultured onto selective medium (MacConkey agar and Pseudomonas agar base) incubated at 37 °C for 24 hours and observe the non-lactose fermenting colonies and sub cultured onto tryptic soya agar plate to observe the pigmentation. Suspected colonies were picked up and subjected to further identifications based on colonial and cellular morphology, pigment production detection of fruity smell, oxidase test and Gram staining. Cultivation on laboratory media and all biochemical tests were performed according to (Quinn et al., 2002) and (Shukla and Mishra, 2015). Oxidase test, catalase test, arginine hydrolysis test, gelatin liquefaction, Indol, methyl red and urease test identified by API 20 typing system to confirm isolates (Cheesbrough, 2000).

III. Serological identification:

Serotyping of the isolated P.aeruginosawas applied by using slide agglutination technique (specific 4 polyvalent and 16 monovalent antisera) according to the recommendation of the manufacturer’s protocol (Bio-Rad®, France) according to Glupczynskiet al. (2010). The division of P. aeruginosa to groups based on O antisera ofP.aeruginosa, depend on the International Antigen Typing Scheme (IATS) according to Legakiset al. (1982).

IV. Antibiotic Sensitivity Test: The antimicrobial sensitivity test was performed as reported byFinegold and Martin (1982) by disc diffusion method. Different antimicrobials were used such as En: Enrofloxacin (5µg),Nor: norfloxacin(10 µg), Tl: tylosin (15 µg),Fl: florphenicol (30µg), Do: doxycycline(30µg),Amp: ampicillin(10 µg),Aml: amoxicillin(10 µg), CTX: cefotaxime (30 µg), E: Erythromycin (15µg), CIP: Ciprofloxacin (5µg) and T: Oxytetracycline (30µg). The interpretation of the measured zone was done according to CLSI (2018).

V. Multiplex PCR:

1 -Extraction of DNA: samples was performed byQIAamp DNA Mini kit (Qiagen, Germany, GmbH) with modifications from the manufacturer’s recommendations,as in the kit.Oligonucleotide Primer. Primers were supplied from Metabionas in table (1).

2 - PCR amplification: For multiplex PCR.

3 - Analysis of the PCR Products:The products of PCR were separated by electrophoresis and the data analyzed through computer software, Primers sequences, target genes, amplicon sizes and cycling conditions for conventional PCR.

Table 1: Target genes,amplicon sizes, primers sequences, and cycling conditions for conventional PCR.

VI. Pathogenicity of P. aeruginosa:

To study the Pathogenicity of P. aeruginosa isolates from diseased ducklings on newly hatched ducklings with dose of infection of 0.2ml of1x10⁷ cfu/ml s/c.

VII. Experimental trials:

It was carried out at Animal Health Research Institute, provisional lab in Kafr el-Sheikh governorate. seventy ducklings one day old were placed into isolation units,feed and water were provided ad libitum, 10 ducks were sacrificed and examined bacteriologically to be free from P. aeruginosa,and the other ducklings were divided to 3 groups; first group as a control group, second group was infected (injected with 0.2 ml of1 x 10⁷ colony-forming units/bird)without treatment and the third group was infected with P. aeruginosaas in second groupand treated with the most effective antibiotic according to sensitivity test ,Florphenicol, for 5 days, symptoms, postmortem lesions andmortalities were recorded for 4 weeks.Mortality was recorded daily, and the ducks that died were necropsied and yolk sacs and liver were cultured. After 14 days, the remaining ducks were euthanatized and necropsied. Bacterial isolates from yolk sacs and liver were identified by using the API 20 system to confirm that it was as the challengedisolated bacterial. All bacterial isolates were identified morphologically, culturally, biochemically and serologically for P. aeruginosa.The challenge bacterial isolates produced somewhat different and often distinctive postmortem lesion.

RESULTS

In this study, from a total of 120 samples were collected from diseased and freshly dead ducklings,20 samples were positive for P. aeruginosa with percentage of 16.66%

Symptoms of examined duckling:

The diseased or freshly dead ducklings showed depression, emaciation, ocular and or nasal discharges,diarrhea and sometimes enlargement of hock joint with lameness

Postmortem lesions of examined duckling:

The diseased or freshly dead ducklings showed airsacculitis, congested liver, catarrhal enteritis and sometimes presence of gelatinous material in the hock joints

Table 2: result of serotyping of isolatedP.aeruginosa

Total No. of samples were 120 for each type.

Table 3: Susceptibility of isolatedP.aeruginosa (20 isolates) to antimicrobial agents.

Photo (1):Agarose gel electrophoresis of multiplex PCR forvirulence genestoxA(396 bp), lasI(606bp) and oprL(504bp) P. aeruginosa

Photo details

Lane L: 100 bp ladder as molecular size DNA marker.

Lane P: Control positive genes.

Lane N: Control negative.

Lanes 1 to10: Positive for 3 genes.

Table 4:Result of multiplex PCR for virulence genes toxA(396 bp), lasI (606bp) and oprL(504bp) P. aeruginosa

Photo 2:Agarose gel electrophoresis of multiplex PCR forAB resistance genes represented by qnrS(417 bp) blaCTX(593 bp) and mexR (637 bp)of P. aeruginosa.

Photo details

Lane L: 100 bp ladder as molecular size DNA marker.

Lane P: Control positive genes.

Lane N: Control negative.

Lane 1 to 10: Positive for 3 genes except no 4 negative forblaCTX.

Table 5:Result of multiplex PCR for AB resistance genes represented by qnrS (417 bp) blaCTX (593 bp) and mexR (637 bp)of P. aeruginosa.

Table 6:Symptoms of Pseudomonesaeruginosachallenged ducklings.

Table 7:Post mortem lesions in weekly sacrificed ducklings.

Table 8: P.aeruginosare-isolation rate from challenged ducklings.

DISCUSSION

Pseudomonas infection was considered an extensive economic problem in poultry farms, especially P. aeruginosaShahatet al. (2019) which causing a high mortality in birds (Elsayedet al., 2016). The complications caused by P. aeruginosa in birds have appeared in the form of respiratory signs, septicemia, keratitis and sinusitis (Hai-ping, 2009).

In our study, the prevalence of Pseudomonesaeruginosa was 16.66% which was higher than that recorded by Asawy and  El-Latif (2010), Hamzaet al.(2019) and Odoi, Hayford (2016) that was (15.8%), 10% and (9.7%) respectively. The affected ducklings showed emaciation, ocular and or nasal discharges, diarrhea and sometimes enlargement of hock joint with lameness, these symptoms nearly similar to that recorded by (Qureshi et al., 2010).Virulent isolates of this bacteria cause dehydration, diarrhea, septicemia, dyspnea, and death as reported by(Harbottleet al., 2007)Regarding to lesions showed on freshly dead ducklings were airsacculitis, congested liver, catarrhal enteritis and sometimes presence of gelatinous material in the hock joints  P. aeruginosa causes sinusitis, respiratory infection,keratoconjuctivitis, keratitis and septicemia and responsible for septicemia, pyogenic infections, endocarditis and lameness De Voset al. (2009). Infections may also occur through contaminated vaccines, skin wounds and antibiotic solutions or syringe used for injection and it may be systemic affecting tissues, multiple organs or localized inair sacs or infraorbital sinus producing swelling of the wattles, head, sinuses and joints in affected birds. P. aeruginosawere isolated from birds and poultry farms all over the worledSams (2001). P.aeruginosa is gram-negative, motile, non-spore forming rods (Elsayedet al., 2016). It is characterized by producing of watery soluble green pigment with a specific fruity odor. (Barnes, 2003) reported that P. aeruginosa produces cheesy deposits and dyspnea. Congestion of internal organs, peri-hepatitis and pericarditis were reported by (Kheir El din and Awaad1986).The identification of these strains should be considered during microbiological examination. In this study, identification showed typical colorless colonies on MacConkey agar media and green-blue color colonies for Pseudomonas spp. on Pseudomonas agar media similar to Haleemet al. (2011), also the morphological features with gram stain showed a gram-negative rods of pseudomonas spp. these findings were supported by Tripathiet al. (2011).

The of bacteriological examination for samples showed a green-blue color colonies with an odor likesweet grape were clear on Pseudomonas agar and didn’t ferment lactose sugar in MacConkey agar. Different biochemical tests were used to identify p. aeruginosa which showed a clear positive result for catalase test, oxidase test, Citrate reaction, arginine hydrolysis (gives brown color) and gelatin liquefaction but is negative to indole production, methyl red reaction and VogesProskauer test. P. aeruginosa produces pyocyanin and pyoverdin pigments, grows well at 42˚C and 4˚C and gives red butt and slant without H2S production on triple sugar iron agar. But the biochemical scheme cannot separate other species due to the high resemblance among the results of isolates so further identification was done by serological test and PCR to reach to accurate species.

Result of serological identification of suspected isolatesof P. aeruginosawereexplained in table (2) showed that serologically identified isolates into 16 isolates of polyvalent I group Iand 4 isolates of polyvalentII group J

The highest rate of isolation was from cloacal swabs, so fecal matter considered as the most dangerous source for spreading of P. aeruginosainside ducks farms, then the second rate of isolation was from heart and liver and these indicate the ability of P. aeruginosa to cause septicemia,  respiratory infections, septicemia and mortalities and these agree whith(Emanet al., 2017), and the last rate of isolation was from yolk sac and this indicate that P. aeruginosacan be transmitted vertically from diseased dam.

P. aeruginosawas listed as the head of most three frequent Gram-negative pathogens and is linked to the worst visual diseases. Its outbreak varies from 2 to 100% (Saadet al., 2017). Serotypes O10 H, O6 G, O11 E, and O2 G E of P. aeruginosawere detected by Shahatet al. (2019).

In table (3): The interpretation of antimicrobial resistance of P. aeruginosa isolates according to CLSI (2018).

Susceptibility of P. aeruginosa for different antimicrobials demonstrated that an obvious resistance was noted against Erythromycin, Oxytetracycline, Ampicillin and Amoxicillin (95%) and was followed by Tylosin(90%) and Doxycycline and Cefotaxim (85%) and the highest sensitivity was observed againstFlorphenicol(80%), Ciprofloxacin (55%),Enrofloxacin      (50%) andNorfloxacin (45%), so the most influential antibiotics was Florphenicol(80%) and it used for treatment in experimental design. Shahatet al. (2019) recorded that antimicrobial resistance is important problems confronting the world and it is elevating in developing countries. Therefore, it's important to detect P. aeruginosa quickly and identify its susceptibility pattern; this may avoid useless antibiotic treatment which presents antibiotic-resistant pathogens (Hamisiet al., 2012). The antimicrobial susceptibility reported that the identification of P. aeruginosa with traditional methods takes a long time to perform.Shahatet al. (2019) detect that Antimicrobial agents Resistance P.aeruginosa isolates where Intermediate Sensitive to Sulphamethazole, Gentamycin, Erythromycin, Tetracycline, Ciprofloxacin, Amoxicillin, Ampicillin, Streptomycin, Nalidixic acid, Norfloxacin. Atlas and Synder (2006). Reported that antibiotic sensitivity tests for all isolates were resist to ceftiofur,sulfisoxazole, lincomycin, bacitracin, oxytetracycline, penicillin,naladixic acid, erythromycin, and tetracycline, varied to other antibiotics, sensitive to gentamicin.  The isolates were identified by biochemical tests as urease production test, motility test, catalase test, oxidase test, citrate utilization test, triple sugar iron agar test, indole test, nitrate reduction test, gelatinase liquefaction, oxidative-fermentative test, haemolysin production, alkaline protease production, lecithinase production and Sugar Fermentation. In Ghana a study carried out show that P. aeruginosaall isolated from poultry were susceptible to antibiotic levofloxacin from 20 to100% and 75% were intermediate susceptibility to aztreonam. and resistance tocarbapenems, cephalosporins, penicillins,monobactam, quinolones and aminoglycoside and β-Lactamase encoding genes as (blaIMP, blaVIM) not detected in any isolates and the class 1 integron carry resistant genes detected in 89.4% of the multi-drug resistant strains Odoi (2016) Identified blaVIM gene in P. aeruginosa from poultry resembled corresponding regions in its clinical isolates of P. aeruginosa andthese isolates were resistant to all β-lactam tested, as, imipenem, meropenem, aztreonam, and ceftazidimeBassettiet al. (2013) and Zhang et al. (2017). In Nigeria P. aeruginosawas resist to tobramycin, β-lactams,nitrofurantoin, tetracycline and sulfamethoxazole-trimethoprim, while ofloxacin, ertapenemand imipenemwere effective against the bacterial pathogens Anioketteet al. (2016). In Pakistan, a study investigated the causative bacteria for necropsy in chicken, about 28% prevalence were P. aeruginosaand it was 100% resistant towards meropenem,colistin, erythromycin, ciprofloxacin and ceftriaxone, while 60% was sensitivit against ampicillin, cefoperazone, ceftazidime,sulbactam and rifampicin. Isolates were multidrug resistance to other antibiotics Sharma et al. (2017). Ahmed (2016) and Tartor and Elnaenaeey (2016) who mentioned that P. aeruginosa was highly resistance to Tetracycline, Erythromycin and Ampicillin,Sulphamethazone, Erythromycin, Ampicillin, Tetracycline, Amoxicillin and Erythromycin followed by Nalidixic acid (57.1%), Streptomycin (42.9%). Abd El-Gawadet al. (1998) reported that P. aeruginosa isolates of chickens were sensitive to Tetracycline. Abdel-Tawabet al. (2016) found that P.aeruginosa isolates were resistant to Nalidixic acid (80%). Mohammad (2013) recorded a high sensitivity with Ciprofloxacin and Norfloxacinbut low sensitivity of P. aeruginosa to Ciprofloxacin and Norfloxacin was recorded by Abd El-Tawabetet al. (2014). Abd El-Gawadet al. (1998) and Kurkureet al. (2001) reported a high sensitivity to antibiotic Gentamycin (88.6% and100% respectively). These variations among the results may be attributable to the difference in many conditions surrounding hatcheries or may be a result of hyper-mutation which occurred frequently in P.aeruginosa strains and leading to the development of various antimicrobial resistance as reported by Zhenget al. (2019). Antibiotic-resistant bacteria (ARB) can easily spread alongside the food chain and cause most of public health hazards (Da Costa et al., 2013, FAO, 2015 and WHO, 2015).

PCR method has been used to provide a specific, rapid, simple, and vastly identifiction of P. aeruginosa whichhas got an enormous numbers of some extracellular virulence factors and cellular components which implicated in pathogenesis Qin et al. (2003) and (Habeebet al., 2012). Molecular examination of P. aeruginosa serologically identified ten isolates in this study for detection of virulence genes toxA gene, lasI gene and oprL gene of P. aeruginosa using multiplex PCR.    

In photo No (1) and table No (4) Result of multiplex PCR for virulence genesrepresented bytoxA gene at 396 bp, lasI gene at 606 bp and oprL gene at 504bp of  P. aeruginosa show that all isolates where positive for the 3 examined virulence genes and these indicate that all isoletes were highly virulent strains.P. aeruginosa has virulence repertoire such as lipopolysaccharide, elastase, alkaline proteases, pyocyanin, pyoverdin, hemolysins, phospholipase C and rhamnolipids. Some factors are coordinated by a global regulatory system activated by autoinducers involved (lasI) gene (Habeebet al., 2012). Also some genes asexoS, exoT, exoU, and exoY genes which regulate the action of P. aeruginosa secretion system which injects toxic effectors proteins into the cytosol of host cells and accompanied by inferior clinical outcomes and elevated mortality rates (Hauser, 2009). P. aeruginosa uses the virulence factor exotoxin A to inactivate eukaryotic elongation factor 2 in the cell, such as the diphtheria toxin does, hence eukaryotes can't synthesize protein and necrotize (Emanet al., 2017). The powerful toxinsof bacteria released during bacteremia continu after P. aeruginosa killed by antimicrobial (Kirienkoet al., 2015). The main troublesome characters of P. aeruginosa is a minimal susceptibility to many types of antibiotics, making it a very hard bacteria to be eliminated and this due toP. aeruginosa genome contains the largest known resistance island genes (Khattabet al., 2015). The important reason for antimicrobial resistance was impermeability which belongs to the outer membrane lipoprotein (oprL gene) that implicated in efflux transport systems and its effects on cell permeability (De Voset al., 1997). Antibiotics are profusely administered for therapeutic and prophylaxis purposes in veterinary field (Dandachiet al., 2018).

Concerning the results of virulence factors we found that the detection of oprL gene in all isolates (100%) confirmed the existence of P aeruginosa DNA whichconsiderd a specific marker for molecular detection of P.aeruginosa and encodes a protein in the inner and outer membranes, which is essential for the invasion of epithelial cells (De Voset al., 1997 and Shahatet al., 2019), the same result obtained by Hassan (2013) and in efflux transport systems affecting cell permeability so there is a strong relation between detection of oprL and phenotypic antibiotic resistance that reported by Qin et al. (2003) and Laveniret al., (2007). In this study, the incidence rate of toxA gene was 71.42%, as shown in similar results of toxA reported by Qin et al. (2003) and Laveniret al. (2007). Khan and Cerniglia (1994) showed that 96% of tested P.aeruginosa isolates contained a toxA gene. Furthermore, the exoS and lasB genes were detected in five isolates of P.aeruginosa (71.42% for each of them) and this percentage was nearly similar to Tartor and El-naenaeey (2016) who found that the colossal majority of P. aeruginosa isolates showed exoS gene (78.6%). The higher percentage was recorded by Nikbinet al.(2012) who detected lasB in all strains of P.aeruginosa (100%). The mentioned virulence genes in this work such as, toxA, exoS and lasB were coordinated by a critical global regulatory systems consisted of transcriptional activator protein (LasR) and Pseudomonas autoinducer, (PAI), the central gene responsible for activation of this system was putative autoinducer synthase (lasI) (Habeebet al., 2012). The lasI gene which is a quorum sensing Regulation gene was detected.Venturi (2006) reported that the lasI is not detected in any Pseudomonas spp. otherwise P. aeruginosa strain. Percentage of lasI gene was less than that was detected by Alshalahet al. (2017) who succeeded in the amplification of lasI gene in all clinical isolates of P.aeruginosa. In addition to, Nikbinet al. (2012) whom explained that the possession of P. aeruginosa for several virulence genes make it a reason for various levels of virulence and pathogenicity.

In photo No (2) and table No (5) Result of multiplex PCR forAB resistance genes represented by qnrSgene at 417 bp,blaCTX geneat 593 bp and mexR gene at 637 bpof P. aeruginosa show that all isolates where positive for the 3 examined AB resistance except isolate No 4 were negative forblaCTX and these indicate that all isolates were highly AB resistance exept isolate No 4 was not haveblaCTX resistance gene and this results differ withShahatet al. (2019)  which detect virulence and disinfectant resistance genes in P.aeruginosa isolates such as (oprL, toxA, exoS, lasB and lasI) which result in amplicons 504bp, 396bp, 118bp, 1220bp and 606bp respectively, and cleared that oprL gene was disclosed in all P. aeruginosa isolates with percentage of 100%, while other genes were detected with the same percentage 71.4%, which it was Serotypes of P. aeruginosa Group P. aeruginosa O10 H P. aeruginosa O6 G P. aeruginosa O11 E P. aeruginosa O2 G and found also resistant genes in P.aeruginosa isolates (no.7 isolates) OprL gene 7 (100%) toxA gene 5 (71.42%) lasI gene 5 (71.42%) lasB gene 5 (71.42%) exoS gene 5 (71.42%) qacA/B gene 1 (14.28%) qacC/D gene 1 (14.28%) qacED1 gene 7 (100%).

Regarding the results of our study in tables (6, 7),The infected ducklings showedgeneral signs of illness (decreased appetite and loss of body weight), respiratory distress, diarrhea and sometimes enlargement of hock joint with lameness, the severity of symptoms appeared in the 2^ndchallenged groups in the first week and began to decline after treatment with florphenicol in the 3^rd group from the 2^nd week without mortalities, however these symptoms continued in the non- treated group till the3rd week with mortality 10% which had lesions of air-sacculitis, congested liver, congested spleen, pericarditis and peri-hepatitis.

In table (8)re-isolation rateof P. aeruginosa from challenged ducklings was negative in group1 and gradually decreased in group 2 due to normal immunity and also decreased in group 3 but it more decreased than group 2 due to use of effective antibiotic in treatment in the third group, so use of effective antibiotic after sensitivity test was important for rapid cure of diseased ducks and effectively decrease rate of shedding of P. aeruginosa to surrounding environment and control disease. The prevalence of P. aeruginosawas 16.66% which was higher than that recorded by Asawy and  El-Latif (2010), Hamzaet al. 2019 and Odoi(2016) that was (15.8%), 10% and (9.7%) respectively. The affected ducklings showed emaciation, ocular and or nasal discharges, diarrhea and sometimes enlargement of hock joint with lameness, these symptoms nearly similar to that recorded by (Qureshi et al., 2010). The lesions showed on freshly dead ducklings were air-sacculitis, congested liver, catarrhal enteritis and sometimes presence of gelatinous material in the hock joints.

CONCLUSION

Our study proved that virulence genes owned by the P. aeruginosa confirming its pathogenicity for ducks, especially in the presence of oprL gene which plays a great role in antimicrobial resistance, so biosafety was recommended for hatcheries and farms, hygiene, cleaning and disinfection will reduce P. aeruginosa spreading in the farms.

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