AssiutUniversity web-site: www.aun.edu.eg

PREVALENCE, PATHOGENICITY AND ANTIBIOGRAM SENSITIVITY OF PSEUDOMONAS AERUGINOSA ISOLATED FROM DISEASED CHICKENS

HEBA BADR; HEBA ROSHDY; ABD EL-HAFEZ S. ABD EL-HAFEZ and                              EMAN M. FARGHALY   

Reference Laboratory for Veterinary Quality Control on Poultry Production, Animal Health Research

 Institute, Ministry of Agriculture, Nadi El-Seid Street, Dokki P.O. Box246, Giza 12618, Egypt.

Received: 1 September 2016;       Accepted: 31 October 2016

INTRODUCTION

Pseudomonas aeruginosa is one of the species belonging to genus Pseudomonads which included in Pseudomonaceae family. It is gram negative bacteria, moving with polar flagella and produces various fluorescent pigments (Tamer et al., 1989).

P. aeruginosa is the representative of the genus with the highest importance in avian pathology (Fodor, 2007). It is ubiquitous and often found in soil, drinking water, and humid environments (Mohamed, 2004; Mena and Gerba, 2009). The infection may occur through skin wounds or contaminated vaccines, morbidity and mortality due to Pseudomoniasis ranged between 2 to 100%, but more commonly about 2-12% with greatest losses in very young birds (John Barnes, 1997). The mortality rate by P. aeruginosa is higher than other gram negative pathogens because it has ability toproduce several extracellular products that after colonization can cause extensive tissue damage and invaded blood and

Corresponding author: Dr. HEBA BADR

E-mail address: drheba_badr@yahoo.com

Present address: Reference Laboratory for Veterinary Quality Control on Poultry Production, Animal Health Research Institute, Ministry of Agriculture, Nadi El-Seid Street, Dokki P.O. Box246, Giza12618, Egypt.

dissemination. Among these extracellular products are alkaline protease, elastase, haemolysins, phospholipase "C". Thus virulence of this organism appears multifactorial, its cellular products and extracellular products ensure its ability to infect most hosts (Herbert, 2003and Ali et al., 2009). In experimental trial, P. aeruginosa was foundto be highly virulent to the young chickens (1 - 10 day old) and less virulent to chickens of 11 - 20 days, while older chickens (>20 days) were found to be resistant to the infection (Kebede, 2010). Naturally P. aeruginosa is resistant to many widely used antibiotics which resulted from an impermeable outer membrane and the production of extracellular polysaccharides (Quinn, 1992). Some authors reported that the pathogenicity of P. aeruginosa in poultry was associated with septicemic and respiratory infections, sinusitis, keratitis, keratoconjunctivitis, and high embryonic death in hatcheries (Hartl et al., 1997; Hussein et al., 2008 and Hai-ping, 2009). Many researchers have made attempts to develop molecular methods especially PCR for the detection of P. aeruginosa) Nikbin et al., 2012).P. aeruginosa was detected using sequence-specific target; the outer membrane protein (OprL) gene locus (Abdullahi et al., 2013). This study was conducted to isolate P. aeruginosa from diseased chickens and apparently healthy chicks, to investigate the presence of organism in chicken and determine the phenotypic and genotypic character of isolates then detection of its pathogenicity in vivo in chicken embryos and one day old chicks.

MATERIALS AND METHODS

1-Sample collection:

A total of 200 samples (150 samples collected from diseased and dead chicken and 50 yolk samples of apparently healthy one day old chicks) were examined. Collected samples included tracheal and cloacal swabs from diseased chickens and internal organs (heart, liver, lungs, intestine and/or bone marrow) from freshly dead one, in addition to the yolk sacs of one day old chicks. All samples used were collected under aseptic conditions and safety precautions to prevent cross contamination according to (Middleton et al., 2005) as in Table (1). The examined samples from farms of different Governorates of Egypt were submitted to the Reference laboratory for veterinary quality control on poultry production (RLQP).

Table 1: Sources and numbers of examined samples.

2- Bacterial Isolation:

Bacteriological examination of 200 chicken samples was done according to Shukla and Mishra, (2015). The samples were collected from internal organs (heart, liver, spleen, and bone marrow) of 150 diseased and dead chicken that showed sever congestion in the internal organs, turbidity in the pericardium, pneumonic lungs and 50 yolk sac from apparently healthy newly-hatched chicks. The samples were inoculated in buffered pepton water and incubated aerobically for 24 hours at 37°C. A loopfull of inoculated broth was streaked onto different selective media (pseudomonas agar, Trypticase soya agar and MacConkey agar) and incubated aerobically for 24-48 hours at 37°C; pigment production and lactose fermentation were detected respectively. The plates containing characteristic colonies of P. aeruginosa (large, irregular, translucent and produced a greenish diffusible pigment and characterized by its fruity smell) were selected and the Gram staining test was performed then subjected to biochemical identification according to (Buxton and Fraser 1977). API 20 was used as a confirmatory biochemical test. Bacterial isolates were confirmed by the api 20 E identification system (BioMérieux®, France).

3- Antimicrobial sensitivity test:

The antibiogram of P. aeruginosa isolates were done by disc-diffusion test according to Koneman et al. (1997) against 13 antibiotics (Oxoid) and interpretation according to the Clinical and Laboratory Standards Institute/ Formerly National Committee for Clinical Laboratory Standard CLSI/NCCLS, (2009) as shown in Table (2).

Table 2: The interpretation of Ps. aeruginosa sensitivity test according to (CLSI/NCCLS, 2009).

4- Molecular identification:

Conventional PCR Assay for confirmation of Pseudomonas aeruginosa isolates. DNA was extracted from isolates by QIAamp DNA Mini Kit, (Qiagen, Germany, GmbH) Catalogue no.51304. PCR were performed on extracted DNA by using specific primer tooprL gene (Metabion, Germany) were amplified according to references mentioned in Table (3). Primer were utilized in a 25- µl reaction containing 12.5 µl of Emerald Amp Max PCR Master Mix (Takara, Japan), 1 µl of each primer of 20 pmol concentrations, 4.5 µl of water, and 6 µl of template. The reactions were performed in a Biometra T3 thermal cycler. The product of PCR was separated by electrophoresis on 1% agarose gel (Applichem, Germany) in 1x TBE buffer at room temperature using gradients of 5V/cm. For gel analysis, 15 µl of the products was loaded in each gel slot. A 100 bp DNA Ladder (Qiagen, Germany, GmbH) was used to determine the fragment sizes. The gel was photographed by a gel documentation system (Alpha Innotech, Biometra) and the data was analyzed through computer software.

Table 3: Oligonucleotide primers sequence encoding to oprL gene for P. aeruginosa and the size of amplified products required for detecting the tested gene.

5- Pathogenicity tests:

A-    Chicken embryo: one hundred and forty five, 7 days old fertilized SPF eggs were used, five from them taken at random and examined bacteriologically to ensure that they were Pseudomonas free. The one hundred and forty eggs were divided into 14 groups: each of the first thirteen groups consisting of ten eggs were inoculated via yolk sac route by 0.1 ml of 24 hour broth culture contain 14x10⁷ viable cell of P./ml (Saad et al., 1981), while The last group consisting of ten eggs were kept as negative control.

B-    Chicks: one hundred and forty five, 3- days-old chicks were divided into 14 groups as follow: thirteen infected groups (ten chicks per group) were inoculated subcutaneously0.1 ml of 24 hour broth culture of the isolated organism and one group (ten chicks) as control negative while five chicks taken at random a day before infection and examined bacteriologically to ensure that they were P. free (Awaad et al., 1981). Birds were fed rations of antibiotic-free and supplied with water adlibitum with 24 hours of light daily.

RESULTS

Clinically diseased cases showed respiratory distress and some cases showed diarrhea in addition to mortality. Postmortem examination of the dead birds showed septacimea, fibrinous pericarditis, perihepatitis, airsaculitis and some cases showed enteritis. In the present study bacteriological examination of the 200 collected samples revealed that 13 samples were positive for P. aeruginosa with a percentage of 6.5% after confirmed of 18 suspected isolates by api 20E (Table, 4 and Fig.1). Out of 150 samples from diseased and dead chicken, 11 samples with a percentage of 7.3% (11/150) were positive for P. aeruginosa and 4% (2/50) from one day old chicks.

In our study the conventional PCR for oprL gene which had done from the enriched isolated strains revealed that all the isolates were confirmed by the polymerase chain reaction (Fig. 2).

Table 4: Incidence of P. aeruginosa.

*Percentage according to total number of each source of sample.

** Percentage according to total number of the samples.

Fig.  (1): Api 20E strip show positive P. aeruginosawith identification number 2202000.

Fig. (2):  Amplification of the oprL gene of P.aeruginosa for the 18 isolates, positive amplification appeared at 504bp lane 21 negative control, lane 12 the positive control (ATCC 9027) and lane 11 the ladder 100+ (Qiagen) .

In the present study, as shown in Table (5) antimicrobial sensitivity test of 13 P. aeruginosa isolates showed high resistance (100%) to Neomycin, Nalidixic acid and Lincomycin followed by Trimethoprim-sulfamethoxazole, Chloramphenicol then Tetracyclin and Doxycyclin. While high sensitivity percentage 100% to Levofloxacin, 92.3% to Norfloxacin, 84.6% to Ciprofloxacin, 77% to Colistin sulphate and Gentamicin but 61.5% to streptomycin.

Table 5: Results of antimicrobial sensitivity tests.

In the present investigation pathogenicity tests revealed that the chicken embryos showing 100% mortality between 2–3 days post- inoculation with formation of caseated material and congestion but in the chicks some groups developed clinical signs after 24-72 hour post-inoculation (P-I) they showed sleepy appearance, closed eyes, sitting on hocks, some birds laid on one side and exhibited convulsions in the legs and head and diarrhoea. The mortality rate reached to 100% in two groups within 24 hour (P-I), 80% in four groups within 72 hrs, 60% mortality in four groups within 72 hrs and one group showed 40% mortality within 72 hrs. While, only one group did not show any mortality rate. Gross lesions revealed sever general congestion of the carcasses combined with haemorrhage in body cavity, peticheal haemorrhages on liver and spleen with congestion of them and pericarditis, lungs were pneumonic, congestion and swollen of kidneys with deposition of ureats in the urters. Also enteritis, enlargement of the gall-bladder, congestion of thigh muscle, caseated material in the abdominal cavity and unabsorbed congested yolk sacs were present. Control group showed no clinical signs or lesions.

DISCUSSION

Pseudomonas infections of birds are of great importance because epidemics may spread rapidly through poultry flocks causing mortality in all ages Shukla and Mishra (2015). Pseudomonas aeruginosa has been isolated from chicks with omphalitis and is mentioned as an opportunistic pathogen that is able to produce a localized or systemic disease in newly hatched chicks. The ability of Pseudomonas spp. to cause infection of yolk sac of a chick is enhanced by its ability to degrade the proteins found in the yolk, providing the opportunity for other bacteria to multiply (Walker et al., 2002). In the present study bacteriological examination of the 200 collected samples revealed that 13 samples were positive for P. aeruginosa with a percentage of 6.5% and confirmed the isolation by api 20E. Out of 150 samples from diseased and dead chicken, 11 samples with a percentage of 7.3% (11/150) were positive for P. aeruginosa and 4% (2/50) from one day old chicks. These results agreed to great extend to that obtained by many author as Choudhury et al. (1993) who isolated P. aeruginosa in a percentage of 4.75%, Younes et al. (1990) who isolated P. aeruginosa from 20 dead chickens with percentage of4.9%. Chakrabarty et al. (1980) isolated P. aeruginosa with incidence of 8% from 100 chicken suffering from respiratory symptoms while Awaad et al. (1981) isolated P. aeruginosa with an incidence of 2.9% from an outbreak in a broiler flock. Similar results were reported by Mrden et al. (1988) who recovered P. aeruginosa from dead broiler with an incidence of 3.6%. Also Mohamed, (2004) recorded that P. aeruginosa was found in 3.3% of examined diseased and dead broilers while in baby chicks P. aeruginosa was isolated from 17.6%. Al-Adl, (2014) isolated seventeen isolates of P.aeruginosa (4.57%) from diseased, dead and apparently healthy at different ages of broiler chicken. Hussein et al. (2008) tested 140 diseased or dead broiler chicks (yolk sac samples) and found P. aureuginosa at 5 samples with percentage 2.6%. On the other hands, reported that the organism was isolated at the rate of 20% at kena Governorate by El-Bakry, (1983) and Hassan, (2013). Shukla and Mishra (2015) isolated P. aeruginosa from apparently healthy and clinically diseased broilers with percentage of 12% and 30% respectively. The conventional PCR for oprL gene which had done from the enriched isolated strains revealed that all the isolates were confirmed by the polymerase chain reaction. These results are in correlated with Malorny et al., 2003 who mentioned that in vitro amplification of DNA by PCR method is a powerful tool in microbiological diagnostics. Also, many authors as (Achtman et al., 1986; Caugant et al., 1985; Whittam and Wilson, 1988) reported that the application of PCR as an additional diagnostic tool is even more important in light of the fact, that the designation of a serogroup does not reflect the virulence of that strain.

The results of antimicrobial sensitivity test of 13 P. aeruginosa isolates showed high resistance (100%) to Neomycin, Nalidixic acid and Lincomycin followed by Trimethoprim-sulfamethoxazole, Chloramphenicol then Tetracyclin and Doxycyclin. While high sensitivity percentage 100% to Levofloxacin, 92.3% to Norfloxacin, 84.6% to Ciprofloxacin, 77% to Colistin sulphate and Gentamicin but 61.5% to streptomycin. Al-Adl, (2014) reported that P. aeruginosa isolates were highly sensitive to Colstin sulphate (76.5%) and Norfloxacin (52.9%) while Gentamicin and Ciprofloxacin gave 23.5% and 17.6% respectively, while resistant to lincomycin, Naldixic acid, Streptomycin, Chloramphenicol and Doxycyclin. Kim et al. (1982) mentioned that all strains of P. aeruginosa were susceptible to Gentamicin and Colistin. Also, Walker et al. (2002) showed that the isolates were resistant to lincomycin, naladixic acid, and tetracycline with varied in sensitivity to other antibiotics, but all isolates were sensitive to gentamicin, Mohamed, (2004) showed that the isolated P. aeruginosa was highly sensitive to the norfloxacin, chloramphenicol and streptomycin but moderate sensitive to Gentamicin.  Moreover, Abd El-Gawad et al. (1998) reported that P. aeruginosa isolates from chickens were highly sensitive to Neomycin, Colistin and Tetracyclin.

In the present investigation pathogenicity tests correlated to that obtained by Mohamed, (2004) who conducted the experimental infection in eggs by isolated P. aeruginosa; showed deaths of all embryos inoculated via yolk sac route, 80% mortality of 3-days old chicks' inoculated subcutaneously and appeared the same clinical signs and gross lesions as our study. Similar to that reported by Awaad et al. (1981) who recorded 100% mortality to the inoculated chicks. Also, Abd-EL Gwad et al. (1998) stated that the experimental infection in 3-day old chicks by subcutaneous route inoculation was highly effective with mortality rate 95% and the gross lesions of inoculated birds showed congested liver, spleen and kidneys. Al-Adl (2014) Showed 100% mortality rate in the two groups inoculated with P. aeruginosa isolates within time 24 to 48 hrs. and stated the same clinical and gross lesion as our result. While, Walker et al. (2002) induced five isolates of P. aeruginosa by inoculated it in five groups of 10 One-day-old chicks via yolk sac. Virulence varied greatly among the isolates, resulting in mortality rates from 0 to 90%. The challenge isolates produced different and often distinctive postmortem lesion patterns.

CONCLUSION

Our results focusing on the P. aeruginosa infection present in the chicken farms and study the Pathogenicity of this isolates in chicken embryos and baby chicks so good hygiene measures especially in hatcheries is fundamental for Pseudomonas control. Also the use of suitable antibiotic in the day old chicks could help in reduce flock mortality. A strict antibiotic policy and establishment of infection control programs will help to lower the incidence of resistance in P. aureginosa.

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