DIARRHEIC SYNDROME IN BROILER AND SOME WILD BIRDS CAUSED BY ESCHERICHIA COLI

Assiut University web-site: www.aun.edu.eg

DIARRHEIC SYNDROME IN BROILER AND SOME WILD BIRDS CAUSED BY ESCHERICHIA COLI

GHADA, O. EL-DEMERDASH ¹; FATMA AMER²AND HEBA ROSHDY²

¹Animal Health Research Institute, FayoumBranch, Agriculture Researches Center, Egypt

²Reference Laboratory for Veterinary Quality Control on Poultry Production, Animal Health

Research Institute, Ministry of Agriculture, P.O. Box 246 – Dokki, 12618 – Giza, Egypt

Received:13March 2021;     Accepted:30 March 2021

INTRODUCTION

Wild birds is important vectors and reservoirs for fecal pathogens in coastal areas.As vectors of many diseases has taken big interest recently, Alsothese birdshave migratory behavior causes dissemination of multi-resistant (MR)bacteria through

Corresponding author:HebaRoshdy

E-mail address:roshdy2019 @yahoo.com

Present address: Reference Laboratory for Veterinary Quality Control on Poultry Production, Animal Health Research Institute, Ministry of Agriculture, P.O. Box 246 – Dokki, 12618 – Giza, Egypt

colonized or infected with resistant bacteria (Guenther et al.,2011;Oteo2018; Arnoldet al.,2016). From bad habits of human help in attracted wild birds to garbage, manure, untreated sewage so those birds carry many pathogens like SalmonellaentericaE.coli, and Campylobacterspp(Moore et al., 2002; Fogartyet al.,2003; Waldenströmet al., 2003)E.coliThe importance as that it found in food, and environment it also harm animal and human as it is opportunistic bacteria (Benskinet al., 2009; Lisa et al., 2013).

The nature of life of crows and egret birds as one of wild birds which living near villages and towns, it disseminateE.coli through feases to the environment causes spread of infection to animals, birds, human through biological or mechanical way(Clark,2003; Hbalck,2004;Mbangaet al., 2015) especial if its aggregation is found near the domestic rearing which causes many economic losses through dissemination of pathogens and also act as carrier and transporter to infection between animals birds and human (Ishii et al.,2007;Maysaet al.,2013). Also its droppings contain nutrient matters that attracts flies which help in transfere microorganisms (Johnson etal., 2007). Recent studies have Proved that wild bird and rooks shedding bacteria which resistant to antibiotics (Hasanet al., 2015; Jamborovaet al.,2017; Keyaet al., 2019). The molecular differentiation of different E. coli strains could give guidance for epidemiological studies of sources of infection and disease transmission. A random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) is quicker and more effective procedure to differentiate variant isolates of E. coli. The distinctive DNApatterns generated by RAPD for each E. coli isolate reflects genetic diversity present in a bird species (Gomes et al., 2005). The aim of this present work was to characterize and investigate the Prevalence and characterize the E. coli isolates from crows, egret birds and broilers chicken (serologically, biochemically, detection of antimicrobial sensitivity to different antimicrobial agents, and detection of some virulence genes of E.coli using PCR technique, and detection of some antibiotic resistance genes in E. coli isolates by PCR technique.

MATERIALS AND METHODS

1. 1.    Samples Collection and Preparation:

A total of 150fecal   swabs were collected fromcrows,egret birds anddiarrhatic fecal swabs from broilerchicken (50 each) and submitted to the Central Laboratory for Veterinary Quality Control on Poultry Production, Dokki and Fayoum to be checked for the presence of E. coli infection. The samples were collected from (Giza, Fayoum) governorates. All samples were collected without any contamination by sterile cotton swabs then inoculated in test tube then rapidly transported in ice box to the laboratory. According to) Middletonet al., 2005).

2. Bacteriological examination:

All samples were examined bacteriologically for the presence ofE.coli. Isolation and identification ofE.coliwere done according to (Leeet al., 2008). Where, all the collected samples were pre-enriched in buffered peptone water (Oxoid) and incubated at 37˚C for 24 hrs under aerobic conditions. Then a loopful from each broth culture was inoculated onto blood agar, MacConkeys^, agar (Oxiod), XLD agar (Oxiod) and Eosin methylene blue agar plates(Oxiod) and incubated at 37ºC for 24 hours. The growing surface colonies were picked up, surfaced and further biochemically tested for growth on triple sugar iron agar and lysine iron agar, citrate utilization, urease production, and indole fermentation were done.

3.Serotyping of E. coli isolates:

E.coli isolates were serotyped by slide agglutination test according to (Leeet al.,2009) using standard E. coli antisera (Sifin and Denka Seiken Comp.).

4. Antibiotic sensitivity tests:

The antibiogramof some E. coli isolates was done by disc-diffusion methodaccording to (Konemanet al., 1997) against (ten)antimicrobials (Oxoid^®), and the zones of inhibition were measured and interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines(CLSI/NCCLS, 2017).The used antibiotics were Amoxicllin+ Clavulanic acid(Am+CL, 20-10µg), Chloramphenicol (C³⁰,30µg), Ciprofloxacin (CF⁵, 5µg), Gentamicin (G¹⁰, 10µg),Nalidixic acid (NA³⁰, 30µg),Nitrofurantoin (F³⁰⁰, 300 µg),Norfloxacin (NX¹⁰, 10 µg),Trimethoprim-sulfamethoxazole(SXT,1.25-23.75µg),Tetracycline(T³⁰,30µg) and Streptomycin (S^10, 10 µg).

5. Detection of virulence and antibiotic resistance genes insome E. coliisolatesby PCR technique:

5.1. Extraction:

DNA of enriched isolates was extracted using commercially available kit, QIAamp DNA Mini Kit, Catalogue no.51304.

5.2. Amplification

Pho-sxt1-sxt2-hyl-eae genes amplification wereamplified according to references mentioned in Table (1).

5.3. Analysis of the PCR Products

The products of PCR were separated by electrophoresis on 1% agarose gel (Applichem, Germany, GmbH) in 1x TBE buffer at room temperature using gradients of 5V/cm. For gel analysis, 15 µl of the PCR products were loaded in each gel slot. A 100 bp and 100 bpplus DNA Ladder (Qiagen, Germany, GmbH)were 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 1:Oligonucleotide primers sequences used for amplification of DNA for the detection of E. coli.

phoA: Alkaline phosphatase.hly: alpha-haemolysine. eaeA: Attachment and Effacement, Stx1: shiga- toxin 1, Stx2: shiga-toxin 2.aadA1: (aminoglycoside 3''-adenylyltransferase activityantibiotic resistance genes).

RESULTS

1. Isolation rateof E.coli recovered fromcrows,egret wild birdsandchicken broiler feces in different governorates:The isolation rates of E.coliwere reported to be higher inFayoum Governorate in (crows, 48%, broiler chicken, 40% and egret wild birds 28%) than Giza Governorate were E.coli was isolated from crows, egret wild birds and broiler chicken is an incidence of 20%, 12% and 20% respectively.

Table 2:Isolation rates of E.coli recovered from crows, egret birds and Chicken broiler samples in different governorates.

Percentage according to total number of the   examined samples in each governorates.

2. Serotyping Results of E. coli isolated from crows feces, egret birdfeces and chicken broiler feces:

The most commonly detected E.coliserogroups isolated were from crows (O78, O91, O145, O127, O158, O119,O125 and O55), egret birdfeces (O78, O158, O125, O119, O91 and O44) and chicken broiler (O78,O125 and O158).

Table 3:The serotypes of E. coli isolated from crows feces, egret birdfeces and chicken broiler feces:

3. Antimicrobial resistance of E.coli isolated from crows feces, egret wild birdfeces and chicken broiler feces:

The results resistance of the testing of E.coli isolates recovered from crowsfeces,egret feces and chicken broiler feces. Against 10 antimicrobial drugs. It is evident that the highest resistanceswere recordedagainst Streptomycin (83.4 %, 66.4%and 42.8%respectively).

Table 4:Interpretation of antibiotic resistance test of some E.coliisolates.

Am+CL= Amoxicillin + Clavulinic acid, C= Chloramphenicol, CIP= Ciprofloxacin, GM= Gentamicin, NA=Nalidixic acid, F=Nitrofurantoin, NX= Norfloxacin, S= Streptomycin, SXT= Trimethoprim-sulfamethoxazole, T= Tetracycline. *(calculated according to the No. of tested E.coli isolates). R=Resistance, I= Intermittent, S= sensitivity.

4. Detection of virulence genes and antibiotic resistance genesof some E.coliisolated from crows feces, egretwild birdfeces and chicken broiler feces by PCR:

To determine the virulence and antibiotic resistance profile of some isolated E.colion a molecular aspects, PCR was performed for related genes, (phoA,Stx2) virulence genes were detected in tested samples and not detected (hly- eaeA and sxt1) virulence genes. On the other handaadA1antibiotic resistancegeneswasharbored by some E.colistrains in table (5-6) and photo (1), (2), (3), (4)& (5).

Table 5:Result of virulence and antibiotic resistance genes of some E.coliisolates.

Table 6:Incidencesof virulence and antibiotic resistance genes of E.coli isolated from wild birds andbroiler chicken by PCR.

Photo (1):Agarose gel electrophoresis of PCR products after amplification of (PhoA)gene at (720) bp amplified product. All tested isolated from Crows are positive (1-12).

Photo (2):Agarose gel electrophoresis of PCR products after amplification of (sxt2)gene at (779) bp amplified product.Tested isolated from Crows are positive:O78 (2)-O91-O125.

Photo (3):Agarose gel electrophoresis of PCR products after amplification of (aadA1)gene at (484) bp amplified product7 out of 12. E.coliCrowsisolates are positive: O55-O119-O158-O78, (2)-O145-O125.

Photo (4):Agarose gel electrophoresis of PCR products after amplification of (PhoA)gene at (720) bp amplified product.All tested isolated fromchicken broilerfeces are positive (1-7). {O158- O125 (3) – O78 (2)} expect (5) {O78} negative and four E.coliegret wild birdfecestested isolatesare positive (8-11) {O119-O78- O125-O158}.while (12-13) negative, {O44-O91}.

Photo (5):Agarose gel electrophoresis of PCR products after amplification of (aadA1)gene at (484) bp amplified product.Two E.coli isolates from egret wild birdfeces(O78-O158) are positive.

DISCUSSION

Wild birds act as vectors of many diseases not affect only birds, animals but also human (Ghariebet al., 2013; Badoueiet al.,2016). The nature of birds, their ability to cover vast distances within a relatively short period of time, their residence near livestock areas, farms, waste disposal sites, and human habitats made them important vectors of some zoonosis (Gioiaet al., 2016and Oravcova, 2016). The data can be used to monitor trends in the occurrence of pathogenic strains, because multiple serogroups are associated with disease, especially O1, O2 and O78 among many others (Dziva and Stevens, 2008).

In Tables (2), the isolation rate of E.coli was reported to be highly in (crows, Egret wild birds and broiler chicken). Where percentage of the isolation rate was 48%, 28%, 40%, 20% and 12%.Respectively.  The variation in E. coli prevalence rates may be attributed to the species of wild bird examined, localities and bird feeding habits. This result was agree with that described by (Magda et al., 2013and Arujiet al., 2004) who isolated itfrom wild birds percentage of 21.6% and 14.52% respectively. Concerning examination of broilers, were positive for E. coli. Nearly similar findings were reported by (Ahmed,2011and Mona et al., 2013).

It was observed that several serotypes were recovered from crows (O78, O91, O145 and O125), egret birdfeces (O78, O158, O125, O119, O91 and O44) and chicken broiler (O78, O125 and O158). Table (3), These results agreed with (Lin et al., 2011 and Hanaaet al.,  2017, El-Sheshtawyet al.,2005 and Maysaet al.,2013)who isolated E. coli from wild birds in Egypt, nearly the same serotypes with a predominance of O78 have been identified (Reda, 2013).

Table (4), illustrated resultsof resistance of some testing E.coli isolates recovered from crowsfeces, egret wild birds feces and chicken broiler feces, against 10 antimicrobial drugs. It is evident that the highest resistance was recorded against Streptomycin (83.4 %, 66.4%and 42.8%). These results nearly similar to (Macielet al., 2017) who Showed thatE.coli isolates resistant to streptomycin, doxycycline, and Chloramphenicol.The resistance of microorganisms to antibiotics due to inactivation, drugmodification, alteration in metabolic pathway, alteration of target site development of new genes and reduced drug accumulation (Blair et al.,2014 and Prudenet al., 2013).

Studies have reported that the environment imposes its own selection on the population of E.coli following fecal deposition from its primary habitat within the intestine of animals (Jang et al., 2017).

Table (5-6) illustrated the resultsof (PhoA) gene. Encodes for a hydrolase enzyme which is responsible for removing phosphate groups from molecule.Alkaline phosphatase (phoA) gene has been used in PCRs for common E. coli strains detection, demonstrating high specificity (huet al.,2011; KeXinet al., 2009).

In the present study, E. coli virulence genes, (stx2₎ known to be associated with human disease was detected in bird fecal samples. (stx2₎was detected more frequently, while none of the isolates from these birds were found to be positive for (stx1).which nearly agreed with (Kobayashi etal., 2009,Persad, et al.,2014, Sancheset al.,2017 and Ahmed et al ., 2018).

In contrast, other researchers could not detect (stx1 or stx2) in wild birds.Considering that stx-2 toxin is more toxic than stx-1 and is often associated with wild birds and chicken Similar results were obtained by (Sancheset al.,2017). Found no stx1 samples among fecal samples from gulls, pigeons, and chickens that were, obtained by (Koochakzadehet al., 2015 and Ahmed, 2011). Other researchers could not detect (eaeA and hly) gene in wild birdssimilar results were obtained by (Mona et al., 2013 andIndranil, et al., 2004). Meanwhile these results disagreed with others who found no eaeAand hly gene detected in broiler chickens (Shimaa, 2013).

Antimicrobial resistance has been known as an emerging worldwide problem in both human and veterinary medicine, and antimicrobial use is considered the most important factor for the emergence, selection, and distribution of antimicrobial-resistant bacteria (Mohammed et al., 2014). the current study, we also screened the isolates for the presence of selected antimicrobial resistance genes, including those for streptomycin (aadA1), The prevalence of these genes was generally higher in the present study than in previous studies (Dehkordiet al.,2014., Marcelinoet al., 2019 andKaret al., 2020). And these results differ from (Momtazet al., 2012).

CONCLUSION

These findings show that wild birds, may constitute an environmental carrier of these pathogens representing a source of infection for other birds, livestock, and humans.  Wild birds may spread pathogens over a wide range, thus enhancing their carrier role. Further investigations should continue to characterize the antibiotic resistance genes and the epidemiology link between poultry and human. Biosecurity on the poultry farms should be the first line of defense against infectious diseases.

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