PREVALENCE OF VIRULENCE FACTORS AND ANTIBIOTIC RESISTANCE GENES IN SHIGA TOXIN-PRODUCING ESCHERICHIA COLI ISOLATED FROM QUAILS

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

PREVALENCE OF VIRULENCE FACTORS AND ANTIBIOTIC RESISTANCE GENES IN SHIGA TOXIN-PRODUCING ESCHERICHIA COLI ISOLATED FROM QUAILS

GHADA ALLAM ABD EL –DAYEM ¹; AHMED H. RAMADAN ² and

HANAA S. ALI ³

¹ Poultry Diseases Department, Animal Health Research Institute (AHRI) (Mansoura Branch), Agriculture Research Center (ARC), Dokki, Giza, Egypt.

² Bacteriology Department,Animal Health Research Institute (AHRI) (Mansoura Branch),

 Agriculture Research Center (ARC), Dokki, Giza, Egypt.

³ Pathology Department Animal Health Research Institute (AHRI) (Mansoura Branch),

Agriculture Research Center (ARC), Dokki, Giza, Egypt.

Received: 17 October 2020;     Accepted: 28 October 2020

INTRODUCTION

Quail farming is considered as one of the  most  alternative   sources   for  meat to

Corresponding author: AHMED H. RAMADAN

E-mail address: ahmedhegazy711@yahoo.com

Present address: Bacteriology Department,Animal Health Research Institute (AHRI) (Mansoura Branch), Agriculture Research Center (ARC), Dokki, Giza, Egypt;

overcome protein shortage in Egypt. This bird species is characterized by rapid growth, short generation, low feed intake, nutritious meat, higher egg production potentiality, and less susceptible to bacterial diseases as compared to other poultry species (Yusuf et al., 2016). Avian pathogenic Escherichia coli (E. coli) strains are responsible for many cases of colibacillosis, yolk sac infection, cellulitis, and omphalitis in quails (Salehi and Ghanbarpour, 2010). Based on the virulence factors, E. coli has been classified into different pathotypes: enterotoxigenic E. coli (ETEC) enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC), diffusely adherent E. coli (DAEC), enteroaggregative E. coli (EAEC), and enterohemorrhagic or Shiga toxin-producing E. coli (EHEC/STEC). The latter is most commonly associated with outbreaks of foodborne diseases(Bandyopadhyay et al., 2011), hemorrhagic diarrhea, hemorrhagic colitis, hemolytic uremic syndrome, and thrombotic thrombocytopenic purpura (Shahreza et al., 2017). STEC pathogenicity depends mainly on many virulence genes such as Shiga toxin type 1 (stx1), stx2, eaeA,and hlyA. The eaeA gene encodes intimin, which enables intimate attachment of bacteria to the lining epithelia of the intestine (Ateba and Mbewe, 2014). The hlyA gene encodes enterohemolysin, which causes hemolysis thereby facilitating iron uptake by bacteria to maintain their survival in the gut (Dontorou et al., 2003).

Antibiotic resistance constitutes a major threat to human and animal health, particularly in developing countries, where antibiotics administrated without regulation from authorities (Spellberg and Gilbert, 2014; El-Domany et al., 2017; El-Adawy et al., 2018). Another major cause of higher pathogenicity for STEC strains is abundant antibiotic resistance to a large variety of most commonly used antibiotics including aminoglyco­sides, penicillin, streptomycin, cephalosporins, sul­fonamides, tetracycline, and quinolones (Wang et al., 2016).

Some published reports investigated STEC in many poultry species such as chicken, duck, quails, and goose (Farooq et al., 2009; Dutta et al., 2011; Sekhar et al., 2017). However, little is known regarding the pathogenicity, virulence, and drug resistance genes of STEC in quails. Therefore, the present study aimed to determine the incidence of Shiga toxin-producing E. coli and the prevalence of their virulence and antibiotic resistance genes in quails. We also investigated the pathogenicity of STEC on the experimentally infected quails.

MATERIALS AND METHODS

Samples collection and culture

A total number of 150 quails were used in the present study. We collected 450 samples as follows: 50 cloacal swabs from 50 healthy quails and 400 samples from heart blood, liver, lung, and intestine of 50 freshly dead birds and 50 diseased birds.Samples were first inoculated into buffer peptone water (with incubation at 37˚C for 18 -24 h under aerobic conditions) and then cultured on 5% sheep blood and MacConkey agar (Merck, Germany) media (with incubation at 37 °C for 24 h). Suspected colonies of E. coli were re-cultured on blood agar plates and EMB agar (Merck, Germany), and colonies with green metallic sheen were selected as E. coli. These colonies were further confirmed by the following biochemical tests: urease, indol, methyl red, lactose and glucose fermentation (using TSI medium), Voges Proskauer, citrate, and lysine decarboxylase (Quinn et al., 2002).

Pathogenicity tests

The pathogenicity of E. coli isolates was tested by congo red (Berkhoff and Vinal, 1986) and hemolysis production (Livezey and Zusi, 2007) tests. To determine congo red binding activity, E. coli isolates were cultured on tryptic soy agar supplemented with 0.02% Congo red (Sigma, USA) and 0.15% bile salt (Difco, USA) at 37°C for 24 h and red colonies (positive) were selected. To determine hemolysis production, E. coli isolates were grown on tryptose blood agar (Difco) plates supplemented with 5% defibrinated washed sheep blood at 37°C for 24h and then suspected colonies were examined for hemolytic activity (Livezey and Zusi, 2007).

Serological identification of E. coli

E.coli isolates were serotyped in Animal Health Research Institute, Dokki, Giza using polyvalent and monovalent diagnostic E. coli antisera (Denka Seiken Co. Ltd,Japan) as previously described (Edwards and Ewing, 1972).

Antimicrobial drug sensitivity test

It was performed by disc diffusion method using Muller-Hinton agar using 7 antibiotic disc belongs to six different antimicrobial classes includ­ing sulfamethoxazole (100 μg/disk), norfloxacin (10 μg/disk), tetracycline (30 μg/disk), neomycin (30 μg/disk), doxycy­cline (30 μg/disk), amoxicil­lin (10 μg/disk), and colistin sulphate, (30 μg/disk) (Oxoid, Basingstoke, UK). Interpretation of the results was done following Clinical and Laboratory Standards Institute Guidelines CLSI. (2011).

Polymerase chain reaction (PCR)

DNA extraction:

DNA extraction from samples was performed using the QIAamp DNA Mini kit (Qiagen, Germany, GmbH) with modifications from the manufacturer’s recommendations. Briefly, 200 µl of the sample suspension was incubated with 10 µl of proteinase K and 200 µl of lysis buffer at 56^OC for 10 min. After incubation, 200 µl of 100% ethanol was added to the lysate. The sample was then washed and centrifuged following the manufacturer’s recommendations. Nucleic acid was eluted with 100 µl of elution buffer provided in the kit.

Oligonucleotide Primer. Primers used were supplied from Metabion (Germany) are listed in table (1).

For stx1, stx2 duplex PCR, primers were utilized in a 50- µl reaction containing 25 µl of Emerald Amp Max PCR Master Mix (Takara, Japan), 1 µl of each primer of 20 pmol concentration, 13 µl of water, and 8 µl of DNA template. The reaction was performed in an Applied biosystem 2720 thermal cycler.

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, 20 µl of the uniplex PCR products and 40 µl of the duplex PCR products were loaded in each gel slot. Generuler 100 bp ladder (Fermentas, Thermo) 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 1: Primers sequences, target genes, amplicon sizes and cycling conditions.

Pathogenicity of E.coli isolates to quails

A total of 95 one-day-old Japanese quails (Coturnix japonica) were used in this experiment. Five birds were randomly sacrificed for the bacteriological examination which proved that they were free from bacterial pathogens. The iron cages were fumigated with formaldehyde and KMnO4 (2:1) fifteen days before the arrival of birds. The cages were kept dry, clean, disinfected, and well ventilated during the entire experimental periods. Incandescent lights were used to brood baby birds at 34°C up to 7 days of age and the temperature was gradually reduced to 26°C by 21 days of age after which, no supportive heat was provided.

Quail chicks were randomly assigned into 3 groups GA, GB, and GC (n = 30 birds/group). Each group subdivided into 2 replicates of 15 birds   for each.: GA non-infected, non-treated, GB infected with E. coli O125 at the age of 7 days, and GC infected with E. coli O125 at the age of 7 days and medicated with neomycin (20%) 1 g/liter for 5 successive days at the age from 9 to 13 days. The E. coliO125 (Shiga toxin-producing E.coli) used in the experimental infection was previously isolated from diseased quails. Birds in GB and GC were orally challenged with 1 ml of saline containing 10⁸cfu/ml E. coli O125 in the back of the oral cavity using a sterile syringe at the age of 7 days (Cao et al., 2013).

The birds in the challenged groups were observed daily after the challenge for any symptoms and deaths throughout the period of the experiment (4weeks). Dead birds were necropsied immediately after the detection of their death and macroscopical lesion scores were registered. For bacterial re-isolation, swabs from the lung, heart, liver, and intestine were collected from 4 sacrificed birds in each group at the 1^st, 2^nd, and 3^rd weeks after the challenge. In addition, tissue samples from these organs were fixed in 10% neutral buffered formalin for histopathological examination. The fixed tissues were dehydrated in methanol, cleared in xylene, embedded in paraffin, sectioned at 4 µm, and stained with hematoxylin and eosin (Bancroft and Gamble, 2008). All tissues were microscopically examined and the histopathological alterations were blindly graded.

RESULTS

Clinical signs and post mortem findings in field cases

The symptoms of colibacillosis were declined food intake and body weight, weakness, ruffling of feathers, and yellowish diarrhea. While, the post mortem examination for dead and diseased birds showed congestion of liver, spleen, kidney, and lung, pericarditis, perihepatitis, air sacculitis, congestion and hemorrhage in the intestinal mucosa, and some cases showed catarrhal enteritis.

 Out of 450 examined samples, E. coli was identified in 20.67% (93/450) of the total examined samples based on morphological and biochemical characteristics. Out of 93strains, ten strains (20%) were isolated from cloacal swabs of healthy birds, 38 strains (19%) from diseased birds and 45 strains (22.5%) from freshly dead. Congo red media was used for differentiation between pathogenic and nonpathogenic E. coli. Out of 93 identified strains, 40(43.01%) were pathogenic (table2). Regarding to hemolysis on blood agar all isolated strains were non hemolytic.

The recovery rates of the pathogenic E. coli from dif­ferent quail samples were 13%, 8%, 6%and 11% form liver, heart blood, lung and intestine, respectively (fig1). E. coli isolates were serotyped into 7 serotypes including O125, O20, O78, O55, O44, O114,and O35 the most prev­alent serotypes were O125, O20 and O78 with an incidence of 22.5%, 20% and15 % respectively (table3).

The antimicrobial susceptibility pattern of (40) pathogenic E. coli strains isolated from cloacal swabs and internal organs of the examined quail revealed variable results in susceptibility and zones of inhibition to the different antimicrobial drugs which commonly used in quail treatments. E. coli isolates demonstrated high rates of resistance to sulfamethoxazole, tetracycline (100%for each) and amoxicillin (75%). Conversely, neomycin, norfloxacin and colistin showed the lowest resistance rate against E. coli isolates (15%, 20% and 25% respectively) Table (4).

Virulence and antibiotic resistance genes:

In this study, the presence of 4virulence genes, including stx1, stx2, eae and hly were verified by multiplex PCR analysis. Among the virulence genes detected, stx2 was detected in (3/8) isolates and eae was detected in (2/8) isolates whereas none of the isolate was carrying stx1 or hly gene. Also, multiplex PCR was used for the detection of anti­biotic resistance genes including tetA and sul1. The antibiotic resistance genes profile revealed the presence of tetA and sul1 in all examined isolates table (5).

 Results of experimental infection:

Clinical signs:

No clinical signs were observed in the uninfected-untreated group (GA) while in infected groups (B&C) clinical symptoms appeared within 48 hours following experimental E .coli infection. The clinical symptoms observed were depression, loss of appetite, ruffled feather, emaciation, conjunctivitis, rhinitis, and severe watery diarrhea. Within 24 hours following initiation of neomycin treatment, infected birds treated with neomycin (GC) improved clinically after 3-days treatment period and clinical signs disappeared within 5 to 7 days following treatment. In contrast, the incidence of clinical disease in surviving infected-untreated birds (GB) was estimated as 20-50%, seven days following infection.

Mortalities: In E. coli challenged groups, mortalities started at the 3^rd day post-challenge and were (40%) while in group(C) treated with neomycin reduced into (23.33%) (Table 6).

Gross lesions: Dead andsacrificed birds of infected-untreated group post infection were represented tracheitis with purulent exudates in the tracheal lumen and bronchi, air-saculitis on both the thoracic and abdominal air-sacs, hyperemia of intestinal mucosa, congestion of liver, pericarditis. While infected and treated groups with neomycin showed less severe lesions .Seven days following initiation of treatment lesions virtually disappear.

Re-isolation of E .coli:

All quails in uninfected-untreated group (GA) were negative for reisolation of the challenge bacteria. E.coli could be recovered from tissues in some birds which infected and treated with neomycin in the drinking water (GC) with re-isolation rate of 21.1%. While infected-untreated group (GB) had a higher re-isolation rate of E.coli (62.2%) from different organs. (Table7).

Histopathological finding:

Histopathological examination of the lung in GA showed normal lung histological structure (Fig. 3A). However, GB showed severe histopathological changes including congestion, hemorrhage (arrow), perivascular edema, bronchopneumonia (arrowhead), mild sero-fibrinous air saculitis (Fig. 3B). Sero-fibrinous exudate extended to interlobular pulmonary septae. GC demonstrated mild congestion of blood vessels (arrow) without exudate in bronchi or air saculitis (Fig. 3C). Livers in GA showed normal histological structure (Fig. 4A). In contrast,GB exhibited focal necrotic hepatic cells intermingled with microsteatosis and leukocytic infiltration in the hepatic parenchyma (Fig. 4B). Necrotic areas demarcated from adjacent degenerated hepatic cells by fibrous tissue containing erythrocytes and mononuclear cells (arrow, Fig. 4B). The majority of portal areas exhibited numerous bile duct with mononuclear cell infiltration. On the other hand, GC demonstrated mild vacuolation in few hepatocytes (arrow) and the majority of the hepatic parenchyma was normal (Fig. 4C). Hyperplastic Kuffer cells and mild interstitial lymphoid aggregation could be seen in livers of this group. Hearts in GA displayed normal histological architecture (Fig. 5A). However, Heart in GB revealed sero-fibrinous pericarditis (arrow) with edema and mononuclear cell infiltration (Fig. 5B). Meanwhile, the pericardium and myocardium restored their normal morphologic picture in GC (Fig. 5C). Histopathological examination of the intestine in GA showed normal histological structure (Fig. 6A). In contrast, the intestine inGB revealed necrotic and sloughed villi (arrow), intestinal exudate, and mononuclear cell infiltration in intestinal crypts (Fig. 6B). On the other hand, GC showed hyperplastic goblet cell (arrow) and proliferative intestinal crypts (arrowhead) to replace sloughed portions (Fig. 6C).

Table 2: Prevalence of E.coli in examined samples collected from quail.

(%^*) No of E.coli positive samples/No.of examined samples.

(%**) No. of E.coli positive congo red / No. of Escherichia coli positive samples

Figure 1: Prevalence of E. coli in different organs of the examined quails

Table 3: Serogrouping of 40 E.coli isolates recovered from examined samples.

Table 4: Results of antimicrobial susceptibility test for 40 E. coli isolates.

Figure 2: Agarose gel electrophoresis of PCR amplified products of sxt1 &sxt2 (A), eae (B), and hly (C) virulence genes and sul1 (D), tetA (E) resistance gens. LaneL: DNA molecular size marker (100 bp), lanes 1- 8 E. coli isolates from quail organs. The size in base pairs (bp) of each PCR product is indicated on the right of the bands.

Table 5: Virulence and antibiotic resistance genes in 8 E. coli isolates.

Table 6: The Mortality rate in experimentally infected quail.

^*post challenge

Table 7: Reisolation rate of E. coli from dead and scarified experimentally infected quail.

^*post challenge     ** Dead and scarified birds

Fig.3: Photomicrograph of sections from lung of quail stained with H&E X120. (A) Histological picture of lung show normal picture in G1 (control group); (B) Severe congestion, hemorrhage, edema and bronchopneumonia in G2; (C) Mild congestion of pulmonary blood vessels.

Fig.4: Photomicrograph of sections from liver of quail stained with H&E X400. (A) Histological picture of liver show normal picture in G1 (control group); (B) focal necrotic hepatic parenchyma encircled by fibrous tissue and inflammatory cells in G2; (C) Mild vaculation of a few hepatic cells.

 Fig.5: Photomicrograph of sections from heart of quail stained with H&E X100. (A) Histological picture of heart show normal picture in G1 (control group); (B) serofibrinous pericarditis containing extravasted erythrocytes and leukocytes inG2; (C) apparently normal myocardium.

 Fig.6: Photomicrograph of sections from intestine of quail stained with H&E X100. (A) Histological picture of intestine show normal picture in G1 (control group); (B) Partial necrosis and sloughing of superficial tips of intestinal villi beside intestinal exudate inG2; (C) Hyperplastic goblet cell and proliferative intestinal crypts.

DISSCUSION

Based on morphological, biochemical and serological characteristics, the overall prevalence of E. coli was 20.67% (Table 2). This recovery rate was close to that obtained by Ibrahim (2019) (which was 28%) but higher than that reported by Farghaly et al. (2017) (8.3%) and lower than that found by Prihtiyantoro et al. (2019) (55.36%). The recovery rate of E. coli isolates differs according to the source of the examined samples. Indeed, our results revealed a higher incidence of isolates from freshly dead quails (22.5%) than diseased quails (19%) and healthy quails (20%) (Table 2). Noteworthy, these healthy quails act as asymptomatic carriers for E. coli and can be considered a possible source of colibacillosis to humans (Kabir, 2010). These carriers also increase the incidence of antimicrobial-resistant bacteria (Pruden et al., 2013).

Congo red media was used for differentiation between pathogenic (pink/brick red colonies) and nonpathogenic E. coli (Singh and Gupta, 2001). The prevalence of the pathogenic strains was 43.01% with the following distribution: 20% from healthy birds, 42.11% diseased birds, and 48.89% recently dead birds (Table 2). Similarly, Azeem Riaz et al. (2016) also reported that 40% of avian E. coli iso­lates were Congo red positive. Regarding hemolysis on blood agar, all isolated strains were non-hemolytic. Similarly, Eid et al. (2016) did not find any E. coli isolates positive for hemolysis on 5%sheep blood agar.

The recovery rates of the pathogenic E. coli from dif­ferent quail samples were 8%, 13%, 6%, and 11% from heart blood, liver, lung, and intestine, respectively (Fig.1). The dissemination of E. coli into these organs agreed with the postmortem lesions including fibrinous perihepatitis, pericarditis, enteritis, and pneumonia. This indicates that E. coli can lead to septicemia which subsequently can cause death (Kabir, 2010).

The pathogenicE. coliisolates were serotyped into seven O serotypes including O125, O20, O78, O55, O44, O114, and O35 with prevalence rates of 22.5% (9/40), 20% (8/40), 15% (6/40), 12.5% (5/40), 7.5% (3/40), 7.5% (3/40), and 5% (2/40), respectively. The prevalence of the untyped strains was 10% (4/40). Consistent with these findings, other Egyptian researchers also found similar serotypes in diseased quail with a predominance of  O125 (Farghaly et al., 2017; Ibrahim, 2019). Other serotypes of E. coli such as O9, O42, and O88 were isolated from dead Japanese quails in other countries (Roy et al., 2006).

The misuse of antibiotics in animal farms induced the development of antibiotic-resistant strains that resulted in inefficient control of bacterial diseases (Darwish et al., 2013). The antimicrobial susceptibility pattern of pathogenic E. coli strains (n = 40) isolated from cloacal swabs and internal organs of the examined quail revealed variable results in susceptibility and zones of inhibition to the different antimicrobial drugs which commonly used in quail treatments (Table4, Fig.2). E. coli isolates were not 100% sensitive to any of the tested antimicrobial drugs. E. coli serotypes showed high sensitivity to neomycin (85%), norfloxacin (80%), and colistin (70%). These results are consistent with those obtained (Farghaly et al., 2017) (Ibrahim et al., 2019) (Ibrahim, 2019). E. coli isolates showed high resistance against sulphamethaxole, tetracycline (100% for each) and amoxicilin(75%). These findings were in agreement with (Youssef and Mansour (2014); Farghaly et al. (2017)). While another study documented lower resistance rates among E. coli isolated from quail against sulfamethoxazole (39.28%), and tetracycline (35%) (Ibrahim, 2019).

Shiga toxins play an important role as virulence factors in the pathogenicity and severity of STEC (Ojo et al., 2010). The presence of stx1, stx2, eae, and hly virulence genes was determined by PCR in 8 pathogenic E. coli isolates including all detected serotypes. Stx2 and eae genes were detected in (3/8) and in (2/8) of examined isolates. Both virulent genes were detected in isolates 2 and 3. Whereas, none of the 8 isolates contained stx1 or hly gene (Table5, Fig.3). Similarly, Himi et al. (2015) also did not find stx1 in all examined E. coli serogroups. However, Prihtiyantoro et al. (2019) detected both sxt1 and sxt2 in 90% and 10 % of E. coli isolated from quails. The stx2 was detected in O125, O55, and O78 serotypes. Eid et al. (2016) also detected stx2 in the same serotypes isolated from broilers. Stx2 is 1000 times more cytotoxic than stx1 and therefore it is associated with many diseases in humans (Mir et al., 2016). We also found another virulent gene, eae in O125 and O55 E. coli with an incidence rate of 25%. In agreement, Eid et al. (2016) also detected eae in 33.3% of E. coli isolates. Taken together, both O125 and O55 E. coli carried the two virulent genes eae and stx2. Dipineto et al. (2014) also detected both genes in E. coli isolated from quail. On the other hand, we and Ezzat et al. (2018) did not find the hly gene in the examined E. coli isolates. In contrast, Dutta et al. (2011) detected hly in 7 E.coli strains out of 10 samples (70%).

The possibility of the transmission of antibiotic resistance genes to humans should be considered when agricultural products, particularly chickens, are infected with bacterial strains that are resistant to most antibiotics(Manges et al., 2007).Herein, we detected two antibiotic resistance genes, tetA, and sul1, among the 8 E. coli isolates with a prevalence rate of 100%, for each (Table5, Fig.3). These findings suggest that the results of the antibiotic disk diffusion study were generally compatible with the PCR findings for the identification of the related antibiotic resistance genes. These findings agree with Ammar et al. (2015) who detected sul1 in 100% and Ibrahim et al. (2019)who detected tetA at an incidence rate of 91.18% among the examined E. coli isolates. In contrast, Younis et al. (2017) had reported a relatively lower prevalence of these antibiotic resistance genes, sul1 87%, and tetA 60%, among E. coli isolates.

In the present study, O125 and O55 E. coli harbored virulence and antibiotic resistance genes. This could explain the severe pathogenicity of these stains and underline the role of quails in transmission of antibiotic resistance bacteria to humans. Previous studies also reported the presence of virulent and antibiotic resistant E. coli strains in poultry meat (Ammar et al., 2015; Himi et al., 2015; Eid et al., 2016; Prihtiyantoro et al., 2019; Mousavi et al., 2020).

Experimental infection of quails was conducted to evaluate the pathogenicity of an isolated virulent field strain of E. coli (O125 STEC) and to assess the efficacy of neomycin in controlling the adverse effects of colibacillosis. No clinical signs were observed in the uninfected-untreated group (GA) while in infected groups (GB and GC) clinical signs appeared within 48 hours following experimental E. coli infection. GB quails showed typical symptoms of E.coli O125 similar to those described by Shen-Orr et al. (2002), which were depression, loss of appetite, ruffled feather, emaciation, conjunctivitis, rhinitis, and severe watery diarrhea. These symptoms were relieved 3 days post-treatment with neomycin (GC) and clinical signs completely disappeared after 5 to 7 days of treatment. This agreed with the results obtained by Abeer et al. (2019). In contrast, the incidence of clinical disease in surviving infected-untreated birds (GB) was estimated as 20-50%, seven days following infection.

The overall mortality rate was 40% and 23.33% in GB and GC, respectively (Table6). The highest death rate was noticed in GB in the 1^st week after the challenge. Post-mortem lesions in dead and sacrificed birds of GB included tracheitis with purulent exudates in the tracheal lumen and bronchi, thoracic and abdominal air-saculitis, hyperemia of the intestinal mucosa, congestion of the liver, and pericarditis. Similar post-mortem lesions recorded by Ameh et al. (2011). These multiple lesions in different organs may be attributed to the ability of virulent E. coli O125 to penetrate the mucosal barrier of the respiratory tract and circulate in the blood to disseminate throughout the body (Norhan et al., 2014). While neomycin-treated group (GC) exhibited less severe lesions which were completely disappeared on the 7^th day of neomycin treatment. However, there were still mild lesions in some organs, which may be due to the long period needed by the inflamed tissues to heal. This result was in agreement with that recorded by Fernandez et al. (1998).

No E. coli isolates were re-isolated from all quails in GA over the three time points (1^st, 2^nd, and 3^rd-week post-challenge). However, GB had a higher re-isolation rate of E .coli (80%, 62.5%, and 33.3%) at the three time points, respectively (Table7). E. coli was also recovered from GC but with lower re-isolation rates of 50% and 33.3%at 1^st and 2^nd w post-challenge. These results agreed with those recorded by Abeer et al. (2019). The presence of E. coli 2 w post-treatment explained by Toutain et al. (2002) who mentioned that infection caused inflammation and production of exudate and other debris, which inhibit penetration of the antibiotic or complete bacterial destruction.

The histopathological changes recorded in the lung, liver, heart, and intestine in the present study came in parallel with that recorded by Ghosh et al. (2006), Manimaran et al. (2003); Kilany et al. (2018) who reported that chicks experimentally infected with E. coli showed sloughing in (epithelial lining), infiltration of mononuclear cell and congestion of blood vessels in lung, liver, heart, and intestine. Improvement in the histopathological lesions of examined organs in the treated group was similar to that reported by El-Sawah et al. (2018).

CONCLUSIONS

The presence of shiga toxin-producing E. coli in quails alarms the public health as quails may serve as a vector for transmission of STEC to the environment and humans. Therefore, eating raw or undercooked quail meat could result in STEC-associated diseases such as foodborne diseases, hemorrhagic diarrhea, hemorrhagic colitis, hemolytic uremic syndrome, or thrombotic thrombocytopenic purpura. Moreover, STECshowed a notable resistance to several antimicrobial drugs that commonly used in poultry production in Egypt. So, further focus has to be given to biosecurity controls in quail farms to minimize infection and subsequently reduce the spread of antibiotic resistance genes between different bacterial populations.

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