Indian Journal of Animal Research

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Indian Journal of Animal Research, volume 57 issue 4 (april 2023) : 499-504

Studies of Virulence Factors of Avian Pathogenic Escherichia coli in Avian Colibacillosis by In vitro Method and PCR

Ayushi Singh1, Daljeet Chhabra1, Rakhi Gangil1,*, Rakesh Sharda1, Ravi Sikrodia1, Sachin Audarya1
1Department of Veterinary Microbiology, Nanaji Deshmukh Veterinary University, College of Veterinary Science and Animal Husbandry, Mhow-453 446, Madhya Pradesh, India.
Cite article:- Singh Ayushi, Chhabra Daljeet, Gangil Rakhi, Sharda Rakesh, Sikrodia Ravi, Audarya Sachin (2023). Studies of Virulence Factors of Avian Pathogenic Escherichia coli in Avian Colibacillosis by In vitro Method and PCR . Indian Journal of Animal Research. 57(4): 499-504. doi: 10.18805/IJAR.B-4329.
Background: Avian colibacillosis is considered as major cause of morbidity and mortality in poultry. It is a common bacterial disease of poultry and many virulence factors of E. coli are associated with the disease. The current study was aimed to investigate the presence of some virulence factors of E. coli isolated from the cases of colibacillosis.

Methods: In present study, total 150 samples (liver, heart, lungs, air sacs and feaces) of chicken exhibiting pathological conditions of colibacillosis were collected from various poultry farms (organized and backyard) situated in and around Mhow and Indore cities. E.coli was isolated and identified from the samples on the basis of cultural characteristics and biochemical test. All E. coli isolates were further subjected to evaluate the presence of virulence factors such as biofilm production, haemolysis, invasiveness and molecular detection of fimH and stx1 gene.

Result: Out of these 51.33% of incidence of E. coli was recorded. E. coli O84 and O149 serotypes were found most prevalent. Out of 77 isolates, 46 (59.7%) and 45 (58.4%) were positive for biofilm formation by tube method and modified CRA method, respectively. All E. coli isolates were showing invasiveness in congo red binding assay while none of the isolates was found haemolytic. Molecular detection revealed the presence of fimH (508bp) gene in 33.3% of tested samples while stx1 gene could not be detected in any isolates.
Poultry is an important source of egg and meat worldwide and the consumption of poultry products is continuously increasing due to rising human population. India ranks 3rd in egg production and 4th in chicken production in the world (Singh, 2018a).  The high consumption of poultry products also necessitates great care in safeguarding the industry against intimidating factors in terms of public health also. There are many bacterial and viral diseases that affect the poultry production and leads to great economic loss in industry. Among them, avian colibacillosis is a common bacterial disease of poultry characterized by air sacculitis, pericarditis, perihepatitis, peritonitis, osteomylitis or yolksac infection. The clinical signs of disease are reported as depression loss of appetite, dropped wing closed eyes, weight loss, decrease egg production, etc.

Escherichia coli is the most important pathogen causing primary and secondary bacterial infection such as colisepticaemia, coligranuloma, omphalitis and Hjarre’s disaese. Although the majority of E.coli strains remain commensal in avian gastrointestinal tract and other surface mucosa but certain strains are capable of causing disease and these are designated as avian pathogenic E. coli (APEC). Colisepticemia is a serious life-threatening condition, which is associated with acute onset of very generalized clinical signs of sickness, such as listlessness, depression, weakness, loss of appetite, and sudden death of birds (Kuznetsova et al., 2020).

Serotyping is useful tool for epidemiological studies. Immune response in poultry against APEC is directly related to “O” antigens (Nolan et al., 2013). Several virulence factors have been associated with the pathogenicty of APEC including haemolysin, adhesin (fim), toxins, invasins, capsule as well as lipopolysaccharide complex (Ewers et al., 2003; Saha et al., 2020). However, several studies have demonstrated that all virulence factors are rarely present in same isolate. Keeping these views, this study was carried out to investigate the presence of some virulence factors such as haemolysin, adhesin, invasiveness, fimbrial adhesin (fim) and heat stable toxins (stx) of E. coli isolated from poultry with confirmed cases of colibacillosis.
The work was carried out in the Department of Veterinary Microbiology, College of Veterinary Sciences and A.H., Mhow during May 2018 to June 2019.
 
Sample collection
 
A total number of 150 samples (liver, heart, lungs, air sacs and feaces) were collected from chickens exhibiting different pathological conditions like perihepatitis, pericarditis, enteritis  and air sacculitis from various organized poultry farms and backyard poultry situated in and around Mhow and Indore cities (Fig 1). All samples were collected aseptically in sterile containers and kept at 4°C till further use.

Fig 1: Pericarditis in poultry.


 
Isolation and identification of E. coli
 
Samples were enriched in brain heart infusion (BHI) broth with incubation at 37°C for 24 hrs followed by subculture on nutrient agar, MacConkey agar and selective media eosin methylene blue (EMB) agar. The organisms were confirmed on the basis of bacterial morphology, cultural characteristics and biochemical tests (Cheesbrough, 1994).
 
Serotyping of E. coli
 
The identified E. coli isolates were sent to National Salmonella and Escherichia Centre, Central Research Institute, Kasauli (H.P.) for serotyping of somatic (O) antigen.
 
Haemolysis
 
Haemolytic activity of E. coli was demonstrated on 5% defibrinated sheep blood agar plate as per the method of Shiva Shankar et al., (2010). The plates were exhibiting “greening” or clearing of the agar around areas of bacterial growth as an indication of alpha or beta hemolytic activity (Rodriguez-Siek et al., 2005).
 
Biofilm formation
Tube method
 
Tube method of biofilm formation was followed as per Dhanawade et al., (2010). All the samples were inoculated in tryptose soya broth (TSB) and after 12 hrs incubation 50% media were replaced with the TSB containing 0.25% sucrose followed by staining with 0.1% safranin. A visible film lined the inner wall and bottom of the tube was considered positive for biofilm formation. The amount of biofilm formation was scored as weak, moderate and strong.
 
Modified congo red agar method
 
Congo red agar was prepared by using blood agar, glucose and congo-red dye as described by Nachammai et al., (2016). Black colonies with a dry crystalline consistency indicated biofilm production; non-biofilm producers usually remained pink.
 
Congo red dye binding assay
 
The test was performed to determine invasiveness of pathogen as per method described by Yadav et al., (2014). The isolates were streaked on tryptone soya agar media containing 0.03% congo red dye, and incubated for 24-72 hrs at both 37°C and 25°C. Appearance of intense orange or red colonies was recorded as a positive reaction. Negative colonies did not bind the dye and remained white or grey.
 
Molecular detection of Virulence genes (fimH and Stx1)
DNA extraction
 
DNA was extracted from 18 randomly selected E. coli isolates. Bacterial pellet was formed from 1.5 ml of an overnight bacterial culture of each isolate and DNA was extracted from each pellet as per manufacturer instructions supplied with the kit (HipurA Bacterial genomic DNA purification kit, Hi Media). DNA was stored at -20oC till further use.
 
Polymerase chain reaction for FimH and Stx1
 
The PCR amplification was carried out for fimH gene and stx1 gene of E. coli with specific published primers (Fernandes et al., 2011; Eid et al., 2016). The nucleotide sequences of the forward and reverse primers for amplification of 508 bp product of fimH gene were as - forward (5’-TGCAGAACGGATAAGCCGTGG-3’) and reverse (5’- GCAGTCACCTGCCCTCCGGTA-3’) (Table1). The nucleotide sequences of the forward and reverse primers were stx1 F (5’- CAGTTAATGTGGTGG CGAAG-3’) and stx1 R (5'- GCAGTCACCTGCCCTCCGGTA-3') for amplification of 180 bp product of stx1 gene. The PCR reaction was optimized for both amplification by adding 2.5 μl of 10X PCR buffer (without MgCl2), 2 μl of 25 mM Mgcl2, 1.0 μl of forward and reverse primer (20 pm/μl) each, 1.0 μl of dNTPs mix (10mM each), 0.2 μl Taq DNA polymerase (5 units/ μl), 5 μl of extracted DNA (150-200 ng) and the reaction was made up to 25 μl using nuclease free water. The amplification cycles for fimH gene were initial denaturation at 95oC for 3 min, 35 cycles of denaturation at 95oC for 45 sec, annealing at 60oC for 45 sec, extension at 72oC for 1 min and final extension at 72oC for 10 min. Annealing temperature for stx1 gene was 51oC for 30 sec and rest of the cycles were same as fimH gene The negative control consisted of sterile water instead of DNA template. The amplified products were electrophoresed in 1.5% agarose gel and visualized in gel documentation system (Syngene, USA). A 508 bp amplified product of fim H gene was sequenced commercially. The presence of fimH gene of E. coli was confirmed through NCBI blast GenBank database.
Avian colibacillosis contiues to be a major problem of morbidity and mortality in poultry and chickens. E.coli is very diverse species of bacteria found naturally in the intestinal tract of poultry. Several structures and products of E.coli have been found to contribute in virulence of the strain, such as capsule, pilli, haemolysin etc. This investigation was associated with the determination of many virulence factors of E.coli strains isolated from colibacillosis cases of poultry.

In present study, out of 150 samples, 77 (51.33%) E. coli isolates were recovered from colibacillosis cases on the morphological and cultural characteristics of E. coli were confirmed on the basis of previous observations recommended by Edwards and Ewing (1972). The biochemical behaviour of the isolates in accordance of Edwards and Ewing (1972) and Sahoo et al., (2012). All E. coli isolates were exhibited purple-black colonies with dark centre metallic sheen on EMB agar (Fig 2). The incidence of E. coli was found 44.44% and 61.66% in organized farms and backyard poultry, respectively (Table 1). Several studies also recorded higher rates of E.coli in poultry (Peer et al., 2013; Ammar et al., 2014). However, in a study 24% of incidence of E.coli were revealed from diarrhoic and non- diarrhoeic poultry samples (Debbarma et al., 2019). Young aged broilers have high affinity to disease in comparison to older chickens (Radwan et al., 2014). Kumar and Gupta (2019) also recovered 106 E.coli isolates from broilers suffering from colibacillosis. There are many different predisposing factors which may increase the affinity of poultry to colibacillosis such as immunosupression due to respiratory viral infections of chickens, production stress and close confinement. In present study, backyard poultry highly affected than organized poultry farm. It may be because of bad hygenic measusres and poor management of poultry.

Table 1: Incidence of E. coli in chicken.



Fig 2: E. coli on EMB agar showing metallic sheen.



All isolates were serotyped from National Salmonella and Escherichia Centre, Central Research Institute, Kasauli, (H.P.). Out of 77 isolates 69 could be typed in different serogroups such as O84 and O149 serogroups were found most prevalent (15.58% each) followed by O120 (11.68%), O9 and O145 (10.38%), O20 (9.09%), O83 (7.79%), and O2 and O119 (2.59%, each) while remain 8 isolates could not be typed (Fig 3). In this study 10 different serotypes were identified from 69 E.coli isolates. E.coli O2 is reported most prevalent serotype associated with colibacillosis worldwide. In accordance to many studies, serotype O2 also recovered from poultry colibacillosis (Eid et al., 2016, Kumar et al., 2019 and Ibrahim et al., 2019). Our results revealed lower percentage of E. coli O2 serotype which is similar to findings of Eid et al., (2016), Shiva Shankar et al., (2010). The serogroup O11 was recorded by Wani et al., (2004) and Serogroup 09 was also found in the reports of Panda et al., (2010) and Sahoo et al., (2012). Similar to our findings serogroups O2, O9, O11, O20, O83, O119, O120 were reported by Rodriguez-siek et al., (2005). According to Ewers et al., (2007) the occurrence of a specific serotype and its role in disease production depends upon the health status of the birds, climatic conditions, geographical situation and management strategies. The variations are found in serotype prevalence from time to time and from region to region. However, serogroups do not reflect the virulence of the strains and therefore serotyping cannot be used as a useful diagnostic tool. It may be used only to characterize strains.

Fig 3: Frequency of ‘O’ serotypes of E. coli isolates.



E. coli produce many virulence factors that may contribute to its pathogenicity in extra-intestinal infections (Sharma et al., 2007). These virulence factors enable some members of the normal flora to elicit an infection by overcoming the host defence mechanisms (Emody et al., 2003). In the present investigation, none of the 77 E. coli isolates produced haemolysis on 5% defibrinated sheep blood agar. Various studies reported the avian E. coli as non-hemolytic and independent of haemolytic activity (Eid et al., 2016), which corresponds with our findings. However, some reports suggested that E.coli has the ability to produce haemolysin (Zahid et al., 2016). The production of different types of hemolysin has been frequently contributed to E. coli from intestinal and extraintestinal diseases. It causes the release of the Ferro from cells, providing iron for the bacteria (Salehi et al., 2007). But Sharada et al., (1999) also reported that avian E. coli to be pathogenic need not to be hemolytic. Shiva Shankar et al., (2010) reported only 1.52% E.coli isolates positive by qualitative test for hemolysis.

Biofilms are the surface bound colonized bacteria surrounded by exopolisaccharide matrix (Donlan and Costerton, 2002). Besides having zoonotic potential, avian pathogenic E. coli isolates are difficult to eradicate because of constant faecal excretion by the host and the ability of bacteria to form biofilms (Bauchart et al., 2010). Biofilms are of particular interest in the poultry industry and public health as these films can harbour pathogenic microorganisms. It confers bacteria with greater resistance to surface cleaning and disinfection since antimicrobial entry and diffusion into the biofilm matrix then become difficult (Skyberg et al., 2007). In the present study, a total 59.7% and 58.4% of E. coli isolates were found positive for biofilm formation by tube method and modified congo red agar method. These results were in agreement with the Skyberg et al., (2007) and Rodrigues et al., (2018) who reported biofilm formation in 55.8 and 55.2 percent E. coli isolates, respectively. In present study, the percentages of biofilm formation by two methods were close. However, contrast to our findings, lower percentage of isolates positive for biofilm production by CRA was reported by Deka (2014) and Dhanawade et al., (2010). Singh (2018b) reported a very high percentage of isolates positive for biofilm production by CRA (70.37%) than by tube (33.33%) method. Several factors have been implicated in the development of a model biofilm system, such as media composition, temperature, presence of antimicrobial agents, the causal organism, quantity of inoculum, hydrodynamics forces and characteristics of the substrata (Donlan and Costerton, 2002).

The Congo red binding ability is used as a phenotypic marker of colisepticemic (invasive) and non-colisepticemic E. coli in poultry and also as an epidemiological marker for discrimination of pathogenic strains from the commensals (Yadav et al., 2014). In present study, all 77 E. coli isolates were found positive for congo red dye binding assay. Several studies also reported 91.7% to 100% of E. coli positive for this assay (Shiva Shankar et al., 2010; Yadav et al., 2014; Eid et al., 2016), which is in agreement with present study. Among many tests, Congo red binding is commonly used as markers of hydrophobicity and has been linked directly to virulence and pathogenicity (Qadri et al., 1988).

Infection ability of E. coli strains is facilitated by a broad variety of virulence factors coded by their virulence-associated genes (VAG). Among them host tissue colonization is thought to be initiated by fimbrial adhesins. Type 1 fimbriae are most commonly encountered in avian pathogenic E. coli. These are encoded by a fim cluster gene which required for its biosynthesis (Pusz et al., 2014). FimH are claimed to be responsible for the first step in the colonization and this indicates that fimH have important role in the pathogenesis of avian colibacillosis (Roussan et al., 2014). Out of 18 tested samples, 33.3% isolates were exhibit 508 bp of amplified product of fimH gene while all tested samples found negative for stx1 gene (Fig 4). Serotype O83, O120, O149, O11 and UT (Untyped) were revealed the presence of fimH gene. Amplified fimH gene sequence was confirmed by blasting in NCBI GenBank.

Fig 4: Results of PCR showing fimH gene. Lane 1: 100 bp ladder Lane 2: Positive control Lane 3-5: Positive sample Lane 6: Negative control



In this study 33.3% of type 1 fimbrial adhesion gene (fimH) were reported in tested samples, which is similar to the findings of Mbanga and Nyarari (2015). Other workers have reported higher occurrences of the fimH gene (Van der westhuizen and Bragg, 2012). In present study, stx1 gene could not be detected, which agree with reports of Janben et al., (2001) and Wani et al., (2004), but disagree with findings of Eid et al., (2016) who detected stx1 gene in 100% of E. coli isolates. These finding revealed that stx1 is not the responsible factor for virulence of E. coli in tested samples.

This study conclude that avian colibacillosis prevalent in Mhow and Indore region. Congo red binding activities, biofilm production and presence of fim H gene were found responsible virulence factors associated with the colibacillosis in this study. Haemolysis is not the essential virulence factor for pathogenecity of E.coli. Multiple virulence factors of APEC can be responsible for colibacillosis but these all are not present in same isolate.
The authors are thankful to Dean, College of Veterinary Science and Animal Husbandry, Mhow to provide the necessary funds and facility to conduct the research.

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