Prevalence, pathology and molecular characterization of Escherichia coli in chickens

Introduction

The Indian poultry sector is a rapidly growing industry that has transitioned into a highly organized sector since the 1960s. Currently, India ranks 3 rd in egg production and 8 th in poultry meat production globally. Poultry products are valued for their high protein, vitamin, and mineral content, as well as their affordability and religious acceptance.However, the intensive practices used to meet high demand have made poultry increasingly susceptible to infectious and metabolic diseases. Key management factors for health include a disease-free environment, proper sanitation, and adequate nutrition. Stressors like unhygienic conditions, overcrowding, and temperature extremes often lead to immunocompromise, allowing bacterial and viral infections to cause significant economic losses. Avian Colibacillosis, caused by Avian pathogenic Escherichia coli (APEC), is a major global threat to the industry. Its most severe form, Colisepticaemia, is a systemic infection affecting multiple organs. Other manifestations include respiratory issues like air sacculitis, and reproductive infections such as salpingitis and egg peritonitis. These conditions result in high flock mortality and decreased egg production, leading to substantial financial impacts on both broiler and layer operations. Avian Colibacillosis is caused by Escherichia coli, a Gram-negative bacterium and normal inhabitant of the poultry intestinal and respiratory tracts. Disease occurs when specific pathogenic strains, categorized as Avian Pathogenic *E. coli* (APEC), exploit host stressors such as immunosuppression or concurrent infections. APEC, a member of the extra-intestinal (ExPEC) group, causes significant morbidity, mortality, and economic losses, particularly in young chicks.

It often acts as an opportunistic pathogen triggered by environmental or viral factors. Transmission occurs horizontally through contaminated environment, feed, and water, or vertically via eggs. APEC also poses zoonotic risks and serves as a major reservoir for antimicrobial resistance.

Materials and Methods

The present study was undertaken for a period of 9 months from November 2021 to July 2022 in 20 poultry farms and the prevalence of avian Colibacillosis were recorded based on typical gross and microscopic lesions in chickens of Durg, Raipur, Rajnandgaon and Surajpur districts of Chhattisgarh.

Pathological examination Gross pathology

Whole carcasses collected from different poultry farms were routinely subjected to detailed and systemic necropsy examination, giving special attention to lesions in liver, heart, lungs, spleen, intestine, oviduct and yolk sac.

Histopathology

Tissue samples from various organs (liver, heart, lungs, spleen, intestine, and oviduct) of birds suspected of Colibacillosis were collected in 10% buffered formalin for histological examination. Specimens were sectioned to 0.5mm, washed overnight, and dehydrated through a graded acetone series. Following clearing with benzene and paraffin embedding, 3-5μm sections were cut, deparaffinized in xylene, and stained with Haematoxylin and Eosin (H&E) before mounting with DPX (Culling, 1963) [10] . Finally, the stained sections were examined under light microscopy.

Isolation and identification of E. coli

Isolation of bacteria: Swab samples from the internal organs of dead birds were inoculated in nutrient broth and incubated at 37°C for 24 hours. The samples were then streaked onto MacConkey agar (MCA) and incubated for another 24 hours at 37°C. Pink colonies from MCA were subcultured on Eosin Methylene Blue (EMB) agar. Isolates displaying a metallic sheen on EMB were identified as E. coli and confirmed via biochemical characterization. Pure colonies were then subcultured on nutrient agar slants, incubated, and preserved at 4°C under sterile liquid paraffin for further study.

Molecular Characterization of E. coli

Samples for PCR

The isolates initially showing cultural features, morphological and biochemical characteristics pertaining to E. coli were subjected to PCR using avian pathogenic E. coli specific tsh gene primer (Subedi et al., 2018) [39] .

Extraction of DNA from E. coli isolates

Isolation of DNA for the PCR assay was prepared by heat lysis method proposed by (Choudhari et al., 2020) [8] [45,27] .

Identification of APEC by PCR targeting tsh gene APEC-specific primers targeting a 153 bp E. coli region were used (Ewers et al., 2004) [17] . DNA was amplified via PCR following the protocol by Chaudhari et al. (2017) [7] . The 10 µl reaction mixture included 2 µl DNA, 0.4 µl of each primer, 5 µl TaqMixture, and 2.2 µl water. Thermal cycling involved initial denaturation at 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s, with a final extension at 72°C for 7 min.

Products were stored at 4°C.

Agarose gel electrophoresis

Following PCR, amplified products were visualized via 1.5% agarose gel electrophoresis. Briefly, a gel casting tray was prepared with a comb and leveled. Agarose (0.75g) was dissolved in 50ml 1X TAE buffer, melted, and cooled before adding 5μl Ethidium bromide. The solution was poured into the tray and solidified for 30 minutes. After removing the comb, the gel was placed in an electrophoresis chamber filled with 1X TAE buffer, ensuring wells faced the cathode. PCR products (8μl) mixed with loading dye (2μl) and a 100bp DNA ladder were loaded into the wells.

Electrophoresis was performed at 75V for approximately 45 minutes. Finally, the gel was visualized using a Gel Doc System.

Nucleotide Sequencing

Representative positive PCR products of the tsh gene were sequenced by Barcode Biosciences (Bengaluru) to generate consensus sequences. The resulting data, received as electropherograms and text files, were analyzed using NCBI BLAST to confirm specificity. Sequences were then compared with existing E. coli nucleotide data in GenBank (Dozois et al., 2000) [14] .

Phylogenetic analysis

The partial tsh gene sequences of APEC were compared with GenBank sequences. Nucleotide identities were aligned using MEGA 11 Clustal W and the Neighbour-joining method. Based on maximum identity scores, sequences were aligned to generate distances and construct a phylogenetic tree. [6] . Conversely, several studies reported significantly higher rates: Khaton et al. (2008) [25] found (83(-87%, while others recorded prevalence between (52.26% and 75.2% ( [3,4,13,29,46] . Jamoh et al. (2018) [23] reported up to 87.5% in broilers and 76.04% in layers. In India, Dadheech et al. (2016) [11] and Panchal et al. (2020) [31] also noted high prevalence, with the latter attributing a 71.38% rate to poor management. In contrast, Giasuddin et al. (2002) [22] , Islam et al. (2004) [22] , Omer et al. (2010) [30] , and Surjagade et al. (2020) [40] reported much lower rates (3.65% to 6.56%), suggesting that superior hygienic practices reduce infection levels.

Results and Discussion

Pathological examination Gross pathology

In this study, 250 post-mortem examinations were performed, identifying 47 cases with lesions suggestive of Colibacillosis. Necropsies revealed E. coli infections such as Colisepticaemia, Egg Peritonitis, and Omphalitis. Gross lesions were primarily observed in the liver, heart, spleen, lungs, intestine, oviduct, and yolk sac, varying by disease condition.

Colisepticaemia

In the present study Colisepticaemia was observed in 59.57% of Colibacillosis affected chickens. Generally young broiler and layer chickens showed the characteristic lesions of Colisepticaemia. Liver, heart, lungs, spleen and intestine were carefully examined in Colisepticaemia affected birds.

a) Liver

The most consistent lesions in Colibacillosis-affected birds were observed in the liver. These included fibrinous perihepatitis characterized by white to yellowish exudate (Fig. 3 and Fig. 4), necrotic foci, and ascites (Fig. 5). Mild cases showed hepatomegaly, congestion, and hemorrhages. [33,42,26,35,2] and pigeons (Dutta et al., 2013) [15] . Generalized visceral congestion likely resulted from bacterial endotoxins and vascular damage (Srinivasan et al., 2014) [38] .

b) Heart

Most chickens with Colisepticaemia exhibited typical fibrinous pericarditis. The pericardium was thickened, covered in thick fibrinous exudate, and adhered to the chest cavity. These findings align with observations by Khaton et al. (2008) [25] , Tonu et al. (2011) [42] , Shah et al. (2019) [35] , and Surjagade et al. (2020) [40] . Similar lesions were reported by Nakamura et al. (1985) [28] in natural Colibacillosis outbreaks, while Ahmad et al. (2022) [2] also noted adhesive fibrinous perihepatitis and pericarditis in affected broilers.

Lungs, spleen and Intenstine: Affected birds showed splenic enlargement, oedema, congestion, and multifocal necrotic foci (Fig. 7), with whitish fibrinous exudates in severe cases (Fig. 8). These observations align with previous reports (Nakamura et al., 1985; Srinivasan et al., (2014) [28,38] . Lungs exhibited congestion, haemorrhages, oedema, and focal fibrin deposits (Fig. 9), consistent with findings by Gangane et al. (2006), Khaton et al. (2008) [25] , and Kumar et al. (2013) [26] . Most birds displayed intestinal congestion, haemorrhages, and enteritis with thick yellowish exudate (Fig. 10 and Fig. 11). Severe cases presented fibrinous exudates on the gastrointestinal serosa and mesentery. These intestinal lesions correlate with studies by Khaton et al.

(2008) [25] , Tonu et al. (2011) [42] , Srinivasan et al. (2013) [38] , and Surjagade et al. (2020) [40] , who noted similar mucinous exudate and congestion.

Colisepticaemia

Histopathological analysis of the liver revealed thickened capsules with fibrin deposits and heterophil infiltration, characteristic of fibrinous perihepatitis (Fig. 12). Some instances also exhibited moderate fatty changes and Kupffer cell hyperplasia (Fig. 13). These findings align with observations by Jamoh et al. (2018) [23] and Surjagade et al.

(2020) [40] . Fibrinous perihepatitis may result from bacterial endotoxins and vascular injuries, which increase vascular permeability and allow fibrinogen leakage into tissues (Truscott et al., 1974; Vegad, 2007) [43,44] . Additionally, hepatic sinusoid dilatation, hepatocyte degeneration, Kupffer cell hyperplasia, congestion, and hemorrhages have been reported (Hooda et al., 2011) [21] . In the heart, microscopic changes included pericardial thickening with fibrinous exudate, cellular infiltration, and necrosis (Fig. 14,15). These findings align with the observations of Shrinivasan et al. (2014) [38] and Singh et al.

(2018) [23,36] . The spleen showed congestion, haemorrhages, lymphoid depletion, focal necrosis, and reticulo-endothelial cell proliferation (Fig. 16). These findings align with observations by Kumar et al. (2013) [26] and Singh et al.

(2018) [23,36] , who also noted lymphocyte depletion in lymphoid organs during E. coli infection. Lungs showed congestion, haemorrhages, and exudation in parabronchi, with dilated septae and perivascular exudate (Fig. 17). These lesions align with the findings of Abalaka et al. (2017) [1] and Shah et al. (2019) [35] . The intestine exhibited enteritis with mucosal epithelial desquamation and infiltration of heterophils and lymphocytes (Fig. 18). Degenerative changes, cryptal gland necrosis, and severe mononuclear cell infiltration were also observed (Fig. 19). Additionally, necrotic villi were heavily infiltrated with inflammatory cells. These findings align with reports by Jan et al. (2018) [24] and Singh et al. (2018) [23,36] . Molecular detection of E. coli isolates PCR of APEC targeting tsh gene DNA from 24 EMB-positive E. coli isolates was screened for the APEC virulence gene tsh via PCR using specific primers (Table 1). Results showed that 17 isolates (73.91%) were positive, producing a 153bp amplicon (Fig. 20). This indicates a high prevalence of APEC in the Durg district, consistent with findings by Delicato et al. (2002) [12] , Ewers et al. (2004) [17] , and Chaudhari et al. (2017) [7] . Similar high detection rates were reported by Jan et al. (2018) [24] , who found 80% positivity for tsh. Located on the ColV plasmid, tsh facilitates early-stage infection and visceral lesion development in chickens (Dozois et al., 2000) [14] . Previous studies also link tsh presence to high-virulence APEC strains.

Nucleotide Sequencing

Nucleotide sequencing of positive PCR product of

Conclusions

Over all prevalence of Colibacillosis in poultry farms was reported as 18.8% in Chhattisgarh. Highest prevalence was observed as 25% in Durg district followed by 20.37% in Raipur, 14.5% in Rajnandgaon and 12% in Surajpur districts of Chhattisgarh. Gross lesions in Colibacillosis affected chickens comprised of fibrinous perihepatitis, fibrinous pericarditis, retained unabsorbed yolk in chicks, rupture of ovarian follicles and watery to yellowish yolk materials in peritoneal cavity in layer birds. Microscopic examination revealed fibrinous perihepatitis and pericarditis along with infiltration of inflammatory cells, degenerative, necrotic changes in liver, heart and spleen along with lymphocytic depletion in spleen. Partial genome sequence of APEC showed 98.23% similarities with isolates of France and Norway which showed that they had a common ancestor.

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