Year : 2018, Volume : 42, Issue : 1
First page : ( 51) Last page : ( 57)
Print ISSN : 0250-4758. Online ISSN : 0973-970X. Published online : 2018 March 1.
Article DOI : 10.5958/0973-970X.2018.00009.3

Etiopathological studies on low pathogenic avian influenza and molecular detection of concurrent infections in broiler chicken

Chaudhari Sonal V.³, Joshi B.P.^3,*, Joshi C.G.^1,3, Shah R.K.^1,3, Bhanderi B.B.^2,3, Ghodasara D.J.³

³Department of Veterinary pathology, College of Veterinary Science and Animal Husbandry, AAU, Anand-388001, Gujarat, India

¹Department of Animal Biotechnology, College of Veterinary Science and Animal Husbandry, AAU, Anand-388001, Gujarat, India

²Department of Veterinary Microbiology, College of Veterinary Science and Animal Husbandry, AAU, Anand-388001, Gujarat, India

*Corresponding author: e-mail: b4bpjoshi@gmail.com

Received:  26  July,  2017; Accepted:  29  January,  2018.

Abstract

The present study was carried out to investigate the mortality in broiler chicken due tolow pathogenic avian influenza (LPAI) and concurrent infections of infectious bronchitis (IB) virus, avian mycoplasma and Escherichia Coli. The study comprised of flock wise mortality, gross and histopathological examination of trachea, bronchi, lung and air sacs and molecular detection of LPAI (H9N2), IB virus, Mycoplasma Gallisepticum (MG) and Mycoplasma Synoviae (MS) and avian pathogenic E. coli (APEC) from pooled samples of bronchi containing caseous plugs from thirty broiler flocks. The mortality among these flocks ranged between 1.26 to 9.08 percent with an average of 3.20 percent per flock. Gross and histopathological lesions were predominantly found in trachea, bronchi, lungs and air sacs. All the flocks (100%) were found positive for LPAI (H9N2) virus as well as for pathogenic E.coli, whereas IB virus, M. gallisepticum (MG), M. synoviae (MS) were detected form 6 (20%), 11 (36.67%), and 3 (10%) flocks, respectively. Molecular characterization of E. coli for the detection of virulence genesrevealed 27 (90%) cases positive for presence of iss gene followed by 25 (83.33%) for vat gene, 15 (50%) for tsh gene and 10 (33.33%) for papC gene. It was concluded that among different respiratory pathogens, mixed infections of LPAI, E. coli and M. gallisepticum were consistent in tracheo-bronchitis with caseous plugs, leading to increased mortality in the broilers.

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Keywords

Broilers, LPAI, PCR, Respiratory infections.

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INTRODUCTION

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MATERIALS AND METHODS

Gross and histopathology

The study was carried out in thirty broiler flocks belonged to different broiler farms and received for post mortem diagnosis at Department of Pathology, Veterinary College, Anand. The pathological study comprised information related to strength of the flock, age, clinical signs, mortality pattern and gross as well as microscopic lesions. Regular necropsies were carried out and flocks showing caseous tracheo-bronchitis were selected for study. Detailed post mortem examination was carried out and gross pathological lesions were recorded. During necropsy, the tissue pieces of organs including trachea, bronchi with caseous plug, lung, and air sacs were collected in 10% neutral buffer formalin.

Molecular detection

For molecular identification of LPAI (H9N2), IB virus, M. gallisepticum, M. synoviae and E.coli, the pooled bronchial tissues including caseous plugs (three samples were pooled from each flock) were collected at the time of post mortem examination in cryovials and stored at −20°C after adding RNA later® solution for RNA stabilization and storage till processing. Frozen tissues were thawed and processed for extraction of the RNA and DNA required for PCR identification. Viral RNA was extracted from all the different pooled samples of bronchial tissue containing caseous plugs by TRIzol-chloroform method. After extraction of viral RNA, Reverse Transcriptase- Polymerase Chain Reaction (RT-PCR) was carried out following two steps method, i.e., synthesis of cDNA from RNA and then PCR with H9 and N2 and IBV specific primers with amplicon size of 287, 310 and 143bp respectively⁶,⁷. DNA was extracted from all the pooled samples of bronchial tissues containing caseous plugs by ultraclean tissue and cell DNA isolation kit. After extraction of bacterial DNA, polymerase chain reaction was carried out with M. gallisepticum (mec2) and M. synoviae (16Sr RNA) specific primers with amplicon size of 302 and 207 bp and for avian pathogenic E.coli virulence marker genes especially iss, papC, tsh and vat forward and reverse primer with amplicon size of 174, 201, 153 and 168 bp respectively⁸,⁹-¹⁰.

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RESULTS

Flock wise mortality

Studies on the flock wise mortality among thirty broiler flocks having total population of 1,67,060 birds of second to fifth week of age showed mortality between 1.26 to 9.08 percent. Broiler chicks as early as day 12 showed clinical signs like respiratory difficulty with gasping and anorexia with gross lesions of caseous tracheo-bronchitis. Majority of the flocks have reported mortality between 12 and 30 days of age. The week wise average mortality was 3.76, 3.60, 2.73 and 3.06 per cent respectively during second, third, fourth and fifth week of age. The mortality was mainly observed during third and fourth weeks of age with higher occurrence during summer and winter months.

Pathological lesions

Gross pathological lesions were predominantly found in organs of respiratory system i.e. in trachea, bronchi, lungs and air sacs. The tracheal lesions observed were severe congestion, excessive mucous exudate, presence of fibrino- necrotic or caseous plug in the tracheal bifurcation and extending in the bronchi (Fig. 1). The lung lesions were mild to marked oedema and congestion. In some of flocks affected during early age, air sacs revealed thickening with fibrin deposition.

Microscopic changes were consistently seen in the trachea and bronchi and were variable from mild to severe. In many cases trachea and bronchi showed congestion along with infiltration of heterophils and lymphocytes in the mucosa and submucosa and presence of fibrinonecrotic masses in the lumen. The tracheal lesions revealed loss of cilia and hypertrophy of mucous glands in early stages of the disease followed by degeneration and denudation of epithelium with infiltration of heterophils and lymphocytes (Fig. 2). In some cases, deposition of fibrinonecrotic mass in the tracheal lumen was observed. The section of bronchi of affected birds showed presence of fibrino necrotic masses in the bronchial lumen which were occluding the air passage (Fig. 3). In addition, epithelialdenudation, infiltration of heterophils and lymphocytes and mild to moderate congestion were also evident in the bronchial mucosa (Fig. 4). The microscopic lesions observed in the lungs were vascular engorgement and haemorrhages, smooth muscle hypertrophy and infiltration of lymphocytic mononuclear cells in the parabronchi. In some cases, lumens of parabronchi were filled with fibrinous exudate. Air sacs showed congestion, presence of fibrinous exudate and lymphocytic infiltration (Fig. 5).

Molecular detection of LPAI (H9N2), IBV, MG, MS and E. coli

All the 30 pooled samples (three samples were pooled from each flock) of bronchi containing caseous plugs were tested along with the positive control for the presence of LPAI (H9 and N2), IBV, MG, MS and avian pathogenic E.coli using specific primers. All the 30 samples (100%) were found positive for LPAI (H9N2) (Fig. 6) as well as for pathogenic E.coli where as IBV, MG and MS were detected from 6(20%), 11(36.67%) and 3(10%) flocks respectively (Table 1; Fig. 7,8,9). In addition to above it was also seen that out of 30 broiler flocks all the 30 (100%) were positive for LPAI+APEC, 13 flocks (43.33%) positive for only LPAI + APEC without complications of IBV + MG + MS; seven flocks (23.33%) positive for LPAI + APEC + MG; four flocks (13.33%) positive for LPAI + APEC + IBV + MG; three flocks (10%) positive for LPAI + APEC + MS and two flocks (6.67%) positive for LPAI + APEC + IBV.

Detection of virulence genes in E. coli by PCR (iss, pap C, tsh and vat)

Molecular characterization of E. coli was carried out to detect presence of four virulence genes i.e. iss, papC, tsh and vat. DNA was extracted from tissue samples (bronchial caseous plugs) and subjected to PCR with gene specific primer pairs. Out of 30 flocks positive for E. coli, the highest number (27) were found positive for iss gene followed by vat (25), tsh(15) and papC gene (13) (Table 2; Fig. 10). Among these, 18 were found carrying two virulence genes out of four. There were seven samples which were positive for three genes while five were having all the four virulence genes in their genome. There was no sample having only one gene of virulence during the study.

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DISCUSSION

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ACKNOWLEDGEMENTS

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Figures

Fig. 1.: Bronchial lumen occluded with caseous mass (arrow) in flock affected with LPAI TopBack

Fig. 2.: Section of trachea showing marked congestion, denudation of epithelium, infiltration of heterophils and lymphocytes.H&E ×120 TopBack

Fig. 3.: Section of bronchi showing fibrinonecrotic masses in the bronchial lumen occluding the air passage. H&E ×50 TopBack

Fig. 4.: Section of bronchi from LPAI affected bird showing congestion, epithelial denudation and deposition offibrinonecrotic mass in the bronchial lumen.H&E ×120 TopBack

Fig. 5.: Section of air sac showing presence of fibrinous exudate and moderate to severe infiltration of heterophils and lymphocytes.H&E ×120 TopBack

Fig. 6.: Gel image showing amplified product with H9 and N2 specific primers. L: 100 bp ladder, H9: +ve control for H9, N2: +ve control for N2, and S: Test sample. TopBack

Fig. 7.: Gel image showing amplified product with IBV specific primers. L: 100 bp ladder, P: +ve control for IBV, N: -ve control for IBV, and 1 to 6: Test sample TopBack

Fig. 8.: Gel image showing amplified product with M. gallisepticum (MG) specific primers. L: 50 bp ladder, P: +ve control for MG, N: -ve control for MG, and 1 to 5: Test samples TopBack

Fig. 9.: Gel image showing amplified product with M. synoviae (MS) specific primers. L: 50 bp ladder, 1 to 3: Test sample TopBack

Fig. 10.: Gel image showing amplified product of E.coli virulence gene. L: 100 bp ladder, iss: +ve control for issgene, pap C: +ve control for pap C gene, tsh: +ve control for tsh gene, vat: +ve control for vat gene and S: test sample. TopBack

Tables