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Pathological and Molecular Investigation of Avian Influenza (ai) in Layer Chickens from Field Outbreaks

T Bari

MAHNA Khan

EH Chowdhury

MT Islam

M Pervin

SA Happy

Abstract/ Executive Summary:

Influenza A viruses infects a variety of animals including human, pigs, horses, marine mammals and birds. All currently known Influenza A subtypes are found in birds and are mostly asymptomatic in aquatic birds. Diagnosis of avian influenza require reference laboratory and appropriate technology for effective investigation and management. In this study gross, microscopic and molecular investigation was carried out in layer chickens naturally infected with Avian influenza (AI). AI viral RNA was extracted from the formalin fixed tissue sample obtained from Field Diseases Investigation Laboratory (FDIL), Manikganj. At time of necropsy the suspected birds (N=10) were sunk in 10% neutral buffered formalin. Trachea (10mg) from the suspected birds were collected in appendorf tubes and viral RNA were extracted using commercial kits. Representative tissues were fixed in 10% neutral buffer formalin for histopathological examination. Viral RNA from confirmed cases of AI was obtained from Bangladesh Livestock Research Institute (BLRI) (A/chicken/Bangladesh/BL 84T/2007/(H5N1). Viral RNA from both the sources were used in molecular detection of AI using one step Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). Results of this study showed high morbidity and mortality in layer chickens in the affected farms. All the chicken in the affected farms were destroyed immediately after outbreak, hence accurate morbidity and mortality rate were not obtained. Results of gross examination revealed hemorrhagic and cyanotic shanks, comb and wattle. Hemorrhages and congestion were common in upper respiratory tract and in the internal organs. The histopathologycal study revealed the presence of wide spread hemorrhages in trachea, lungs, spleen, liver, kidney, skin and intestine. The death of affected chickens as seen is natural outbreak could be due to respiratory failure as there were massive congestion, haemorrhages and exudation in lungs. When tested by RT-PCR, extracted RNA from BLRI were positive for matrix protein (MP) gene and H5 gene, but did not amplify MP gene and H5 gene of H5N1 AI from formalin fixed tissue RNA. The RT-PCR protocol we adopt can be used to diagnose AI with the RNA obtained from fresh tissues. RNA extracted from formalin fixed tissues can not be used in RT-PCR detection of AI viruses from field cases. However, Proteinase K treatment of extracted RNA from formalin fixed tissue could have improved physiology of Viral RNA and be used in RT-PCR detection of MP and H5 genes of AI viruses, required further investigation.

Keywords: RT-PCR, Avian influenza, Histopathology, Viral RNA

Location: Manikganj

Start Date: 00-01-2008

End Date: 00-12-2008

Program Area: Animal Health and Management

Commodity: Chicken

Objectives:

To investigate gross and microscopic changes in tissues to understand the mechanism of death following AI outbreaks and
To adopt molecular protocol (RT-PCR) to diagnose AI from field outbreaks.

Methodology:

A total of 10 dead layer birds from different field outbreaks submitted to FDIL, Manikganj during the period from January to December 2008 constitute the materials of this study. Extracted viral RNA from confirmed cases of AI from field cases was obtained from Bangladesh Livestock Research Institute (A/chicken/Bangladesh/BL 84T/2007/(H5N1) and used as positive control for RT-PCR detection of AI virus. A detail necropsy was carried out of suspected birds (n=10) sunk in 10% neutral buffered formalin. Necessary precaution was taken not to get infected during study procedure at FDIL, Manikganj. The comb, wattle, legs, shanks, beaks were examined carefully and changes were noted. After a systemic dissection changes in the internal organs were recorded and representative samples were collected for histopathological study. Following necropsy the comb, wattle, trachea, lungs, liver, brain, spleen, heart, kidney, spleen, caecal tonsils, intestines, proventriculas, shanks and egg follicles were collected and fixed in 10% neutral buffered formalin. After fixation the tissues were processed, sectioned and stained as per standard procedure. All the samples were studied under low (10x) and high power (40x, 100x) microscopic field and captured images of notable changes. The extracted RNA from formalin fixed tissues and RNA obtained from known case of AI from BLRI was used in RT-PCR. The RT-PCR condition for the amplification of MP gene of AI virus was adopt with following cycling conditions. A reverse transcription was carried out for 45min at 45°C. In amplification reaction an initial denaturation was set at 95°C for 4min. The 40 cycles of amplification reaction in a thermocycler consisting of denaturation for 60secs at 95°C, annealing for 60secs at 45°C and extension for 3min at 72°C. The final elongation was set at 72°C for 10min and the reaction was held at 4°C. The RT- PCR condition for the amplification of H5 gene of AI virus (Lee et al. 2001) was set at 45°C for 45mins (reverse transcription) and 95°C for 3 min (initial denaturation). The thermal profile of 35 cycles of amplification reaction consisting of 95°C for 30secs, 50°C for 40secs and 72°C for 40secs. The final elongation was set at 72°C for 10mins and the reaction was held at 4°C. The amplified PCR products were electrophoresed in 1.5% agarose gel, stained with ethidium bromide and examined against UV light using an image documentation system (Photo Doc, Labortek, Germany).

Source of Information: Bangladesh Vet J (2009) 43(1-4):40-51

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

This study adopt an RT-PCR protocol for the detection of MP gene and H5 gene of AI viral RNA. This study provide evidence that the RT-PCR protocol we adopted to detect MP and H5 genes of highly pathogenic avian influenza virus of AI (viral RNA obtained from BLRI) successfully amplify both the genes and this RT-PCR protocol can be used to detect field outbreaks of AI. Viral RNA extracted from formalin fixated tissues failed to amplify any of the MP and H5 genes. Formalin fixation killed RNA viruses, led chemical modification of the RNA and enabled cross-linking of nucleic acids and proteins. Formalin fixation results in an approximately 2 log10 reduction in detection limit of genomic RNA in compared to fresh tissues. However, proteinase K digestion (24h) could improve extraction of amplifiable RNA from formalin fixed tissues. In this investigation the suspected tissues was sunk in 10% buffered neutral formalin in order to avoid viral contamination, collected upper respiratory tract and extracted viral RNA 24hors after the fixation. From formalin fixed sample we were able to extract amplifiable RNA but failed to amplify viral genome in RT-PCR protocol. This could be due to the damaging effect of formalin on to the viral RNA or starting with very low concentration of viuses in the samples. However, proteinase K digestion can improve the genomic RNA extraction procedure from the formalin fixed tissues. It needs to improve RNA extraction procedure from formalin fixed sample in order to carry out RT-PCR protocol for the detection of AI viruses.

Knowledge Management: Journal