Biosurveillance of avian influenza and Newcastle disease viruses in the Barda region of Azerbaijan using real time RT-PCR and hemagglutination inhibition

Mahira Vatani,Shalala Zeynalova,Fizuli Guliyev,Bahruz Abbasov

doi:10.3389/fmicb.2015.01128

Abstract

The Azerbaijan State Veterinary Control Service (SVCS) has conducted active serological surveillance for avian influenza (AI) in poultry since 2006, when the first outbreak of AI H5N1 occurred in Azerbaijan. Samples are collected from September to May annually and tested using a hemagglutination inhibition (HI) assay to detect antibodies against H5 AI viruses. HI testing is also performed for Newcastle disease virus (NDV) upon request, but since this method cannot distinguish between natural infections and immune responses to vaccination, all positive results require follow-up epidemiological investigations. Furthermore, blood collection for the surveillance program is time-intensive and can be stressful to birds. In order to improve the national surveillance program, alternative sampling and testing methodologies were applied among a population of birds in the Barda region and compared with results of the national surveillance program. Tracheal and cloacal swabs were collected instead of blood. Rather than testing individual samples, RNA was pooled to conserve resources and time, and pools were tested by real-time reverse transcription polymerase chain reaction (rRT-PCR). Environmental sampling at a live bird market was also introduced as another surveillance mechanism. A total of 1,030 swabs were collected, comprising tracheal, and cloacal samples from 441 birds and 148 environmental surface samples from farms or the live bird market. During the same time, 3,890 blood samples were collected nationally for the surveillance program; 400 of these samples originated in the Barda region. Birds sampled for rRT-PCR were likely different than those tested as part of national surveillance. All swab samples tested negative by rRT-PCR for both AI and NDV. All blood samples tested negative for H5 by HI, while 6.2% of all samples and 5% of the Barda samples tested positive for exposure to NDV. Follow-up investigations found that positive samples were from birds vaccinated in the previous month. This study demonstrated that taking swabs was quicker and less invasive than blood collection. Results of rRT-PCR testing were similar to HI testing for H5 but also ruled out infection with all influenza type A viruses and not just H5. In addition, rRT-PCR testing was able to rule out active infections with NDV.

Highlights

Influenza A virus subtype H5N1, known as highly pathogenic avian influenza (HPAI) or H5N1, is a virus that infects birds and can cause serious illness and death in humans (Liu et al, 2005; Alexander and Capua, 2009; Belser et al, 2011)
The specific goals of this study were to use reverse transcription polymerase chain reaction (rRT-PCR) to test tracheal and cloacal swab samples for AI and Newcastle disease (ND) viral RNA; to screen samples for the influenza A matrix gene so as to detect all subtypes known to be pathogenic to humans; to implement sample pooling as a way to reduce the costs of testing; and to introduce environmental sampling in live bird markets as a way to increase the chances of detecting disease in an area where species of birds from many locations are commingled
The results from the rRT-PCR testing conducted in this study aligned with the results of the State Veterinary Control Service (SVCS) hemagglutination inhibition (HI) testing program for H5, which has not detected any birds infected with AI H5 since 2006

Summary

Introduction

Influenza A virus subtype H5N1, known as highly pathogenic avian influenza (HPAI) or H5N1, is a virus that infects birds and can cause serious illness and death in humans (Liu et al, 2005; Alexander and Capua, 2009; Belser et al, 2011). After its re-emergence in 2003 and 2004, the virus spread from Asia to Europe and Africa, where it has since resulted in over 800 human infections and 400 deaths (World Health Organization [WHO], 2005; Gilsdorf et al, 2006; Belser et al, 2011). Migratory birds can spread H5N1 and other avian viruses over long distances (OIE World Organization for Animal Health, 2012). The circulation of H5N1 in poultry threatens public health, since this strain has the potential to cause serious disease in people and is capable of mutating into a form that is more transmissible among humans, increasing the risk of a pandemic and mass mortality event

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