EPIDEMIOLOGY OF AVIAN INFLUENZA IN DOMESTIC POULTRY AND WILD BIRDS IN THE TEMA METROPOLIS FENTENG DANSO EDWARD University of Ghana http://ugspace.ug.edu.gh SCHOOL OF PUBLIC HEALTH COLLEGE OF HEALTH SCIENCES UNIVERSITY OF GHANA, LEGON EPIDEMIOLOGY OF AVIAN INFLUENZA IN DOMESTIC POULTRY AND WILD BIRDS IN THE TEMA METROPOLIS By FENTENG DANSO EDWARD THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF MASTERS OF PHILOSOPY IN APPLIED EPIDEMIOLOGY AND DISEASE CONTROL NOVEMBER, 2010 University of Ghana http://ugspace.ug.edu.gh ii DECLARATION I hereby declare that this dissertation except for references to other peoples work which have been duly cited, this work is the result of my own research and that this dissertation has neither in whole nor in part been presented for any other degree. Candidate ……………………………….. FENTENG DANSO EDWARD Academic Supervisors ……………………………….. DR. WILLIAM KWABENA AMPOFO (Virology Dept., Noguchi Memorial Institute for Medical Research) ………………………………. PROFESSOR BAWA AWUMBILA (Dept. of Animal Science, University of Ghana, Legon) University of Ghana http://ugspace.ug.edu.gh iii DEDICATION This work is dedicated to my dear wife Kate Fenteng Danso and my lovely children, Kwame Fenteng Danso and Abena Asantewa Fenteng Danso. University of Ghana http://ugspace.ug.edu.gh iv ACKNOWLEDGEMENT My special thanks go to my academic supervisors; Dr. William Kwabena Ampofo and Professor Bawa Awumbila for their guidance throughout the research. I am grateful to the School of Public Health (SPH), Veterinary Services Directorate (VSD) and Centers for Disease Control and Prevention (CDC), Atlanta, USA for offering me the golden opportunity to pursue the post graduate programme in Applied Field Epidemiology and Disease Control. My sincere gratitude goes to Professor Edwin Afari, Dr. Agyen Frimpong, Mr. David Mensah, Dr. E. B. Koney, Dr. Michael Aryee, Dr. Samuel Sackey, Dr. Priscilla Awo Nortey, Dr. Richard Suu-ire, Dr. Joseph Awune, Dr. Owusu Darlington, Dr. Andy Kwabena Alhassan, Dr. Nathaniel Yebuah, Dr. Mark Hansen, Dr. Abuh Joseph, Dr. (Mrs.) Benita Anderson, Miss Cedonia Luuse, Mr. Kofi Bonney, Miss Ivy Asante, Mrs. Beatrice Amanquah, Miss Veronica Mensah, Mr. Joseph Asare Yirenkyi, Tema Metropolis Veterinary staff, staff of Wildlife Division of Forestry Commission at Sakumono Ramsar site, National Influenza Center team members of the Noguchi Memorial Institute for Medical Research and all people who in various ways made it possible for me to go through this work. My warm thanks to United States Naval Medical Research Unit #3 for all their support. University of Ghana http://ugspace.ug.edu.gh v ABSTRACT BACKGROUND Avian influenza (AI) is an infectious disease of birds caused by influenza type A viruses. Migratory waterfowl - most notably wild water fowls are the natural reservoir of all influenza A viruses. There are 16 subtypes of influenza A viruses, of which H5 and H7 subtypes are the most pathogenic In April 2007, the first outbreak of HPAI was reported in Ghana in a small scale poultry farm at Kakasunanka, near Michel Camp in the Tema Metropolis. There were subsequent outbreaks of the AI virus at Adjei Kojo in Tema Metropolis, Sunyani Municipality in the Brong Ahafo and Aflao in Ketu South District in the Volta regions in the same year. All infected poultry farms were stamped out. This study sought to determine the current profile of Avian Influenza viruses in domestic commercial poultry, backyard poultry, live bird markets and wild birds in the Tema Metropolis over a one year period and covering the dry and wet seasons. METHODS From May 2009 to March 2010, we administered a semi-structured questionnaire to poultry farmers and conducted a cross sectional study on 1282 field samples involving fresh faeces, tracheal and cloacal swabs from domestic poultry, live bird markets and wild birds from 16 communities in the Tema Metropolis. These samples were then University of Ghana http://ugspace.ug.edu.gh vi subjected to real-time Reverse Transcriptase- Polymerase chain reaction analysis for Influenza A virus. RESULTS All the 1282 avian samples tested, were negative for Influenza A viruses. However, Newcastle disease virus was detected in 8% (5/63) of the farms where birds sampled showed respiratory and nervious signs. Commercial farms accounted for 5%, backyard for 2% and live birds market 1%. Also, adherence by farmers to good poultry management practices and proper bio-security measures was found to be low. CONCLUSIONS There was no evidence of circulation of AI H5N1 among domestic poultry and wild birds in the study population, between May 2009 and March 2010. This negative result for AI virus in the study shows that measures taken by poultry farmers and other stakeholders were probably effective. However, VSD should conduct further education of farmers on good poultry practices and bio-security. University of Ghana http://ugspace.ug.edu.gh vii Table of Contents DECLARATION ....................................................................................................................... ii DEDICATION ......................................................................................................................... iii ACKNOWLEDGEMENT ......................................................................................................... iv ABSTRACT ............................................................................................................................... v LIST OF TABLES .................................................................................................................... x LIST OF FIGURES ................................................................................................................ xi LIST OF ABBREVIATIONS................................................................................................. xii CHAPTER ONE ........................................................................................................................ 1 1.1 INTRODUCTION.................................................................................................. 1 1.2 PROBLEM STATEMENT ..................................................................................... 3 1.3 JUSTIFICATION ................................................................................................. 4 1.4 GENERAL OBJECTIVE ........................................................................................... 5 1.5 SPECIFIC OBJECTIVES ......................................................................................... 5 1.6 RESEARCH QUESTIONS....................................................................................... 5 CHAPTER TWO ....................................................................................................................... 6 LITERATURE REVIEW ................................................................................................... 6 2.1 AVIAN INFLUENZA DISEASE .......................................................................... 6 2.2 HIGHLY PATHOGENIC AVIAN INFLUENZA H5N1 OF ASIA LINEAGE ...... 7 2.3 EMERGENCE OF HPAI H5N1 IN POULTRY IN SOUTHEAST ASIA............. 8 2.4 ECONOMIC CONSEQUENCES OF HPAI ............................................................ 9 2.5 DESCRIPTION OF AVIAN INFLUENZA VIRUS ............................................ 9 2.6 EPIDEMIOLOGY OF AVIAN INFLUENZA ........................................................ 12 University of Ghana http://ugspace.ug.edu.gh viii 2.7 GEOGRAPHICAL SPREAD OF HPAI H5N1 OUT OF SOUTHEAST ASIA 15 2.8 OUTBREAKS OF HPAI H5N1 SINCE 2006 AND THE CURRENT SITUATION .................................................................................................................... 17 2.9 MAJOR OUTBREAKS OF HPAI H5N1 IN WILD BIRDS ............................... 19 2.10 AVAIN INFLUENZA AND WETLANDS ........................................................... 21 2.11 WILDLIFE CONSERVATION IMPLICATIONS .............................................. 22 2.12 CLINICAL PRESENTATION OF AI IN POULTRY ......................................... 23 2.13 PATHOLOGY OF AI ............................................................................................ 25 2.14 DIFFERENTIAL DIAGNOSIS OF AI FROM OTHER DIEASES .................. 26 2.15 LABORATORY DIAGNOSTIC PROCEDURES OF AVIAN INFLUENZA .... 26 2.16 CONTROL MEASURES AGAINST HIGHLY PATHOGENIC AVIAN INFLUENZA (HPAI) ..................................................................................................... 32 2.17 VACCINATION .................................................................................................... 34 2.18 BIO-SECURITY MEASURES ........................................................................... 37 CHAPTER THREE ................................................................................................................. 38 METHOD ......................................................................................................................... 38 3.1 STUDY AREA ......................................................................................................... 38 3.2 STUDY DESIGN ................................................................................................ 41 3.4 SAMPLING PROCEDURE..................................................................................... 43 3.5 MOLECULAR DETECTION .................................................................................. 43 3.6 DATA PROCESSING AND ANALYSIS .............................................................. 46 3.7 ETHICAL CLEARANCE ......................................................................................... 47 3.8 LIMITATIONS ........................................................................................................ 47 CHARPTER FOUR ................................................................................................................. 48 RESULTS ........................................................................................................................ 48 University of Ghana http://ugspace.ug.edu.gh ix 4.1 DESCRIPTIVE CHARACTERISTIC OF BIRDS ............................................ 48 4.2: CLINICAL FINDING ......................................................................................... 55 4.3 MANAGEMENT PRACTICES ............................................................................... 58 4.4 BIO-SECURITY MEASURES ............................................................................ 60 4.5 LABORATORY RESULTS .................................................................................. 63 CHAPTER FIVE ..................................................................................................................... 65 DISCUSSIONS .............................................................................................................. 65 CHAPTER SIX ....................................................................................................................... 69 CONCLUSIONS AND RECOMMENDATIONS ......................................................... 69 6.1 CONCLUSIONS ..................................................................................................... 69 6.2 RECONMMENDATIONS ....................................................................................... 71 REFERENCES ........................................................................................................................ 73 APPENDIX I ........................................................................................................................... 90 University of Ghana http://ugspace.ug.edu.gh x LIST OF TABLES 1: Major outbreaks of highly pathogenic avian influenza H5N1 in wild birds……...20 2: PCR primer and probe sequences…………………………………………………45 3: Descriptive characteristic of birds………………………………………………...50 4: Descriptive characteristic of birds (common/scientific names)…………………...51 5: Descriptive characteristic of birds (source)……………………………………….54 6: Mortality rates (May 2009-March 2010)………………………………………….57 7. Poultry management practices in the Tema Metropolis (May-March 2010)……..59 8. Bio-security practices by poultry farmers in the Tema Metropolis……………….62 9. Disposal of dead birds in the Tema Metropolis (May 2009-March 2010)……….62 10: Results of RRT-PCR for Influenza A ………………………………....................64 University of Ghana http://ugspace.ug.edu.gh xi LIST OF FIGURES 1: Avian Influenza viral features………………………………………………………11 2: Avian Influenza transmission cycle…………………………………………………14 3: H5N1 outbreaks in poultry and wild birds since 2003………………………………16 4: Map of the first outbreak areas for HPAI in the Tema metropolis, Ghana………….39 5: Sampling sites in the Tema metropolis……………………………………………....40 6: Active avian influenza surveillance diagram……………………………...................41 7: Total stock of birds per farm………………………………………………………...52 8: Detection of Newcastle virus RNA from field samples……………………….........64 University of Ghana http://ugspace.ug.edu.gh xii LIST OF ABBREVIATIONS AI Avian Influenza AIV Avian Influenza virus O C Degree Celsius CDC Centers for Disease Control and Prevention Ct Cycle Threshold DEFRA Department for Environment, food and Rural Affairs DIVA Differentiating Vaccinated from Infected Animals DNA Deoxyribonucleic acid dNTPs Deoxynucleoside triphosphate ELISA Enzyme Linked Immunosorbent Assay EU European Union FAO United Nations Food and Agriculture Organization HA Haemagglutinin HI Haemagglutination Inhibition HPAI Highly Pathogenic Avian Influenza HPAIV Highly Pathogenic Avian Influenza Virus LPAIV Low Pathogenic Avian Influenza Virus l micro liter ml milliliters University of Ghana http://ugspace.ug.edu.gh xiii NA Neuraminidase NMIMR Noguchi Memorial Institute for Medical Research OIE International Organization for Epizootic RNA Ribonucleic Acid RRT-PCR Real Time Reverse Transcriptase Polymerase Chain Reaction RT-PCR Reverse Transcriptase Polymerase Chain Reaction SARS Severe Acute Respiratory Syndrome USAID United States Agency for International Development VSD Veterinary Services Directorate WHO World Health Organization University of Ghana http://ugspace.ug.edu.gh 1 CHAPTER ONE 1.1 INTRODUCTION Fowl plague which is now known as highly pathogenic avian influenza was first recognized as an infectious disease of birds in Italy, in 1878 (Perroncito, 1878). In 1901, Centanni and Savonuzzi, identified a filtrable agent as responsible for causing the disease, and in 1955, Schäfer characterized these agents as influenza type A viruses. In 1997, there was an outbreak of AI in Southern China (Hong Kong) that affected 1.4 million chickens and eighteen people of whom six persons died (Kaye and Pringle 2005). With the assumption that the human infection came from poultry, all birds were culled. Five years after the 1997 China outbreak, the disease resurfaced again in China and by 2005 most Asian countries had experienced outbreaks although they were successfully managed. In spite of these successes in control, the AI virus appears to have become endemic in most of the outbreak countries. The Asian experienced is similar in African countries that have experienced AI outbreaks including Egypt, South Africa, Nigeria, and Benin. In April 2007, the first outbreak of AI was reported in Ghana at a small – scale poultry farm at Kakasunanka, near Michel Camp in the Tema Metropolis. This was despite several biosecurity measures such as a ban on importation of poultry and poultry products from Southeast Asia which affected European and African countries, disinfection of farm workers and provision of equipments and vehicles to curtail the entrance of the disease University of Ghana http://ugspace.ug.edu.gh 2 into Ghana. AI however, appeared in Sunyani in the Brong Ahafo region and Aflao in the Volta region. The economic losses due to AI can be described through the following: Compensation to poultry farmers whose birds are affected, high cost of equipments and disinfectants. Also, is the cost of human resources needed to control the outbreak and losses in international trade in terms of ban on poultry and poultry products. Following the outbreak of AI in Ghana, the Government of Ghana paid compensation to poultry farmers for the birds that were culled. The compensation ranged from 70% to 90% of the market prices for day-old chicks, broilers, cockerels and layers, while table and fertile eggs were paid at rates of 50% and 60% of market prices respectively. In all, a total of 13,391 birds were affected and 36,376 birds were destroyed. The Government paid an amount of 160,000 US dollars as compensation to affected farmers (VSD. Annual reports 2007, 2009). The cost of Veterinary interventions and public education on prevention and control of the disease was estimated at 2 million US dollars. Much of this financial support came from donor partners including USAID and FAO (VSD Annual report 2009). The purpose of this study therefore was to determine the current profile of avian influenza viruses in domestic poultry and wild birds in the Tema Metropolis during the period May 2009 to March 2010. University of Ghana http://ugspace.ug.edu.gh 3 1.2 PROBLEM STATEMENT The highly pathogenic avian influenza A (H5N1) outbreak in Asia, Europe, Near East and Africa is not expected to disappear in the short term. It is possible that H5NI virus infections have become endemic among domestic birds in certain areas and that sporadic human infections resulting from direct contact with infected poultry or wild birds will continue to occur. Migratory water fowls, most notably wild ducks, are natural reservoirs of avian influenza and are most resistant to Al infection. Live bird markets, movement of poultry and poultry products, and movement of poultry farm workers have probably all played an important role in the spread of the disease in Africa. The H5N1 virus was detected for the first time in Ghana, on a small –scale poultry farm at Kakasunanka, near Michel camp in the Tema Metropolis in April 2007. The second outbreak also occurred in the Sunyani Municipality in May 2007 and the third outbreak occurred in the month of June 2007 at Aflao. Subsequently, these areas were declared free of H5N1 in September, 2007. However, the current profile of Avian Influenza viruses in domestic commercial poultry, backyard, live bird markets and wild birds in the Tema Metropolis where an outbreak had occured is not known. Also, bio-security practices among backyard and commercial poultry farmers in the Tema Metropolis are yet to be reviewed to determine current risk status for AI infection. University of Ghana http://ugspace.ug.edu.gh 4 1.3 JUSTIFICATION Following the arrival of the HPAI disease into Ghana, it is important to determine the profile of avian influenza viruses in domestic poultry and wild birds. The H5N1 virus, which has spread from Asia to Europe and Africa, poses a real public health threat as H5N1 can occasionally infect humans. In Ghana, backyard poultry and wild birds may be acting as a silent reservoir for the H5N1 virus and other low pathogenic avian influenza viruses. The concern is greatest in rural areas, where traditional free-ranging ducks, chickens and wildlife mingle frequently with each other, sharing the same source of water and feed. The experience of the 2008 outbreak of HPAI in Togo has shown the ability of the H5N1 virus to persist discreetly among traditional farms (scavenging poultry) where chicken mortality is common and usually goes unreported (FAO EMPRES, 2008). The role of domestic birds and wild birds in the maintenance and spread of H5N1 and other influenza A viruses has not been investigated in Ghana, though other countries such as China and Nigeria have also experienced outbreaks of HPAI have conducted such studies. In Ghana, outbreaks of H5N1 were recorded in 2007 at Kakasunanka and Adjei Kojo all in Tema Metropolis of the Greater Accra region. The metropolis is notable for its significant poultry production and it is also a major port city in Ghana. University of Ghana http://ugspace.ug.edu.gh 5 Hence, there is the need to determine the current profile of AI infection in commercial birds, backyard birds, live bird markets and wild birds in Tema Metropolis. 1.4 GENERAL OBJECTIVE To determine the current profile of Avian Influenza viruses in domestic commercial poultry, backyard poultry, live bird markets and wild birds in Tema Metropolis between May 2009 and March 2010 covering the dry and wet seasons. 1.5 SPECIFIC OBJECTIVES 1. To determine current profile of avian influenza virus infection in domestic poultry (commercial poultry, backyard poultry and live bird markets) and wild birds in the Tema Metropolis. 2. To assess Bio-security procedures and practices on commercial poultry farms, backyard farms and live bird markets in the Tema Metropolis. 1.6 RESEARCH QUESTIONS 1. Is the AIV that was detected in poultry in Tema, in April, 2007 still circulating in domestic poultry, backyard birds and wild birds in the Tema Metropolis? 2. Are the bio-security processes and procedures for HPAI in Tema Metropolis adequate? University of Ghana http://ugspace.ug.edu.gh 6 CHAPTER TWO LITERATURE REVIEW 2.1 AVIAN INFLUENZA DISEASE Avian influenza (AI) is a disease of viral etiology that ranges from a mild or even asymptomatic infection to an acute, fatal disease of chicken, turkey, guinea fowl, and other avian species, especially migratory waterfowl (Alexander 1982, Hinshaw and Webster 1982, Beard 1989, Webster et al 1992, Easterday et al 1997, Stalknecht and Brown 2007). Wild water birds have been identified as natural reservoir host of avian influenza viruses. Generally, the infection is asymptomatic as influenza A virus strains of low pathogenicity co-exist in almost perfect balance in wild water birds (Webster et al 1992, Alexander 2000).Recently, avian influenza has acquired world-wide attention when a highly pathogenic strain of the subtype H5N1, which probably arose before 1997 in Southern China, gained enzootic status in poultry throughout South East Asia. The H5N1 virus had traversed interclass barriers (Perkins and Swayne, 2003) and had been transmitted from birds to mammals (cats, swine, and humans). Although not an entirely unprecedented event (Koopmans et al 2004, Hayden and Croisier 2005), the substantial number of documented cases in humans, associated with severe disease and several fatalities raised serious concerns about a pandemic potential of the H5N1 strain (Klempner and Shapiro 2004; Webster et al 2006). There are several further lines of evidence suggesting that the H5N1 virus has acquired increased pathogenic potency for several mammalian species. Justifiably, this has caused world-wide public health concern (Kaye and Pringle 2005). University of Ghana http://ugspace.ug.edu.gh 7 2.2 HIGHLY PATHOGENIC AVIAN INFLUENZA H5N1 OF ASIA LINEAGE According to the International Organization for Epizootics (OIE) 2008, more than sixty countries in Asia, Europe and Africa‟s HPAI H5N1 infection in domestic poultry, captive and wild birds are linked to the Asian lineage virus. By November 2005, over 200 million domestic birds had died from the disease or been slaughtered in attempts to control its spread in western Eurasia and Africa. Also, by March 2008, the World Health Organisation had confirmed more than 370 human cases of H5N1 with a case fatality rate of 60% (World Health Organisation 2008). Sporadic deaths of wild birds due to H5N1 have been reported since 2002 and the first outbreak involving a large number of wild birds was reported in May 2005, in Qinghai province, China (Chen et al. 2005; Liu et al. 2005). Between 2002 and 2008, the virus has infected a wide range of wild bird species (Olsen et al. 2006; USGS National Wildlife Health Center 2008; Lee 2004), but which species are important in H5N1 HPAI movement and whether the virus will become enzootic in wild bird populations is still unknown (Brown et al, 1998). The H5N1 virus has also infected a limited number of domestic, captive and wild mammals, including captive Tigers (Panthera tigris), Leopards (Panthera pardus), domestic pigs in southeast Asia, domestic cats and a wild Stone Marten Martes foina in Germany. These cases were the result of „spillover‟ infection from birds. There is no known reservoir of HPAI H5N1 virus in mammals and there remains no sound evidence that the virus can be readily transmitted from mammal to mammal (Perkins and Swayne 2003). University of Ghana http://ugspace.ug.edu.gh 8 2.3 EMERGENCE OF HPAI H5N1 IN POULTRY IN SOUTHEAST ASIA Highly Pathogenic Avian Influenza H5N1 first received widespread recognition following the 1997 outbreak in poultry in Hong Kong with subsequent spread of the virus to humans. During that outbreak, 18 human cases were reported and six patients died. The outbreak was effectively controlled through slaughtering of all domestic chickens in infected farms as well as those held by wholesale facilities and vendors in Hong Kong. A precursor to the 1997 H5N1 strain was identified in Guangdong, China, where it caused deaths in domestic geese in 1996 (Webster et al. 2006). Between 1997 and 2002, different reassortments (known as genotypes) of the virus emerged, in domestic goose and duck populations, which contained the same H5 HA gene but had different internal genes (Guan et al. 2002; Webster et al. 2006). In 2002, a single genotype emerged in Hong Kong and killed captive and wild water birds in nature parks. This genotype spread to humans in Hong Kong in February 2002 (infecting two, killing one). Between 2003 and 2005, the HPAI H5N1 spread in an unprecedented fashion across southeast Asia, affecting domestic poultry in Vietnam, Thailand, Indonesia, Cambodia, Laos, Korea, Japan, China and Malaysia. In April 2005, the first major outbreak in wild birds was reported. Some 6345 wild birds were reported dead at Qinghai Lake in central China. Species affected included Great Black-headed Gull (Larus ichthyaetus), Bar- headed Goose (Anser indicus), Brown-headed Gull (Larus brunnicephalus), Great Cormorant (Phalacrocorax carbo) and Ruddy Shelduck (Tadorna ferruginea) (Chen et al. 2005; Liu et al. 2005). University of Ghana http://ugspace.ug.edu.gh 9 2.4 ECONOMIC CONSEQUENCES OF HPAI Outbreaks of highly pathogenic avian influenza can be catastrophic for single farmers and for the poultry industry of an affected region as a whole. Economical losses are usually only partly due to direct deaths of poultry from HPAI infection. Measures established to prevent further spread of the disease levy a heavy toll. Nutritional consequences can be equally devastating in developing countries where poultry is an important source of animal protein. Once outbreaks have become widespread, control is difficult to achieve and may take several years (WHO 2004). This is illustrated by the Southeast Asian outbreaks which resulted in high avian mortalities, resulting in the destruction of over one hundred and fifty million (150,000,000) birds accounting for losses in revenue estimated at over ten billion US dollars (Diouf 2005). This was a serious setback for the agricultural development. There was associated poverty for many rural farmers who depended solely on small scale backyard poultry farming for household income. 2.5 DESCRIPTION OF AVIAN INFLUENZA VIRUS Influenza viruses are spherically or longitudinally shaped enveloped particles with an eight-fold segmented, single-stranded RNA genome of negative polarity. Influenza viruses belong to the Orthomyxoviridae family and are classified into types A, B or C based on antigenic differences of their nucleo- and matrix proteins. Avian influenza viruses (AIV) belong to type A (Sidorenko and Reichl 2004, Center for Infectious Diseases Research and Policy 2007). University of Ghana http://ugspace.ug.edu.gh 10 The main antigenic determinants of influenza A and B viruses are the haemagglutinin (HA) and the neuraminidase (NA) transmembrane glycoproteins, capable of eliciting subtype-specific and immune responses which are fully protective within, but only partially protective across, different subtypes. On the basis of the antigenicity of these glycoproteins, influenza A viruses currently cluster into sixteen HA (H1 - H16) and nine NA (N1 - N9) subtypes. These clusters are substantiated by phylogenetic analyses and deduced amino acid sequences of the HA and NA genes, respectively (Fouchier et al 2005). The conventional nomenclature for influenza virus isolates requires connotation of the influenza virus type, the host species, the geographical site, serial number, and year of isolation. For influenza virus type A, the haemagglutinin and neuraminidase subtypes are added in brackets. One of the parental avian strains of the current outbreaks of H5N1 of Asian lineage was isolated from a goose in the Chinese province, Guangdong. Accordingly, it is designated A/goose/Guangdong/1/96 (H5N1) (Class et al 1998) while the isolate originating from the first-documented human case of Asian lineage H5N1 infection from Hong Kong (Xu et al 1999) is referred to as A/HK/156/97 (H5N1). The Haemagglutinin, a glycosylated and acylated protein consisting of 562 - 566 amino acids, is incorporated in the viral envelope. The globular head of its membrane-distal, knob-like external domain is associated with binding to cellular receptors composed of oligosaccharides which terminally carry derivates of neuraminic acid (Watowich et al 1994). The exodomain of the second transmembrane glycoprotein, the neuraminidase (NA), exerts sialolytic enzymatic activity and liberates virus progeny captured at the University of Ghana http://ugspace.ug.edu.gh 11 surface of infected cells during egress. This function prevents viral aggregation during egress, and possibly also facilitates the drifting of the virus through the mucus layers of the targeted epithelial tissues leading to viral attachment (Matrosovich et al 2004). This renders the neuraminidase an interesting target of antiviral agents (Garman and Laver 2004). Mutually attuned and co-ordinated actions of the antagonistic glycoprotein species HA and NA of a viral strain are pivotal for effective attachment and release processes of the virions (Wagner et al 2002). Figure1. Avian Influenza Viral features. Source: Hoffmann 2006 NA HA University of Ghana http://ugspace.ug.edu.gh 12 2.6 EPIDEMIOLOGY OF AVIAN INFLUENZA Wild aquatic birds, notably members of the orders Anseriformes (ducks and geese) and Charadriiformes (gulls and shorebirds), are carriers of the full variety of influenza virus A subtypes, and thus, most probably constitute the natural reservoir of all influenza A viruses (Webster 1992, Fouchier et al 2003, Krauss et al 2004, Widjaja et al 2004). While all avian species are thought to be susceptible, some domestic poultry species - chicken, turkey, guinea fowl, quail and pheasants - are known to be especially vulnerable to the sequelae of infection. Avian influenza A viruses generally do not cause disease in their natural hosts. Instead, the viruses remain in an evolutionary stasis, as molecularly signalled by low N/S (non- synonymous vs. synonymous) mutation ratios indicating purifying evolution (Gorman et al 1992, Taubenberger et al 2005). Host and virus seem to exist in a state of a meticulously balanced mutual tolerance, clinically demonstrated by absence of disease and efficient viral replication. Large quantities of virus of up to 10 8.7 x 50% egg-infective dose (EID50) per gram faeces can be excreted (Webster et al 1978). When transmitted to highly vulnerable poultry species, usually mild, if any, symptoms ensue. Viruses of this phenotype are referred to as low pathogenic (LPAIV) and, in general, only cause a slight and transient decline in egg production in layers or some reduction in weight gain in fattening poultry (Capua and Mutinelli 2001). However, strains of the subtypes H5 and H7 carry the potential to mutate to a highly pathogenic form after transmission and adaptation to the new poultry hosts. The highly pathogenic forms of H5 and H7 or of other subtypes had not been previously observed in wild birds (Webster 1998). Therefore, University of Ghana http://ugspace.ug.edu.gh 13 one may conclude that highly pathogenic forms are artificial, made possible as a result of man-made interference with a naturally balanced system. Once Highly Pathogenic Avian Influenza Virus (HPAIV) become established in domestic poultry, a highly contagious disease results and wild birds are no longer an essential factor for the spread (Swayne and Suarez 2000). This might have changed fundamentally since early 2005, when a large outbreak of the Asian lineage H5N1-related HPAI was observed among thousands of wild aquatic birds in a nature reservation at Lake Qinghai in the North West of China (Chen et al 2005, Liu et al 2005). As a result of this, further spread of this virus towards Europe during 2005 may have been initiated (OIE 2005). Infected birds excrete virus in high concentration in their faeces and also in nasal and ocular discharges. Once introduced into a flock, the virus is spread from flock to flock through direct contact or by usual methods involving the movement of infected live birds or illegal trade or their unprocessed products, contaminated equipment, egg flats feed trucks. Unintended mechanical passing-on of virus through human movements probably has been the main factor in the spread of HPAIV (Stegaman et al 2003). Airborne transmission may occur if birds are in close proximity and with appropriate air movement. Birds are readily infected via instillation of virus into the conjunctival sac, or the tracheal. Preliminary field and laboratory evidence shows that Avian Influenza virus can be recovered from the yolk and albumen of eggs laid by hens at the peak of the disease. The possibility of vertical transmission is unresolved, however, it is unlikely that infected embryos could survive and hatch. Attempts to hatch eggs in disease cabinets from a broiler breeder flock at the height of disease failed to result in any Avian University of Ghana http://ugspace.ug.edu.gh 14 Influenza-infected chickens. However, broken contaminated eggs could be the source of virus infection to chicks after they hatch in the same incubator (Hoffmann et al 2006). Figure 2. Avian Influenza Transmission Cycle. Source: Hoffmann 2006 University of Ghana http://ugspace.ug.edu.gh 15 2.7 GEOGRAPHICAL SPREAD OF HPAI H5N1 OUT OF SOUTHEAST ASIA In July 2005, Russia reported its first outbreaks of H5N1 in domestic flocks in six regions of western Siberia and dead wild birds were reported in the vicinities of some of these outbreaks. Kazakhstan reported its first outbreak in August 2005 in domestic birds. In the same month, 89 wild birds described as migratory species were reported infected at two lakes in Mongolia (OIE 2005). Europe reported its first outbreaks in October 2005 when H5N1 infection was detected in domestic birds in Romania and Turkey. In the same month, Romania reported sporadic cases in wild birds as did Croatia and European parts of Russia. In November, the virus spread to domestic birds in the Ukraine, and the Middle East reported its first case: a flamingo kept as a captive bird in Kuwait. During December 2005, two outbreaks were reported in European Russia in wild swans (species unreported) in regions near the Caspian Sea. In the first half of 2006, the spread of HPAI H5N1 continued across Europe (Sabirovic et al. 2007; Hesterberg et al. 2009) and the Middle East and into Africa. Between January and May, infection was reported in 24 European countries with the majority of cases occurring in February and March in wild birds. During the same period, outbreaks were reported across central Asia and the Middle East, affecting domestic birds in Azerbaijan, India, Bangladesh, Pakistan, Iran and Iraq, with Azerbaijan also reporting infected wild birds. The first reported outbreak in Africa occurred in January 2006 in poultry in Nigeria, and by the end of April 2007, eight other African nations had reported outbreaks:Burkina Faso, Cameroon, Djibouti, Egypt, Ghana, Cote d'‟Ivoire, Niger and University of Ghana http://ugspace.ug.edu.gh 16 Sudan (OIE 2008). By May 2006, reports of outbreaks in Europe, the Middle East and Africa had for the most part decreased in frequency. Small numbers of cases of infection were reported in Hungary, Spain and the Ukraine in June; Pakistan and Russia in July; and one case was identified in a captive swan in Germany in August. Egypt was exceptional, continuously reporting outbreaks throughout 2008. It is also considered likely that outbreaks continued in poultry in Nigeria (UN System Influenza Coordinator & World Bank 2008). Throughout the time HPAI H5N1 was spreading across central Asia, Europe, the Middle East and Africa; it maintained a stronghold in poultry in Southeast Asia. In 2006, outbreaks were reported in Cambodia, China, Hong Kong, Indonesia, Korea, Laos, Malaysia, Myanmar, Thailand and Vietnam (Figure 3: H5N1 outbreaks). Figure3:H5N1outbreaks University of Ghana http://ugspace.ug.edu.gh 17 2.8 OUTBREAKS OF HPAI H5N1 SINCE 2006 AND THE CURRENT SITUATION 2.8.1 GLOBAL HPAI H5N1 SITUATION Compared with 54 countries reporting 1,470 outbreaks to the OIE in 2006, thirty countries reported 638 outbreaks in 2007 (OIE 2008). In 2007, six European countries (Poland, Hungary, Germany, the United Kingdom, Romania and the Czech Republic) reported sporadic and relatively isolated outbreaks in poultry that were quickly controlled. Outbreaks in domestic birds were also reported in European parts of Russia and in Turkey. Infected wild birds were reported in Germany, France, the United Kingdom and the Czech Republic; and birds at a rehabilitation centre were affected in Poland. In the Middle East and central Asia, poultry outbreaks occurred throughout 2007. Some 350 outbreaks were reported from Egypt and Bangladesh alone. Poultry (and in some cases captive birds) were also affected in India, Kuwait, Saudi Arabia, Pakistan, Afghanistan and Israel with most outbreaks occurring between February and April, and again between October and December. Again, as in 2006, poultry outbreaks continued across Southeast Asia. Sporadic cases in wild birds were reported in Japan and Hong Kong SAR. In January and February 2008, a small number of wild bird cases were detected in the United Kingdom; large numbers of poultry outbreaks occurred in India and parts of Southeast Asia; and the virus was considered to be enzootic in poultry in Egypt, Indonesia and Nigeria; and possibly enzootic in Bangladesh and China (UN System Influenza Coordinator & World Bank 2008). Globally, 63 countries have reported avian influenza outbreaks since 2006 to March, 2010. University of Ghana http://ugspace.ug.edu.gh 18 2.8.2 AFRICA HPAI H5N1 SITUATION In Africa, HPAI H5N1 was reported in domestic birds in Nigeria, Egypt, Togo, Ghana and Benin; and is considered to have become endemic in Egypt (OIE 2008; UN System Influenza Coordinator & World Bank 2008). Also, in the African Union/Interafrican Bureau for Animal Resources progress report from May 2007 to February, 2009, it was reported that eleven African countries namely; Benin, Burkina Faso, Cameroon, Cote d‟Ivoire, Ghana, Niger, Nigeria, Togo, Egypt, Djibouti and Sudan had been infected since the emergence of AI virus in domestic Africa. So far, there have been 52 reported H5N1 human cases with 23 fatalities in Africa. From March, 2009 to March, 2010, only Egypt reported outbreaks in domestic poultry. 2.8.3 GHANA HPAI H5N1 SITUATION On April 24 th , 2007, Ghana reported her first outbreak of HPAI H5N1 virus infection on a small-scale poultry farm situated at Kakasunanka near Michelle camp in the Tema Metropolitan area of the Greater Accra region. This virus was detected by the using of the rapid test kit for influenza type A viruses. The Emergency Preparedness Team of VSD immediately moved to the outbreak area and stamped out all in-contact birds of the infected poultry farm. Further tests conducted at Noguchi Memorial Institute for Medical Research (NMIMR) and international reference laboratory for HPAI at the Instituto Zooprofilattico Spermantalle delle Venezie, Padova, Italy and the United States NAMRU-3 in Cairo, Egypt, also confirmed the presence of H5N1 virus. An active search for HPAI virus was conducted in the Tema Metropolis and as a result, three positive University of Ghana http://ugspace.ug.edu.gh 19 cases were detected on three farms at Adjei Kojo. Ban on movement and sale of poultry and poultry products in the outbreak area was placed. A total of 13,391 birds were affected and died naturally. Further more, 36,376 apparently healthy birds were destroyed on the infected farms. On May 15, 2007, a second outbreak of HPAI H5N1 virus was detected on a backyard farm at New Dormaa in the Sunyani Metropolis of the Brong Ahafo region. The third outbreak was reported at Aflao in the Ketu South district of the Volta region on the June 13, 2007. The source of introduction of HPAI H5N1 virus into Ghana has not been traced. However, according to Mabbett (2007), the virus strain in Ghana was between 98.8% and 99.6% similar to other isolates from Burkina Faso, Cote d‟Ivoire, Nigeria and Sudan. 2.9 MAJOR OUTBREAKS OF HPAI H5N1 IN WILD BIRDS Prior to HPAI H5N1, reports of HPAI in wild birds were very rare. Species of wild birds, especially water birds, are susceptible to infection by the HPAI H5N1 virus. Close contact between poultry and wild birds can lead to cross-infection, from poultry to wild birds and from wild birds to poultry. Additionally, species that live in and around poultry farms and human habitations may serve as “bridge species” that could potentially transmit the virus between poultry and wild birds either by direct contact between wild birds and poultry kept outside or by indirect contact with contaminated materials. While there is no sound evidence that wild birds have carried the virus long distances on migration (Feare and Yasué 2006), analysis of genetic sequences and other largely indirect evidence suggests that wild birds are likely to have contributed to spread (Chen et al. 2005).The following table (Table 1) summarises the known major outbreaks of HPAI H5N1 in wild birds. University of Ghana http://ugspace.ug.edu.gh 20 Year Month(s) Location(s) Description of affected birds 2005 April Qinghai Lake in central China 6345 waterbirds, the majority of which were Great Black-headed Gulls Larus ichthyaetus, Bar- headed Geese Anser indicus and Brown-headed Gulls Larus brunnicephalus August Lake Erhel & Lake Khunt in Mongolia 89 waterbirds including ducks, geese and swans October – November Romania & Croatia Over 180 waterbirds, mainly swans 2006 January Coastal area in the vicinity of Baku, Azerbaijan Unspecified number of birds reported to the OIE as “various migratory birds” January – May 23 countries in Europe including Turkey and European Russia Most cases occurred in ducks, geese and swans but a wide variety of species was infected including other waterbirds and raptors February Rasht, Iran 153 wild swans May Multiple locations in Qinghai province, China Over 900, mainly waterbirds, and mostly Bar- headed Geese Anser indicus May Naqu, Tibet Over 2,300 birds – species composition unclear but 300 infected Bar-headed Geese Anser indicus were reported June Lake Hunt in Bulgan, Mongolia Twelve waterbirds including swans, geese and gulls 2007 June Germany, France and the Czech Over 290, mainly waterbirds, found mostly in Germany Table 1. Major known outbreaks of highly pathogenic avian influenza H5N1 in wild birds Source; OIE Disease Information Reports and German Friedrich-Leoffler Epid. bulletins University of Ghana http://ugspace.ug.edu.gh 21 2.10 AVAIN INFLUENZA AND WETLANDS Given the ecology of the natural hosts of LPAI viruses, it is unsurprising that wetlands play a major role in the natural epidemiology of avian influenza. As with many other viruses, avian influenza virions survive longer in colder water (Liu et al. 2003; Stallknecht et al. 1990), and the virus is strongly suggested to survive over winter in frozen lakes in Arctic and sub-Arctic breeding areas. Thus, as well as the waterbird hosts, these wetlands are probably permanent reservoirs of LPAI virus (Rogers et al. 2004; Smith et al. 2004) (re-)infecting water birds arriving from southerly areas to breed (shown in Siberia by Okazaki et al. 2000 and Alaska by Ito et al. 1995). Indeed, in some wetlands used as staging grounds by large numbers of migratory ducks, avian influenza viral particles can be readily isolated from lake water (Hinshaw et al. 1980). An agricultural practice that provides ideal conditions for cross-infection and thus genetic change is used on some fish-farms in Asia: battery cages of poultry are placed directly over troughs in pig-pens, which in turn are positioned over fish farms. The poultry waste feeds the pigs, the pig waste is eaten by the fish or acts as a fertiliser for aquatic fish food, and the pond water is sometimes recycled as drinking water for the pigs and poultry (Greger 2007). These kinds of agricultural practices afford avian influenza viruses, which are spread via the faecal-oral route, a perfect opportunity to cycle through a mammalian species, accumulating the mutations necessary to adapt to mammalian hosts. Thus, as the use of such practices increases, so does the likelihood that new influenza strains infectious to and transmissible between humans will emerge (Culliton 1990; Greger 2007). As well as providing conditions for virus mutation and generation, agricultural practices, University of Ghana http://ugspace.ug.edu.gh 22 particularly those used on wetlands, can enhance the ability of a virus to spread. The role of Asian domestic ducks in the epidemiology of HPAI H5N1 has been closely researched and found to be central not only to the genesis of the virus (Hulse-Post et al. 2005; Sims 2007), but also to its spread and the maintenance of infection in several Asian countries (Shortridge et al 1998). Typically this has involved flocks of domestic ducks used for „cleaning‟ rice paddies of waste grain and various pests, during which they can potentially have contact with wild ducks using the same wetlands. Detailed research (Gilbert et al. 2006; Songserm et al. 2006) in Thailand has demonstrated a strong association between the HPAI H5N1 virus and abundance of free-grazing ducks.(Gilbert et al. 2006) concluded that in Thailand “wetlands used for double-crop rice production, where free-grazing duck feed year round in rice paddies, appear to be a critical factor in HPAI persistence and spread”. 2.11 WILDLIFE CONSERVATION IMPLICATIONS Prior to the outbreak of HPAI H5N1, reports of HPAI in wild birds were very rare. The broad geographical scale and extent of the disease in wild birds is both extraordinary and unprecedented, and the conservation impacts of HPAI H5N1 have been significant. It is estimated that between 5-10% of the world population of Bar-headed Goose Anser indicus died at Lake Qinghai, China in spring 2005 (Chen et al. 2005; Liu et al. 2005). At least two globally threatened species have been affected: Black-necked Crane, Grus nigricollis in China and Red-breasted Goose, Branta ruficollis in Greece. Approximately 90% of the world population of Red-breasted Goose is confined to just five roost sites in Romania and Bulgaria, countries that have both reported outbreaks, as also have Russia University of Ghana http://ugspace.ug.edu.gh 23 and Ukraine where these birds also over-winter (Bird Life International 2007). However, the total number of wild birds known to have been affected has been small in contrast to the number of domestic birds affected, and many more wild birds die of commoner avian diseases each year. Perhaps a greater threat than direct mortality has been the development of public fear about waterbirds resulting in misguided attempts to control the disease by disturbing or destroying wild birds and their habitats. Such responses are often encouraged by exaggerated or misleading messages in the media. Currently, wildlife health problems are being created or exacerbated by unsustainable activities such as habitat loss or degradation, which facilitates closer contact between domestic and wild animals. Many advocate that to reduce risk of avian influenza and other bird diseases, there is a need to move to markedly more sustainable systems of agriculture with significantly lower intensity systems of poultry production. These need to be more biosecure, separated from wild waterbirds and their natural wetland habitats resulting in far fewer opportunities for viral cross-infection and thus pathogenetic amplification (Greger 2007). There are major animal and human health consequences (in terms of the impact on economies, food security and potential implications of a human influenza pandemic) of not strategically addressing these issues. However, to deliver such an objective in a world with an ever-growing human population and with issues of food- security in many developing countries, will be a major policy challenge (Sonaiya 2007, Roland-Holst et al., 2008). 2.12 CLINICAL PRESENTATION OF AI IN POULTRY Following an incubation period of usually 3 to 5 days but rarely up to 21 days, depending upon the characteristics of the virulence of the infecting virus, species affected, sex and University of Ghana http://ugspace.ug.edu.gh 24 age of the bird, the clinical presentation of avian influenza in birds is variable and symptoms are fairly unspecific (Elbers et al 2005). Therefore, a diagnosis solely based on the clinical presentation is impossible. The symptoms following infection with low pathogenic AIV may be as discrete as ruffled feathers, transient reductions in egg production or weight loss combined with a slight respiratory disease (Capua and Mutinelli 2001). Some low pathogens (LP) strains such as certain Asian H9N2 lineages, adapted to efficient replication in poultry, may cause more prominent signs and also significant mortality (Bano et al 2003, Li 2005). In its highly pathogenic form, the illness in chickens and turkeys is characterised by a sudden onset of severe symptoms such as depression, ruffled feathers, cyanotic comb and wattles, haemorrhages on the shanks and mortality can approach 100 % within 48 hours (Swayne and Suarez 2000). Spread within an affected flock depends on the form of rearing: in herds which are litter-reared and where direct contact and mixing of animals is possible, spread of the infection is faster than in caged holdings (Capua et al 2000). Also, many birds die without premonitory signs so that sometimes poisoning is suspected in the beginning (Nakatami et al 2005). It is worth noting, that a particular HPAI virus isolate may provoke severe disease in one avian species but not in another. In industrialised poultry holdings, a sharp rise followed by a progressive decline in water and food consumption can signal the presence of a systemic disease in a flock. In laying flocks, a cessation of egg production is apparent. Individual birds affected by HPAI often reveal little more than severe apathy and immobility (Kwon et al 2005). Oedema, visible at feather-free parts of the head, cyanosis University of Ghana http://ugspace.ug.edu.gh 25 of comb, wattles and legs, greenish diarrhoea and laboured breathing may be inconsistently present. In layers, soft-shelled eggs are seen initially, but any laying activities cease rapidly with progression of the disease (Elbers et al 2005). Nervous symptoms including tremor, unusual postures (torticollis), and problems with co- ordination (ataxia) dominate the picture in less vulnerable species such as ducks, geese, and ratites (Kwon et al 2005). During an outbreak of HPAI in Saxonia, Germany, in 1979, geese compulsively swimming in narrow circles on a pond were among the first conspicuous signs leading to a preliminary suspicion of HPAI. 2.13 PATHOLOGY OF AI Birds that die of peracute disease may show minimal gross lesions, consisting of dehydration and congestion of viscera and muscles In birds that die after a prolonged clinical course, petechial and ecchymotic haemorrhages occur throughout the body, particularly in the larynx, trachea, proventriculus and epicardial fat, and on serosal surfaces adjacent to the sternum. There is extensive subcutaneous oedema, particularly around the head and hocks. The carcass may be dehydrated. Yellow or grey necrotic foci may be present in the spleen, liver, kidneys and lungs. The air sac may contain an exudate. The spleen may be enlarged and haemorrhagic (Perkins and Swayne 2003) Avian influenza is characterised histologically by vascular disturbances leading to oedema, haemorrhages and perivascular cuffing, especially in the myocardium, spleen, lungs, brain and wattles. Necrotic foci are present in the lungs, liver and kidneys. Gliosis, vascular proliferation and neuronal degeneration may be present in the brain (Perkins and Swanyne 2003, Kwon et al 2005, Brojer et al 2009). University of Ghana http://ugspace.ug.edu.gh 26 2.14 DIFFERENTIAL DIAGNOSIS OF AI FROM OTHER DIEASES clinically, the less severe forms of AI may be confused with many other respiratory or enteric diseases in poultry (Elbers et al 2005). However, in the laboratory, AI can only be differentiated from other acute poultry respiratory diseases such as Newcastle, infectious laryngotracheitis, duck plague, acute fowl cholera and other septicemia diseases, by serological tests (Agar gel-diffusion test) or virus isolation and molecular detection by the RT-PCR (Animal health advisory leaflet 8; 1996). Avian Influenza should be suspected in any disease outbreak in poultry that persists despite the application of preventive and therapeutic measures for other diseases (Elbers et al 2005).Newcastle disease which has very similar signs and lesions as AI is characterized by the sudden onset of watery discharge from the nostrils, labored breathing, facial swelling, paralysis, trembling and twisting of the neck. Mortality ranges from 10 to 80% depending on host immunity. It causes also, drastic reduction in egg-laying and production of soft-shelled eggs. Lesions of Newcastle disease include hemorrhages of peyers patches in the intestines, hemorrhages in the ovaries, proventiculus, intestine lining and caecal tonsils. 2.15 LABORATORY DIAGNOSTIC PROCEDURES OF AVIAN INFLUENZA 2.15.1 COLLECTION OF SPECIMENS Specimens should be collected from several fresh carcasses and from diseased birds of a flock. Ideally, adequate sampling is statistically backed up and diagnosis is made on a flock basis. When sampling birds suspected of HPAI, safety standards must be observed to avoid exposure of the sample collectors to potentially zooanthroponotic HPAIV University of Ghana http://ugspace.ug.edu.gh 27 (Bridges et al 2002). Guidelines have been established by the CDC (CDC 2005).For virological assays, swabs obtained from the cloacal and the oropharynx generally allow for a sound laboratory investigation. The material collected on the swabs should be mixed into 2-3 ml aliquots of a sterile isotonic transport medium containing antibiotic supplements and a protein source (e.g. 0.5 % [w/v] bovine serum albumin, up to ten percent of bovine serum or a brain-heart infusion).At autopsy, carried out under safe conditions and avoiding spread of disease, unpreserved specimens of brain, trachea/lung, spleen and intestinal contents are collected for isolation of the virus.For serological purposes, native blood samples are taken. The number of samples collected should surffice detection with a 95 % confidence interval for a parameter with a prevalence of 30 % (CDC, 2005). 2.15.2 TRANSPORTATION OF SPECIMENS TO THE LABORATORY Swabs, tissues and blood should be transported chilled but not be allowed to freeze. If delays of greater than 48 hours are expected in transit, these specimens should be frozen and transported on dry ice. In all cases, transport safety regulations should be punctiliously observed to avoid spread of the disease and accidental exposure of personnel during transport. It is highly advisable to contact the assigned diagnostic laboratory before sending the samples and, ideally, even before collecting them. 2.15.3 DIRECT DETECTION OF AVIAN INFLUENZA VIRUS INFECTION University of Ghana http://ugspace.ug.edu.gh 28 Basically, there are two (parallel) lines of diagnostic measures that attempt to (a) isolate and subtype the virus by classical methods (OIE Manual 2005) and (b) molecularly detect and characterize the viral genome (Ng et al, 2005). Conventionally, AI virus is isolated by inoculation of swab fluids or tissue homogenates into 9- to 11-day-old embryonated chicken eggs, usually by the chorioallantoic sac route (Woolcock et al 2001). Depending on the pathotype, the embryos may or may not die within a five-day observation period and usually there are no characteristic lesions to be seen in either the embryo or the allantois membrane (Mutinelli et al 2003b). Eggs inoculated with HPAIV-containing material usually die within 48 hours. The presence of a haemagglutinating agent can be detected in harvested allantoic fluid. Haemagglutination (HA) is an insensitive technique requiring at least 10 6.0 particles per ml. If only a low virus concentration is present in the inoculum, up to two further passages in embryonated eggs may be neccessary for some LPAIV strains, in order to produce enough virus to be detected by HA. In the case of HPAIV, a second passage using diluted inoculum may be advantageous for the optimal production of haemagglutination (Woolcock et al 2001). Haemagglutinating isolates are antigenically characterised by haemagglutination inhibition (HI) tests using (mono-) specific antisera against the 16 H subtypes and, for control, against the different types of avian paramyxoviruses which also display haemagglutinating activities. The NA subtype can be subsequently determined by neuraminidase inhibition assays, again requiring subtype-specific sera (Aymard et al 2003). In case isolates of the H5 or H7 lineages are encountered, their intravenous University of Ghana http://ugspace.ug.edu.gh 29 pathogenicity index (IVPI) needs to be determined to distinguish between LP and HP biotypes (Allan et al 1977). This is achieved by inoculation of ten 6-week old chickens with the egg-grown virus isolate (0.1 ml of a 1 in 10 dilution of allantoic fluid containing an HA titre greater than 1 in 16). The chickens are observed over a period of ten days for clinical symptoms. Results are integrated into an index which indicates a HPAI virus when values greater than 1.2 are obtained. Alternatively, a HPAI isolate is encountered when at least seven out of ten (75 %) inoculated chickens die within the observation period (Hoffmann et al 2006). The classical procedures can lead to a diagnosis of HPAI within five days but may demand more than a fortnight to rule out the presence of AIV. In addition, high quality diagnostic tools (SPF eggs, HA- and NA-subtype specific antisera) and skilled personnel are a prerequisite. Currently, cell culture applications for the isolation of AIV that can achieve the sensitivity of embryonated hen eggs require the use of Biosafety Level 3 laboratory facilities. A more rapid approach, especially when exclusion of infection is demanded, employs molecular techniques, which could also follow a cascade style: the presence of influenza A specific RNA is detected through the reverse transcription-polymerase chain reaction (RT-PCR assay) which targets fragments of the M gene, the most highly conserved genome segment of influenza viruses (Fouchier et al 2000, Spackman et al 2002), or the nucleocapsid gene (Dybkaer et al 2004). When a positive result is obtained, RT-PCRs amplifying fragments of the haemagglutinin genes of subtypes H5 and H7 are then University of Ghana http://ugspace.ug.edu.gh 30 conducted to detect the presence of notifiable AIVs (Dybkaer et al 2004, Spackman et al 2002, Oraveerakul et al 2004). Then, a further molecular diagnosis of the pathotype (LP versus HP) is feasible after sequencing a fragment of the HA gene spanning the endoproteolytic cleavage site. Isolates presenting with multiple basic amino acids are classified as HPAI. Various techniques have been developed for the detection of Asian lineage H5N1 strains (Collins et al 2002, Payungporn et al 2004). Non-H5/H7 subtypes can be identified by a canonical RT-PCR and subsequent sequence analysis of the HA-2 subunit (Phipps et al 2004). There are also specific primers for each NA subtype. A full characterisation might be achievable within three days, especially when real time PCR techniques are used (Perdue 2003, Lee et al 2004). However, DNA chips could also be used for the typing of AI viruses (Li 2001, Kessler et al 2004). An exclusion diagnosis is possible within a single working day. The disadvantages of molecular diagnostics is the cost of equipment, reagents and consumables, although, if available, many samples can be analysed by less personnel in shorter times in comparison to virus isolation in eggs. However, it is a well established fact that each PCR or hybridisation reaction, in contrast to virus isolation in eggs, harbours an intrinsic uncertainty related to the presence of specific mutations in a given virus at the binding sites of primers and/or probes, which might produce a false negative results. Thus, a combination of molecular (e.g. for screening purposes) and classical methods (e.g. for final characterisation of isolates and confirmation of diagnosis of an index case) may help to counterbalance the disadvantages of the two principles. University of Ghana http://ugspace.ug.edu.gh 31 Rapid assays have been designed for the detection of viral antigen in tissue impression smears and cryostat sections by use of immunofluorescence, or by antigen-capture enzyme-linked immunosorbent assay (ELISA) and dip-stick lateral flow systems in swab fluids. So far, these techniques have been less sensitive than either virus isolation or PCR, and therefore might be difficult to approve for a legally binding diagnosis, especially of an index case (Davison et al 1998, Selleck 2003, Cattoli et al 2004). 2.15.4 INDIRECT DETECTION OF AVIAN INFECTION VIRUS INFECTION Serology on a herd basis may be useful for screening purposes (Beck et al 2003). For the detection of AIV-specific antibodies in serum samples from birds, or in egg yolk in the case of laying flocks, the haemagglutination inhibition (HI) assay using reference subtype antigens still represents the gold standard test. Group-specific antibodies (influenza virus type A) against the nucleocapsid protein can also be detected by agar gel immunoprecipitation and by enzyme-linked immunosorbent assays (ELISA) (Meulemans et al 1987, Snyder et al 1985, Jin et al 2004). Competitive ELISA formats allow the examination of sera of all bird species, independent of the availability of species-specific conjugates (Shafer 1998, Zhou et al 1998). An ELISA format for the detection of H7- specific antibodies has been reported (Sala et al 2003), but there is no commercial assay presently available for the detection of H5-specific antibodies in avian sera.Subtype- specific antibody kinetics depend on the viral strain characteristics and, primarily, on the host species. In gallinaceous birds, AIV-specific antibodies reliably become detectable during the second week following exposure; antibodies in egg yolk are detectable after a University of Ghana http://ugspace.ug.edu.gh 32 delay of a few days (Beck et al 2003). The production and detection of antibodies in Anatidae species are much more variable (Swayne and Suarez 2003). 2.16 CONTROL MEASURES AGAINST HIGHLY PATHOGENIC AVIAN INFLUENZA (HPAI) Due to its potentially devastating economic impact, HPAI is subject world-wide to vigilant supervision and strict legislation (OIE Terrestrial Animal Health Code 2005). Measures to be taken against HPAI depend on the epidemiological situation of the region affected. In the European Union (EU) where HPAIV is not endemic, prophylactic vaccination against avian influenza is generally forbidden. Thus, outbreaks of HPAI in poultry are expected to be conspicuous due to the clinically devastating course of the disease. Consequently, when facing such an outbreak, aggressive control measures, e.g. stamping out affected and contact holdings, are put in place, aiming at the immediate eradication of HPAI viruses and containing the outbreak at the index holding (OIE 2008). For these purposes, control and surveillance zones are erected around the index case with diameters varying from nation to nation (3 and 10 kilometres, respectively, in the European Union). The quarantining of infected and contact farms, rapid culling of all infected or exposed birds, and proper disposal of carcasses, are standard control measures to prevent lateral spread to other farms (OIE – Terrestrial Animal Health Code 2005). It is pivotal that movements of live poultry and also, possibly, poultry products, both within and between countries, are restricted during outbreaks. University of Ghana http://ugspace.ug.edu.gh 33 In addition, control of H5 and H7 subtypes of LPAI in poultry, by testing and culling of acutely infected holdings, may be advisable in non-endemic areas in order to reduce the risk of a de novo development of HPAIV from such holdings. Specific problems of this eradication concept may arise in areas with a high density of poultry populations (Marangon et al 2004, Stegemann et al 2004, Mannelli et a 2005) and where small backyard holdings of free roaming poultry prevail (Witt and Malone 2005). Due to the close proximity of poultry holdings and intertwining structures of the industry, spread of the disease is faster than the eradication measures. Therefore, during the Italian outbreak of 1999/2000 not only infected or contact holdings were destroyed, but also flocks with a risk of infection within a radius of one kilometre from the infected farm were pre-emptively killed. Nevertheless, eradication required four months and demanded the death of 13 millions birds (Capua et al 2003). The creation of buffer zones of one to several kilometres around infected farms completely devoid of any poultry was also behind the successful eradication of HPAIV in the Netherlands in 2003 and in Canada in 2004. So, not only the disease itself, but also the pre-emptive culling of animals led to losses of 30 and 19 million birds, respectively. In 1997, the Hong Kong authorities culled the entire poultry population within three days (on the 29 th , 30 th , and 31 st December; 1.5 million birds). The application of such measures, aimed at the immediate eradication of HPAIV at the cost of culling also non- infected animals, may be feasible on commercial farms and in urban settings. However, this will afflict the poultry industry significantly and also prompts ethical concern from University of Ghana http://ugspace.ug.edu.gh 34 the public against the culling of millions of healthy and uninfected animals in the buffer zones. Such measures are most difficult to implement in rural areas with traditional forms of poultry holdings where chickens and ducks roam freely and mingle with wild birds or share water sources with them. Moreover, domestic ducks attract wild ducks and provide a significant link in the chain of transmission between wild birds and domestic flocks (WHO 2005). These circumstances may provide the grounds for HPAI viruses to gain an endemic status. Endemicity of HPAI in a certain region imposes a constant pressure on poultry holdings. As the above mentioned restrictions can not be upheld over prolonged periods without vital damage to a country's poultry industry or, in the developing world, leading to a serious shortage of protein supply for the population, other measures must be considered. Vaccination has been widely used in these circumstances and may also be a supplementary tool in the eradication process of outbreaks in non-endemic areas. 2.17 VACCINATION In the veterinary medicine, vaccination pursues four goals: (i) protection from clinical disease, (ii) protection from infection with virulent virus, (iii) protection from virus excretion, and (iv) serological differentiation of infected from vaccinated animals (DIVA principle). In the field of influenza vaccination, neither commercially available nor experimentally tested vaccines have been shown so far to fulfill all of these requirements (Lee et al University of Ghana http://ugspace.ug.edu.gh 35 2005). The first aim, which is the protection from clinical disease induced by HPAIV, is achieved by most vaccines. The risk of infection of vaccines with and excretion of, virulent field virus is usually reduced but not fully prevented. This may cause a significant epidemiological problem in endemic areas where exhaustive vaccination is carried out: vaccinated birds which appear healthy may well be infected and excrete the field virus 'under cover' of the vaccine.The effectiveness of reduction of virus excretion is important for the main goal of control measures, that is, the eradication of virulent field virus. The effectiveness can be quantified by the replication factor r0. Assuming a vaccinated and infected flock passes on the infection on average to less than one other flock (r0 < 1), the virulent virus is, on mathematical grounds, prone to be extinguished (van der Goot et al 2005). When dealing with vaccination against the potentially zoonotic H5N1 virus, reduction of virus excretion also reduces the risks of transmission to humans, since a significant dose of virus seems to be required to penetrate the species barrier between birds and humans. Also, a DIVA technique allows the tracing of field virus infections by serological means in vaccinated birds. Various vaccine concepts have been developed. Most are still based on inactivated, adjuvant whole virus vaccines which need to be applied by needle and syringe to each animal separately. Inactivated homologous vaccines, based on the actual HPAI strain, induce proper protection but do not allow a distinction between vaccinated and infected birds serologically.Since the vaccine is made from the current HPAI virus, there is an inherent delay before such vaccines can be used in the field. University of Ghana http://ugspace.ug.edu.gh 36 Inactivated heterologous vaccines, in contrast, can be used as marker vaccines when the vaccine virus expresses the same HA- but a different NA-subtype compared to the field virus (e.g. H5N9 vaccine vs. H5N2 HPAI). By detection of NA subtype-specific antibodies, vaccinated and infected birds can be distinguished (Cattoli et al 2003). However, these methods can be laborious and may lack sensitivity. Nevertheless, such vaccines can be kept in vaccine banks comprising several H5- and H7-subtypes with discordant NA subtypes. Reverse genetics will greatly aid in producing vaccines both for veterinary and medical use with the desired HxNy combinations in a favourable genetic background (Liu et al 2003, Neumann et al 2003, Lee et al 2004, Chen et al 2005, Stech et al 2005). Currently, inactivated heterologous vaccines have been used the H5N1 hot spots of South East Asia as well as in Mexico, Pakistan and Northern Italy ( Garcia 1998, Swayne et al 2001). As an alternative DIVA system for use with inactivated vaccines, the detection of NS-1 specific antibodies has been proposed (Tumpey et al 2005). These antibodies are generated at high titres by naturally infected birds, but at considerably lower titres when inactivated vaccines are used. Recombinant live vector-engineered vaccines express an H5 or H7 HA gene from the backbone of viruses or bacteria capable of infecting poultry species (e.g. fowl pox virus [Beard 1991, Swayne et al 1997+2000c], laryngotracheitis virus [Lueschow et al 2001, Veits et al 2003] or Newcastle Disease virus [Swayne 2003] among others). Being live vaccines, mass application via water or sprays is often feasible. While allowing for a clear-cut DIVA distinction, a pre-existing immunity towards the vector virus, however, will grossly interfere with vaccination success. Some field experience with fowl pox recombinants has been collected in Mexico and the U.S. University of Ghana http://ugspace.ug.edu.gh 37 Finally, successful use of recombinantly expressed HA proteins and of DNA vaccination using HA-expressing plasmids has been experimentally proven (Crawford et al 1999, Kodihalli et al 1997). Vaccination has been used on a nationwide scale in several countries in South East Asia (Normile 2005). 2.18 BIO-SECURITY MEASURES The aim of a bio-security programme is to reduce sources and causes of contamination to enable the supply of a healthy, safe and reliable product (Artois et al 2009). According to the Department for Environment, Food and Rural Affairs (Defra 2007), guidelines, Institute de Selection Animale (ISA) of Netherlands outlines the following, bio-security programme for effective control of poultry diseases. These include a buffer zone and clean area. Persons entering clean area should at least change shoes and clothes. Also, equipment entering the clean area should be cleaned and disinfected. Again, all materials entering this zone should be stored for at least two days in a clean, dry and rodents freed room. Visitors entering poultry premises should fully understand proper hygienic procedures and records must be kept of all visitors. Farm workers must wash their hands thoroughly with soap after any farm activity. Also, care should be taken at all times to protect the health and safety of farm workers and visitors (OIE Guidelines). Poultry houses must be cleaned and disinfected after emptying them. Footbaths should be replenished with additional disinfectant daily to maintain sufficient depth and the whole contents of the bath renewed once soiled or at least twice a week (Defra 2007). University of Ghana http://ugspace.ug.edu.gh 38 CHAPTER THREE METHOD 3.1 STUDY AREA The study took place in the Tema Metropolis, located along the eastern coast of Ghana covering an area of 396 square kilometers. It lies within the coastal savannah zone and has a vegetation of grassland and shrub land. Tema metropolis is flat, rising from the coast to 35 meters above sea level. There are a few inselbergs that do not rise more than 65 meters above sea level. Average rainfall is 700 millimeters. It is boarded on the north by Ashaiman, to the south by the Atlantic Ocean, to the East and West by Dangbe West District and Accra Metropolitan Area respectively. Tema has an estimated total population of 511,459 people according to the 2000 census and less than 10% of the population lives in rural communities (Ghana Statistical Services 2000). Also, it is the major industrial zone of Ghana. It has several communal natural resources such as the Chemu and Sakumono lagoons. Poultry production in Tema Metropolis could be classified into three categories according to installed capacity, marketing system and level of integration of its operations. These are commercial farms, semi-commercial farms and backyard/village poultry producers. It has an estimated total poultry population of 696,694 according to 2009 VSD annual report. These are made up of 534,852 layer chicken, 100,586 broiler chicken, 40,492 local chicken and 11,930 cockerels. The rest are 4,434 ducks, 2,920 turkeys and 1,480 guinea fowls. The metropolis also, has two poultry hatcheries and four commercial feed University of Ghana http://ugspace.ug.edu.gh 39 processing plants namely; Ghana Agro Food Company, Afariwaa Farms, Central Feed Mills and Glamour Farms. On animal health services delivery, the study area has one government veterinary hospital and two private veterinary hospitals that provide regulatory, field and clinical services. Figure 4. Map of first outbreak areas for HPAI in Ghana (source VSD, 2007 reports)4646444555/llkun9 890u University of Ghana http://ugspace.ug.edu.gh 40 # # # # # # # # # # # # # # # #PARAGON KATAMANSU ZENU GBETSILE TEMA COMM 1 MARKETKUBEBRO ASHIAMAN MARKET ADJEI KOJO SAKOMONO TEMA COMM 9 MARKET KAKASONANKA KPONE SAKI GOLF CITY 4 0 4 8 Miles N EW S Sampling Sites Ghdist.shp Tema metropolis Figure 5. Sampling sites in Tema Metropolis (Source, VSD, 2009) Commercial poultry and backyard poultry were sampled from Kakasunanka, Bethlehem, Golf City, Serbrepor, Gbetsile, Adjei Kojo, Michelle camp, Saki, Kpone, Kubekro, Zenu and Katamansu. Live bird markets in this study were located at Tema community one, Tema community nine and Ashiaman. Again, all wild birds sampled came from Sakumono Ramsar site. The reasons for the choice of Tema Metropolis were that it had experienced an outbreak of Avian Influenza in April 2007 which affected four commercial farms at Kakasunanka University of Ghana http://ugspace.ug.edu.gh 41 and Adjei Kojo. Thirteen thousand, three hundred and ninety one (13,391) birds were affected and thirty six thousand, three hundred and seventy six (36,376) birds were destroyed. The outbreak necessitated the institution of further control measures such as quarantine, disinfection and temporal ban of movement and sale of birds and poultry products in and out of the affected areas. The situation was brought under control by September, 2007 when Tema was declared free of the H5N1 virus. The choice of Tema was also informed by the fact that it is a port city with various commercial activities. 3.2 STUDY DESIGN The study type was a descriptive and cross sectional using active avian influenza surveillance approach. Thus, the study obtained tracheal specimens from commercial poultry, backyard poultry and live bird markets while cloacal and fresh feaces were obtained from wild birds and ducks for reasons of these being the natural reservoir of avian influenza viruses (Webster 1992, Fouchier et al 2003, Krauss et al 2004, Widjaja et al 2004). The study also employed both qualitative and quantitative methods of data collection. The qualitative method was used to capture information that was not catered for in the quantitative technique. It was also used to validate findings obtained through quantitative method. Figure 6: Active Avian Influenza Surveillance tle 1. Poultry : commercial and backyard 2. Wild birds 3. Live bird market Collection of trachea for poultry, cloacal swabs or fresh feaces for wild bird RRT-PCR for Influenza A virus detection RT-PCR for Newcastle virus detection University of Ghana http://ugspace.ug.edu.gh 42 3.3 SAMPLE SIZ SAMPLE SIZE DETERMINATION Using the following formula, the sample size was calculated in Epi info version 3.4.1 at 95% confidence level, and prevalence of Avian Influenza among domestic poultry, back yard poultry and wild life birds of 0.5 N=z 2 p(1-p) d 2 Where N= sample size z= risk of Type 1 error= 1.96 (at 95% confidence level) p= prevalence of Avian Influenza= 0.5 (assumed as no previous study to provide reference) d= precision= 5% Minimum sample size= 384. Commercial poultry, backyard poultry and live bird markets accounted for 384 birds each while 121 wild birds were sampled. This was in accordance with the recommendations made by Beal (1983) and Wildlife Health Center and Cooperative Extension, that for wildlife flocks of more than 2000 wild birds at 95% confident level the sample size should be a minimum of 58 birds. University of Ghana http://ugspace.ug.edu.gh 43 3.4 SAMPLING PROCEDURE A simple random sampling procedure was used to select communities in the metropolis. Also, using criteria for eligibility based on birds whether apparently healthy or with respiratory signs or gastroenteritis or nervous illness, were conveniently selected for tracheal swabbing, cloacal swabbing, collection of fresh faecal and identification in the case of commercial poultry, backyard poultry and the live bird markets. With regards to the wild birds, mist nets were used to capture wild birds. The captured ones were then marked to avoid recounting upon recapture of the same bird. Cloacal swabbing or collection of fresh faeces and identification were made before release of the birds. In this study, data were collected by reviewing available farm records. Poultry farm owners, poultry farm workers, poultry traders and staff of Wildlife Division of Forestry Commission were interviewed and a semi-structured questionnaire was administered for data collection. All specimens were put in vials containing viral transport medium (VTM) and transported on ice in cool boxes for laboratory analyses at the Virology Department of the Noguchi Memorial Institute for Medical Research. 3.5 MOLECULAR DETECTION 3.5.1 RNA EXTRACTION Ribonucleic acid was extracted from samples with the QIAamp viral RNA mini kit 250 (Qiagen, GmbH). Briefly, 560 µl of prepared buffer (viral lysis buffer plus RNA carrier) was added to 140 µl of trachea, cloacal and faecal samples obtained from domestic poultry and wild birds in the Tema Metropolis. It was then mixed by pulse vortexing for 15 seconds and then left to stand at room temperature for 10 minutes. Also, it was mixed University of Ghana http://ugspace.ug.edu.gh 44 by pulse vortexing with 560 µl of absolute ethanol. Then, 630 µl of the mixture was clarified by centrifugation at 8000 xg for 1 minute and repeated. The filtrate tube was discarded and column tube placed in a new collection tube. Then, 500 µl of wash buffer 1 was added and spun at 8000 xg for 1 minute. Again, the filtrate tube was discarded and the spin column placed in a new collection tube. Five hundred µl of wash buffer 2 was added and spun at 14000 xg for 3 minutes. Then, the filtrate was discarded and a new collection centrifuged at 14000 xg for 1 minute. Finally, the RNA was eluted in 40 µl twice with a final product of 80 µl. This was stored in a -70 0 C ultra low freezer thermo scientific at the P3 laboratory corridor, Noguchi Memorial Institute for Medical Research and 8 µl of RNA was used as template for the real time RT-PCR. 3.5.2 REAL-TIME REVERSE TRANSCRIPTASE-POLYMERASE CHAIN REACTION (RRT-PCR) The Qiagen one step RT-PCR kit (Protocol: Influenza A Matrix gene AB17300 RT-PCR Spackman et al., 2002) was used with a 20 µl reaction volume under the following amplification cycling conditions; 0.8 µl of Qiagen one-step enzyme (Qiagen GmbH), 10 pmol of each primer, 0.3 µM probe, 0.8 µl of deoxynucleoside triphosphate (dNTPs), 1 µl magnesium chlorite (MgCl2), 4 µl of 5X buffer of Qiagen kit, 1 µl of ROX dye working dilution (1:100) and 6.5 U of RNase inhibitor. The RT step conditions for forward and reverse primer set were 30 minutes at 50 0 C and 15 minutes at 94 0 C. A two-step PCR cycling protocol was used for the matrix gene primer set as follows; 45 cycles of 94 0 C for 0 seconds and 60 0 C for 20 seconds. The H5 University of Ghana http://ugspace.ug.edu.gh 45 PCR was as follow; 94 0 C for 0 seconds, 57 0 C for 20 seconds and 72 0 C for 5 seconds for 40 cycles. Primers used for this amplification are listed in table 2. Table 2. PCR primer and probe sequences for Influenza A virus detection Specificity Primer/Probe Sequence”(5‟-3‟) Influenza A virus M + 25 AGA TGA GTC TTC TAA CCG AGG TCG M - 124 TGC AAA AAC ATC TTC AAG TCT CTG M + 64 FAM-TCA GGC CCC CTC AAA GCC GA-TAMRA Avian H5 H5+1456 ACG TAT GAC TAT CCA CAA TAC TCA G H5-1685 AGA CCA GCT ACC ATG ATT GC H5+1637 FAM-TCA ACA GTG GCG AGT TCC CTA GCA-TAMRA Avian H7 H7+1244 ATT GGA CAC GAG ACG CAA TG H7- 1342 TTC TGA GTC CGC AAG AC TAT TG H7 + 1281 FAM-TAA TGC TGA GCT GTT GGT GGC A-TAMRA FAM, 6-carboxyfluorescein; TAMRA, 6-carboxytetralrhodamine. Source; Spackman et al 2002 RRT-PCR was performed by an advanced pathogen identification device called Applied Biosystems 7300 Real Time PCR System. Results of RRT-PCR were determined by the device autoanalysis software with a positive control cycle threshold (Ct) at 20.45 and a negative control Ct at undetectable (table 10) University of Ghana http://ugspace.ug.edu.gh 46 3.5.3 CONVENTIONAL RT-PCR FOR NEWCASTLE DISEASE VIRUS (NDV) Conventional RT-PCR NDV was performed as described in the field and laboratory manual (chapter 11.3). Briefly, the reaction conditions used were as follow; 50 0 C for 30 minutes and 94 0 C for 15 minutes with annealing temperature of 94 0 C for 30 seconds, 55 0 C for 1 minute and 68 0 C for 1 minute on a 35 cycle run with a final extension of 68 0 C for 7 minutes. Fragment size at 270bp (Capua and Alexander. Avian Influenza and Newcastle Disease. A field and laboratory Manual. Chapter 11.3). Conventional RT-PCR NDV was by Applied Biosystems 2720 Thermal Cycler. Primers used for this cycle were as follow; Primer Forward: NOH-For 5‟ TAC ACC TCA TCC CAG ACA GG 3‟ Primer Reverse: NOH-Rev 5‟ AGT CGG AGG ATG TTG GCA GC 3‟ 3.6 DATA PROCESSING AND ANALYSIS Data collected was checked for accuracy, completeness and consistency. The data was coded and fed into SPSS statistic version 17.0 software programme. Also, cleaning of data was ensured by double entry and reconciliation of these entries. Simple frequencies, percentages, graphs and tables were then generated. University of Ghana http://ugspace.ug.edu.gh 47 3.7 ETHICAL CLEARANCE Scientific review of the study protocol was sort from the Scientific Technical Committee of the Noguchi Memorial Institute for Medical Research. Contacts were made with Tema Metropolis veterinary officer, Wildlife Division of Forestry Commission; poultry farm owners, local bird owners and poultry traders in the selected communities of the metropolis for their permission and informed consent (Appendix I). 3.8 LIMITATIONS Financial constraints were the major challenge. Also, late arrival of materials and reagents resulted in the delay in completing the study on scheduled. Again, lack of prevalence data on avian influenza disease in Ghana was a limitation as that could have affected the sample size. University of Ghana http://ugspace.ug.edu.gh 48 CHARPTER FOUR RESULTS 4.1 DESCRIPTIVE CHARACTERISTIC OF BIRDS A total number of one thousand two hundred and eighty two different birds were sampled from 16 different places within 2.5 kilometers to18 kilometers of the Tema Metropolis. Out of these, 384 samples were from Commercial poultry farms, 393 samples were from Backyard farms, 384 samples were from Tema Community One, Tema Community Nine and Ashiaman live birds markets and 121samples were from wild birds at Sakumono Ramsar site (Table 3). For commercial poultry, tracheal samples were obtained from layer chicken (Gallus gallus domesticus). In the backyard and live bird markets, tracheal swabs from local fowls (Gallus gallus domesticus), guinea fowls (Numida meleagris), pigeons (Family Columbidea), turkeys (Meleagris gallopavo) and cloacal swabs from ducks (Anas platyrhynchos domesticus) were sampled. For the wild birds, cloacal swabs were obtained from white-faced tree duck (Dendrocygna viduota), black-winged stilt (Himantopus himantopus), white-backed stilt (Himantopus himantopus meleaurus), black tern (Chlidonias niger), quails (Coturnix coturnix), prantcole (Glareola nordmanni), little egret (Egretta garzetta), sand plover (Charadrius mongolus), spur-winged plover (Vanellus spinosus), ringed plover (Charadrius hiaticula), village weaver (Ploceus cucullatus), pygmy cormorant (Phalacrocorax pygmaeus) and goliath heron (Ardea goliath) (Table 4). University of Ghana http://ugspace.ug.edu.gh 49 About, 91% (349/384) of birds sampled from commercial poultry farms were females while 9% (35/384) were males. The sex distribution of the backyard poultry was found out to be at 72% (283/393) for females and 28% (110/393) for males. Again, the study observed 69% (265/384) females and 31% (119/384) males in the case of the poultry birds in live bird markets. However, all wild birds sampled at the Sakumono ramsar lagoon, sex identification was not done. This was due to the fact that there was no expert to determine the sex of the wild birds. We found out that 15% of the farms sampled had grower age range (less than 18 weeks for commercial birds and less than 24 weeks for backyard birds) and 85% had adult birds (more than 18 weeks for commercial birds and more than 24 weeks for backyard birds) at the time of sampling. University of Ghana http://ugspace.ug.edu.gh 50 Table 3. Descriptive characteristics of Birds Count Percent Flock Grouping Commercial 384 30% Backyard 393 30.6% Live Bird Market 384 30% Wild Bird 121 9.4% Total 1282 100% Age Range Grower 192 15% Adults 1090 85% Total 1282 100% Male Female Sex Commercial 9%(35/384) 91%(349/384) Backyard 28%(110/393) 72%(283/393) Live Bird Market 31%(119/384) 69%(265/384) Wild Bird Unknown Unknown *Wild birds could not be sexed University of Ghana http://ugspace.ug.edu.gh 51 Table 4. Descriptive characteristic of birds (common/scientific name) System of Production Common Name Scientific Name Commercial Poultry Broiler and Layer Chicken Gallus gallus domesticus Backyard Poultry Local fowls Gallus gallus domesticus Ducks Anas platyrhynchos domesticus Guinea fowls Numida meleagris Turkey Meleagris gallopavo Live Bird Market Local fowls Gallus gallus domesticus Ducks Anas platyrhynchos domesticus Guinea fowls Numida meleagris Turkey Meleagris gallopavo Doves/Pigeons Family Columbidea Wild Birds White-faced tree duck Dendrocygna viduota Black-winged stilt Him antopus himantopus White-backed stilt Himantopus himantopus melanurus Black tern Chlidonias niger Quails Coturnix coturnix Pranticole Glareola nordmanni Little egret Egretta garzetta Sand plover Characdrius mongolus Ringed plover Charadrius hiaticula Spur-winged plover Vanellus spinosus Village weaver Ploceus cucullatus Pygmy cormorant Phalacrocorax pygmeaus University of Ghana http://ugspace.ug.edu.gh 52 Note: (1) Live bird markets in this study were located at Tema community one, Tema community nine and Ashiaman. (2) Wild birds were sampled from Sakumono Ramsar site (3) Commercial poultry and backyard poultry were sampled from Kakasonanka, Bethlehem, Golf city, Serbrepor, Gbetsile, Adjei Kojo, Michelle camp, Saki, Kpone, Kubekro, Zanu and Katamansu In reviewing the total stock of birds per farm, the study found that in the one hundred and five (105) farms sampled, eight percent (8%) had less than ten birds, while 56.8% farms kept between ten (10) and ninety nine (99) birds. Also, 18.2% of the farms in the study area had a stock populations of between one hundred (100) and nine hundred and ninety nine (999) birds. Farms found to be keeping more than thousand (>1000) birds accounted for seventeen percent (17%) of the total number of farms sampled, (Figure 7). Total Stock of Birds per Farm 0 10 20 30 40 50 60 <10 10.-99 100-999 >1000 Number of Birds per Farm P e rc e n ta g e Figure 7: Total stock of birds per farm University of Ghana http://ugspace.ug.edu.gh 53 The flocks of birds were established from hatcheries in Accra, Dormaa Ahenkro, Kumasi and Tema. Also, day old chicks were imported from Holland and Belgium. Tema accounted for sixty three farms representing 60% of the source of birds introduced into the metropolis, followed by Accra, Holland, Belgium, Kumasi and Dormaa Ahenkrom in that order. Also, wild birds accounted for 16.2% of the number sampled. (Table 5) This profile shows that the source of the birds is from within Tema. This scenario also indicates that major prophylactic measures would probably have to be targeted within Tema. From Table 5. it was observed that most of the birds were slaughtered for food (50.5%) while a significant number (33%) were sold as live birds in the markets. This informs us that a potential source of spread of Avian Influenza could be from the market since birds bought for home consumption were invariably immediately prepared for consumption. But this could be a source of infection to humans since handling, slaughter of poultry are