Epidemiology of Influenza Virus H5n1 in Islamabad Capital Territory by Zahida Fatima (2005-Va-246) a Thesis Submitted in the Pa

EPIDEMIOLOGY OF INFLUENZA VIRUS H5N1 IN ISLAMABAD CAPITAL TERRITORY

ZAHIDA FATIMA

(2005-VA-246)

A THESIS SUBMITTED IN THE PARTIAL FULFILLMENT OF THE

REQUIREMENTS FOR THE DEGREE

DOCTOR OF PHILOSOPHY

EPIDEMIOLOGY AND PUBLIC HEALTH

UNIVERSITY OF VETERINARY & ANIMAL SCIENCES, LAHORE

(2015)

The Controller of Examinations,

University of Veterinary and Animal Sciences,

Lahore.

We, the supervisory committee, certify that the contents and form of the thesis, submitted by ZAHIDA FATIMA, Regd. No. 2005-VA-246 been found satisfactory and recommend that it be processed for the evaluation by the External Examiner(s) for the award of the degree.

PROF. DR. MUHAMMAD ATHAR KHAN ______

SUPERVISOR

DR. KHALID NAEEM ______

CO-SUPERVISOR

PROF.DR. MANSOOR UD DIN AHMAD ______

MEMBER

PROF.DR. KHUSI MUHAMMAD ______

MEMBER

DEDICATED

MY LATE FATHER

MAY HIS SOUL

REST IN BEST PEACE

(AAMEEN)

i ()

In the name of Allah the most magnificent and the most beneficent.

All praise for ALLAH All Mighty who has the control and command of each and every

thing.

It is He who has sent down to you, [O Muhammad], the Book; in it are verses [that are]

precise - they are the foundation of the Book - and others unspecific. As for those in whose

hearts is deviation [from truth], they will follow that of it which is unspecific, seeking

discord and seeking an interpretation [suitable to them]. And no one knows its [true]

interpretation except Allah. But those firms in knowledge say, "We believe in it. All [of it]

is from our Lord." And no one will be reminded except those of understanding.

ACKNOWLEDGEMENTS

First of all I would like to thanks Allah Almighty and like to express my sincere and humble gratitude to Almighty whose blessing, help and guidance has been a real source of all my achievements in my life.

This dissertation would not have been possible without the guidance and the help of several individuals who in one way or another contributed and extended their valuable assistance in the preparation and completion of this study.

I would like to admit that I completed this project due to my loving parents who pray for my success and my husband, sisters and brothers for their efforts, love, support and encouragement.

I wish to express my heartiest gratitude to my supervisor and my great mentor DR. MUHAMMAD ATHAR KHAN who introduced me to the new dimensions of knowledge of Epidemiology and public health and for his full co-operation throughout the course work and research work. I find it hard to imagine that anyone could be a more sincere, kind and better research advisor than what he has been to me throughout my phD.

I’ve been most fortunate to have the guidance of PROF .DR. MANSOOR Ud DIN AHMAD, Department of Epidemiology and Public Health, University of Veterinary Sciences Lahore. I would like to record my gratitude for his kind supervision as a member in my supervisory committee, sympathetic attitude, constructive advices, and scientific discussions.

And heartily thankful to Dr Khalid Naeem,CSO,ASI, NARC Islamabad, who helped a lot in all the aspects of research work, without his generous cooperation it was not possible for me to continue. He is the person who directed me in the true sense towards adopting the subject of Epidemiology as my future endeavors of my professional life.

I wish to pay my gratitude to DR. KHUSHI MUHAMMAD, Department of Microbiology, University of Veterinary and Animal Sciences, Lahore, as member in my supervisory committee for his support, encouragement, ever helping behavior and valuable guidance.

iii

I wish to express my sincere thanks to all my supervisory committee.

It was a great honor for me to work under the guidance of Dr Muhammad Hassan Mushtaq and his sincere efforts for me.

Above all, I must acknowledge NRLPD, Animal health, NARC, my work place that offered a scientific environment for carrying out this work.

Special thanks to Zubair Anwar, Amjad Khan,Luqman Sohail, Dr. Jaamal, Dr. Asad Shah, Dr. Anum Hadi and my colleagues at Animal health who are always great help and source of encouragement and moral support throughout my PhD.

ZAHIDA FATIMA

iv DEDICATION (I)

ACKNOWLEDGMENT (iii)

LIST Of FIGURES (vi)

LIST Of TABLES (vii)

LIST OF ABBREVIATIONS (viii)

TABLE OF CONTENTS

S. NO. CHAPTERS PAGE NO.

1 1 INTRODUCTION 8 2 REVIEW OF LITERATURE 51 3 MATERIALS AND METHODS 69 4 RESULTS 92 5 DISCUSSION 101 6 SUMMARY 103

7 LITERATURE CITED

v LIST OF FIGURES

TITLE PAGE FIGURE NO. NO.

2.1 Important events of avian influenza in Pakistan 13

2.2 Country distribution of H5N1 in poultry in 1996 15

2.3 Epidemiological process in Asian H5N1 epidemics 18

2.4 Emergence of A/goose-guangdong/1/96-like H5N1 virus 19

2.5 Transmission pattern of influenza virus 21

2.6 Vehicles of transmission of AIV 22

4.1 Trends of mortality, morbidity and case fatality in layers 71

4.2 Trends of mortality, morbidity and case fatality in breeder 73

4.3 Rapid response team working in HPAI infected form 74

4.4 Trends of mortality, morbidity and case fatality in broiler 75

4.5 Spatial distribution of outbreaks in study areas 77

4.6 GMT of H5, H7, H9 87

vi LIST OF TABLES

TABLE NO TITLE Page #

2.1 Major outbreaks in 2006 16

2.2 Inhibitors of AI 38

3.1 Data of administrative units of study area 51

3.2 Census of birds in study area 52

3.3 No. of samples taken from LBM 59

4.1 Outbreaks in layers in 2006-2008 70

4.2 Outbreaks of H5N1 72

4.3 Outbreaks in broiler birds 74

4.4 Case control age and case description 76

4.5 Univariate analysis of associated risk factors 78

4.6 Serology of H5N1 81

4.7 Serology of H7 83

4.8 Serology of H9 85

4.9 Samples taken from LBM 89

vii

LIST OF ABBREVIATION

AIV AVINA INFLUENZA VIRUS

HA HAEMAGGLUTININ

NS NON STRUCTURAL

NEP NUCLEAR EXPORT PROTEIN

LPAIV LOW PATHOGENIC AVIAN INFLUENZA VIRUS

HPAIV HIGH PATHOGENIC AVIAN INFLUENZA VIRUS

ICT ISLAMABAD CAPITAL TERRITORY

LBM LIVE BIRD MARKET

RT-PCR REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION

NP NUCLEOPROTEIN

NARC NATIONAL AGRICULTURAL RESEARCH COUNCIL

WHO WORLD HEALTH ORGANIZATION

CDC CONVENTIONAL DENDRITIC CELLS

NOS NITRIC OXIDE SYTHETASE

TIV TRIVALENT INACTIVATED

NI NURAMINIDASE INHIBITION

HI HAEMAGGLUTINATION INHIBITION

PCR POLYMERASE CHAIN REACTION

GMT GEOMETRIC MEAN TITER

SAARC SOUTH ASIAN ASSOCIATION FOR REGIONAL COOPERATION

SARS SEVERE ACUTE RSPIRATORTY SYNDROM

viii

CHAPTER 1 INTRODUCTION

Influenza remains the most studied viruses in the scientific community throughout world

(WHO, 2013) Avian influenza viruses have been isolated are found almost everywhere in the wild bird population, but most importantly in wild aquatic birds. The virus is the cause of disease in poultry at various levels of severity. There are two pathogenic strains, depending on the severity of diseases caused by virus, albeit viruses may switch between the two types due to mutation or re assortment of genes. The term “influenza” refers to as epidemics of acute, fast circulating respiratory or oral infections in human beings caused basically by various types of

Orthomyxoviruses (Anonymous, 2006). The AIV viruses have been isolated from multiple hosts like avians, equines, swines, seals and human beings and many other type of hosts (Webster et al., 2008). Infection of AI viruses in poultry causes very high economic losses to the poultry farmers in Pakistan. One of the AIV types (H5N1) has been reported from many Asian, African and European poultry producing countries and this virus type has caused significant number of human infections most of which have been reported as fatal.(WHO,2013). The scientists suggest that this AIV type has the potential to mutate and emerge into a new form by developing necessary receptors for causing human to human infections by breaking species barriers.

(Kilbourne et al., 2006; Lindstrom et al., 2004). Although sharing many symptoms with other respiratory infections,AIV influenza in addition causes unanticipated onset of three-day fever, including muscle pain and a degree of weakness or dizziness with the severity of other symptoms in the humans. In poultry farming, domestic poultry infection by avian influenza viruses (AIV)

Introduction generally bring forth syndromes which range from general infection to respiratory abnormalities and loss in egg production to severity in infection with almost 100% mortality (Bahlet al., 1977)

1.2 Types of Influenza

The Influenza virus is divided into type A, B and C based on of host relationship in terms of virus antigenicity. An influenza virus is spherical or longitudinal in shape. These particles are enveloped. The viral genome is segmented and single stranded, negative sense RNA. All the virus types are differentiated on the basis of antigenic differences in their matrix and nucleoproteins. Amongst these AIV belongs to type A. It contains eight segments coding 11 proteins (Lamb et al., 1982).

1.2.1 Subtypes of Influenza

Haemagglutinin (HA) and the neuraminidase (NA) transmembrane glycoproteins are main determinants of influenza A and B viruses’ antigenicity. These can provoke a subtype-speciﬁc immune response. Influenza A virus gather into sixteen H (H1 to H16) and nine N (N1 to N9) subtypes (Tong et al., 2012).

1.3 Natural hosts

Wild birds, specifically species in order Charadriiformes (e.g tern, gulls) and anseriformes (e.g swans, ducks, geese), are reckoned the natural reservoir of subtypes of AIVs. These are widely distributed in these bird species all across the globe (Stallknecht 1990).

In general natural hosts do not exhibit clinical signs of disease because of AIV type A viruses.

Instead the viruses remain in an evolutionary equilibrium (Gorman 1991; Taubenberger 2005).

Host and virus exists in a very precise and unbiased mutual tolerance, as shown in the absence of

Introduction disease clinically, and efficient viral replication (Webster 1978). Often mild symptoms are noticed upon virus transmission to vulnerable poultry species. Viruses with such phenotype are called to be low pathogenic (LPAIV), causing a small and temporary decrease in production of eggs or decrease in weight gain among broiler birds (Capua and Mutinelli 2001). Whenever transmitted and adapted to the new hosts, the strains of subtypes H5 and H7 are likely to change into highly pathogenic form. Origin of HPAI H5 and H7 or other subtypes hasn't been reported in wild birds (Scholtissek et al., 1998).

Once HPAIV phenotype appears in domesticated birds, it can easily be spread from poultry into the wild birds and vice versa. The susceptibility of wild birds towards HPAIV varies from species to species, age and viral load in the environment. Before viruses of Asian lineage (H5N1

HPAI) emerged spread of HPAIV in the wild birds was episodic and curtailed to a specific region (Becker 1966), so wild birds were not considered as important spreader of of HPAIV at that time (Swayne and Suarez 2000a; Stallknecht et al., 1990a).

1.4 Influenza Virus Serotypes

Type A influenza viruses can infect a variety of mammals and bird species, when in a particular host species is known to be specie specific. The H1N2 virus isolated from a turkey breeder flock, in which a fanciful drop in egg production was observed. Sequence analysis of the isolate showed it was mutant virus having viral genes of swine, human and avian origin influenza. A swine influenza virus was reported from pigs in Indiana, USA, having same gene characteristics.

Another report stated that the influenza viruses can move across species and cause an epidemic.

The diagnosticians are well acquainted that the variation of influenza viruses can pave the way for distinguishing new isolates. In a study from 1993 to 2000, gallinaceous birds, specimens

Introduction from environment and waterfowls in the live bird markets (LBMs) and non-LBM regions in the northeastern United States were taken with an objective of checking presence of AIV was conducted. Pathogenic properties of AIV subtype especially those of H5 and H7 subtypes and a possible association between LBM and rest of LBM infections were investigated. The AIV subtypes H5 and H7 have been proved low pathogenic in the chickens. They suggested that a low pathogenic virus (H5 or H7) may alter into highly pathogenic by mutation of acquired additional basic amino acids (Panigrahy et al. 2002).

In a study presence of LPAI H7N3 virus on a two-age broiler breeder farm in Abbotsford, British

Columbia (BC) was reported. The older flock subjects showed 0.5% mortality over 72 hrs that resolved during the following week. Ten days after this a younger flock in an adjacent barn suspiciously showed 25% mortality in 48 hr. A LPAI (H7N3) virus isolates from the older flock, and younger flock showed that younger flock had an additional 21 base insertion at the hemagglutinin-cleavage site. This mutation suggested transformation of LPAI into HPAI form some point between the infection in first and second sheds (Sharkey et al. 2008).

1.5 Virus Mutation

Point mutations result in antigenic drift in mammalian influenza viruses in the HA and/or NA genes that causes minor antigenic changes in the coding proteins. Antigenic shift on the other hand is caused by genetic reassortment between the gene segments of the two viruses that infect same cell. The genetic reassortment leads to the accumulation of new HA and/or NA antigens in a virus population which may have chances to cause pandemic situation (Webster et al., 1983).

Pandemics are due to novel virus subtypes of influenza A created by antigenic shift and the annual epidemics of influenza A and B viruses are because of antigenic drift. Degree of infection

Introduction of influenza depends on the HA cleavage by particular host proteases, whereas NA is responsible for virions released from the surface of infected cell and also protects from agglutination of newly formed virus (Zambon1999). The transmission of influenza A viruses within interspecies, which circulates in wild aquatic birds every now and then results in the influenza epidemics in mammals, including human beings (Beare and Webster., 1991). This suggests that the receptor- binding specificity of the HA is changed soon after the transmission of an AIV to humans and pigs (Beare and Webster., 1991). Lin et al. (1998) reported that the domain of transmembrane is required for apical transport constituted the residues most conserved among HA subtypes.

Kawaokaet al. 1984 reported that the reassortants of AIV carrying human influenza virus (IV)

HA didn’t replicate in the ducks, although the two mutations in the receptor-binding site of a human HA allows the virus to replicate in the ducks. Clements et al. 1989 reported that an AI

A/Pintail/Alberta/119/79 (H4N6) virus was mated with the wild-type human influenza

A/Washington/897/80 (H3N2) virus ensuing reassortant influenza A virus. Buranathai et al.,

2007) from one chicken isolate and the two additional human isolates and sorted the whole genome of three human H5N1 isolates, along with the HA and NA genes. These isolates were got from Thailand during AI pandemics from year 2003 to 2004. These viruses had more avian- specific leftovers than Hong Kong H5N1 viruses of 1997, conferring that the virus probably have evolved to be more efficient in transmission in various species of birds. In 2002, H5N1 virus isolates from migratory population of birds and indigenous waterfowl from Hong Kong Parks had evident antigenic drift as they were highly pathogenic for ducks (Lipatov et al., 2004; Sturm-

Ramirez et al., 2005). Viruses isolated were responsible for two human cases in 2003 in Hong

Kong which were similar in antigenicity and molecularity (Lipatov et al., 2004; Peiris et al.,

2001). Strains of H5N1 from the healthy ducks of Southern China from 1999 to 2002 were found

Introduction antigenically same to precursor Influenza A/G/Gd) 96 virus (Chen et al., 2009). Healthy ducks had a vital role in making of virus stains a reason for the outbreaks in South-east Asia since

2003. The recent H5N1 virus evolution is considered to be linked with elevating virulence and spreading range of hosts which now constitutes wild birds, poultry and mammals.

Kida et al (1994) reported that influenza A virus which are pandemic arose from genetic reassortment between human and avian virus. Pigs are considered to create such reassortants as middle host. They studied the growth potential of growth in 42 strains of influenza virus (IV) in pigs. Out of which 38 were from birds, having 27 with non-human-type hemagglutinins. They observed that the AIVs with or without non-human-type HA transmits to pigs, thereby increasing the chances of involvement of their genes into humans.

1.6 AIV Transmission

Conceptually AIV can be transmitted via five different routes

1. Contact with AI diseased birds.

2. Contact with AIV respiratory secretions or feces contaminated utensils.

3. Entry and exit of people with AIV contaminated shoes or clothing.

4. Water contaminated with AIV.

5. Movement of AIV through air AIV may be dispersed by air through aerosols, dust and

feathers. It happens especially during the depopulation of AIV infected birds,feed or

eggs.(Sharkey et al., 2008).

Introduction

1.7 OBJECTIVES AND STATEMENT OF PURPOSE

The main goal of my research studies was to gain better understanding of the HPAIV H5N1 outbreaks occurred first time in the history of Pakistan through retrospective investigations.

Although information regarding molecular analysis and mapping of the reported outbreaks both at commercial and backyard farms is available, but data on epidemiological findings of these outbreaks during the period 2006-2008 along with risk factors quantification and live bird market surveillance in study area has not been analysed. Moreover almost all the previous reports are from foreign countries, whereas very less information regarding Pakistan and Islamabad to be more specific is available. Keeping the above reports and demographics in view, the present study has been designed to focus on the following key objectives:

1- To investigate the outbreaks of HPAIV H5N1 from 2006-2008 at Islamabad Capital

Territory. This will lead to gather information regarding the distribution, trend and

pattern of disease.

2- To identify and quantify the risk factors linked with HPAI virus H5N1 by a cross

sectional survey in different ecological zones of ICT.

3- To record the persistence, perpetuation and / or circulation of HPAI H5N1 in the live bird

markets in and around ICT areas.

CHAPTER 2 REVIEW OF LITERATURE

The oldest evidence of avian influenza were found in 1878 when pathogenic avian influenza was primarily an poultry infection in Italy, and it was called ‘fowl plague’. It was referred as an infection of virulent serotype Influenza A virus of Orthomyxoviridae family. It causes outbreaks in poultry and human infections in Asian, African, and European countries (Alexander, 1986). In

1901, two scientists Centanni and Savonuzzi recognized a filterable organism which was causing the infection, but after 1955 Schäfer characterized this agent as influenza A virus (Alexander,

1986, Feldmann et al., 2000).There were different strains which were discovered by different scientists but the most virulent strain of the bird flu virus is called H5N1. The first outbreak of this HPAI virus subtype H5N1 was first identified in Vietnam in late 2003 (DAH 2005b). After the emergence of HPAI H5N1, this has attracted substantial public attention because it was the causative agent in both animals and humans. Since 2003, HPAI virus subtype H5N1 has affected the animals of specific geographic area and becoming a persistent potential pandemic threat to animals and sporadic cases of infection in humans (DAH (2007). The cases of avian influenza have been reported from, Holland, Belgium, Mexico, Pakistan, USA, Egypt, Algeria, China,

Bangladesh, Bhutan, Austria, Cameron, Cambodia, Burkina Faso , Australia and many more.

Among different strains, H5 type causes highly contagious diseases and is responsible for recent out breaks in different countries like Vietnam, Pakistan, Thailand , Taiwan, South Korea, Japan and Hong Kong, etc and killing both birds and humans and effecting the survival and economy of livestock farmers.

2.1: Brief history of avian influenza virus H5N1

8 Review of Literature

Due to its pathogenicity and subsequent effects on the economy, avian influenza virus has been the focus of research by various scientists. Some of the important studies are following:

Sarwar et al (2013) studied the surveillance of the AIV in LBM’s of Lahore. For this purpose

1500 cloacal swabs were collected from seven live bird markets of Lahore. The samples were tested for virus isolation in chicken embryos. Allanto-amniotic fluid (AAF) of each of the embryos was used for Haemagglutination (HA) test. Eighteen (18/1500) samples were positive for HA activity. AIV Antigen Rapid Test Kit differentiated 4 HA virus as AIV and other 14 as

Newcastle disease virus. Haemagglutination Inhibition test also proved four HA positive isolates as AIV, while remaining fourteen as NDV, using specific sera. All the AI virus isolates were confirmed by RT-PCR by a universal nucleoprotein (NP) primer set specific to the virus NP gene and serotype specific primers (H9 and N2) for its sub typing as H9N2.

Zahida et al (2012) explained that every year during extreme months of winter, December and

January, the migratory birds from Siberia travel to Indus Valley and other parts of Indo-Pakistan subcontinent. These migratory birds include, ducks, geese, swans and the water fowls. These birds are known to carry all avian influenza viruses and by shedding virus, they are assumed to be vector. Samples were taken from migratory and non-migratory wild birds on lakes, zoo and live bird market. Samples were processed in the NRLPD, National Agricultural Research

Council (NARC), and Islamabad for virus isolation, HI and RT-PCR for the recognition of H3,

H5, H7 and H9 serotypes of AIV type A. It was concluded that migratory birds flying over the

Pakistan territory were negative against H3, H5 and H7. However, positive samples for H9 serotype were found in backyard poultry.

Abdul Ahad et al (2013) stated that among the different subtypes of avian influenza virus (AIV)

H9 and H7 is able to infect poultry and human. In some countries these viruses have been

Review of Literature isolated from the occupational hazard group of people, like veterinarian, poultry attendant and poultry retailers. Therefore, the objective of this project was to find the seroprevalence of H9 and

H7 AIV subtype in different occupational people who are directly or indirectly involved with the poultry industry. Antibodies to H9N2 and H7N7 avian influenza virus were measured by modified horse RBC hemagglutination inhibition (HI) test using receptor destroying enzyme

(RDE) treated sera. Total 465 human sera samples were analyzed who were directly exposed to poultry industry and 25 samples were taken as control that was not exposed to poultry industry.

The highest (85.7%) seroprevalence against H9 was recorded in vaccinators and the lowest

(30.4%) was recorded in veterinarians. On district wise the highest (82.1%) seroprevalence against H9 was observed in Toba Tek Singh district and the lowest (9.7%) was observed in the

Islamabad. In case of H7 AIV subtype the highest (44.4%) seroprevalence was recorded in lab technicians and the lowest seroprevalence (11.1%) was recorded in butchers. By district wise the highest (57.9%) seroprevalence against H7 was recorded in Haripur district and the lowest

(4.6%) was recorded in Gujranwala district.

Capua and Alexander (2009) observed that influenza viruses which infect birds can be categorized into 2 types. These avian viruses were only limited to H5 and H7 strain, but not all

H5 and H7 viruses grounds HPAI. HPAI viruses were seldom separated from wild birds, but for low pathogenic avian influenza viruses; surveillance studies showed high isolation rates. These viruses affect different species of birds around the world and primarily the infection depends upon the level of contact of the poultry with feral birds (birds that have been escaped from domestication and have managed to establish breeding populations in the wild). In addition to this spread of secondary infection, it is also associated with human involvement with either of transfer of feces from birds having infection to birds which are susceptible to infection, or by

Review of Literature bird or bird product movement, moreover wild birds could also be the reason. In present time, occurrence of HPAI appears to have elevated, particularly in heavily populated poultry areas of

Pakistan, Netherlands, Italy and Canada. To reduce this outbreak, thousands of birds were killed to keep outbreaks restrained. Since the 1990s, avian influenza become capable of explaining into

2 subtypes because of their epidemiology and causing diseases in birds all across the globe. The

LPAI H9N2 virus has becoming endemic in birds in many Asia countries but occurrence of

H9N2 has been overshadowed by the H5N1 HPAI. It has been spreading in wild birds as well as in poultry, or both all over Asia, Africa and Europe causing in the death of hundreds of millions of birds and therefore becoming an important zoonotic menace.

Takano et al., (2009) explained that found that the spreading of H5N1 strain of avian influenza is highly pathogenic and it is also a serious threat to human health. In 2009 Indonesia experienced the highest human infections and deaths, moreover 24 H5N1 viruses were isolated from poultry birds during 2003 to 2007. These isolated viruses were characterized on the basis of their evolutionary development. According to the previous studies Indonesian viruses were divided into class 2.1.1-2.1.3, but interestingly the evolutionary and phylogenetic analyses of these viruses showed a new sublineage that did not relaetd to any of the present species.

Wang et al., 2008 reported that HPAI H5N1 virus has clean up the Europe across the world and this provides the opportunity for the scientists to debate on the role the migratory birds play in transport of virus since the first outbreak in migratory birds of Lake Qinghai, 2005. In 2006, six novel strains were isolated by another huge outbreak of Lake Qinghai. Whole-genome sequencing of these six isolates were carried out and their evolutionary history showed that

QH06 viruses were the derivatives of the lineages of Lake Qinghai, 2005. Out of these six, five were closest to the strain A/Cygnus olor/Croatia/1/05, and the sixth one belongs to the strain

Review of Literature

A/duck/Novosibirsk/02/05, which was isolated from flyway. Finally it was concluded that QH06 and QH05 viruses are alike this was proved with the help of antigenic analysis. These QH06 viruses of Lake Qinghai move back through migratory birds, though didn’t exclude the possibility of local transport of viruses of Lake Qinghai.

Uchida et al. (2008) described a detailed molecular epidemiological analysis on HPAIV of the

H5N1 subtype affecting poultry and wild birds from 2004 to 2007 in Thailand. After sequencing and phylogenetic analysis, it was found that viruses belongs to the clades 1 and it was also shown in HA phylogenetic tree thatclade 2.3.4 circulates in Thailand.

Naksupan et al. (2008) described the complete avian virus sequence in aquatic residents’ birds in

Thialand. It was shown that 20 amino acids were deleted in neuraminidase and a five amino acid deleted in the nonstructural protein. Hence, this was categorized as genotype Z.

Leong et al. (2008) studied that avian influenza (HPAI) H5N1 virus subtype was first detected in

1996 in Guangdong and China. Since 2003, occurance of H5N1 was reported in different parts of

Asia, Middle East, Europe, and Africa. This disease causing strain is becoming a serious problem for the animals and for the human health.

Keawcharoen et al. (2008) reported that wild birds were the real cause of infection and they were responsible for the spread of HPAIV (H5N1) outbreaks across the world. When wild birds were infected, it was found that mallards, Eurasian pochards, tufted ducks released more virus than common teals, pigeons, and gadwalls; yet only tufted ducks and, to a lesser degree, pochards will die. So some species of wild ducks, particularly mallards, can carry HPAIV (H5N1) for a longer time period and that others, particularly tufted ducks, will act as a mutants. In 2008 Pakistan occurrence of H5N1 on commercial level especially in farms of Sindh (Karachi) was reported

(WHO, 2006).

Review of Literature

Tiensin et al. in 2007 reported the occurrence of different sorts of domestic birds infections in

Thailand which occurred in the year of 2004-05. H5N1 strain of avian influenza viruses were also found in mammalian species and in humans and infections were mainly found in backyard poultry and ducks. In 2004 infection of H5N1 occurred worldwide, especially throughout the

Thailand; most outbreaks occurred in the, the south of the Northern Region, Central Region and the Eastern Region. Later on in 2005, the H5N1 outbreaks persisted and showed a huge collection of pattern in four provinces in the south of Northern Region and in one province in the

Central Region four. Outbreak of this virus caused serious social and economic threats to the social community, the poultry industry, human health, and the farmers' livelihood. After important steps were taken, the incidence of the outbreaks dropped significantly in 2005.

Fig:2.1: Important events in the history of avian influenza of Pakistan

Review of Literature

In any developing country the poultry sector is the most important sector for the better economy and agricultural sector of the country. It produces a large number of employments. Its contribution to agricultural growth is 4.8% and to livestock growth 9.8%. The sector has shown a growth of 8–10% annually which shows its potential (Abbas et al., 2012).But in 2006 the first outbreak of avian influenza H5N1 strains in poultry birds were reported across Pakistan. This was the first time when Pakistani scientist reported the occurrence of avian influenza virus strain

(WHO, 2006). Due to this virus different cities and different areas of the country were affected and it also affects the humans and disease in them. In 2007 different Pakistani scientists reported that H5N1 strain is found in commercial, poultry Of the North West Frontier. Later on, 12

December 2007, report was published by Pakistan that there was more H5N1 occurrence in commercial poultry in North West Frontier and Punjab provinces. After that, in same year

Pakistan informs WHO of 8 people in the NWFP and was tested in laboratory and found positive for H5N1 subtype and these are the first suspected human cases ever reported in Pakistan. In

Pakistan first outbreak of avian influenza H5N1 strain in poultry was reported during 2006. This was the first time when Pakistani scientist reported the occurrence of avian influenza virus strain

(WHO, 2006).

Review of Literature

Fig 2.2 Map showing country distribution of H5N1 hemaglutinin clades reported from poultry and wild bird’s since 1996

Review of Literature

Table 2.1: Major Outbreaks In Different Years

Review of Literature

2. 2: Molecular epidemiology of avian influenza and H5N1 avian influenza

The epidemiology of AIV is very complex involving different free living, captive, feral, domesticated birds as well as different wild and domesticated mammalian hosts. Class Aves contain 29 order, 187 families, 2000 genera spanning over 9600 species, on the basis of difference in anatomy, behavior, ecology and physiology (Gill, 1995). The most common subtypes H3, H4 and H6 isolated from Anseriformes H3, H9, H11 and H13 subtypes from

Charadriiformes (Stallknecht and Shane, 1988; Manvell et al., 2000). But in contrast to LPAI virus, HPAIV have rarely been isolated from free living birds, which were free living (US

Geological Survey 2006). H5N1 HPAIV has been isolated from the birds of strigiformes

(owl),phoenicopteriformes (flamingoes), Falconiformes (falcons, eagles, and hawks), (US

Geological Survey, 2006).

Review of Literature

Fig 2.3 Epidemiological process involved in Asian H5N1 epidemic

Review of Literature

HPAIV of subtype H5N1 spreads across a large region (for example a continent), or even worldwide for example in many Asian and African countries. Genetic analyses suggested that the

4 gene segments of Gs/GD/1/96 found in avian influenza H5 subtype were closely linked to viruses extracted from migratory birds and poultry across Eurasia, birds in Hokkaido, Japan.

According to the studies of different years it was stated that these gene segments had similar time of emergence in poultry in south of China.

Fig 2.4 The emergence of the A/Goose/Guangdong/1/96-like H5N1viruses from the natural gene pool (a) and the interaction between the low pathogenic natural gene pool and the highly pathogenic gene pool in poultry

2.3: Genesis of avian influenza H5N1 subtype:

Genetic analysis helps to alter virus`s virulence and make it possible for it to cross the specie barrier. These changes are likely to occur in an epidemiological setup where there is regular interface between various reservoirs than confined areas. For Influenza A viruses H5N1 subtype,

Review of Literature host range is wide and infects animals including pigs (Scholtissek et al., 1998), humans (Webster et al., 1981b), dogs (Crawford et al., 2005), sea mammals (Webster et al., 1981b), birds and horses (Sovinova et al., 1958). In case of aquatic birds, virus replication occurs in the gastrointestinal tract, resulting in the excretion of high titre of virus in feces. So fecal-oral route transmission helps in spreading of viruses (Webster et al., 1978). But this avian virus generally does not cause disease in their natural hosts. Virus and host exist in a balanced mutual tolerance, explained by non attendance of clinical disease and efficient viral multiplication (Webster et al.,

1978).

Study of each gene segment in AIV has shown 2 lineages, geographically take apart the

American, the Eurasian (Donis et al., 1989), signifying that birds move from Southern to

Northern Hemispheres is very important in the AIV transmission. Influenza viruses in water birds emerge to have reached a finest state of adjustment, in which no selective advantage by amino acid changes (Gorman et al., 1991). This data, along with the actual influenza virus infected water birds hardly show any signs of disease, show that evolutionary equilibrium has been achieved by influenza virus in these birds, which are the natural reservoir. Wild water birds such as orders Charadriiformes (gulls and shorebirds) and Anseriformes (ducks and geese) carrys of the full range of influenza virus A subtypes and are the natural reservoir hosts of AIV. In these species no or little disease is caused by infection because influenza A viruse coexist in perfect equilibrium with the host (Webster et al., 1992, Alexander, 2000).

Fig 2.5 Possible transmission pathway for avian influenza

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2.4: Deaths rate due toH5N1 subtype

H5N1 subtype of avian influenza spread to human beings during 1997 and was first identified in

Guangdong, China, in 1996, and when a considerable deaths in geese were caused then it attains a very little attention. Later on it was discovered that the virus found in goose have the internal gene segments and this was also found in quail (A/Quail/HK/G1/97 [H9N2]). In addition to this it also contains neuraminidase gene segment from a duck virus (A/Teal/HK/W312/97 [H6N1]) as it was the indicator of presence of avian influenza virus and this virus killed 6 of 18 infected persons. Now this influenza virus strain H5N1 have acquired the extraordinary capability to 21

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infect human beings and cause disease that affect the different types of proteins and cause

neurodegenerative diseases with increase in death rate in waterfowl, ferrets and mice in nature

and transmitted among cats and other felid species.(Hye-Ryoung et al ., 2012).

Fig 2.6: Mode of transmission: Various vehicles of infection transfer

Direct Contact Indirect Contact

People’s body, clothes, Infected domestic poultry, shoes goggles, mobile phones etc (Chicken, ducks, turkeys)

Motorbike,

Bicycle, Feed trucks, litter trolleys Infected farm WildBirds

Water fowls, quails, pigeons sparrows, crows etc

Poultry flock Manure, feces

Water ponds

LPAIV circulates in a stable form in wild water birds (Webster et al., 1992) and the rate of

infection depends on faeco-oral transmission routes. This virus can be transmitted directly from

host to host, and also indirectly spread through fomites and water polluted with virus but in case

of mammals (humans, swine, and horses) transmission occur through the aerosols. Virus can be

transmitted from wild birds to domestic birds because they move freely and share water supply

Review of Literature or food with wild birds that is contaminated by oral fecal secretions of carrier wild birds which are infected (Capua et al., 2003; Henzler et al., 2003).

According to one of the studies, it has been observed that AIV subtype H5N1 can be protected or maintained in its original stage in environmental ice for a long time (Li et al., 2004), and these antique viruses and their genotypes might be recycled from this source (Rogers, Starmer, and

Castello, 2004). So H5 and H7 subtypes of avian virus are the basis of infection in poultry and this will develop highly pathogenic biotypes. Illegal trade and transport of infected poultry or exotic birds is another mode of transmission for H5N1. In countries, where the demand for chicken is very high, then in spite of known H5N1 virus this poultry birds are illegally sent to other areas and countries. According to the current studies, transmission to humans can also be done through the children because of their weak immune system and acquired infection more rapidly than older age groups, and it also contribute more in spread of virus. Moreover, children spend enough time in areas where they regularly contact with other people for example, in play grounds and daycare centers, and this transmission is more frequently occur through the close contact. In addition to this human risk for infection of H5N1 has been strongly linked by handling with poultry but not usually with exposure to pigs which are infected (Smith GJ et al.,

2006).

2.5: Live Bird Markets (LBMs)

Live bird markets are a permanent structure in which birds are housed until they are sold in many developing countries. Multiple suppliers of all features of farms make potential source of avian influenza virus (Senne et al., 2003). Studies in Hong Kong, China, Indonesia, USA, showed that

LBMs can be source of AIVs, including HPAI H5N1. It has been associated with also human

Review of Literature infection (Wang et al., 2006, Kung et al., 2007, Cardona et al., 2010). Continuous trafficking of birds into, out of markets provides chance for entry, maintenance and spread of Avian Influenza

Viruses. Majority of the studies focused on testing live birds rather than environmental sites of

LBMs (Cardona et al., 2010). However in a study in New York H7N2 virus was isolated from walls, floors, drain of poultry areas of LBMs (Trock et al., 2008). In another study done in Hong

Kong LBMs showed that H9N2 was isolated in higher rates from poultry drinking water from fecal droppings of birds (Leung et al., 2007).

2.6: Wild Bird Surveillance for the Avian Influenza Virus

Feral birds are considered as a primary reservoir for all known hemagglutinating (H1-16) and neuraminidase (N1-N9) subtypes of AIV (Webster, 1992). Naturally the disease has been reported in free living birds which are represented in more than 90 species of birds in 12 orders

(Hanson, 2002). Majority of these species of birds are linked with aquatic habitats in the order of anseriforms (geese, ducks and swans) and charadriiforms (terns, shorebirds, gulls) are thought to be the most pivotal pool for AI viruses. The birds belonging to these two orders are miscellaneous with a wide variety of habitat types ranging from tiny freshwater marshes to saline water habitats. Prevalence of AI viruses goes to the peak in North American anseriform population occurs in late summer or early fall and is related with premigration state (Hinshaw et al., 1985). This climax is due to marshalling of young susceptible birds throughout the Canada and the north of United States. At this time rates of AI infection can shoot up to more than 30% in juvenile ducks (Hinshaw et al., 1985). Little information is known about influenza virus in the birds belonging to charadriiforms but the data shows that AI virus infection in these species differs temporally and genetically from which was found in anseriforms. This is the unique place in the world where consistent AI virus isolations have been demonstrated; in general prevalence

Review of Literature rate from these species in other places or times either very minimum or zero (Stallknecht and

Shane, 1988). The transmission and carrying of AIV in feral birds are based on oro-fecal route transmission via contaminated water (Hinshaw et al., 1980). AIV primarily replicates in the intestinal tract (Slemons and Easterday, 1977) of ducks and high concentration of the viruses shed in the feces (Webster et al., 1978). In an experiment it showed that Muscovy duck experimentally infected duck shed 6.4 gram of feces per hour which contains a viral titre of

7.8 1X10 mean embryo infectious dose (EID50)/ gram (Webster et al., 1978). Viral shedding in ducks can be longer as it is shown in experimentally infected Pekin duck, which shed virus more than four weeks (Hinshaw et al., 1980). In the year 2002, HPAI H5N1 cause significant mortality in captive and wild aquatic birds in Hong Kong (Kung et al., 2007). Since 2002, HPAI

H5N1 cause mortality in wild birds continued (US Geological Survey, 2006) and the spread of the virus to Europe and Africa presumed that distant range spread of these viruses occurred by migratory birds (Normile, 2006). Avian influenza has occurred on many occasions in the past in this country (Pakistan), but the outbreaks of HPAI H5N1 have occurred repeatedly during 2006,

2007 and 2008.

2.7: Immune response to AIV H5N1 subtype:

During 21st century in 2009, new influenza A/ H1N1 virus of swine origin causing first pandemic in the world were reported. Besides H5N1, H7N7, and H9N2 of avian origin were also reported to transmit sporadically from animal to human being (de Wit et al., 2008). AIV infection is first confronted by innate immune system. Initially viral RNA is recognized by pattern recognition receptors. It includes retinoic acid inducible gene -1 (RIG-1) (Pang and

Iwasaki, 2011). Viral RNA binds with toll like receptors (TLR7) which provoke production of cytokines and type I IFN (Lund et al., 2004). IFN-α, IFN-ß have strong antiviral activity. Viral

Review of Literature replication and protein synthesis are blocked by this interferon (Sato et al., 1998). Role of alveolar macrophage in influenza virus infection: Soon after infection, alveolar macrophage activate and phagocytose (apoptotic) influenza virusinfected cell and confine virus disseminate

(Tumpey et al., 2005; Kim et al., 2008). Once macrophage activated it produces TNF α, nitric oxide synthase 2 (NOS2) and leads to influenza virus mediated damage (Jayasekera et al., 2006;

Lin et al., 2008). Blood derived macrophage infected with influenza H5N1 produce more cytokines in contrast to alveolar macrophages (van Riel et al., 2011).

2.8: Role of Dendritic cells in influenza specific immunity

In influenza virus infections dendritic cells (DC) are regarded as professional antigen presenting cells. The conventional DCs (cDC) are located beneath respiratory epithelium. It can detect and opsonize virion during infection (Hintzen et al., 2006, GeurtsvanKessel and Lambrecht, 2008).

Viral proteins are degraded by dendritic cells and subsequently MHC class I or class II molecules present the epitopes. Viral epitopes are degraded by proteasome and liberated into cytosol. Subsequently it is transported to endoplasmic reticulum along with MHC class molecules. For MHC II presentations viral antigens are degraded by lysosome in endosome

(CD11+ DC). Adaptive Immune System: Influenza virus elicits virus specific antibody (Mancini et al., 2011). Surface viral proteins HA and NA are important as these produce protective immunity. Antibodies against HA prevent the attachment of virus into host cell. They can neutralize the virus. It also facilitates phagocytosis by expressing Fc receptors in the cell.

Antibodies against NA have a potential protective role against AIV infection. Antibodies bind with NA inhibit the spread of virus to the adjacent cell. It also facilitates antibody dependent cell mediated cytotoxicity (Capua and Alexander, 2002). Antibodies against nucleoprotein (NP) may contribute to protect against avian influenza infection. Exact role of antibodies is yet to be

Review of Literature elucidated. It is believed that it can provoke complement associated lysis of infected cell.

Cellular immunity: CD4, CD8 and T regulatory cells are activated by AIV infection. APCs associated with class II MHC molecules present viral peptides to CD4 cells. Infected cells are lysed by some CD4 cells (Soghoian and Streeck, 2010). IL-4, IL-13 are produced by Th2 cells which promotes B cell response (Lamb et al., 1982). IFN-γ, IL-2 are produced by Th1 cells which leads to cellular immune response. Influenza virus infections activate CD8+ cells and more to site of infection. Influenza virus infected cells are recognized there by CD8 cells and inhibit the production of progeny virion. Perforin and granzyme mediate the lysis of infected cells. Permeability of infected cells is increased by perforin and subsequently granzyme leads to apoptosis of the cell (Metkar et al., 2008; Regner et al., 2009).

2.8.1: H9N2 AIV infections in Human Beings

H9N2 is one of the major subtypes of avian influenza virus which is prevailing among domestic poultry since 1990s. Three human infections of H9N2 AIV have been reported in Hong Kong between 1999 and 2003. In a sero survey from 150 blood donors in Hong Kong, three individuals had neutralizing antibodies against H9N2. The capacity of H9 HA surface glycoprotein to bind with the avian and human being receptors highlighting it may lead to influenza pandemic (Peiris et al., 2001). Analysis of 75 human serum samples in USA revealed that 93.3%, 5.3% and 1.3% individuals had <1:10, 1:10 and 1:20 antibody titer against H9 avian influenza virus (Kayali et al., 2008). In China, highest antibodies against H9 were reported in poultry retailers (15.9%) as compared to other worker groups associated with poultry industry (Wang et al., 2009). In

Xinjiang and Liaoning provinces of China, 1.7 and 1%, respectively seropositivity against H9 virus was recorded when sera of poultry workers were analyzed through HI test using horse

RBCs. A sero survey conducted on (n=300) exposed individuals with respiratory signs and

Review of Literature healthy unexposed individuals in Fars province of Iran revealed higher prevalence of H9N2 in exposed individuals i.e. butchers and veterinarians (Hadipour, 2010; Hadipour et al., 2011). A seroprevalence of 85.7% and 30.4% against H9 was recorded for vaccinators and veterinarians, respectively in Punjab province of Pakistan. The higher prevalence rate in vaccinators may be attributed to their longer duration of exposure as compared to veterinarians. By district wise the highest seroprevalence (82.1%) was recorded in Toba Tek Singh whereas the lowest (9.7%) was recorded in Islamabad (Ahad et al., 2013)

2.9: Control Strategies

Hence from the above mentioned studies and observations it is confirmed that AIVs specially

H5N1 subtype cause serious deadly problems in birds and the mammals, so there is an immense need of implementing various control strategies to protect the animal and human health. Some of the important control strategies include public awareness, farm bio-security, vaccination, culling of AI suspected flocks; proper disposal of dead/ moribund birds, avoidance of human/ poultry contact with the sick bird’s, surveillance and tracing of new outbreaks, capacity building in terms of AI control trained manpower, strengthening of laboratory diagnostic facilitates and coordination between various Government agencies and the farmers.The main target of adoption of a vaccination policy is either to control or to prevent the rate of infection in poultry.

Nevertheless, there is a need to improve efforts in order to stop further spread of infection because it will also cause heavy economic losses.

2.10: Economical Affect

Loss in poultry production due to disease causing viruses will lead to the great loss in economy of the country (Barrett et al., 1999). Avian virus caused the deaths of millions of birds that were

Review of Literature infected with the disease. This disease was rapidly increasing among birds and this will lead to the great loss in poultry industry particularly in Asian countries. This loss in poultry industry will lead to the loss of farmers and there will be low economic level (Dolberg, et al.,

2005).Generally, the greatest losses have occurred during epizootics of HPAI in commercially reared poultry under intensive production systems. There was a huge loss due to these outbreaks in the form of high morbidity and mortality losses, depopulation and disposal costs, indemnities paid for elimination of marketable birds and quarantine and surveillance costs.In U.S, during the year 1992-1995, there was a great loss of $1 million ($10 million adjusted to 2001) due to the

HPAI outbreaks.

Thailand is high poultry-producing country in the area with a considerable amount of worldwide trade system. According to Oxford Economic Forecasting, in 2004, Thailand, the total

GDP loss due to economic losses in poultry farms prior to the HPAI outbreaks was US $1.2 billion. Ban on export of poultry farms have damaged the industry, suppliers, feed mills farm workers, hatcheries, and traders. Furthermore, it also affects the humans and causesmuch different type of diseases and deaths.

In Pakistan a few poultry farms in the provinces of NWFP and Punjab were diagnosed as infected with AIV H5N1 virus, and as a consequence to infection confirmation there were high morbidity and mortality in the infected flocks. All the housed birds at the affected farms were killed and disposed of under the advice of state veterinarians. The infection and depopulation caused heavy economic losses to farmers of commercial and breeding flocks. There were additional losses on cost of medication, isolation, quarantine, disinfection etc. The panic of bird flu had its greatest economic impacts on the farm workers and farm owners (some going out of business due to loss of their total investments) as the public panic arose and it stopped to use

Review of Literature poultry and its products. This situation caused the closure of over 70 percent of commercial broiler and layer farms in the provinces of NWFP and Punjab. Many of the breeding flocks although not infected with H5N1 flu virus, had to be culled due to economic pressure as the sale of progeny chicks dropped to its minimum level and the price of day old chicks was also the lowest in the past about 5 years. However, the public consumer of chicken meat and eggs also paid the price of Flu panic. After regaining the trust of consumer after reports on the absence of bird flu, it has become difficult to find poultry and its products on reasonable price, the formula of demand and supply affecting the economics of households – due to short supply of chicken and eggs in the market presently the poultry and eggs are being sold at very high price for the last over 4 months (Live bird at over R. 90/ a kg as against about Rs. 50-60 a kg in routine normal situation). All this calls for an effective AI control strategy through the cooperation of farmers, diagnostician, epidemiologists, public health and planning bodies, government etc. This is also important in the context of world trade of poultry as any threats to the human/animal health lead to economic impacts on poultry producers and consumers. In addition to above economic stresses to the farmer, labor working at the affected farm also faced embarrassment of continuity of employment, layoffs, and chances of getting infected with AI.

2.11: Biosecurity

Biosecurity is a way of avoiding contact between microbes and its various hosts.

Operational biosecurity does not cost too much, however its practice helps in maintaining the healthy flocks at farms. Biosecurity measures can be used in commercial production units and also on rural backyard or very small-scale farms production units. For small-scale production units, biosecurity consists of various measures mainly aimed at keeping the microbes and deadly pathogens away from the farm premises and flocks. It is important that the virus may get entry in

Review of Literature a poultry farm through by following practices; arrival of one or more domestic birds, even if those are apparently healthy, human beings (family members or relatives, visitors, “paravets”, vaccination crews, middlemen, people delivering medicines, feeds or those collecting litter) reaching the farm after been on some other farm, in a LBM, at a slaughterhouse, in a laboratory.

That had previously been infected or contaminated. All the poultry dealing personnel’s may carry the virus on their, shoes, boots, clothes, vehicles, on the egg crates, cages. Introduction of other animals housed at a farm with infected birds. Dogs fetching dead birds from other diseased farms. Wild birds when migrate from one region to the other may pollute the farm through contact with the domestic poultry or through their infected feces dropped on the ground or in water bodies. The ducks going and coming back from rice fields and any domestic poultry that must find food outside the farm, having contact with the contaminated water ponds or with the infected manure may also carry the pathogens. These principles may however be difficult to follow in these cases and in such situations efforts be made to keep such birds separated from other type of poultry. Biosecurity principles are also helpful in rural areas. At this level, rural development agencies can be instrumental in enhancing the benefits of keeping birds in a fenced farms where external factors are minimized, birds are safer from the scavengers. Restricting access to a poultry farm by using special and large fences and enclosures helps to prevent exposure of virus to the other clean farm areas and the outside environment. Access to a poultry farm is restricted to people known by the farmer, people who do not own birds, and do not take part in events where birds gather.

2.11.1: Preventive measures

Keep the farm area clean from waste. When the owner or worker needs to be present at to chickens e.g. collecting eggs, change the bedding litter, feeding or watering chores or the repair

Review of Literature of fencing material, a change of dress and shoes must be practiced. Dirty clothes used by the farm keepers should be washed with properly and dried in the sun. Washing of hands are washed with soap before entering the caged area must be practiced. Tools like shovels, feeding scoops, brooms and feeding pans used in the caged areas should be kept clean and disinfected. All biological wastes should be properly discarded. Most preventive and a good biosecurity practice is to always wash with chlorinated and detergent added water before entering or leaving the enclosure. Sick and dead chickens should be discarded and community health officials and veterinarian be informed of illness or mortality at the poultry farms or in individual cages of the birds. Transportation of live birds to the farm can be a significant risk – not only the farmer should be aware of the better price obtained, but also that vehicles, equipment, cages and feed may be contaminated when returning or entering the farm. Newly obtained equipment must be disinfected prior to use. Newly-purchased birds should be housed separately for at least two weeks, monitored for the presence of any pathogens or their antibodies, and vaccinated/ treated with appropriate medicine under the advice of an avian veterinarian before permitting them to mingle with birds already present in the farm. Metal cages can be easily cleaned, washed and disinfected. The construction of poultry sheds is very crucial for the biosecurity practices. The farm buildings are washable, air controlled, vermin proof and must have adjustable and cleanable feed and water supply systems.

2.12: Vaccines and Vaccination

2.12.1: Vaccine considerations

Vaccination, against various virus entities induces resistance in vaccinates against infection / disease caused by those viruses, eliminating the possibility of recurrence of infection

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(Martin et al., 2006). However, considering economic and ethical concerns over depopulation and culling of millions of infected birds especially in case of LPAI vaccination has been used as an important preventive tool in poultry against various AIVs. Strategic vaccination against H5N1 virus of poultry is being practiced in Pakistan. Uncontrolled LPAI virus may allow the emergence of HPAI virus types as has been observed in USA (Howse et al., 1999, Leslie et al.,

1999, Henson et al., 2000). There exists a difference of opinion on the use of AIV vaccine in birds as a tool to control the disease. It may be noted that in the field of veterinary medicine vaccination is used for obtaining the following goals:

(i) Prevention of clinical disease

(ii) Protection from infection with virulent virus

(iii) Protection from virus excretion in the flock environments

Neither commercially available nor experimentally tested influenza vaccines have been shown to meet all the requirements of successful immunization. Despite all concerns over their efficacy, scientific facts are that inactivated vaccines lead to reduced flock’s susceptibility to AIV infection, and to reduced quantity of virus shed post-challenge, transmission and disease losses.

The regulations preventing vaccination against H5 or H7 LPAI have had the effect of promoting circulation of LPAI virus in the commercial poultry and also in the live poultry markets which occasionally lead to emergence of HPAI viruses (Henson et al., 2000). Killed, virus vaccines against influenza have been used successfully in a variety of avian species. Since the birds are susceptible to all 16 HA subtypes of influenza viruses, preventive vaccination prior to an outbreak is not of any practical use. However, when a subtype is identified in a poultry flock and the biosecurity measures appear to be inadequate in that event controlled vaccination is an effective tool to reduce the susceptibility of poultry and to help bring the infectious outbreak

Review of Literature under control. Sturm et al. 2005 reported that vaccination of chickens with a commercially available killed H5N2 product has been evaluated as an additional tool to enhanced biosecurity measures and intensive surveillance for control of HPAI subtype H5N1 disease in Hong Kong in

2002.

2.12.2: Mortality due to H5N1:

In December 2002 to January 2003, H5N1 disease was found in water fowl in two recreational parks, wild water birds, five chicken farms and at several poultry markets. In at least two farms, infection spread to the recently vaccinated sheds with low rate mortality due to H5N1 in the chickens between 9 and 18 days post-vaccination (PV). After this intensive monitoring by virus culture on these farms there was no such evidence of asymptomatic shedding of the virus, and it shows that this vaccine can interrupt virus transmission under field conditions (Normile,

2006).Lee et al. 2004 observed that the AIV vaccination does not always prevent infection; the clear benefit of vaccination is in its ability to prevent disease and to reduce the amount of virus circulation in the environment. Thus, use of vaccination is important component of control program used for the H5 subtype. However, the long-term use of vaccination without other disease prevention and eradication measures may lead to the selection of the antigenically divergent strains, which could compromise the value of vaccination. Currently vaccination is an important tool of control programs and can be called second line of defense (Cox et al., 1998).

AIV vaccination against H7N3 and H9N2 viruses has been successfully used in poultry flocks in

Pakistan.

2.12.3: Humans

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De la Barrera and Reyes-Tera´n (2005) reported that the vaccines are universally regarded as the most important medical intervention for preventing influenza and reducing its health consequences during a pandemic. In the past, however, vaccines were never available early enough and in sufficient quantities to have an impact on morbidity and mortality in humans during pandemics. There are two approaches for the prevention of influenza:

The live, attenuated, cold-adapted and intranasally administered genes from the target AI virus, with the alteration init’s HA connecting peptide if necessary and transfecting an appropriate cell line (Webster etal., 1978). The next step is to take these plasmid-derived influenza virus vaccines through clinical trials to address crucial questions such as number and quantity of doses and the role of added adjuvants. Trivalent inactivated (TIV) vaccines are produced from the viruses grown in embryonated hen eggs and are of three types: whole-virus,

“split-product”, or subunit “surface-antigen” formulations. Trivalent vaccines contain 15 mg each of two A subtypes (H1N1 and H3N2) and one B strain. TIV obtained relatively specific strain response of humoral, with a reduced efficacy against antigenically drifted viruses, and are ineffective against unrelated virus strains. The influenza A virus components of annual influenza vaccines are typically derived from the egg grown reassortant viruses that have the relevant HA and NA genes of the antigenically relevant strain, and the six remaining gene segments from

A/Puerto Rico/8/34 (H1N1). Vaccine production against these disease causing avian influenza viruses is complex because of the requirement for high biosafety containment facilities. This process requires large number of eggs and many companies lack the flexibility to respond rapidly to a pandemic event. In addition, highly pathogenic H5 and H7 viruses cannot be grown in large quantities because they are lethal to the chicken embryos.

2.12.4: Use of AIV vaccines

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OIE recommend the use of OIE-approved HPAI vaccines, and several such vaccines are now commercially available. If used as per FAO/OIE recommendations, these vaccines provide excellent protection against clinical disease in chickens by reducing the bird mortality and production losses. Vaccination of poultry also reduces the virus pool contaminating the environment and thereby the risk of infection to poultry and humans. HPAI-vaccinated birds are not debarred from the trade, although precise measures must be practiced to make sure that the vaccine is being applied properly and its effect is monitored effectively. The vaccination strategy aims at promoting production capacity in quality control of vaccine at the national level, and it will ensure that vaccines are applied with a specific objective linked to post-vaccination monitoring and surveillance. The strategy will also toil with the private sector to ensure that sufficient vaccines are available before launching a vaccination program. Currently, Peoples

Republic of China, Vietnam, Pakistan and Indonesia are the only countries using vaccination as part of their HPAI control strategy. Without the application of monitoring systems, strict biosecurity and depopulation in the face of infection, there is the possibility that these viruses could become endemic in the vaccinated flocks. Long-term circulation of the virus in a vaccinated population may result in antigenic and genetic changes in the virus as has been observed in Mexico. Live conventional influenza vaccines against any subtype and any form are not recommended in any of the hosts.

2.13: Antiviral Therapy

Antiviral therapy against influenza viruses is aimed at disruption of itsreplication and production of progeny virion for which antiviral agents such as Oseltamivir(Tamiflu),Ribavarin,

Amantadine, etc are used. Ludwig et al., (1998) observed that the clinically approved anti- influenza drugs available now a days are aimed against essential viral protein functions.

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Adamantane compounds, such as rimantadine and amantadine, have a type-specific inhibitory effect on type-A, but not on type-B influenza viruse. These agents block the activity of ion- channel of the viral M2 protein, which is mainly required during virus entry in the initial phase of the replication. However, amantadine-sensitive influenza A virus strains rapidly develop resistance in-vitro and in-vivo (Galbraith et al., 1969). Two products of modern drug design,

Oseltamivir and Zanamivir, block the receptor-destroying activity of the viral NA protein, thereby preventing release of virus release from the cell membrane. Both the drugs efficiently inhibit influenza virus A and B strains in clinical trials and are recognized and being marketed commercially in many countries. However, escape from the selective pressure of NA inhibitors has been seen in cell cultures and patients, as a result of virus mutational changes although on a quite smaller scale than that observed with adamantane compounds. Ongoing studies are attempting to find new anti-influenza drugs that target the virus replication cycle at its various stages.However, so far none has been approved for its clinical use. All these approaches have the major disadvantage that they attack a viral function and/or structure. Hence, even though strongly conserved elements will not change easily, the virus will eventually adapt to and escapes from the selective pressure exerted by the antiviral therapy. Rawlinson (2001) observed that development of new, effective drugs and modification and potentiation of pre-existing drugs or regimens is involved in antiviral therapy. The neuraminidase inhibitors: zanamivir and oseltamivir are new agents against influenza virus which attack specific viral processes and have least side effects on the host functionality. Galbraithet al., 1969 reported that the influenza neuraminidase enzyme is alluring target for antiviral intervention. Its active site is protected in all strains and is pivotal to viral replication. They suggested that the concept of neuraminidase

Review of Literature inhibition such as of the oral agent R0640796 (GS4104) is most probable to lead to a major breakthrough in controlling of influenza virus infection (Garman et al., 2004)

Table 2.2 Inhibitors of influenza virus

Inhibitor Effect on viral replication Action

Sialic-acid analogs Attachment of the virion to cell Block the receptor-binding

surface receptors pocket of HA

Quinone- and Acquirement of the fusion-active Inhibit the low-pH-dependent hydroquinone-derivatives conformation of HA conformational change of HA

Anionic polymers Insertion of the fusion peptide Prevent the hydrophobic

into the cell membrane membrane attachment

M2 inhibitors (amantadine Early release of the viral genome Inhibit early H+ influx into the and rimantadine) into the cytoplasm and late virion and late H+ efflux from

induction of HA fusion transport vesicles

conformation

Inhibitors of the viral Transcription of viral mRNAs Inhibit the endonucleolytic(‘cap-

RNA-polymerase subunit, snatching’)

PB2 activity of PB2

Neuraminidase inhibitors Detachment of the virion from Inhibit the cleavage of the

Review of Literature zanamivir, oseltamivir the cell glycosidic bond of terminal

(Tamiflu) sialic-acid residues on receptor

determinants

Synthetic capped RNA Transcription and replication Inhibit viral polymerase activity and

phosphorothioate oligonucleotides

Inhibitors of host-cell Endocytosis Block the PKC activity required functions Protein kinase C for a late endosomal process (in

(PKC) inhibitors (e.g. particular the PKCβII isoform) bisindolylmaleimide)

Protease inhibitors Processing of the HA0 precursor Inhibit proteolytic cleavage of

(exogenous/endogenous) into HA1 and HA2 molecules HA

H+-ATPase inhibitors pH-dependent activation of the Inhibit the acidification of

HA fusion activity endocytotic vesicles

Ribavirin Transcription of viral mRNAs Inhibits cellular synthesis of the

7mGPPPcap of mRNAs

ERK = extracellular-signal-regulated kinase

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MAPK= mitogen-activated protein kinase.

During the year 2001 orally given NA inhibitor RWJ-270201 was tested in parallel with oseltamivir and zanamivir against a panel of AIVs to inhibit its NA activity and replication in tissue cultures. The agents were then checked for mice safety against lethal H5N1 and H9N2 virus infection. In-vitro, RWJ-270201 was observed very successful against all NA subtypes. NA inhibition by RWJ-270201 (50% inhibitory concentration, 0.9 to 4.3 nM) was better than that by oseltamivir and zanamivir carboxylate. RWJ-270201 obstructed the replication of both Eurasian and American lineage AIVs in MDCK cells. Mice given 10 mg of RWJ-270201 per kg of body weight per day were completely safe from lethal threat from influenza A/Hong Kong/156/97

(H5N1) and A/quail/Hong Kong/G1/97 (H9N2) viruses. Both RWJ-270201 and oseltamivir had substantially declined virus concentrations in the mouse lungs at daily dosage levels of 1.0 and

10 mg/kg and provided safety barrier from future viral spread to the brain. At the start of the treatment, 48 hr after exposure to H5N1 virus, 10 mg of RWJ-270201/kg/day reduced 50% mortality in mice. These findings indicated that RWJ-270201 is almost as effective as either zanamivir or oseltamivir against AIVs.

Natalia et al. 2005 reported the divergence of amantadine-resistant mutants among AI A viruses with sporadic possibilities (H5, H6, H7, and H9 HA subtypes). Drug-resistant variants were not found among 1979–83 isolates, whereas 31.1% of H5 and 10.6% of H9 strains from South-east

Asia isolated in 2000–04 carried mutations in M2 protein. In Northern America, resistant variants appeared among H7 viruses, out of which 16.4% were tested. H6 viruses were sensitive to amantadine. These recordings instigated concerns about the control of sporadic influenza, also worries that forthcoming pandemic virus will be resistant to amantadine and the urge to

Review of Literature supervise the drug usage in poultry. To prospectively detect amantadine-resistant influenza when amantadine was given for influenza A outbreak control. The design used was proposed clinical examination and virus culture of all new respiratory illnesses during the span of amantadine prophylaxis deliverance. Residents of a veteran's hospital and their spouses acted as participants.

Nasopharyngeal and throat virus culture was used for virus isolation. All doctors with positive result developed respiratory symptoms while receiving amantadine prophylaxis. Schilling et al.

(2004) reported that the amantadine prophylaxis was delivered back to back in 9 of 14 wards to all healthy residents for about 14 to 31 days in order to control influenza epidemics during

December 9, 1993, to January 28, 1994. Amantadine treatment was simultaneously given to 29 patients. Between December 3, 1993, and January 22, 1994, 68 culture-positive cases of influenza A were detected. 20 subjects were getting amantadine prophylaxis therapy.

Amantadine effectiveness was tested on 16 residents, out of which 12 residents had amantadine resistant strains. Four of the 12 were not delivered any antiviral treatment. Commencement of illness ranged from 1 to 22 days after amantadine prophylaxis was started to be given as treatment. Two ribonucleic acid (RNA) mutations in the gene coding the M2 protein transmembrane region were observed. Isolates of strains from two roommates, one on amantadine treatment for 18 days and had no antiviral delivered had same RNA sequences.In a study Ward et al. (2005) reported recent inter-species transmission of AI has the menance of pandemic influenza in the lime light. Oseltamivir (Tamiflu) was observed to be successful in the treatment and prevention of epidemic influenza in adults, adolescents and the children (>1 year), albeit oseltamivir is not approved for prophylactic use in children, but is known to be effective.

Oseltamivir is also active against AIV strains. It is indicated evidently that small doses or shorter runs of chemoprophylaxis other than those approved may not be successful and may confer

Review of Literature emergence of viral resistance. Study showed that 5-day courses of 150 mg doses daily two times and for 6 weeks 75 mg of dose twice daily for prophylatic measure were well put up with as the approved dose regimens. To develop use of antiviral medications, actions are required to collect, examine and report outcome from use for chemoprophylaxis of pandemic influenza during the first-wave outbreaks.

Whitley et al. (2001) determined the performance, safety and sustainability of oseltamivir in kids with influenza. In this randomized, double blind, placebo-controlled study, children up to 12 years with fever (>=100 oF, 38 Co) and a past of cough or coryza for 48 hr duration either got delivered with oseltamivir 2 mg/kg/dose or placebo dose twice daily for 5 days. Oseltamivir therapy was broadly well put up with, albeit linked with an excess frequency of emesis (5.8%).

Anonymous (2006) reported that amantadine and the neuraminidase inhibitors: zanamivir and oseltamivir respectively are recently accepted in France for to avoid or treat influenza.

Performance of antiviral drugs has not been studied in comparative randomised trials in which complexities of death and influenza were elementary outcome measures. A review of 20 comparative random trials involving about 2500 healthy individuals showed that amantadine decreased the frequency of flu-like syndromes by about 7% in absolute terms (26.3% versus

33.1% with placebo). Oseltamivir and Zanamivir have only revealed to decline the density of serologically affirmitive episodes of influenza. In a random placebo mediated experiment of oseltamivir, involving 548 institutionalized subjects more than 65 years of age, over 80% of whom were vaccinated, respiratory tract infections were less common in the oseltamivir group.

Efficacy of antiviral drugs on avian influenza / bird flu was analyzed during a 2003 Dutch pandemic due to a type A/H7N7 virus. Among the 38 exposed and treated people, about 3% developed symptoms, as compare to 10% of 52 exposed persons who reprimanded treatment.

Review of Literature

The low statistical power and the lack of randomization rule out any firm conclusions on preventive effects. The 3 antiviral drugs have different figures of detrimental effects and drug interactions. Amantadine bears a risk of, atropinic, neuropsychological and dopaminergic harmful effects, and can communicate with drugs that have similar effects. Zanamivir brings danger of life-threatening bronchospasm. Oseltamivir was accepted comparatively recently and its full presence of detrimental effects is not yet known; mainly there appears to be mild gastrointestinal disturbances.

2.14: Public Awareness

After going through all the available information on the virus, its potential to mutate, presence of multiple types, capability to infect various types of avian and mammals including humans, the non-availability of the stable vaccine against the contagium and its potential for the high morbidity and mortality in man and animals; it is important to take steps for the public awareness on this disease in view of diverting panic in the masses. It may be remembered that the zoonotic and deadly occurrence of disease in poultry flocks are the two major problems, which lead to panic in public upon occurrence of AIV infection.The following strategies can be practiced for making public aware of the facts on Avian influenza /Bird flu:

1. A chapter on the important zoonotic diseases like avian influenza be made part of the

curriculum at secondary and higher secondary levels to make the public aware about the

behavior of those diseases in the population. Public at large be educated on the

importance of public health and hygiene, use of antiseptics and sanitation at homes, all

such education be aimed at prevention of infectious diseases.

2. Consumer education on the purchase of good raw meat and eggs and its handling and

cooking is very helpful in disease control.

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3. The poultry framers are educated on the importance of culling and killing of the infected

flock rather than marketing of such problematic flocks.

4. Programs highlighting the importance of various infectious communicable diseases be

prepared for the education of masses and media on various disease issues be mobilized in

the interest of public awareness. For this purpose the scientists, professionals, farming

community and bio-medical personnel’s be engaged. News and print media should

disseminate information on infectious diseases using articles from renowned

professionals, veterinarians and medical specialists. Similarly, booklets on bird flu and

other zoonotic diseases be prepared and distributed in schools and various public

institutions for the benefit of people.

5. Movies on the signs of bird flu in chickens, isolation and quarantine of sick birds, culling

and disposal process, disinfection of farms premises etc be prepared and shown to people

linked with poultry production and its trade (Kobal et al., 1998)

2.14.1: Public handling of wild birds

As guideline, the public should avoid to handle live or dead wild birds. If anyone observes wild birds showing illness or finds dead birds, local or provincial wildlife and public health authorities should be informed. If contact with wild birds is not avoidable, gloves and doubled plastic bag be used and contact with blood, fluids and feces be avoided. Hands be thoroughly washed with soap and warm water after each such contact.

Safe food handling practices be adopted. Game meat be cooked thoroughly to an internal temperature of approximately 71ºC (160 º F). The transmission of AIV to people from eating undercooked eggs, uncooked poultry or eggs is unlikely. However, proper safe handling practices such as hand washing and keeping egg and poultry products separate from other food

Review of Literature products to avoid cross contamination should be followed. Contaminated surfaces on work places and tools be cleaned thoroughly with warm, soapy water and some household disinfectant be used. Hands be washed for minimum 20 seconds before and after handling raw meat, poultry, seafood or eggs.

2.14.2 Instruction for hunters

Handling or catching sick birds that have died from unknown causes be avoided. Direct contact with blood, respiratory secretions feces, and of all wild birds should be avoided. Eating, smoking or drinking should be avoided while cleaning game. latex gloves or dish gloves or must be worn when handling or cleaning. Gloves, clothing and hands washed with soap and warm water immediately after handling slaughtered birds/ meat. Contaminated surfaces may be thoroughly cleaned (tools and work surfaces) with hot, soapy water and the area disinfected using a household disinfectant. Never use contaminated things but if there are signs of illness after handling birds the health physician must immediately be consulted and informed about contact with the wild birds/ meat, fecal material etc.

2.14.3 Preparing for an Outbreak Preparedness and planning for any expected outbreak for minimizing its adverse are necessary for continued poultry development. This planning should include mechanisms like how to rapidly recognise an illness, initiate, diagnose and plan and implement various control and future preventive strategies. Preparedness requires coordination and evaluation of veterinary service potentials, legal procedure in which these services operate, and the cooperation from farming community. The structure and scope of poultry industry must be observed to determine the factors, which allow entry of virus, its precipitation and spread. An effective public awareness program is critical to muster support for various disease control activities and for avoiding the risk of zoonotic infections.

Review of Literature

Increased surveillance and detection capabilities and preparedness to deal with influenza panic are basic requirements. Public awareness, education and training of veterinarians and para- professionals, state agencies marketers, farmers, poultry transport contractors and egg collectors is necessary to ensure that the infection is either prevented or detected and controlled, in any newly colonized ecosystem (anonymous, 2006).When the migratory avian are thought as risk factors, it is mandatory to identify the migratory habits of different species, their origin, destinations and timing of migration. While disease in wild birds is usually not expected, it is of value to keep wildlife authorities alert and ask them to report unusual deaths in the wildlife.

It is recommended that countries conduct studies for risk assessment of the introduction of control system in order to define the suitable surveillance strategy. In any event, each country will have particular preferences and surveillance systems, e.g. those countries with a less risk factor of becoming effected will seek reach to detailed, up to date information on risks and will focus on the detection of incursions making early warning and surveillance as their priority.

Public awareness campaigns should be undertaken in such a manner that it does not cause undue concerns in the human population. In case of disease suspicion, a sample of all domestic species of bird that die in the restricted area should be investigated and specimens be submitted to laboratories for virus isolation, characterization and reporting. Field surveillance examinations are needed to detect changes in flock health and its growth and production performances.

Trainees should be aware of the potential risk to health of human and should be taken preventive measures like protective goggles or face shield, gloves, mask, rubber gum boots disposable gowns or coveralls, while entering or coming out of the infected farm premises.

A worldwide strategy to prevent spread of H5N1 has been planned by international organizations like WHO and FAO. Core reasons for such strategy are:

Review of Literature

1- HPAI is a worldwide, rapidly spreading highly infectious disease

2-It is zoonotic and transboundary disease.

3- It has emerged and spread because of consequence of globalized poultry trade.

4- It spreads quickly by the migratory birds and flyways and in resting and nesting areas.

5- Influence on the livelihoods of millions of people, especially the poultry farm owners, workers and professional and para-professionals.

6- It harms trade and places the international poultry industry at risk.

7- HPAI viruses are of particular concern as they are very labile and undergo constant genetic changes leading to mutations and gene reassortment, resulting in antigenic shifts.

For poor households depending for their livelihood on poultry, HPAI has caused the loss of income and food security. Important areas of focus at national level, in countries like Pakistan, are outlined below:

 Strengthening the legal and institutional structures to create an conducive environment to

support various control measures for various virus infectious diseases.

 Preventing the entry of HPAI in areas are free from this deadly disease.

 Providing socio-economic impact evaluation on disease control strategies and, when

required, provide an objective assessment of the impacts on different stakeholders.

 Strengthening relation between technical and planning authorities and between

departments of agriculture, livestock, human health, finance and planning for improving

capacity to respond to emergencies, which may arise in the event of diseases like HPAI.

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Notwithstanding the importance of these efforts to be ready for a possible H5N1 pandemic, more structural and long term international efforts are needed to allow for early detection of new influenza viruses or other promising pathogens causing disease in humans. In 2002, WHO

Global Agenda for Influenza Surveillance and Control was adopted which was aimed at strengthening surveillance, improving the knowledge of disease burden, increasing the vaccine use, and accelerating pandemic preparedness (Stohr, 2003). Many South-east Asian countries lack the skill, financial capital, and infrastructure for human and animal disease diagnosis and surveillance. International investments for improving public health care infrastructures and laboratory amenities, and to relocate clinical, epidemiological, and scientific knowledge to these countries are very much needed (Hien et al., 2004). The margin of opportunity in the era of international travel is narrow. Building of local capacity, and less belief on foreign laboratories and expertise, will allow for earlier detection and faster responses to various epidemics such as

AI. Additionally, availability of local clinical, scientific, and laboratory facilitates and expedites in clinical, virological, and epidemiological analyses needed to optimize outbreak control, infection control, and clinical cases management. It also guarantees the apt availability of virus strains to monitor virus evolution and development of vaccines by reference laboratories. Such global investments to improve local infrastructure and expertise will boost the chances of success of containing an influenza pandemic at the source by antiviral prophylaxis and other preventive measures suggested by recent mathematical modeling studies (Galbraith et al., 1969, Welliver et al., 2001)

2.14.4: The case of Pakistan

As a result of an outbreak in Pakistan, following control measures were implemented:

Review of Literature a. Quarantine and isolation of affected farms was practiced. Culling of all birds at farms

tested positive for H5N1 infection under the supervision and instructions of veterinarians

and state authorities was undertaken. b. Culling and disposal of infected flocks was undertaken in a way to avoid contamination

of nearby premises while the bird carcasses were deep buried in soil under the cover of

biosecurity practice. c. The infected farm premises were thoroughly cleaned; the shed corners walls, inside roofs

and other contaminated areas were washed with disinfectant solution and fumigated with

formalin and potassium permanganate. No traffic or visits were allowed in such cleaned

farm premises. d. Strategic vaccination (H5N8) and autogenous isolate H5N1 inactivated vaccines prepared

by local manufacturers was practiced in 15-20 km radius area of the infected farm and in

those areas birds of all kind; breeder, layer and broilers were vaccinated. e. Farmers were advised to implement strict vaccination policy against H7N3, H9N2 and

H5N1 AI viruses. f. This vaccination policy has yielded quite encouraging results as no further H5N1 positive

flocks were observed in Punjab up to October 2006. No human H5N1 positive case was

reported.

Pakistan has to be a part of global and regional strategy for controlling the further spread of this transboundary disease. Training of paramedical, paraveterinarian and public health engineers, the environment protection personnel along with wildlife personnel on prevention and containing of AIV infection is direly needed. Continuous monitoring of the wildlife, commercial and domestic poultry are a basic requirement. Since Pakistan has common borders with India,

Review of Literature

Iran, Afghanistan, China and Russia, it is necessary to devise a regional control strategy in collaboration with these countries. For the time being the AIV/BF is well under control.

However, there is no warranty that the H5N1 virus will not strike again. To deal with situation, continuous professional vigilance and prevention plans are needed.

In summary, the AI control strategies must focus on public awareness, improvement of practice of biosecurity, surveillance for the pathogen and new outbreaks, culling of infected flocks, compensation to the farmers to alleviate their economic stress, vaccine development and monitoring seroconversion following vaccination, and restocking of farms only after proper disinfection.

CHAPTER 3

MATERIALS AND METHODS

Study Area

Being a high density poultry farm region and central live bird market, Islamabad capital territory

(ICT) of Pakistan was selected as study area. It is situated between the province of Khyber

Pakhtoon Khwa (KPK) and Punjab at latitude (33.49)-north and longitude (72.24)-east with altitudes ranging from 457-610 meters. It covers an area of 1165.5 km2 of which 906 km2 is

Islamabad proper. The area is administratively divided into rural and urban segments. Division of rural area was into (12) union councils which were further subdivided into 140 jurisdictions/villages. The name of each union council with number of its respective jurisdictions is mentioned below.

Table 3.1. Data of Administrative Division of Study Area.

Serial # Name of Union Council Number of

Jurisdictions/villages

1. Sihala 20

2. Rawat 14

3. Tarlai Kalan 10

4. Koral 21

5. Charah 03

6. Kirpa 06

51 Materials and Methods

7. Phulgran 16

8. Tumair 11

9. Kuri 10

10. Bhara Kahu 12

11. Shah Allah Ditta 05

12. Sohan 12

Total 12 140

The total population of the area is 950000 of which 66% is urban. Humidity was 55% with an rainfall of 1450mm per year. The minimum and maximum average temperatures are 110C &

290C respectively. Being reminiscent of tropical climate, the area retains mild winters without any snow fall. The total number of broiler, breeder and layer farms in rural area with capacity of birds is given below.

Table 3.2. Census of Poultry Birds in Study Area

Serial # Type of # of Capacity of Birds Total # of Birds

Farm Farms

1. Broiler 100 3000 – 30000 300000 – 1000000

2. Breeder 20 3000 – 30000 600000 – 600000

3. Layer 50 3000 – 30000 150000 – 1500000

Total 170 1050000 – 3100000

Materials and Methods

The birds in above mentioned farms and live bird markets of the area (ICT) constituted the study population for this project. The study was carried out from 2010-2011 under following objectives.

1. Trends and patterns of (HPAIV) H5N1-outbreaks through surveillance.

2. Risk factors associated with (HPAIV) H5N1 outbreaks quantification.

3. Surveillance of live bird markets for detection of (HPAIV) H5N1.

4. TRENDS AND PATTERNS OF H5N1 (HPAIV) STRAIN OUTBREAKS

THROUGH SURVEILLANCE:

Study Design:

A cross-sectional survey was conducted in ICT from April 2006 to june 2008 in order to

assess the trends and patterns of HPAIV H5N1 outbreaks occurred during the period of

2006-2008. We used and analyzed the data collected through an active disease

surveillance by NRLPD, PRI and livestock department of ICT.

Case Definition

Higher incidence of H5N1 virus isolation from the affected farm confirmed by laboratory

diagnosis (Thursfield 2005).

Target Population

Birds in all commercial poultry farms (broiler, breeder, layer) of Pakistan constituted the

target population.

Study Population

All broilers (1000000); breeders (600000) and layers (1500000) in 170 commercial

poultry farms of ICT were selected as study population.

Sampling

Materials and Methods

Selection of sample was carried out by multistage sampling.

First Stage or Primary Sampling

All (12) union councils of ICT were taken as sampling frame whereas one union council

was taken as sampling unit.

Second Stage or Secondary Sampling

All (43) villages/jurisdictions of 4 selected union councils were taken as sampling frame

whereas one village was taken as sampling unit.

Third Stage or Tertiary Sampling

All birds (187520) in 35 commercial poultry farms (broiler, breeder, layer) of 8 selected

villages were taken as sampling frame whereas one bird was taken as sampling unit.

Sample Size

Sample size was calculated according to recommendations of Thrusfield (2005).

2 1.96 푃 푒푥푝푒푐푡푒푑 (1 − 푃 푒푥푝푒푐푡푒푑)⁄ 푛 = 푑2 n = sample size required

Pexpected = prevalence expected d = Required absolute precision

By specifying expected prevalence and desired absolute precision as 5% and 5%, the estimated/ desired sample size was n = 73

Randomization

Out of total 12 union councils, 4 union councils including Kuri, Tarlai Kalan, Sihala and Chirah were randomly selected. From these four union councils, a total of 8 villages (2 from each union 54

Materials and Methods council) including Chak Shahzad, Kuri, Ara, Cherah, Dhok Kazim, Tarlai Kalan, Jandala and Sihala were randomly selected for present study.

Data Collection

Required information about birds (broilers, layers & breeders) of all commercial poultry farms was recorded in Performa designed for present study. One Performa was filled for one form. The data about following variables were recorded.

 Date of outbreak

 Location of farm

 Type of farm

 Capacity of farm

 Type of birds

 Total number of birds

 Age of birds

 Number of infected birds

 Number of culled birds

 History of vaccination

 Management system of farm

Statistical Analysis

The data regarding above variables were pooled in data entry sheet and analyzed for morbidity, mortality and case fatality rates separately for broilers, breeders and layers following the

Materials and Methods recommendations of Thrusfield (2005).descriptive statistics was evaluated using SPSS 20.0 version.

QUANTIFICATION OF RISK FACTORS ASSOCIATED WITH HIGHLY

PATHOGENIC AVIAN INFLUENZA VIRUS (HPAIV) H5N1 OUTBREAKS

A cross sectional epidemiological study was conducted for quantification of risk factors associated with HPAIV H5N1 outbreak.

Target Population

Birds in all commercial poultry farms (broiler, breeder, layer) of Pakistan constituted the target population.

Study Population

Birds in all commercial poultry farms (broiler, breeder, layer) of study area i.e. ICT were selected as study population.

Sample Size

Desired sample size was estimated following the model recommended by Schlesselman (1982) for two sided test of significance. For estimation of sample size, entry of wild/migratory birds in premises of farm was considered as most important hypothesized risk factor. By specifying the values of α (probability of type 1 error), ᵦ (probability of type 2 error), R (the hypothesized relative risk of sufficient biologic importance) and p0 (expected rate of exposure among controls) as 0.05, 0.10, 10 and 0.60 respectively, the desired sample size was n = 31 per group (cases and controls).

Materials and Methods

Selection of Cases/Case Flocks

Flocks affected by HPAIV infection during outbreaks of HPAIV H5N1 period i.e. from 2006 to

2008 were selected from study population. The diagnosis was made by clinical manifestation of the disease and later confirmed by virus isolation techniques done at National Reference

Laboratory ,NARC.

Selection of Controls/Control Flocks

One healthy flock (HPAIV free) was selected against each case flock from study population.

Matching

Each case flock was individually matched to its respective control with respect to various descriptive and demographic characteristics including area/jurisdiction, size of flock and age and type of birds (breeder, layer & broiler).

Minimizing Bias

Flocks effected during the study period i.e. from 2006 to 2008 were selected as cases in order to minimize the chances of bias (selection bias, recall bias).

Informed Consent

Owner of each selected flock was taken into confidence and his consent was obtained before the start of study.

Data Collection

Materials and Methods

Data of each selected case and control flock regarding house system, farm capacity, fences around farm, boundary wall, entry of wild birds, entrance in farm, poultry density in the area, ventilation by cooling pad, all in all out production system, rodent density, backyard poultry at the farm, presence of pet birds and animals at the farm, accessibility of wild birds and animals and disposal of dead birds were recorded in predesigned Performa/questionnaire. One Performa was used for one flock (case or control) for one outbreak.

Statistical Analysis

Odds ratios, their 95% confidence intervals and significance/P values were calculated using

Open Epi software and multivariable logistic regression model was used through SPSS version

20.0 statistical software, for following hypothesized risk factors.

 House system

 Farm capacity

 Fences around farm

 Boundary wall/bared

 Entry of wild birds

 Entrance at farm

 Farm situated in high poultry farm density area

 Ventilation by cooling pad

 All in all out production system

 High rodent density

 Raising of backyard poultry at the farm

 Keeping of pet birds at the farm

Materials and Methods

 Pet animals at the farm

 Accessibility to stray and wild animals

 Disposal of dead birds

SURVEILLANCE OF LIVE BIRD MARKETS FOR DETECTION OF (HPAIV) H5N1

Study population

Birds reared in captivity (broilers, layers, desi birds and pet birds), kept in live bird markets of

ICT, and was presented to sale was study population. Samples from migratory birds near tarbela lake, Ayub National Park and some desi birds kept at poultry and wild life department, NARC were also collected and processed.

Samples Collected

Serum, swabs and tissue samples were collected from all the captive birds of live bird markets in

ICT. Samples were taken randomly from different stalls where different kinds of birds like broilers, layers, breeders and wild migratory birds are kept for sale every week. All the samples from one stall were pooled and were transported in lab in cold conditions via transport media for further procesing.

Table 3.3. Total no. of samples from different live bird markets at ICT.

Month LBLM1 LBM2 LBM3

Serum Swabs tissues Serum Swabs Tissue Serum Swab tissue

Jan 34 22 17 24 26 14 26 16 11

February 29 24 19 20 27 11 32 11 09

Materials and Methods

March 21 25 29 25 22 14 27 24 11

April 22 16 11 33 11 23 33 19 12

May 22 26 09 36 19 08 25 24 13

June 19 23 07 24 13 14 27 14 06

July 21 19 14 24 12 09 23 13 09

August 27 17 03 29 12 07 27 11 19

September 23 18 16 27 17 13 24 19 21

October 17 16 09 23 15 17 21 09 17

November 36 12 03 11 21 11 23 12 03

December 23 29 11 29 19 09 22 15 11

Total 294 247 148 305 214 150 337 173 142

Sample Processing For Virus Isolation:

The collected organs were homogenized by blending 20gm of tissues in 80ml of brain heart infusion (BHI) broth (Oxoid) using a homogenizer (STOMACHER, Biomaster, UK) and centrifuged at 13000rpm for 10 minutes at temperature of 10°C. The swabs dipped in 1ml of BHI broth were spun at 13000rpm for 10 minutes. Supernatants were filtered through 0.2µm syringe filters for both type of samples (Sartorius, Minisart, Germany) and saved at 4°C till used.

Propagation of Isolates In Embryonated Chicken Eggs

For virus isolation the above filtrates were inoculated into the Allantoic cavity of 9-day old embryonated SPF (specific pathogen free) chicken eggs (Senne 1998). The eggs were candled for viability of the embryo, blood vessels and air sacs were marked. Broad end of the eggs were

Materials and Methods disinfected with 70% ethanol. Under the Biosafety Cabinet (BSL 3), a hole was drilled in the middle of the egg shell at the top and with the help of a 1ml disposable insulin syringe, 0.2ml of inoculum was injected through the hole, just penetrating the chorioallantoic membrane right below the air space. The hole was sealed with melted paraffin and the eggs were incubated at

37°C for 48 hours where after these were chilled for 4 hours at 4-8°C (Swayne et al., 1998b).

Sterile forceps was used to break the egg shell and the allanto amniotic fluid (AAF) was aspirated with the help of sterile disposable 3mL syringe. The collected fluid (1ml of aliquot) was preserved in the eppendorf tube and preserved at -200C until use.

Preparation of 5% RBCs Suspension

From the wing vein, five ml of blood was collected in sterile disposable syringe containing few granules of anticoagulant ethylene diamine tetra acetic acid (EDTA). The blood was mixed gently, to avoid clotting, and then centrifuged at 800xg, at 10°C for 10 minutes. The supernatant was then removed with a syringe and to this double quantity of 0.1M PBS (MP Biomedicals,

France) pH 7.2 was added. The RBCs were gently re-suspended in PBS and the procedure was repeated for three times. Finally the clear supernatant was discarded and the volume of the remainder washed RBCs was measured. Five percent RBC solution was prepared by adding 95 mL of volume PBS and 5mL of packed cell volume of RBC. It was stored in a refrigerator and used up to 2 days.

Spot hemagglutination (HA) test

A rapid check for the presence of hemagglutination virus was done by placing a drop of collected allantoic fluid along with a drop of 5% fresh washed chicken RBC. Solution was mixed with the

Materials and Methods help of a stirrer. AAF positive with HA property was indicated by appearance of granular appearance observe behind the light.

Preparation of 1% RBCs Suspension

The procedure was already mentioned above. However finally in the dilution 1mL of packed cell volume was mixed with 99 mL of PBS and thus 1% RBC was prepared. Once 1% chicken RBC is prepared and it was stored at chilling temperature for 4 days (Thayer and Beard, 2008).

Reference antigen and antiserum

Inactivated H5N1 antigen: A/Ck/Scot/59 (Veterinary Laboratory Agency, Surrey, UK)

H5N1 antiserum: A/ Ck/Scot/69 (VLA, Surrey, UK)

Inactivated H7N7 antigen; A/ tky/ Eng/647/77 (VLA, Surrey, UK)

H7N7 serum: A/Tk/Eng/647/77 (VLA, Surrey, UK)

Inactivated H9N2 antigen: A/turkey/ Wisc/66(VLA, Surrey, UK)

H9N2 antiserum: A/Turk/Wisc/1/66 (VLA, Surrey, UK)

Procedures for microplate HI test for identification of H5N1 virus isolates

The “V” bottom shaped 96 well microplates were used to perform HI test. The plates were marked for testing the HA positive allantoic fluid sample using known sera against H5, H7, H9 and Newcastle disease virus (NDV). All the test samples were run in duplicate. Two rows were kept as positive control and two rows were kept as negative control. One row as kept for serum

Materials and Methods and the other row was kept for RBC. A reference antiserum containing 1:64 Hi titre or above was sufficient to identify the virus subtype by HI test. A 25 µL containing 4 HA of each specimen was dispensed to each of 11th well per row (22 wells for duplicate rows). Plate was incubated at

370C for 30 minutes. A 25 µL of 1% chicken RBC was added to all well by multichannel pipettor. For identification of an isolate of H5N1, the HI titre of test sample was compared to reference H5N1 virus to determine the specifity and identical nature of the reactions (Thayer and

Beard, 2008).

Hemagglutination Inhibition Test for detecting antibody titre

To check the antibody titer of each serum sample as per standard protocols earlier developed

Hemagglutination inhibition (HI) assay was used (Beard 1980).

1. The round bottom 96-well microtitration plate with 8 rows and 12 columns of wells

were used. A 25µl of 0.1M PBS (pH 7.2) was added to each of the 1-12 wells of A-H

rows of the microtitration plate.

2. In well 1A to 1H for testifying the samples , twenty five micro liter of each serum was

added. After mixing in each well, with the micropipette, 25µl of each mixture was

transferred from well 1 to 2 and so on. In this way, the sera from A to H were diluted

two fold from well 1 to 12.

3. Then 25µl of 4HAU of antigen was added in the well 2-12 leaving first column serving

as control. The plates were agitated and incubated for 30 minutes at 37ºC.

4. After 30 minutes, 50µl of 0.1% chicken RBCs was mixed in each well. To make sure

even erythrocytes dispersion in the mixture the plate was gently agitated. The plate was

Materials and Methods

then incubated at 37ºC for 30 minutes by which time a clear pattern of button formation

(hemagglutination inhibition) or haziness (hemagglutination) was seen.

For reading of the plate, it was tilted at 450angle for 30 seconds and the last well showing tear- drop formation was considered as highest dilution of antisera positive for HI antibodies. To check the inhibition of hemagglutination the highest dilution of each serum sample was considered as the end point. Titers were calculated as the reciprocal of the last HI positive serum dilution and samples with HI titers of 8 or below were considered negative.

Identification of Influenza Virus By RT-PCR:

World health organization protocol on Animal Influenza Diagnosis and Surveillance (2002) describes the RT-PCR for AI virus which was completed in the following steps:-

1- Extraction of viral RNA from samples

2- cDNA Prepration

3- Further processing on Polymerase chain reaction (PCR)

4- Analysis of PCR products by help of agarose gel electrophoresis

RNA Extraction From Viral Isolates:

Total RNA extraction Kit (Catalog #74105): collection tubes (1.5ml), collection tubes (2 ml), diluted 70% ethanol, buffer RLT, Wash buffer#1, mercaptoethanol, buffer RPE, RNase-free water, RNeasy mini spin columns, sterile 1.5 ml microcentrifuge tubes, adjustable pipettes and tips, microcentrifuge adjustable to 13000 rpm, and mechanical shock given by vortex are needed to extract RNA from the virus isolate.

Materials and Methods

RNA Extraction Procedure:

1. β-mercaptoethanol , 350 µl of lysis buffer, 3.5 µl; 550 µl of 70% ethanol and 200 µl of

the virus suspension were mixed thoroughly and the mixture was pipetted out in the

column and centrifuged at 10,000 rpm for 15 seconds.

2. Collection tube was discarded with the liquid in it and column placed in new collection

tube.

3. 700 ul of wash buffer-1 was added to the column and for 15 seconds it was centrifuged

on 10000 rpm. 500ul of wash buffer RPE was added to the column after transferring it to

the new clean collection tube, again centrifuged at 10 thousand rpm for 15 seconds.

4. The liquid was then removed from the column collection tube and after adding 5 hundred

µl of (RPE) wash buffer to the column it is centrifuged for two minutes at 12 thousand

rpm.

5. The column was then transferred to a 1500 ul micro centrifuge tube and 200 µl of R-Nase

free H2O and was pipetted directly onto the filter of the column allowing it one minute to

react.

6. The material was centrifuged at 10, 000 rpm for 60 seconds. The sample ready for cDNA

synthesis. The isolated viral RNA was stored at -20 oC.

Synthesis of cDNA:

Material:

Sterile 500 ul micro centrifuge tubes; full panel of adjustable pipettes and tips, microcentrifuge adjustable to 13000 rpm, mechanical shaker, water bath/thermocycler for 42ºC and 90ºC incubation, RNase Inhibitor (40 units/µl), viral RNA, ultra pure water, primer ‘Uni12’:

Materials and Methods

AGCAAAAGCAGC (1 µg/µl), 10 mMdideoxynucleotide triphosphate (dNTP) mix, , 5x Reverse

Transcriptase buffer, AMV Reverse Transcriptase (25 units/µl, Life Sciences, Inc, Cat# ARB-46) and ice.

Procedure:

Briefly, the labeling of 500 ul micro-centrifuge tube for each of the RNA was done. Rnase free water was used as the negative control. To 5 µl of negative control and extracted viral RNA

(water blank) 0.5 µl primer was added, and the test was incubated at 72 ºC for 5 minutes, A cocktail of, 2.0 µl of RTranscriptase buffer, 1.5 µl of water, 0.5 µl of RNasin, and 1.0 µl of

Reverse Transcriptase is prepared , 0.5 µl of 10 mMdNTP mix, and 5.5 µl of this cocktail was added to each tube and maintained cooling on ice, Incubation of 10 µl RNA/Primer mix was done for 1 hour at 42ºC, and RT-reaction was stopped due to incubating at 95ºC for 5 minutes.

PCR test:

The following materials are needed for PCR test:

A. AIV subtypes amplified by

(a) Reverse and Forward primers 1 µg/µl

(b) HA-Reverse & HA-1144

B. Amplification done by help of (a) H6: HA-1144 and H6-1480R, (b) H5: HA-1144 and H5-

1735R, (c) Forward and reverse primers at 1 µg/µl, (d) H9: H9-1 and H9-808R

C.Internal positive control was added for the exclusion of contamination chances for the amplification of M-gene by help of reverse and forward primers.

Materials and Methods

D.Sterile 500ul micro-centrifuge tubes, full panel of adjustable pipettes and tips vortex, polymerase chain reaction machine, micro-centrifuge adjustable to 13000rpm,, Taq Polymerase

(5 units/µl, Promega Cat #M1902), 10x PCR Buffer, 25 mMMgCl 2; 10mM d NTP mix, H2O, ice cubes and sterile mineral oil.

Procedure:

Only 1.5 µl for each PCR reaction was taken from the cDNA synthesized and added to 48.5 µl of the master mix, which was prepared depicted under:

A. PCR buffer (5ul), 3 µl of 25 mMMgCl 2, 38.65 µl H2O,0.25 µl of Taq DNA

polymerase, 1 µl 10 mM d NTP mix, 0.3 µl of each reverse and forward primer

(1µg/µl).

B. Primers (1µg/µl) were spun briefly. After addition of mineral oil on top of the micro-

centrifuge tube was amplified in pcr: by treating it at 94 ºC for 2 minutes, denaturing at

94 ºC for 1 minute, annealing at 50 ºC for 1 minute, extension at 72 ºC for 3 minutes,

step 2, was repeated in cycles for 30 times for 8 minutes at 72 ºC and kept at 4 ºC until

further use.

Analysis of PCR product on agarose gel electrophoresis (AGE)

Materials:

Tray caster for agarose gel ,electrophoresis chamber, 10µl adjustable pipette and tips, 1% agarose gel in 1x TBE. 1x TBE Buffer, power supply and electrode leads, hand-held UV light

(302 nm) or a UV-light box, camera and film, molecular weight marker (Low DNA Mass

Materials and Methods

Ladder, Gibco, Cat # 10078- 013) , 30%glycerol, Ethidium Bromide (10 µg/µl), gel loading buffer (GLB, 0.25% BPB and 0.25% XC), and the PCR product containing micro-tubes .

Procedure:

1. The tape from gel frame was removed and the gel was paced into the

electrophoresis chamber; while covering the gel with 1x TBE label 0.5 ml

microcentrifuge tubes, separately.After removing the tape from the gel caster,

agarose gel placed in the electrophoretic caster frame, while agarose gel covered

in buffer.

2. From each PCR tube, 4ul taken and transferred to the corresponding micro-

centrifuge tube, and mixed with 3 micro-liter loading buffer.

3. In first well of the agarose gel 4 micro-liter of weight marker was loaded.

4. In two wells of the gel, 7 micor-liter of negative and positive control was poured

from PCR tubes.

5. The gel was run for 30 to 40 minutes on 120 volt, after closing and attachment of

the power supply.

6. Ultra violet light visualize was used to detect the presence of the PCR product

bands and marker.

7. Photograph of the gel was saved and than analyzed by comparing the PCR

product bands with the molecular weight marker.

CHAPTER 4 RESULTS

Investigating the outbreaks of Highly Pathogenic Avian Influenza (HPAIV) from 2006-

2008 at ICT

Retrospective data was collected from different layer farms in and around Islamabad Capital

Territory Area from March 2006 - May 2007 (Table-1). Total number of birds ranged from

1600-14000. Average flock size was 5097. The outbreaks occured in the age between 4 - 45 weeks. However most of the outbreaks occured during around 45 weeks of age of birds. The morbidity rate ranged from 57–95. Although in most cases outbreak rate was above 70. The mortality rate ranges was 5-43. On an average mortality rate was 24. The highest culled birds in one outbreak were 11600, and the number of birds was also the highest (14000) in that farm. The lowest culled birds were 1040 in an outbreak where the population of the farm was 4000 (Table-

1). In case of percentage, the highest percentage (95.29) of culled birds in an outbreak and the lowest percentage of culled birds were (56.26).

92 Results

Table 4.1: Outbreak of HPAIV in Layer farm during 2006-2008

Sr Date of Total Age of Morbidit Mortali Case No. of % of #1 outbreak no. of birds y rate ty rate fatality culled culling birds in birds weeks 1 13-04-06 5000 12 74 26 35 3700 82.86 2 20-04-06 5000 45 90 10 11 4500 78.80 3 20-04-06 2800 43 78 22 27 2200 62.33 4 21-04-06 14000 16 83 17 21 11600 91.78 5 22-04-06 2000 18 79 21 27 1576 70.24 6 23-04-06 4000 04 62 38 60 2493 82.00 7 24-04-06 1800 43 91 09 08 1652 65.00 8 24-04-06 8400 04 70 30 42 5900 95.29 9 25-04-06 3500 32 82 18 22 2870 80.92 10 01-05-06 1600 45 65 35 54 1040 56.65 11 01-05-06 7000 45 95 05 05 6670 86.28 12 03-05-06 3600 32 81 19 24 2913 63.28 13 03-05-06 6200 36 57 43 76 3512 85.15 14 09-05-06 7500 36 86 14 16 6471 62.65 15 09-05-06 4000 40 63 37 58 2531 74.07 16 12-05-06 6000 38 85 15 17 5109 63.29 17 11-05-07 4750 16 63 37 57 2976 82.86 18 19-05-07 2700 34 74 25 35 2000 78.80 19 22-05-07 7000 17 63 37 58 4430 62.33

Figure1 shows the comparison of morbidity, mortality and case fatality rate in layer farm. It showed that morbidity rate was the highest among the different rate. In most of the outbreak it mortality rate was above 60 and it goes up to 90. Case fatality rate is the lowest among these three rates. However in some instance like in an outbreak it goes up to 76 crossing the morbidity rate. This rate was observed in an outbreak where the age of bird was 36 week and flock size was

3600. The mortality trend is intermingling with the case fatality rate. Highest mortality rate 43 was observed in the same outbreak where the highest fatality rate was observed (Figure 1).

Results

Figure 4.1: Trend of morbidity, mortality and case fatality rare in different outbreak in layer farm

Retrospective data was analyzed in breeder farm from March 2006- May 2007. Flock size of the farms was ranging from 15000-1500 (Table-2). Average flock size of the farms was 4910. The outbreaks occur in the age between 5- 45 week. However most of the outbreak occurs during 40 weeks of age of birds. The morbidity rate ranges from 63 – 96. Although in most cases outbreak rate was above 80. The mortality rate ranges was 4-37. On an average mortality rate was 20. The highest culled birds in one outbreak were 10569, and the number of birds was also the highest

(25000) in that farm. The lowest culled birds were 1200 in an outbreak where the population of

Results the farm was 1500 (Table-2). In case of percentage the highest percentage (95.64) of culled birds in an outbreak and the lowest percentage of culled birds were (62.96).

Table:4.2 Outbreak illustrations by H5N1 in breeder flock

Sr Date of Total Age of Morbidity Mortality Case No. of % of #1 outbreak no. of birds in rate rate fatality culled culling birds weeks birds 1 18-04-06 15000 41 70 29 42 10569 70.46 2 19-04-06 4500 41 82 18 23 3700 82.22 3 20-04-06 2700 40 63 37 58 1700 62.96 4 21-04-06 5300 40 81 19 23 4300 81.13 5 21-04-06 1500 13 80 20 25 1200 80.00 6 24-04-06 2500 13 86 14 16 2148 85.92 7 30-04-06 2500 35 67 33 49 1672 66.88 8 01-05-06 2500 45 96 04 05 2391 95.64 9 02-05-07 4000 27 84 16 19 3350 83.75 10 19-05-07 8600 05 90 10 11 7760 90.23

Figure 2 showed the trends of morbidity, mortality and case fatality rate in breeder flock in different weeks. The highest trend was observed in morbidity rate. In most cases morbidity rate was above 80. However in three instances the morbidity rate was lower than 80. These outbreak occured in 41st, 40th and 35th week respectively. The lowest rate was observed in case of mortality rate among these three rates. The lowest mortality rate was 4 and the highest was 37 which were observed in the age of 45th and 40th week respectively. In case of case fatality rate it was almost near to mortality rate (Figure 2).

Results

Figure 4.2: Trend of morbidity, mortality and case fatality rate in breeder flock

Outbreak data due to H5N1 HPAIV was investigated in the broiler farm from 2006-2008 (table-

3). Total numbers of birds were ranging from 3000-12000. Average flock size was 4928.

Outbreaks occurred during 35-45 days of age chicken. The morbidity rate ranges from 81-88 recorded by Rapid Response Team (Fig-1). The mortality rate ranges was 12-19. The highest number culled birds in one outbreak were 9752. It occurred in the age of 45 days and flock size was 12000. The lowest culled birds were 2640 in an outbreak where the population of the farm was 3000 (table-3). The highest percentage (88.63) of culled birds in an outbreak where the flock

Results size was 3570 and the lowest percentage of culled birds were (81.27) in an outbreak where flock size was 12000.

Fig. 4.3. Rapid Response Team Working on HPAI Virus Infected Flock

Table 4.3: Outbreak illustrations by H5N1 in broiler flock

Sr #1 Date of Total Age of Morbidity Mortality Case No. of % of outbreak number of birds in rate rate fatality culled culled birds days birds birds 1 22-04- 5000 42 87 13 14 4375 06 87.50 2 24-04- 3000 35 85 15 18 2640 06 88.00 3 27-04- 3570 44 88 12 13 3164 06 88.63 4 26-06- 10000 45 83 17 23 8290 06 82.90 5 01-07- 12000 45 81 19 23 9752 06 81.27 6 01-07- 8000 44 82 18 28 6570 06 82.13

Results

Figure 3 showed the trends of morbidity, mortality and case fatality rate in farm in different days.

The highest trend was observed in morbidity rate. In most case morbidity rate was above 80 and

below 90. The lowest rate was observed in case of mortality rate among these three rates. The

lowest mortality rate was 12 and the highest was 19 which were observed in the age of 44th and

45th day respectively. In case of case fatality rate, the highest was 28 and the lowest was 13

(figure-3).

Figure 4.4: Trend of morbidity, mortality and case fatality rate in broiler flock

100

90 80

60 Morbidity Rate% 50 Mortality rate% 40 Case fatality%

0 42 35 44 45 45 44

Results

Identification and quantification of risk factors associated with HPAIV H5N1 by a cross

sectional survey in different ecological zones of ICT

Table no 4.4 represents the detailed description of age and birds number controls matching with

cases in the study area during the 2006-2007 outbreaks due to HPAI H5N1 in poultry

commercial farms.

Table 4.4. Showing the case and control age and birds number description.

Bird population and ages on 70 commercial poultry farms in ICT Pakistan 2006-2007. Variables Type Case farms* (n=35) Control farms (n=35) Mean Min Max Mean Min Max Age of chickens Layers (weeks) 24.5 4 45 25.5 4 47 Breeders(weeks) 25.5 4 45 24.5 6 43 Broilers(days) 40 35 45 38.5 31 46 Number of Birds Layers 5097 14000 16000 6540 12000 16000 Breeders 4910 1500 15000 4723 1350 17000 Broilers 4928 3000 12000 5300 4000 10000 *During the onset of outbreaks

Results

Fig 4.5 Spatial distribution of cases and control in ICT Pakistan during HPAI H5N1 2006-

2007 epidemics.

During the AI H5N1 outbreak in local chicken farms, a case-control study was conducted to identify risk factors that may have contributed to this outbreak (Table-5). A questionnaire was administered by interview to gather the data for this study. Multivariate logistic regression models revealed that movement of people and fomites from live bird markets to farms were important influences on transmission, and that the live bird markets were the probable source of virus for farms.

The housing system was included as dummy variable and the difference in housing system is positive and significant. The difference in the fence around farm is negative and significant indicating that non infected farm have fence around them as compared to infected farms and in other wards the fence reduces the chances of infection and vice versa. The difference in the

Results boundary wall around farm is also negative and significant. The difference in farm entrance is negative and significant. The difference in the farm situated in the high density area is positive and significant indicating that the infected farms are located in the high density area and vice versa. The difference in the farm located in the urban areas is also positive and significant. The difference in the all in all out is negative and non significant. The infected farms have higher rodent density as the difference is positive and significant. The difference in backyard chicken population is positive and significant. The difference in pet keeping is positive and significant.

The difference in farm location is positive and significant (table-4.5).

Table 4.5: Result of univariable analysis by Mantel-Haenszel matched pair analysis of risk factors associated with the risk of AIV H5N1 infection in 2006-2007 epidemics.

Risk Factors Description Case Control Matched OR* CI P-Value farms farms (95%) (n=35) (n=35) Open 34 27 10.074(1.186-85.57) 0.012 House system Semi Open 1 8 <5000 18 29 - 0.008 <10000 13 2 - Capacity of farm <25000 3 4 - <50000 1 0 - No 31 0 9.750(3.85-24.67) 0.000 Fences around farm Yes 4 35 Boundary wall/barbed No 8 0 2.296(1.730-3.049) 0.003 wire Yes 27 35 No 0 32 12.667(4.27-37.52) 0.000 Entry of wild birds Yes 35 3 Single 7 29 0.052(0.015-0.173) 0.000 Entrance at farm Multiple 28 6 Farm situated in high No 0 25 4.50(2.60-7.77) 0.000 poultry density area Yes 35 10 Farm situated in urban No 0 33 18.50(4.80-71.21) 0.000 area Yes 35 2 Ventilation of farm by No 35 35 - using cooling pad Yes 0 0 - Farm has all in all out No 1 0 2.029(1.59-2.57) 0.314

Results production system Yes 34 35 Farm has high rodent No 4 35 0.103(0.041-0.260) 0.000 density Yes 31 0 Raising of backyard No 30 35 0.462(0.355-0.600) 0.020 poultry at the farm Yes 5 0 Keeping of pet birds at No 28 29 0.828(0.247-2.76) 0.759 the farm Yes 7 6 Keeping of pet animals No 18 30 0.176(0.056-0.561) 0.002 at the farm Yes 17 5 Farm is accessible to No 0 35 - 0.000 stray and wild animals Yes 35 0 Farm situated near No 12 28 0.130(0.044-0.385) 0.000 water source Yes 23 7 .00 1 1 - 0.000 Mash 8 30 - Crumbs 25 4 - Home 1 0 - Type of feed Mixed 5 0 2.160(1.660- 0.027 Automatic 2.8170) Method of feeding Mannual 30 35 13 35 0.2710(0.170- 0.000 Well Source of Drinking 0.431) water Bore 22 0 Nipple 3 0 2.09(1.630-2.690) 0.120 Method of Drinking Drinker 32 35 0.012 Is drinking water No 6 0 2.207(1.686-2.888) Disinfected Yes 29 35 Drinking water stored at No 1 0 2.029(1.597-2.578) 0.500 farm Yes 34 35 If Yes, is it properly No 12 0 2.522(1.836-3.464) 0.000 covered? Yes 23 35 N/A 9 30 0.058(0.017-0.194) 0.000 Egg collection system Manual 26 5 Use of disinfectant at No 35 0 - 0.000 the point of entry at the 0 35 farm Yes Mantel-Haenszel matched pair analysis

Objective 3: Serology on H5N1, H9 and H7 in live bird market

Results

A longitudinal cross sectional sero survey for twelve month was done in live bird market to detect antibody titre against H5N1 in the year 2011 (table-5). Sample was taken once in each month. A total 929 of sera samples were analyzed. The highest sample number (88) was taken in the month of April and July. The lowest sera sample (61) was taken in the month of October.

Among the total 929 sera samples, 98 sera sample titre was zero. However geometric mean titre(GMT) of antibody against H5N1 was almost same throughout the year. The range of GMT was 2.65-4.32. In the studied none of the sample had antibody titre above 64. The highest antibody titre (64) with the highest frequency (23) was observed in the sera sample in the month of February (Table-5).

Results

Table- 4.6 Serological evaluation of live bird market samples using HI antigens of AIV

subtype H5N1

Months No. of Samples No. of positive samples under each titer group HI

samples showing 2 4 8 1 32 6 12 2 51 1024 2048 GMT

zero 6 4 8 5 2

titer 6

January 84 12 - 8 1 2 26 ------3.33

8 0

February 81 10 1 4 1 1 - 2 - - - - - 3.34

2 5 7 3

March 73 6 2 5 1 2 7 1 - - - - - 3.63

8 3 2

April 88 15 7 1 - 2 9 1 - - - - - 3.72

2 1 4

May 83 11 - 7 2 2 10 1 - - - - - 3.43

3 0 2

June 70 6 2 1 1 1 - 2 - - - - - 3.57

2 6 4 0

July 88 11 - 1 1 1 17 1 - - - - - 3.72

0 5 6 9

August 83 12 - 1 1 1 25 1 - - - - - 3.97

3 7 4 2

Results

September 74 7 1 1 9 1 9 1 - - - - - 3.13

2 2 3 2

October 61 8 6 1 1 8 10 ------2.65

3 6

November 70 - 1 1 1 9 11 1 - - - - - 3.74

0 0 3 7

December 74 - 4 1 1 2 13 1 - - - - - 4.32

6 0 2 9

Live bird market same sample were evaluated against H7 (table-6). Among the total 929 sera

samples, 357 sera samples titre was zero. Within these 357 sera sample the highest frequency

(36) sera sample having titre zero (0) was observed in the month of August. The lowest

frequency (24) was observed having titre zero (0) was observed in the month of September. The

range of GMT was 1.59-0.87. In the studied none of the sample had antibody titre from above

32. The highest antibody titre (16) with the highest frequency (13) was observed in the sera

sample in the month of in September (Table-6) and the lowest frequency (3) was observed in the

month of April and May (Table-6)

Results

Table 4.7: Serological evaluation of live bird market samples using HI antigens of AIV subtype H7

Month No. of No. of No. of positive samples under each titer group HI

samples samples 2 4 8 1 3 6 12 2 5 1024 2048 GMT

showing 6 2 4 8 5 1

zero titer 6 2

January 84 35 2 2 4 - 0.89

3 0

February 81 30 3 1 6 0.92

3 2

March 73 25 2 1 12 1.19

1 5

April 88 30 2 1 13 3 1.25

5 7

May 83 28 2 1 11 3 1.26

2 9

June 70 32 2 1 5 0.87

0 3

July 88 28 2 1 9 1 1.40

5 6 0

August 83 36 1 1 7 8 1.10

9 0

September 74 24 1 1 9 1 1.59

Results

7 1 3

October 61 25 1 9 8 9 1.44

November 70 35 1 9 9 6 1.14

December 74 29 1 1 8 1 1.44

5 0 2

Live bird market same sample were evaluated against H9 (table-7). Among the total 929 sera

samples, none of the sera samples titre was zero. The lowest antibody titre (4) with the highest

frequency (17) sera was observed in the month of July. The highest antibody titre (2048) with the

highest frequency (13) was observed was observed in the month of September. The highest

antibody titre with the lowest frequency (3) was observed in the month March. The range of

GMT was 6.0-8.17 (table-7).

Results

Table 4. 8: Serological evaluation of live bird market samples using HI antigens of AIV subtype H9

Months No. of No. of No. of positive samples under each titer group HI

samples samples 2 4 8 16 32 64 128 25 512 102 204 GMT

showing 6 4 8

zero titer

Jan 84 5 11 11 11 7 8 10 10 11 7.25

Feb 81 12 12 9 6 9 5 11 9 8 7.07

Mar 73 13 11 8 16 12 10 3 8.17

Apr 88 9 10 19 12 20 12 6 6.36

May 83 20 14 11 12 17 9 7.13

June 70 6 8 9 9 8 8 7 9 6 6.97

Jul 88 1 9 20 12 10 11 9 6.18

Aug 83 1 11 8 13 5 9 7 11 9 6.37

Sep 74 12 17 11 9 12 13 7.70

Oct 61 1 7 12 12 09 11 6.00

Nov 70 7 9 13 11 8 12 10 6.45

Dec 74 11 13 9 13 16 12 7.33

Results

Geometric mean titer (GMT) of three subtypes (H5, H7 and H9) of AIV were compared (figure

4) for each month in the year of 2011. Data revealed that in all cases, antibody titre against H9 was the highest and H7 was the lowest among these three subtypes of AIV. In case of H9 the highest GMT (8.17) was observed in the month of March and the lowest GMT (6.0) was observed in the month of October. GMT against H5 was the highest (4.32) in the month of

December and the lowest (2.65) was observed in the month of October. Antibody titre against

H7 showed that the highest GMT (1.59) was observed in the month of September and the lowest

GMT (0.87) was observed in the month of June (figure 4).

Results

9 8 7 6 5 4 H5 3 H7 2 H9 1

GMTof different three subtype 0

Figure 4.6: Geometric mean titre of three subtypes H5,H7 and H9 in the each month of 2007

Market 1:

Blood samples were collected from (Table-9) all chickens before entering the retail stall and again at slaughter, along with 294 blood samples and 247 tracheal, cloacal and fecal swab samples and 12 water samples taken over the four sampling days. There were 13 tagged chickens infected with H9 virus at the end of Day 1, and a further 13 tagged chickens were found infected with H9 virus on Day 2. No H9 virus was detected in faecal and water samples from this stall until Day 3, when H9 viruses were retrieved from the water trough in the cage with tagged chickens, and the faecal swabs from linked cages and nearby stand-alone cages on days 3 and 4.

Results

On day 4 H9 viruses was also isolated from the water tank which was used for washing the carcasses. Tracheal, cloacal and blood samples were also taken from 10 quail on day 4.

Market 02:

There were 214 swabs (tracheal and cloacal) collected from tagged chickens, plus 164 faecal, 8 tracheal, 8 cloacal samples taken from the stall for three days.

Market 03:

There were 337 swabs (tracheal and cloacal) taken from tagged chicken and 88 faecal swabs collected from untagged birds, 2 tracheal swabs collected from the rooster, and 6 water samples taken in stall 3. Five H9 viruses were isolated from faecal swabs (two on Day 2 and three on Day

3). Twelve Newcastle disease viruses were isolated from faecal swabs (3 on Day1, 5 on Day 2 and 4 on Day 3). Tracheal swabs from the pet rooster were negative for AI and NDV viruses

(table-8).

Results

Table 4. 9: Sampling distribution in three live birds markets

Month LBLM1 LBM2 LBM3

Serum Swabs tissues Serum Swabs Tissue Serum Swab tissue

January 34 22 17 24 26 14 26 16 11

February 29 24 19 20 27 11 32 11 09

March 21 25 29 25 22 14 27 24 11

April 22 16 11 33 11 23 33 19 12

May 22 26 09 36 19 08 25 24 13

June 19 23 07 24 13 14 27 14 06

July 21 19 14 24 12 09 23 13 09

August 27 17 03 29 12 07 27 11 19

September 23 18 16 27 17 13 24 19 21

October 17 16 09 23 15 17 21 09 17

November 36 12 03 11 21 11 23 12 03

December 23 29 11 29 19 09 22 15 11

Total 294 247 148 305 214 150 337 173 142

Sampling from wild life species

Various samples were collected from wild species in the study located in the study premises as depicted in Table 8. We could not isolate any strain of avian influenza virus though Newcastle

(ND) virus was isolated successfully. Details are given in the table below.

Results

Table 10.4. .Detailed sampling, technique and outcomes for the study conducted in wild life species for isolation of avian influenza viral strains in the study premises national parks, backyard poultry, lakes and zoo, s.

Sample Location Type No. of HI Virological source/type of sample antibo evaluation of bird sampl s dies e titres of AIV (GMT = log2)*

H3 H5 H7 H9 AIV Other isolation** isolation**

Pheasantry Mansehra Swab 05 - - - - Nil Nil Hazara University (Ring Necked) Pheasantry Mansehra Swab 06 - - - - Nil Nil Hazara Serum 03 0.0 0.0 0.0 0.0 University (Silver Necked) Pheasantry Mansehra Swab 03 - - - - Nil Nil Hazara Serum 0.0 0.0 0.0 0.0 University 03 (White Turkey) Terbela Haripur FM 46 - - - - Nil Nil Lake (Migratory Birds) Abbotabad Abbotaba Serum 15 0.0 0.0 0.0 4.66 Nil Nil (backyard d

Results poultry) Livestock Islamaba Swab 20 - - - - Nil Nil Research d Serum 40 0.0 0.0 0.0 0.0 Station (Duck) Livestock Islamaba Swab 02 - - - - Nil Nil Research d Serum 04 0.0 0.0 0.0 0.0 Station (Guinea Fowl) Ayub Rawalpin Swab 21 - - - - Nil ND virus National di Park (Mixed wild birds) Livestock Islamaba Swab 11 - - - - Nil Nil Research d Serum 30 0.0 0.0 0.0 0.0 Station (Desi Chicks) Live Bird Islamaba FM 04 - - - - Nil Nil Market d (Grey Partridge) Live Bird Islamaba Swab 05 - - - - Nil Nil Market(Mix d serum 10 0.0 0.0 0.0 0.0 ed wild birds) -, HI not performed; *only ELISA positive samples are serologically evaluated; **virus isolation (for influenza and New Castle Disease) was attempted by egg inoculation.

CHAPTER 5 DISCUSSION

The present study area Islamabad Capital Territory (ICT) holds large number of poultry farms and central live bird markets (LBMs). In Pakistan, all the four poultry production systems identified by FAO exist and are being practiced. Pakistan reported its first case of H5N1 in

February 2006 in the North-West Frontier Province, now called Khyber Pakhtunkhwa (KPK).

By June 2008, 51 outbreaks had been reported to the Office International des Epizooties (OIE), the World Organization for Animal Health, 39 in commercial farms and 12 in back yard poultry. This study was designed to investigate the devastating outbreaks of HPAI H5N1 infections in commercial layer, broiler and breeder poultry farms in and around ICT area from

2006-2007.

Descriptive epidemiological technique was used to investigate the trends and pattern of morbidity, mortality and case fatality during these outbreaks. There are only some published reports on quantification of risk factors and to the authors information it is the first descriptive epidemiological report investigating HPAI H5N1 infections outbreaks 2006-2007 in commercial poultry farms and LBMs of ICT. For this purpose retrospective data was collected from different layer farms (Average flock size=5097) in ICT area from March 2006 to May 2007. Most of the outbreaks occurred in 45 weeks old age birds. The cumulative morbidity and mortality rate ranged 57–95% and 5-43% respectively. The highest culling percentage recorded in layers was

95.29%. It was done to control and contain the disease spread in the area. The movement and sale was banned under the law of Government of Pakistan while in Hong Kong and Thailand culling of birds in infected premises were done as control strategy under intense control

92 Discussion surveillance program against HPAI H5N1 (Sims et al. 2002; Hulse et al. 2005). The Morbidity

(63-96%), mortality (4-37%) and culling percentage (62.96%) in breeder farms while morbidity and mortality rate of 81-88% and 12-19% with mean culling percentage of 81.27% was recorded respectively. The case fatality rate in the present study was much higher as compared to the previously studies (Bulaga et al. 2003) reporting 29% case fatality. The higher morbidity, mortality and case fatality in our study may be attributed to the susceptible production systems in

Pakistan. Density of poultry farms as is in the present study area is also an important factor in spread of disease (Chaudhry et al. 2015). Our findings revealed higher morbidity and mortality rates in layers farms than that of broilers and breeders with the broilers ahead of breeders. These findings are very much aligned with the results of Biswas et al. (2011) who reported the same pattern and trend for HPAI in poultry farms. The highest morbidity rate recorded in the present study for nondescript layer farms were strongly in consent with those of Ayaz et al. (2012). The higher mortality rate in layers may be due to the elongated exposure period as compared to broilers as per reported by (Alexandar et al. 1995). Lowest morbidity rate in breeder farms in the present study were in agreement to the findings of (Biswas et al. 2011). During 2006-2007 outbreaks due to HPAI H5N1, most of the cases were recorded in the months of April, May and

June in layers, breeders as well as broilers farms. This shows the influence of climatic variables and season on occurrence of infection. As previously reported by Halvorson et al. (1983) and

Halvorson et al. (1987) that influenza outbreaks do have seasonal occurrence in the high risk areas. It may be due to the high rate of migratory and wild birds activities mostly in these months as most of birds having breeding season, therefore in search of proper nesting places, in the meanwhile commercial farms remains the best choice for sparrows, pigeons and other wild bird species that may play a vital role in the spread of influenza viruses.

Discussion

Exploring age of birds at the outbreak showed that most of the outbreaks in layers and breeders occurred 5-45 week of their life stage while in broilers mostly outbreaks were recorded during

35-45 day of bird’s age. Its might be due to the increasing susceptibility of birds with increase in age, due to the negative associated impact on immune status and housing stress, ultimately rising the exposure opportunity for viruses (Alexandar et al. 1995).

2. Identifying risk factors associated with outbreaks from HPAI (H5N1) in 2006-2007 in different ecological zones of ICT:

Analytic epidemiological models were used in the present study to divulge the possible potential risk factors associated with HPAI H5N1 2006-2007 outbreaks in commercial poultry farms in ICT and LBMs in Pakistan, so that efficient risk management could be advocated. The use of these epidemiological models proved to be useful in identifying risk factors related with commercial poultry farms being classified as infected by HPAI H5N1 virus in 2006-2007 epidemics in Pakistan. While underreporting and or over reporting (mostly due to perplexity with other infectious diseases) may have happened during these outbreaks, such possibilities didn’t sway the investigated control and case farms here.

Frequent HPAI H5N1 outbreaks in commercial poultry farms occurred in ICT during

2006-2007. In the present study, a total of 47 candidate variables as potential risk factors were studied. Overall the data analysis revealed the biosecurity standards in practice contributed to the spread of HPAI H5N1 in commercial poultry farms in ICT. Farms locality i.e. farms in urban areas were the most significantly associated (OR = 18.50; p<0.05) with the infection. Large number of other factors studied, were involved in the transmission of the HPAI. It may be attributed to the fact as reported previously that distance of poultry sheds <0.5 km from the main road increases the risk of AI infections. Many studies (Ward et al. 2008, Farg et al. 2008)

Discussion conducted demonstrated that closeness to the highways and main roads were being associated with AI. This may also be due to several other factors i.e. wild life population in urban areas, large number of easily affected water sources i.e. Canals, Ponds or water reservoirs in public parks and in general could play eminent role in introduction and transmission of AI as they act as a source of attraction for wild birds (Farg et al. 2008; Si et al. 2013), which might contaminated the environment (Alexander et al. 2000; Biswas et al. 2009). Previous studies also demonstrated that AI introduction, persistence and transmission had been associated with low biosecurity

(Nishiguchi et al. 2007; Abbas et al. 2011, Tonbari et al. 2013), proximity to natural water sources (Ward et al. 2008; Farg et al. 2008; Biswas et al. 2009), from neighboring commercial poultry farms (Mannelli et al. 2006; Nishiguchi et al. 2007), proximity to roads (Trevennec et al.

2011), poultry trading pattern (Abbas et al. 2011; Desvanix et al. 2011) and poultry and human densities and their accessories movement (Mcquestion et al. 2005; Kung et al. 2007; Woo et al.

2008; Loth et al. 2010). The Urban area as a significant risk factor may also be due to poultry and road density here, that had a statistically highly significant correlation with HPAI outbreaks in the poultry farms. These findings were in agreement with the results reported by Yuni et al.

(2010).

Farms having no fences around farm were significantly at higher risk (OR= 9.75; P<0.05) of getting AI. Healthy biosecurity measures like farm fence that can keep rodents, stray and wild dogs and other animals away from the highly susceptible birds prevent mechanical transmission

(Beeler et al. 2009; Songserm et al. 2006), that potentially limits acquaintance with wild birds

(Alexander et al. 2000; Boon et al. 2007). Candidate variables i.e. use of disinfectants; workers wearing disinfected boots and changing cloths prior entering the sheds, footbath at the farm entrance were found significantly associated with AI outbreaks as protective factors (OR=<1;

Discussion

P,0.05). These findings were in consent with the results of (Nishiguchi et al. 2007; Abbas et al.

2011). Wild birds entry into the farm was recorded as a significant risk factor (OR= 12.66;

P<0.05) for the HPAI outbreaks, that may contaminate the sheds directly if allowed access, they act as biological and/or mechanical vectors. They play an important role especially in introducing viruses into new areas (Si et al. 2013; Alexander et al. 2000). Non disinfected water was associated with AI spread and occurrence (OR=2.094; P<0.05). Infected water may be a source of infection into the poultry farms as suggested by research to date that water being contaminated with H5N1 feces could serve as a potential source of infection for poultry farms

(Brown et al., 2007). Method of feeding was found statistically associated (OR=2.16; P<0.27) with higher risk of AI. It may be due to the frequent visits of workers changing feeding trays that may put birds in stress in large commercial poultry farms and also can act as a mechanical source of virus transmission. Other managemental factors reported in a previous studies were also identified and evaluated in this study including the presence of automatic ventilation pads

(Mcquestion et al. 2005), proper dead birds disposal (Mcquestion et al. 2005; Abbas et al. 2011), vehicles entering into the farm premises (Biswas et al. 2008), rodents density on the farm premises (Buscagia et al. 2007). Though several of these factors were strongly associated (P<

0.05) with AI infection in the analysis but did not prove to be risk factors in the final model.

Epidemiological tracing shows that persistent visiting vehicle into the farm premises for various purposes could be the source of new virus introduction into the poultry farms (Biswas et al.

2008; Capua et al. 2003).

The risk of keeping pet birds at farm premises (OR=0.82; P<0.05), raising backyard poultry (OR=0.46; P<0.05) and keeping pet animals at same premises (OR=0.17; P<0.05) were also studied. The association was strongly present but it does not showed direct effect on the

Discussion occurrence of HPAI H5N1 infections in poultry farms, though they had a protective effect that can be seen from there OR values. These findings were in contrast to that of Bavinck et al.

(2009) reporting that these factors as high risk for AI infection poultry farms. Similarly

Backyard poultry was also reported to be an imperative source of persistence and spread of

HPAI (H5N1) in South East Asia (Tiensin et al. 2005). The difference may be attributed to agro ecological, geographical and difference in production systems in Pakistan. Using disinfectants at farm entrance was found as a protective factor against HPAI infections in the poultry farms.

These findings were in line with that of (Brown et al. 2007) but in contrast to the results of

(Henning et al. 2009a). These variations showcase the fact that using disinfectants special care must be taken i.e. proper handling of disinfectants, use of appropriate disinfectant and in proper doses as per manufacturers recommendations.

The commercial poultry farms house system and design was also recorded as a highly significant (OR=10.074; P<0.05) risk factor. Poultry farms having an open house system were

10.07 times at greater risk of HPAI H5N1 infection that those having semi closed housing system. That may be attributed to lot of factors i.e. in open house system it’s difficult to manage the biosecurity levels of such standards as per recommended by OIE. There may be easy access for wild birds, animals and visitors without any restriction. These findings were in accordance to those of (Biswas et al. 2009; Vong et al. 2009 and Brown et al. 2007). Between commercial poultry farms in open house system the short buffer distance also elevates the risk of AI spread as reported by (Abbas et al. 2011). Distance to the nearest infected farm of 1 km is also associated with increases risk of AI. Where open house system is highly susceptible to new viruses and could easily acts as a potential source of AIV spread as also suggested by (Brugh et al. 1987). In such circumstance having an infected poultry farm as a neighbor was a high risk

Discussion factor for HPAI H5N1 infection in the poultry farms studied here. The established risk factors discussed here are inherently behavioral those could be customized by human decisions.

Considering the fact that location of both case and control farms are in the same territory. For risk factors the difference observed here between infected and non infected farms ruled out the impact of geography as determining factor of the infection.

3. Objective (Serology on H5N1, H9 and H7 in live bird market)

In Asian countries LBMs remains the main source of HPAI H5N1 i.e. in Hong Kong

(Shortridge et al. 1977), Vietnam (Guan et al. 2000) and in china (Cameron et al. 2000). LBMs are widespread in developing countries. Mixing domestic poultry different species (water fowl and terrestrial poultry) in LBMs is a common practice in Pakistan. Where domestic and wild birds imported or caught are kept in very close proximity that imposes a higher cross contamination risk. This facilitates virus evolution and dissemination.

The present longitudinal survey was conducted for a period of one year to detect all the

H5, H9, and H7 seasonal patterns and incidence in LBMs. Total of 929 sera samples were taken.

Out of total (n=929) 98 samples were found with zero sero conversions. Geometric mean titre

(GMT) (mean=2.65-4.32) against H5N1 was found same throughout the study period. In

February the highest GMT was recorded. Against H7 GMT ranging (1.59-0.87) was recorded in572/929 sera samples. While GMT against H9 was recorded in 929 samples no zero conversion was recorded against H9 in a single sera sample. The results revealed the highest

GMD against H9 and lowest against H7. Which confirms the circulation of H9 and H5 in LBMs and shows its strong association of outbreaks in commercial poultry farms? Against H9 highest

GMT was recorded in the month of March (8.17) and lowest (6.0) in October. These results shows that LBMs in the study year studied are infected with HPAI H5N1 and H9N2. The

Discussion presence of an elevated core is most likely to pose substantial challenges for the control of either

HPAIV H5N1 or H9N2 provided flow of infected poultry through LBMs. Provided the results they highlight the LBMs role in Pakistan in facilitating the live poultry seasonal movements between different parts of the territory. Similar observations were also put forward by (Soares et al. 2012). LBMs once contaminated may even serve as a reservoir for viruses especially in mix live birds market (Fournie et al. 2011; Fourine et al. 2012) such type LBMs exists in Pakistan.

LBMs contacts with commercial poultry farms in terms of trader movements play a vital role in spread of AI (Davis et al. 2010; Kim et al. 2010).Several studies conducted in recent past recorded LBMs as a probable mechanism for maintenance of infection for longer periods of time that poses additional risk for human exposure as well as disease spread (Bulaga et al., 2003; Choi et al., 2005 and Wang et al., 2006).

No H5N1 virus was isolated though sero conversion against H5 was recorded. H9N2 was isolated both from feacal and water samples. These findings were similar to the findings of (Wan et al. 2008). We could not succeed to isolate any Avian Influenza virus strain from quails. That may be due to the shorter stay of quails at LBMs. These results were in contrast to the study of

(Davis et al. 2010). An elevated percentage of positive nasopharyngeal samples as compared to cloacal swabs for influenza infection are a rise of concern regarding mode of transmission for these viruses. As reported, that several H9N2 virus subtypes can transmit via direct contact of healthy bird with infected ones (Shi et al. 2010; Wan et al. 2008). The high prevalence of subtype H9N2 in LBMs may be of major concern to public health because many H9N2 subtype could cause infection in humans as reported by (Matrosovinch et al. 2001; Wan et al., 2007).

Current surveillance efforts reveal H9N2 as a primary subtype circulating in LBMs (Lee et al.,

2010; moon et al., 2010)

Discussion

The findings of this study will contributes to better understanding of LBMs trading of poultry and partially unveils the role of LBMs in the occurrence and persistence of H9N2 and

HPAI H5N1, thus contributing new prospects for disease control strategies. From a policy point of view, perhaps a strategic response could be made to isolate the live chicken sales and minor poultry (Ducks, geese, fancy birds, imported birds and pet animals). That will have an extra benefit of preventing the cross species infection in poultry.

100

CHAPTER 7

SUMMARY

The poultry sector in Pakistan is the second largest industry that contributes to the national gross domestic products (GDP) and remains a major source of nutrition (protein and energy) for human population in Pakistan. Highly Pathogenic Avian Influenza (HPAI) outbreaks due to

H5N1 virus in poultry have been recorded in over 62 countries, indicating the contagious nature of the disease and its potential to infect various avian species. These HPAI outbreaks in poultry have lead to killing/culling of around 120 million birds in various countries. During 2009, the

Avian Influenza continues to occur in poultry in China, Hong Kong, India, Egypt, Nepal,

Bangladesh and Canada . In Pakistan, an HPAI outbreak due to H7N3 virus was first observed in

1994-95 and those due to H9N2 virus in broiler and layer chickens were recorded between late

1990’s and early 2000. During the period between 2006 and 2008, poultry heavily suffered due to multiple outbreaks caused by H5N1 virus.

The country experienced several and severe HPAI subtype H5N1 outbreaks during 2006-2008 in commercial poultry farms mostly, causing mass economic losses. In Pakistan all the four poultry production system exists being identified by FAO. The present study was conducted in peri- urban areas of ICT Islamabad, capital of Pakistan. The objectives of the present study were to investigate the outbreaks due to HPAIV H5N1 in 2006-2007 in ICT and identify the pattern and trends of these outbreaks. For this purpose descriptive epidemiological study was conducted and data was collected on a predesigned questionnaire regarding farm demography, culling, morbidity and mortality. The result statistical analysis showed a significantly (P< 0.05) higher

101

Summary morbidity, mortality, case fatality and culling rate in layers farms than breeders and broilers respectively. Layers and breeders of old ages were mostly affected with having higher mortality and culling in comparison to younger age layer and breeder commercial farms. The mean morbidity and mortality rates ranged 57–95% and 5-43% correspondingly.

After the HPAIV H5N1 first reported outbreak in Pakistan in 2006 culling strategy was adopted after devastating outbreaks regularly reported from throughout the country. The reasons behind these emerging epidemics were unknown and several hypotheses were given birth after these outbreaks. Knowledge regarding potential risk factors responsible for HPAIV H5N1 epidemics in commercial poultry farms in Pakistan was lacking. Therefore we conducted a longitudinal cross sectional survey (1:1 matched case control study) to identify potential risk factors at farm level responsible for 2006-2007 HPAIV H5N1 infection in poultry in ICT.

Information on farm characteristics, biosecurity practices and farm management were collected.

Logistic regression model on data was used to unveil the potentially associated risk factors with cases (farms confirmed HPAI H5N1 Positive). Several candidate variables were studied and investigated for association. The results multivariable logistic regression showed that farm location such as in urban area (P<0.05: OR=18.50), wild birds entry (P<0.05: OR= 12.66) and farms situated in highly dense poultry populated area (P<0.05:OR=4.50) were found significantly associated with outbreaks of HPAIV H5N1 infection in commercial poultry farms during 2006-2007 epidemics in the study area.

Live bird markets (LBMs) are essential for poultry marketing in developing countries like

Pakistan. One year active disease surveillance for influenza viruses in avian species in LBMs in

ICT area was conducted in 2011. LBMs in Pakistan are typically urban that brings together many avian species produced by different suppliers. Which make LBMs in Pakistan a potential source

102

Summary of HPAIV viruses as well as other emerging poultry pathogens i.e. new castle disease virus,infectious bronchitis etc. The results of the present surveillance data showed that seroconversion against H5N1 and H9N2 is present in LBMs bird species which were isolated from different samples like serum, cloacal, nasal samples and organ samples. This indicates the continuous threat of AIV viruses circulating in the live bird markets set up of Pakistan.

Findings of these studies will help to tailor control and prevention measure against devastating outbreaks in future regarding the local circumstances of commercial poultry farms as well as in LBMs. These studies also succeeded to unveil the true reasons behind these devastating outbreaks and their higher impact on poultry industry. Such type of surveillance programs will be useful in future to investigate several emerging diseases and outbreaks in

Pakistan and other developing countries.

103

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UNIVERSITY OF VETERINARY AND ANIMAL SCIENCES, LAHORE

Epidemiology and Public health

Supervisor: Dr. Muhammad Athar Khan

PhD Scholar: Dr. Zahida Fatima

(Questionnaire) Questionnaire# 1

Investigating the outbreaks of Highly Pathogenic Avian Influenza (HPAIV) from 2006-

2008 at ICT

Interview Date……/…../20……

Owner Name ………………………... Sex M/F

Contact number.……………………….

Farm address…………………………. District ………………………..

Demographic information of flock

 Specie Layers Broilers Breeders

 Date of outbreak specify………………………………..

 Age (weeks/days) specify …………………………………

 No of total birds at farm specify………………………………….

 No of morbid animals specify………………………………….

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 No of died (mortality) specify………………………………….

 No of culled animals specify…………………………………

 Any other information......

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UNIVERSITY OF VETERINARY AND ANIMAL SCIENCES, LAHORE

Epidemiology and Public health

Supervisor: Dr. Muhammad Athar Khan

PhD Scholar: Dr. Zahida Fatima

(Questionnaire) Questionnaire# 2

Study of risk factors associated with highly pathogenic avian influenza virus H5N1

outbreaks at commercial farms of Islamabad Capital Territory

Case Questionnaire Control Questionnaire Dated……/…../201……

Name of Farm...... Location of Farm......

Longitude...... Latitude......

Owners name......

I. Host characteristics

Age of birds………………. Sex……………………………

Type of birds………………Source of birds……………….

Total no of birds…………………………………………......

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II. Farm management and Housing pattern

 Was your farm environmentally controlled a. strictly confined...... b. Not

strictly confined but has protective wall around......

 Open without any protective wall a. Free range smallholding...... b. Other please

specify......

 House system a. Open b. Close c. Semi close

 Capacity of farm a. <5000 b. 10000 c. 25000 d.50000 e.100000

 Fences around farm a. Yes b. No

 Boundary wall/barbed wire a. Yes b. No

 Entry of wild birds a. Yes b. No

 Entrance at farm a.Yes b. No

 Farm situated in high poultry density area. a. Yes b. No

 Farm situated in urban area a. Yes b. No

 Ventilation of far by using cooling pads a. Yes b. No

 Farm having all in all out production system a. Yes b. No

 Rising of backyard poultry at the farm a. Yes b. No

 Keeping of pet birds at the farm a. Yes b. No

 Keeping of pet animals at the farm a. Yes b. No

 Farm is accessible to the stray and wild animals a. Yes b. No

 Disposal of dead birds by a. Burial b. Burning c. Throwing in the open d. Any

other

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 If yes then which kind of water source a. Pond b. River c. stream d. Well

III. Sources of feed and Water

 Type of feed a. Mash b. Crumbs c. Pellets

 Storage of feed at farms a. Yes b. No

 Method of feeding a. Automatic b. Manual

 Source of drinking water a. Well b. Bore c. Ponds d. Rain water

 Methods of drinking a. Nipple b. Drinker

 Is the drinking water disinfected a. Yes b. No

 Is drinking water stored at the farm? a. Yes b. No

 If yes is it properly covered? a. Yes b. No

 Place of storage of water at the farm a. Yes b. No

 Egg collection system a. Automatic b. Manual

IV. Biosecurity at farm

 Display of biosecurity signs a. Yes b. No

 Use of disinfectant at the point of entry of farm a. Yes b. No

 Name of the disinfectant used a. Yes b. No

 Methods of disinfection......

 Methods of disinfection at the farm a. Fumigation b. Chemical disinfectants c.

burning.

 Vehicles can enter at the farm a. Yes b. No

 Is the vehicle disinfected before entry at the farm a. Yes b. No

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 Do the workers change rubber boots or soaked them in water foot baths before

entry in the sheds a. Yes b. No

 Do the workers change clothes before entering of bird shed a. Yes b. No

 Do workers use gloves a. Yes b. No

 Do workers us masks a. Yes b. No

 Do the workers take shower before entering the farm a. Yes b. No

 Do the visitors take the shower bef0re entering the farm. a. Yes b. No

 How frequently unwanted visitors/guest visited at the farm a. not at all b. Less

frequently c. Quite frequent

 Do any vet visited the farm a. Yes b. No

 Does the same veterinarian go to other farm? a. Yes b. No

 Vaccinators/ plumbers/Electrician visited your farm a. Yes b. No

 Is the same vaccinators/ plumbers/ electrician a. Yes b. No

 How many days did you keep free between two batches of birds?

V. Vaccination history

 How many types of vaccines are used at your farm specify......

 Vaccination against avian influenza has been done or not a. Yes b. No

 If yes then what subtype of avian influenza vaccine was used a. H5 b. H 7c. H9

 What is the source of all vaccine a. Local b. Imported

 AI vaccine used was a. Oil based b. Water based

 Route of vaccine a. Oral b. Sub cut c. I.M

 Dose of vaccine a. Single b. Double c. Triple

 How many shots of these vaccines are used? a. Single b. Double c. Triple

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 Name of the manufacturer of the vaccine......

VI. Public health relationship

 Was any worker at the farm become sick during the outbreak? Any respiratory

signs. a. Yes b. No

 If yes what were the signs and symptoms a. Fever b. Headache c. Flu

 Did worker took any treatment a. Yes b. No

 If yes which medicine he took ......

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