IDENTIFICATION OF AVIAN INFLUENZA VIRUS SUBTYPE H9 AT CULLING LAYER IN INDONESIA Repository - UNAIR REPOSITORY
IDENTIFICATION OF AVIAN INFLUENZA VIRUS S UBTYPE H9
AT CULLING LAYER IN INDONESIA
Siti Hajariyah Fahyuna
ABSTRACT
The aims of this research are to identify Avian Influenza Virus subtype H9 from culling layer in Indonesia and to know the percentage of this virus found based on the positive sample that collected from trachea and cloaca swab. The 350 samples of trachea and cloaca swabs from culling layer were examined by isolating on embryonated SAN chicken egg followed by Hemagglutination and Hemagglutination Inhibition using antisera H9 test. The research was conducted at Professor Nidom Foundation laboratory at Surabaya, inside the Biosafety Cabinet (BSC). The surveillance started in January 2018 and the location of sampling was taken from DKI Jakarta, West Java, Yogyakarta, East Java and South Kalimantan at year 2012 throughout 2018. The 350 samples were collected which is 10 samples each year for each regency. The result from Hemagglutinin Inhibition using antisera H9 showed, 3 positive sample in year 2012 (1.7%), 0 samples in year 2013 throughout 2015 (0%), 16 sample from in year 2016 (4.5%), 0 sample in year 2017 (0%) and 3 samples in year 2018 (1.7%) were positive, indicating the presence of potential Avian Influenza virus subtype H9 in culling layer.
Keyword: Identification, Avian influenza, H9, Culling layer, Indonesia
viii
ACKNOWLEDGMENT
I would like to acknowledge my gratitude to Allah subhanahu wa ta’ala for blessing, love, opportunity, health, and mercy to complete this undergraduate thesis. This undergraduate thesis entitled IDENTIFICATION OF AVIAN
INFLUENZA VIRUS SUBTYPE H9 AT CULLING LAYER IN
INDONESIA can be finished. Shalawat and salaam always dedicated to Prophet Muhammad S.A.W., who lead us to the right path and bring us from the darkness to the lightness.
With this opportunity, I would like to convey my heartfelt gratitude to all people who have been always motivating and helping me throughout this research, especially to: Prof. Dr. Pudji Srianto, drh., M.Kes. as the Dean of the Faculty of Veterinary Medicine, Universitas Airlangga, for the given opportunity, so I can carry out the education in Bachelor program Study in the Faculty of Veterinary Medicine, Universitas Airlangga.
The supervisor committee, Prof. Dr. Rahaju Ernawati, M.Sc., drh and Dr. Kadek Rachmawati, drh., M. Kes. as co-supervisor for the time, patience, advice, precious lesson and guidance, that has been given to me until the completion of this thesis. The examiner committee, Head of Assessor Prof. Dr. Chairul Anwar Nidom, drh., Ms and as the research leader for all the support, advice, facility and accommodation in guiding me to complete this thesis, the examiner secretary Dr. Eduardus Bimo Aksono H, drh., M. Kes., and examiner member Adi Prijo Raharjo, drh., M. Kes., for all the guidance and advice that has been given to me.
ix Dr. Kadek Rachmawati, drh., M. Kes., as the academic advisor who has always been patient and giving me courage and support throughout all these semesters thru my thesis.
All the Professor Nidom Foundation (PNF) staff, Mbak Ire, Mbak Anis, Mbak Ana, Mas Khalim and all the other staff that I can't say one by one and research team for their support and help for my research.
To my beloved parents, Haruna Djawaru and Connie Francis Daimboa for their endless love, patient, inspiration, pray, material and moral support throughout my studies. My older brother Fahrun Revak Djawaru, my older sister Mukrima Fauriska Djawaru and my sister-in-law Dian Sandra Dewi who always giving me strength, inspiration, advice and fund me to accomplish my ambitions.
My best colleagues, Shendy Canadya Kurniawan, Nina Sagitha Pratiwi, Reni Ramadhani, and all international class members for the supports and meaningful days that we spent together and thanks to everyone that cannot be mentioned one by one for all the help, encourage, and motivation that given to the author.
My best friend thru thick and thin, Nisha Anggraeny, Rustikanti Ayu Ningrum and Dian Lastriana for the love, support, and motivation.
My laboratory and research mate, Azrina Khalida Imani Abbas, Kartika Buana Sari, Indahsari Ahmed and Nur Shabrina for support, motivation, guidance, and all the hard work we’ve been through together to finish this research.
My research partners who always guide me patiently Ratna, Fariz, Shendy, Mijar, Dhea, Afifatus, Azrina, Eka, Indah, Kartika, Adi, Alut, Balqis, Nur Shabrina
x and Deva for great teamwork and meaningful days that we spent together doing our research.
Surabaya, July 2018 Author
xi
xii
CONTENTS
2.1.6. Clinical sign of Avian Influenza Virus .......................................... 13
3.3.1 Material ......................................................................................... 22
3.3 Material and Equipment of Research ..................................................... 22
3.2 Time and Place of Research .................................................................. 22
3.1 Research Design.................................................................................... 22
CHAPTER 3 MATERIALS AND METHOD .................................................... 22
2.5 Hemagglutinin Inhibition Assay Test ................................................... 20
2.4 Hemagglutinin Assay Test ................................................................... 20
2.3 Antigen and Antibody ........................................................................... 19
2.2.2 Swab Trachea and Cloaca .............................................................. 18
2.2.1 Scientific Classification of Chicken .............................................. 16
2.2. Overview of Poultry ............................................................................. 16
2.1.9. Control and Prevention Avian Influenza ....................................... 15
2.1.8. Different Diagnoses ...................................................................... 15
2.1.7. Diagnoses ..................................................................................... 13
2.1.5. Transmission of Avian Influenza .................................................. 11
ENDORSEMENT FORM .................................................................................... ii DECLARATION ................................................................................................ iii
2.1.4. Cycle Infection ............................................................................. 10
2.1.3. Avian Influenza Virus Subtype H9 ............................................... 10
2.1.2 Characteristic of Avian Influenza ..................................................... 7
2.1.1 Etiology and Morphology Avian Influenza Virus ............................. 6
2.1 Avian Influenza ...................................................................................... 6
CHAPTER 2 LITERATURE REVIEW ............................................................... 6
1.6 Hypothesis .............................................................................................. 5
1.5 The outcome of Research ........................................................................ 5
1.4 The aims of Research .............................................................................. 5
1.3 Theoretical Basis ..................................................................................... 3
1.2 Statement of the Problem ........................................................................ 3
1.1 Background ............................................................................................. 1
CHAPTER 1 INTRODUCTION .......................................................................... 1
IDENTITY ......................................................................................................... iv SUMMARY ....................................................................................................... vi ABSTRACT ...................................................................................................... vii ACKNOWLEDGEMENT .................................................................................. ix LIST OF CONTENTS ....................................................................................... xii LIST OF TABLES ............................................................................................ xiv LIST OF FIGURES ........................................................................................... xv LIST OF APPENDICES ................................................................................... xvi ABBREVATION AND SYMBOLS ................................................................ xvii
3.3.2 Equipment ..................................................................................... 23
xiii
3.5 Data Analysis ........................................................................................ 29
6.1 Conclusion ............................................................................................ 41
CHAPTER 5 DISCUSSION .............................................................................. 36 CHAPTER 6 CONCLUSION ............................................................................ 41
4.4 Graphic of Positive H9 .......................................................................... 34
4.3 Percentage of Positive Sample According to Year ................................. 34
4.2 Hemagglutination Inhibition using Antisera H9 test Positive Result ...... 33
4.1 Hemagglutination Test Positive Result .................................................. 32
CHAPTER 4 RESEARCH RESULT ................................................................. 32
3.6 Research Flow Chart ............................................................................. 30
3.4.9 Hemagglutination Inhibition (HI) Test using Antisera.................... 28
3.4 Research Methods ................................................................................. 23
3.4.8 Retitration of 8 HA Unit Antigen ................................................... 28
3.4.7 Hemagglutinin Assay Test ............................................................. 27
3.4.6 Preparation of 0.5% Chicken Erythrocyte Suspension .................... 26
3.4.5 Harvesting of Embryonated Chicken Eggs ..................................... 25
3.4.4 Inoculation of Embryonated Chicken Eggs .................................... 24
3.4.3 Sampling Handling in Laboratory .................................................. 24
3.4.2 Sample Obtaining and Handling .................................................... 23
3.4.1 Location of Sampling .................................................................... 23
6.2 Suggestion ............................................................................................ 41 REFERENCES .................................................................................................. 42 APPENDICES ................................................................................................... 47
LIST OF TABLES
Table Pages
2.2 Characterize of laying hens .......................................................................... 18
4.1 Hemagglutination Test Positive Result ......................................................... 32
4.2 Hemagglutination Inhibition using Antisera H9 test Positive Result ............. 33
xiv
LIST OF FIGURES
Figures Pages
2.1 Structural of Influenza A virus . ...................................................................... 7
2.2 Gallus gallus domesticus .............................................................................. 16
3.1 Research Flow Chart .................................................................................... 30
4.4 Positive sampel of H9 virus .......................................................................... 35
xv
LIST OF APPENDICES
Pages
1. The titer of HA and HI using antisera H9 test result. ...................................... 47
2. Hemagglutination Assay test schematic ......................................................... 52
3. Hemagglutination Inhibition test using antisera of H9 schematic .................... 54
4. Documentation............................................................................................... 56
xvi
ABBREVATIONS AND SYMBOLS
xvii
ºC = Celcius µ l = Microliter AIV = Avian Influenza Virus EDS = Egg Drop Syndrome ELISA = Enzyme Linked Immunosorbent Assay FAO = Food and Agricultural Organization HA = Hemagglutination HI = Hemagglutination Inhibition HPAI = Highly Pathogenic Avian Influenza
IB = Infectious Bronchitis
IBD = Infectious Bursal Disease
ILT = Infectious Laryngo Tracheitis
IU = International Unit
IUCN = International Union for Conservation of Nature LPAI = Low Pathogenic Avian Influenza M = Matrix M1 = Matrix Protein 1 M2 = Matrix Protein 2 ml = Mililiter mRNA = Messenger Ribonucleic Acid N/NA = Neuraminidase ND = Newcastle Disease NP = Nucleoprotein NS1 = Nonstructural Protein 1 NS2 = Nonstructural Protein 2 OIE = World Organization of Animal Health PA = Polymerase Component PB1 = Polymerase Component 1 PB2 = Polymerase Component 2 PBS = Phosphate Buffer Saline PCR = Polymerase chain reaction pH = Potential of Hydrogen RBC = Red Blood Cell RNA = Ribonucleic Acid Rpm = Rotation per Minute RT-PCR = Reverse Transcription Polymerase Chain Reaction SAN = Specific Antibody Negative ssRNA = Single-stranded RNA WHO = World Health Organization
IR
- – PERPUSTAKAAN UNIVERSITAS AIRLANGGA
CHAPTER 1 INTRODUCTION
1.1 Background
Avian influenza diseases became a special concern as health issues that spread and threaten some countries such as China, Europe, Thailand, Vietnamese and also Indonesia. In Indonesia, it remains enzootic for Avian Influenza Virus. The first outbreaks that happened in Indonesia were H5N1, this influenza was reported in November 2003, and now the virus has spread to 32 out of the 33 Indonesian provinces, affecting both intensively farmed birds as well as backyard chickens.
Influenza viruses are classified into 3 types, those are type A, B and C. Influenza type A is one of the pandemic types, because it can mutate itself, either in the form of antigenic drift or antigenic shift that form new variants which are more pathogenic. AI is an influenza disease that infected poultry, such as chicken, duck, and bird species (Nidom, 2010; Nidom et al., 2012; Neny and Herra, 2014).
Avian Influenza divided into two categories, highly pathogenic avian influenza (HPAI) viruses, and low pathogenic avian influenza (LPAI) viruses. One of HPAI virus is H5N1, the infections are constantly monitored worldwide not only because of the high mortality they produce in poultry (causing great losses of poultry industry) but also because of the spread to humans and fear of a pandemic (Jong and Hien, 2006; Jackwood et al., 2012). And LPAI virus reported as H9N2, even though it is LPAI, it continues to threaten the poultry population worldwide.
H9N2 virus infection in chicken results in mild respiratory sign and egg production losses. These viruses have been reported to cause flu-like disease in human (Tosh, 2008).
In early 2017, Dirjen Peternakan dan Kesehatan Hewan, Kementrian Pertanian, I Ketut Diarmita, gave a statement that AIV subtype H9N2 was detected through a surveillance that conducted by Balai Veteriner Kementrian Pertanian in South Sulawesi, West Java, Bali, Central Java and Yogyakarta. These cases have led to a fall in egg supply by the end of 2017 (Dirjen Peternakan, 2017).
H9 first time isolated were reported in the USA in 1966 (Homme & Easterday, 1970). While in Asian countries, H9N2 is one of subtype AIV that widespread in domestic poultry (Mosleh, et al., 2009) and have been reported in various regions including Hong Kong, mainland China, South Africa, the Middle East, Europe, North America and South Korea (Alexander, 2000; Mo et al., 1997; Xu et al., 2007). Globally, there are two major, distinct gene pools of H9N2 avian influenza viruses: The North American and the Eurasian (Gua et al., 2000; Webster et al., 1992). Eurasian avian influenza subtype H9N2 virus are divided into three distinct sublineages represented by their prototype strain A/Duck/Hong Kong/Y280/97 (Y280-like), A/Quail/Hong Kong/G1/97 (G1-like) and A/Chicken/Korea/38349-p96323/96 (Korean-like) (Li C et al., 2005; Li KS et al., 2003; Guan et al., 2000).
AI subtype H9N2 cause decreased egg production, cost money for vaccination in the commercial poultry farms in Iran, also an increased mortality because H9N2 influenza virus could make chicken more susceptible to secondary infections, especially Escherichia coli infections with a mortality rate of at least 10%. In addition, the trachea or bronchi are easily embolized by mucus when the ventilation is poor, leading to severe respiratory disease and death. Mortality about
20-60% was reported in the affected broiler farms with clinical signs that were characterized as swelling of periorbital tissues and sinuses, typical respiratory discharge and severe respiratory distress (Nili and Asasi, 2003).
A case that reported in human at 1999 was two children were infected with Influenza A subtype H9 virus in Hongkong, then in 2003, there was also a report of a child infected with the same virus (Rahardjo and Nidom, 2004).
Until now the case of H9 virus has not been found in the human in Indonesia but has been found in animals such as laying hens. In some H9 cases in the world, this virus can be transmitted from animal to human. It is necessary to do this research to review the transmission of the H9 virus from laying hens to humans from live poultry market and also to study if H9 virus plays role in the decrease of egg production. This study was conducted to detect specific antigen to antibodies of H9 virus in the culling layers in the markets .
1.2 Statement of Problems
Ba sed on the background above, the problem can be formulated: “Is Avian Influenza Subtype H9 can be detected from the sample that collected in Indonesia from culling layer?”
1.3 Theoretical Basis
AIV has a characteristic the viruses are enveloped, single-stranded, negative-sense RNA viruses of the family Orthomyxoviridae, and divided into types A, B, and C. Recently have been detected there are 18 HA (Hemagglutinin) and 11 NA (Neuraminidase) subtypes of AIV. All AIV subtypes are known infect birds, except for H17N10 and H18N11, which have recently been identified in bats (Webster., et al, 1992; Tong., et al, 2012; Tong., et al, 2013).
It causes a variety of infection in avian and mammals. The H9 virus is widespread in the world and the most prevalent subtype of AI. This subtype was reported in China over the last decade. Although H9 is characterized as LPAI virus, occasional infection of human has caused great concerns in poultry that isolated from domestic birds, however, wildfowl and shorebirds are the natural hosts of AIV and they facilitate the transmission of avian influenza (Ji K et al., 2013; Olsen et al., 2006; Peng et al., 2013)
According to Kepala Sub Pengawasan Obat Hewan Direktorat Jendral Peternakan dan Kesehatan Hewan, Drh. Ni Made Ria Isriyanti, Ph.D., the current condition of the H9 virus is its spread in many provinces in Indonesia such as Java, Sumatera, Kalimantan, Sulawesi, and Bali. With H9N2 positive sample amounted up to 49. The average age of infected chicken is 30
- –60 weeks. Mortality is generally low, but a symptom like decreasing egg production up to 40-60% of the usual production, resulting in significant economic losses for farmers is one of the ways to characterize the H9 virus. (ASOHI, 2017).
The diagnosis of H9 virus in poultry can be done with a test called HA (Hemagglutination) test and will be followed by Hemagglutinin Inhibition test using Antisera H9. HA is used to detect a virus that has haemagglutinin. The presence of hemagglutinin can be seen by its ability to agglutinate erythrocytes of some species, such as poultry, mammals, and human. Antisera test used to see specific antigen H9 in certain organs suspected for AIV (WHO, 2005; Nidom, 2010).
The prevalence of H9N2 virus throughout the world, along with their ability to infect mammals and human, increases concern about their pandemic potential.
Because of the ongoing concern about the transmission of the H9N2 virus to mammals and human, continued surveillance of H9N2 virus from live poultry markets is needed (Fei Fei et al., 2009).
1.4 The Aim of Study
The aims of this study are to identify AIV subtype H9 from culling layer in Indonesia and to know the percentage of this virus found based on the positive sample that collected from trachea and cloaca swab.
1.5 Outcome of Study
The outcome of this study is to identify the AIV subtype H9 in poultry within a certain period of time in several places in Indonesia. Can also assess the development of the virus.
1.6 Hypothesis Based on the theoretical basis, researcher can conclude the hypothesis that
Avian Influenza virus subtype H9 can be detected by HA and HI using antiserum
H9 test
IR
- – PERPUSTAKAAN UNIVERSITAS AIRLANGGA
CHAPTER 2 LITERATURE REVIEW 2.1. AVIAN INFLUENZA 2.1.1. Etiology and Morphology Avian Influenza Virus Influenza viruses belong to the Orthomyxoviridae family and are divided
into types A, B, and C. Influenza types A and B are responsible for the epidemic of respiratory illness that is often associated with increased rates of hospitalization and death. Influenza A is one of the pandemic types because it can mutate themselves, either in the form of antigenic drift or antigenic shift that form new variants which are more pathogenic. All influenza viruses are negative-stranded RNA virus with a segmented genome. Influenza type A and B have 8 genes that code for 10 proteins, including the surface proteins Haemagglutinin (HA) and Neuraminidase (NA). In the case of influenza type A, virus further subdivision can be made into different subtype according to differences in these two surface proteins. Influenza virus A can be found in chicken, duck, goose, eagles, pigeon, pigs, and human. Influenza virus type B and C can be found at human (WHO, 2011; Neny and Herra, 2014; Raharjo and Nidom, 2004).
The two glycoprotein of influenza A virus, HA and NA, play essential roles during virus entry host cells and release from those cells. Influenza A virus attaches to cells through HA binding to the terminal sialic acid of glycoprotein on the surface of respiratory epithelial cells. The host range of influenza A virus is dictated mainly by their affinity for different sialo sides: avian virus preferentially binds to sialic acid linked to galactose via an α2-3 linkage, and human’s virus preferentially bind to sialic acid linked to galactose via an α2-6 linkage. And the
6 external region (exodomain) of the second transmembrane glycoprotein is NA, conducting sialolytic enzymatic and releasing the virus progeny that trapped on the surface of the infected cell. This function prevents the accumulation of virus and may also facilitate the movement of the virus in the mucous membranes of the targeted epithelial tissue. Then the virus will stick to the target. (Websters, 1992; Matrosovich et al., 1997; Rogers et al., 1983; Matrosovich et al., 2004).
2.1.2. Characteristic Avian Influenza
Influenza A has 8 segments (Figure 2.1) that encode for viral genes; Hemagglutinin (HA), Neuraminidase (NA), Matrix 1 (MA1), Matrix 2 (MA2), Nucleoprotein (NP), non-structural protein 1 (NSP1), NSP2/NEP, polymerase protein (PA), polymerase basic protein 1 (PB1), PB2-F2.
Figure 2.1 Structural of Influenza A virus ( Vincent Racanie llo , 2009)Transmembrane protein HA, NA, and M2 are the lipid bilayer of Influenza Virus A's enveloped.
AI viruses are classified into two categories, low pathogenic and highly pathogenic AIV. A virus is defined as HPAI or LPAI by its ability to cause severe disease in intravenously inoculated young chickens in the laboratory, or by its possession of certain genetic features associated with HPAI virus. AIV subtype H5 and H7 are the fully virulent virus found in nature, although there are rare examples of another virus that could technically be considered as HPAI (OIE, 2014).
HPAIV of subtype H5 and H7 have caused severe disease high mortality in poultry. Historically HPAIV H5N1 infection has resulted in the culling or death of more than 500 million poultry in more than 62 countries. During 2016 and January to February 2017, there was no case of H5N1 in humans reported in Indonesia.
HPAIV subtype H5N1 have been found to cause disease in humans since 1997. In March 2017, reported there have been cases of H5N1 in Indonesia that caused the death of 12.136 poultries consists of 1018 free-range chicken (Ayam kampung), 4618 ducklings, 2300 quails, 3985 chicken broilers, 15 turkeys and 200 laying chickens. The World Organization of Animal Health (OIE) recommends the control of HPAIV at poultry source to decrease the viral load in susceptible avian species, thereby decreasing the risk of transmission to humans (OIE, 2009; WHO, 2009; Ditjen PHK, 2017; OIE, 2007).
AIV H9N2 has become widespread among poultry in some areas and also have been detected in wild birds. This virus characterizes as LPAI. Pigs and dogs have recently detected of H9N2 virus. A serological test has been conducted in Bangladesh and China, infection was acquired in macaques in Bangladesh and wild plateau pikas in China, also pikas could be infected experimentally (OIE, 2014).
Influenza virus belongs to the segmented genome, making it easy to mutate. Mutations can occur through two processes antigenic, antigenic drift and antigenic shift. Changes in the surface of HA and NA proteins are mutations occurring on the surface of HA (5 epitopes) and NA (4 epitopes), so that when there is a change in the arrangement of the epitope or even removing the epitope on the surface of HA and NA, the antibodies in the poultry body cannot recognize the virus, or even be overcome with an existing vaccine, this process is called “antigenic drift”. Another type of change is the antigenic shift. “Antigenic shift” is a recombinant activity of two types of influenza A virus that produce new gene segments. This activity leads to antibodies that have been formed in the body cannot neutralize new viruses. The results of this recombinant will result in a new subtype that could pose a pandemic (Dirjen Peternakan, 2014).
AIV can easily die from heat, sunlight, and disinfectant (detergent, ammonium quarter, formalin 2-5%, etc.). Heat can damage the virus infectivity. In the temperature 56°C, AIV can live for 3 hours and 30 minutes for 60°C. Fat solvents such as detergent can damage the double layer of fat in the viral casing.
This virus damage envelope causes the influenza virus to be non-infective again. Other factors are acidic pH, non-isotonic and dry condit ions. Ether compounds or sodium dodecyl sulfate will disrupt the envelope, thus damaging the proteins of haemagglutinin and neuraminidase. Viral carriers come from sick chickens, birds and other animals, feed, chicken manure, fertilizers, transportation equipment, egg trays, and contaminated equipment. Also, the influenza virus can survive in water for up to 4 days at 22ºC and more than 30 days at 0ºC (Dirjen Peternakan, 2014).
2.1.3. Avian Influenza Subtype H9
Decreased egg production is one of resulted from H9 across North Africa, the Middle East, and Asia. Despite being LPAI viruses, these viruses have gained the ability to cause severe respiratory distress accompanied by high morbidity and mortality and a marked reduction in egg production That make some significant . economic losses in the poultry due to moderate to high mortality. The current circulation Eurasian H9 LPAIV has rapidly spread to become the most prevalent LPAIV in domestic poultry since their initial isolation in China during 1994 (Fusaro et al., 2011; Iqbal et al., 2009; Lee and Song. 2013; Zhang et al., 2009).
2.1.4. Cycle Infection
AI subtype H9 infection occurs when the virion spikes with specific receptors located on the surface of the host cell are attached and the virus enters the host cell. The virion will enter the cell's cytoplasm and will integrate its genetic material within the nucleus of its host cell, then the virus can replicate to form new virions and the virus can re-infect adjacent cells. Avian influenza virus can replicate in nasopharyngeal cells and in gastrointestinal cells. This virus can also be detected in the blood, cerebrospinal fluid and feces (Hidaningrum et al., 2016).
The attachment phase is the phase that most determines the viral infection cycle, whether the virus can enter or not into the host cell to continue the replication.
The entry influenza virus A into the host cell is through the major lipid bilayer, which is HA. Through the hemagglutinin spike, the influenza A virus binds to receptors containing sialic acid (SA) present on the surface of the host cell. Specificity of the HA molecules in binding to cell surface sialic acid receptors are different from human and avian. There are two major linkages found between sialic acid and carbohydrates they are bound to in glycoproteins; α (2,3) and α (2,6). In avian influenza viruses can recognize and bind to receptor α (2,3), while influenza viruses in human can recogniz e α (2,6) (Hidaningrum et al., 2016; Samji, 2009).
Upon HA’s spike binding with host cell's sialic acid residues, receptor- mediated endocytosis occurs and the virus enters the host cell in an endosome. With low pH in endosome (5-6), it triggers fusion of the viral and endosomal membrane. The low pH also opens M2 channel. M2 is a type III transmembrane domain from a channel that acts as a protein-selective ion channel. Opening the M2 ion channels acidifies the viral core. This from M1 such that vRPN (PB1, PB2, PA) is free to enter the host cell. The release of vRPN, influenza virus will transcript and replicate itself in host cell’s nucleus. After that, all the viruses have to do is form viral particles and leave the cell. Influenza virus is known as an enveloped virus, so it uses the host cell's plasma membrane to form the viral particles that leave the cell with exocytosis and go on to infect neighboring cells (Samji, 2009).
The damage caused by avian influenza comes from one of the following three processes (1) the direct process of viral replication in cells, tissues and organs, (2) indirect effects of cell mediators such as cytokines, (3) ischemia (insufficient blood supply) due to the presence of blood clots (thrombus) in the heart and blood vessels (Raharjo and Nidom, 2004).
2.1.5. Transmission of Avian Influenza Wild waterfowl are considered the natural reservoir of all influenza A virus.
They have probably carried influenza virus, with no apparent harm, for centuries.
The highly pathogenic avian influenza (HPAI) spreads very rapidly through poultry flocks, causes disease affecting multiple internal organs, and has a mortality that can approach 100%, often within 48 hours (WHO, 2007).
Transmission can occur through direct contact of infected poultry and sensitive birds through the respiratory tract, conjunctiva, mucus, and feces; or indirectly through dust, feed, drinking water, officers, cage equipment, shoes, clothing and vehicles that contaminated with AIV and live chicken infected.
Waterfowl such as ducks and geese can act as carriers without showing clinical symptoms. Direct is presently considered the main route of human infection. The most cases in human that got infected by avian influenza because there are many households keep small poultry flocks, and the poultries can roam freely entering homes or sharing outdoors area where children play. As infected birds shed large quantities of virus in their faces, the environments easily contaminated by the virus.
).
It mostly happens in rural areas (WHO, 2007 Waterfowl usually serves as a source of transmission to a chicken or turkey farm. Transmission vertically or continentally is not known, because there is no scientific or empirical evidence. The incubation period is varying from several hours to 3 days in an individually infected poultry or 14 days in floc.
Migratory birds, humans, and equipment are regarded as risk factors for the entry of the disease. Bird markets and gathering traders also play an important role in the spread of the disease. Viral carriers come from sick chickens, birds and other animals, feed, chicken manure, fertilizers, transportation equipment, egg trays, and contaminated equipment. Humans spread the virus by moving and selling sick or dead birds (Dirjen Peternakan, 2014).
2.1.6. Clinical sign of Avian Influenza Virus
Clinical symptoms seen in chickens HPAI sufferers include, comb, eyelids, feet, and abdomen that is not overgrown feathers look purplish blue (edema and cyanosis), discharge from the eyes and nose, swelling of the face and head, diarrhea, coughing, sneezing, and snoring, those are some clinical sign of neurological from HPAIV. Decreased appetite, decreased egg production and mushy eggshell. The presence of bleeding in the legs of red spots (petechia) or commonly called a foot scrap. No signs are pathognomonic but death occurs quickly (Dirjen Peternakan, 2014; OIE, 2014).
According to Dirjen Peternakan dan Kesehatan Hewan, Kementrian Pertanian, I Ketut Diarmita, H9N2 virus is a type of avian influenza virus that is low pathogenic Avian Influenza (LPAI), although not deadly, it can cause decreased immune to the poultry and damage to some organs. Because it can lower the immune in poultry, it causes the infection along with other infectious diseases such as Newcastle Disease (ND) or better known as Tetelo, Infectious Bronchitis (IB) and Egg Drop Syndrome (EDS) it can lead to decreased egg production (Dirjen Peternakan, 2017).
2.1.7. Diagnoses
LPAIV cannot be diagnosed on bases of the spectrum of clinical signs, whereas it can be diagnosed by comparison of weight gain of the infected birds. But based on Subtain et al (2011) research, all birds were normal in control group while in the infected group most of the birds showed slight depression with low intake of feed and water between 2 to 7 days PI of H9 virus. Among these birds, 4 birds suffered from diarrhea while 3 birds revealed depression on 5th day PI. All the birds were recovered from depression after 7 days PI while diarrhea persisted up to 12th day PI. No mortality was observed among the birds.
Subclinical infections or mild illnesses in poultry and other poultry are common in LPAI viruses. Decreased egg production and quality, the respiratory sign like sneezing, coughing, ocular and nasal discharge and swollen infraorbital tissue, lethargy, decreased consumption feed and water or somewhat increased flock mortality rates seen in chickens and turkeys (OIE, 2014).
AIV subtype H9 is one of LPAI, which result from the same decrease in the feed consumption possible cause of reduction in weight could be effect of viral infection on pancreatic tissue which results in decreased production of pancreatic enzymes essential for efficient digestion (Silvano et al., 1997; Shinya et al., 1995).
While on the gross pathology, all visceral organs were found normal with no abnormal gross changes in the control group while in infected birds only slight hyperemia and congestion was observed in trachea and lungs in two birds each which were slaughtered on 5th and 9th day PI. Kidneys were in six out of 14 birds.
The frequency of changes was 43 % in kidneys while only 10% in trachea and lungs (Subtain et al., 2014).
Isolation virus by inoculation at embryonated chicken egg for detecting a property of red blood cells precipitation by Hemagglutinin Assay (HA) test, or by ELISA (Enzyme-Linked Immunosorbent Assay) for serologic test, if the test is positive, then confirm for subtypes by using serum specific for H9 by Hemagglutination Inhibition test (HI test) to detect the inhibition of red blood cells precipitation. Reverse Transcription Polymerase Chain Reaction (RT-PCR) or genetic sequencing as confirmation test to determine the presence of virus (National Bureau of Agricultural, 2008; Suwarno et al., 2006).
2.1.8. Different Diagnoses
Avian influenza is often confused with Newcastle disease (ND), Infectious Bronchitis (IB), Fowl Cholera, infectious laryngotracheitis (ILT), duck plague, acute poising, bacteria cellulitis and Escherichia coli infections. These diseases are common in HPAI and are mistaken for hemostasis in the wound and comb accompanied by high mortality. Egg Drop Syndrome is one of LPAI different diagnoses for AIV subtype H9 (Dirjen Peternakan, 2014; Werner and Harder 2006; Dirjen Peternakan 2017).
2.1.9. Control and Prevention of Avian Influenza
There are several control and prevention to reduce the virus spreading. H9 is one of LPAI, which mean it won’t harm human as HPAI like H5 or H7. But a good control of the virus can help the farmers from losing more productivity from their poultry.
Based on Kepdirjennak No: 17/Kpts/PD.640/F/02.04 there are 9 strategies to control Avian Influenza biosecurity, selective poultry destruction in infected areas, vaccinations, traffic controls that include strict regulation of live poultry income and expenditure, surveillance and traceability of infection sources, community awareness raising, poultry restocking, stamping out in newly contracted areas and monitoring (Dirjen Peternakan, 2014).
2.2. Overview of Poultry
Wild bird is the main host of AIV, but occasionally, the virus can spread from its natural reservoir to poultry. AIVs in the wild bird is generally poorly adapted to domestic Galliformes (chickens, quail, partridge), but as a condition permit, the virus can be transmitted and adapt to the new host. Mostly wild bird does not show clinical sign infection with AIVs. AIVs known can replicate in cells of both the respiratory and intestinal tracts, but in ducks, they are reported to favor the intestinal tract. Live bird market (LBM) is a potential source of human infection with AIV. In LBM we can find many waterfowl species to bought. These waterfowl species play an important role in AIV transmission and are regardless as a natural reservoir of AIV (Lou et al., 2017; Wang et al., 2017).
2.2.1. Scientific classification of chicken
Figure 2.2 Gallus gallus domesticus (OliBac, 2014) Classification chicken (IUCN, 2003) is: Kingdom : Animalia Phylum : Chordata Class : Aves Order : Galliformes Family : Phasianidae Genus : Gallus Species : Gallus gallus Subspecies : G. gallus domesticus Laying hens are adult female chickens that are kept to be taken the eggs.Females over one-year-old known as hens and younger females as pullets although, in the egg-laying industry, a pullet becomes a hen when she begins to lay eggs at 16 to 20 weeks of age. Laying hens are very efficient to produce eggs and start laying eggs around ± 5 months with 250 eggs each year of production. (Rasyaf, 2008).
A good layer will have a large, smooth, moist, almost white vent. The two small bones at the sides of the vent are called the pubic bones. They should be flexible and wide apart, with at least two finger widths between them (one finger width = ¾ inch). The abdomen should be deep, soft, and pliable without an accumulation of body fat. The depth of the abdomen is measured between the tip of the keel or breastbone and the pubic bones. Laying hens should have a depth of three or four finger widths (2½ to 3 inches). Characterize of laying hens (Mississippi State University, 2015): Comb and Wattles Large, bright red
Head Neat, refined Eye Bright, prominent
Eye ring Bleached Beak Bleached
Abdomen Deep, soft, pliable Pubic bones Flexible, wide apart
Vent Large, moist, bleached Chicken cull is a chicken that is not actually a broiler type but used as a meat-producing chicken derived from laying hens put aside as inferior, deform or productivity down. (Tien R. Muchtadi, et al., 2011). Culling laying hens are laying hens with low egg production of about 20 to 25% at the age of about 96 weeks (Gillespie and Flanders, 2010; Eko, et al. 2012).
2.2.2. Swab trachea and cloaca
The trachea of live birds is swabbed by inserting a dry cotton or polyester swab into the trachea and gently swabbing the wall, and the swab is placed in transport medium (WHO, 2002).
Cloacal swab was doing of lives birds by inserting a swab deeply into the vent and vigorously swabbing in the wall. The swab should be deeply stained with fecal material and is then placed in transport media (WHO, 2002).
Gallinaceous birds typically shed AI viruses in respiratory secretions, so a tracheal or oropharyngeal swab is the primary source of virus detection from chickens and turkeys. The virus replicated itself efficiently in tracheas. While waterfowl typically shed AI viruses in their fecal secretions, so a cloacal swab is the primary source of virus detection (Li et al., 2005).
2.3. Antigen and Antibody Reaction
Antibodies are immunoglobulins formed by body cells (B cells) as receptors for antigenic stimulation. All antibody molecules have four basic polypeptide chains consisting of two heavy chains and two identical light chains linked together with disulfide bonds. The part consisting of amino acids assigned to bind the antigen is called site binding antigen. Antibody titer is the antibody content measured by titration. An antigen is an alien substance that can be recognized and well-bonded by microorganisms such as viruses, parasites, bacteria, and fungi. Part of an antigen that can bind to a receptor such as an antibody called an epitope (Hidaningrum et al., 2016).
Antigen and antibody interactions are divided into two types: interaction of primary antigen-antibody and interaction of secondary antigen-antibody.
Interaction of primary antigen-antibodies is the binding of molecular level antibodies that require indicators for example with enzymes or fluorescein dyes and others. Its testing uses three techniques namely, isotope technique with RIA (Radioimmunoassay), enzyme labeling technique with ELISA and Immunofluorescence technique. interaction of secondary antigens is an interaction that can lead to precipitation and agglutination (Bijanti et al., 2015).
2.4. Hemagglutinin Assay (HA)
The hemagglutination (HA) assay is a tool used to screen cell culture or amnio-allantoic fluid harvested from embryonated chicken eggs for hemagglutinating agents, such as type A influenza. The HA assay is not an identification assay, as other agents also have hemagglutinating properties. Live and inactivated viruses are detected by the HA test. Amplification by virus isolation in embrocating chicken eggs or cell culture is typically required before HA activity can be detected from a clinical sample. The test is, to some extent, quantitative [1 hemagglutinating unit (HAU) is equal to approximately 5-6 logs of virus]. It is inexpensive and relatively simple to conduct. Several factors (quality of chicken erythrocytes, laboratory temperature, laboratory equipment, technical expertise of the user) may contribute to slight differences in the interpretation of the test each time it is run (Killian, 2008).
2.5. Hemagglutination-inhibition Assay (HI)