SEROLOGICAL STATUS OF NEWCASTLE DISEASE IN COMMERCIAL CHICKENS IN PROVINCES OF SOUTHERN VIETNAM

The thesis title “Serological Status of Newcastle Disease in commercial chickens in some provinces of southern Vietnam” was conducted in Venky’s Poultry DiseaseDiagnostic and Nutrition L

MINISTRY OF EDUCATION AND TRAINING

NONG LAM UNIVERSITY DEPARTMENT OF BIOTECHNOLOGY

BACHELOR THESIS

SEROLOGICAL STATUS OF NEWCASTLE DISEASE IN

COMMERCIAL CHICKENS IN PROVINCES

OF SOUTHERN VIETNAM

Student : Nguyen Thi Thoi Course : 2007 - 2011

MINISTRY OF EDUCATION AND TRAINING

NONG LAM UNIVERSITY DEPARTMENT OF BIOTECHNOLOGY

BACHELOR THESIS

SEROLOGICAL STATUS OF NEWCASTLE DISEASE IN

COMMERCIAL CHICKENS IN PROVINCES

OF SOUTHERN VIETNAM

July, 2011

This chapter of acknowledgements has given me a golden occasion to convey myhearty thankfulness to all of them who have directly or indirectly contributed to thisthesis and stretched their helping hands for successful execution of my researchproject

It gives me immense pleasure to express my sincere and heartiest sense ofgratitude, abstruse indebtedness and best regards to Respected Managing Director,Uttara Feeds & Foods Pvt Ltd for giving me permission to do my research and thesisunder his lab

I take the privilege in expressing my deep sense of gratitude and indebtedness to

Dr Karnam Shiv Shankar, Manager of Venky’s Poultry Disease Diagnostic andNutrition Laboratory for noble guidance, close supervision, constructive criticism andconstant encouragement with relevant suggestions during entire period of my studyand research work

I take the opportunity of recording my heartfelt thanks to Dr Le Dinh Don, Head

of Biotechnology Department of Nong Lam University for his awe - inspiring support,constant encouragement, cooperation, precious help and charming loom during entirestudy period

It is my amusing duty to pronounce my heartily thanks to All staff of Venky’sVietnam Co., Ltd for their support, cooperation and timely help during research work

I would like to extend my sincere thanks to all my teachers and friends ofBiotechnology Department of Nong Lam University for their untiring help, neverending support and active cooperation whenever required during the entire study

I found my vocabulary to be exhausted to render my most sincere, respected anddeepest sense of reverence to my beloved parents for their eternal blessing andaffections and innumerable sacrifice, which has brought me up to this stage

Nguyễn Thị Thời

July, 2011

The thesis title “Serological Status of Newcastle Disease in commercial chickens

in some provinces of southern Vietnam” was conducted in Venky’s Poultry DiseaseDiagnostic and Nutrition Laboratory at Research Institute for Biotechnology andEnvironment, Nong Lam University from February to June, 2011

A survey on serological status of Newcastle Disease was carried out in broiler,layers and colour chickens in Vung Tau, Binh Duong, Dong Nai province of southernVietnam A total of 701 samples were collected from broiler, layers and colourchickens farms were screened for presence of antibody titers against NDV by usinghaemaglutination inhibition test Of these, 596 samples (85.0%) were positive forNDV antibody titer (HI titers ≥ log23) while 105 samples (15.0%) were negative.Positive percentage of ND - HI titers of broiler, colour chickens and layers were 77.2,80.0 and 95.5 respectively Statistical analysis by Chi - square test revealed that therewas significant difference (p < 0.05) in the prevalence of positive NDV antisera fromlayers in 6 - 18 weeks group (91.7%) and 19 - 40 weeks group (100%) There wassignificant difference (p < 0.05) in rate of sero - positive colour chickens of differentage groups 71.8%, 76.4% and 86.6% colour chickens had protective HI titers in group

of 1 - 13 days, 14 - 27 days and 28 - 63 days respectively Seroprevalence rate of NDVantibodies of broilers were 69.6% in group of 1 - 13 days, 71.9% in group of 14 - 27days and 84.8% in group of 28 - 42 days but the difference was not significant(p > 0.05) There was no significant difference (p > 0.05) in positive for specificimmunity of serum samples between different regions The result showed that the level

of protection in layers was more satisfactory than that of broiler and colour chickens.Keywords: Newcastle disease virus, Haemagglutination, Haemagglutination inhibition

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS 1

SUMMARY ii

TABLE OF CONTENTS iii

LIST OF ABBREVIATIONS v

LIST OF FIGURES, TABLES vi

Chapter 1 INTRODUCTION 1

1.1 Introduction 1

1.2 Objectives 2

1.3 Performances 2

Chapter 2 LITERATURE REVIEWS 3

2.1 Newcastle disease 3

2.1.1 History 3

2.1.2 Etiology 3

2.1.2.1 Genome of NDV 3

2.1.2.2 Important viral proteins and biologic properties 3

2.1.3 Epidemiology 6

2.1.3.1 Hosts 6

2.1.3.2 Pathogenesis 6

2.1.3.3 Sources of NDV 7

2.1.3.4 Spread of NDV 7

2.1.4 Clinical signs 7

2.1.5 Lesions 8

2.1.6 Immunity 8

2.1.6.1 Cell - mediated immunity 8

2.1.6.2 Humoral immunity 9

2.1.6.3 Local immunity 9

2.1.6.4 Passive immunity 9

2.1.6.5 Immunosuppression 9

2.1.7 Diagnostics 9

2.1.7.1 Serology 9

2.1.7.2 Virus isolation 10

2.1.7.3 Pathogenicity tests 10

2.1.7.4 Molecular techniques 11

2.1.8 Prevention 11

2.1.8.1 Vaccination 11

2.1.8.2 Biosecurity and hygiene 12

2.1.9 Zoonosis 12

2.2 Haemagglutination and hsaemagglutination inhibition test 13

2.2.1 Haemagglutination 13

2.2.2 The haemagglutination inhibition test 14

2.3 Studies about prevalence of antibody against NDV in vaccinated chickens 15

Chapter 3 MATERIALS AND METHODS 17

3.1 Time and place to do thesis 17

3.2 Materials 17

3.2.1 Subject 17

3.2.2 Equipment 17

3.2.3 Reagents 17

3.3 Method of study 18

3.4 Technical 18

3.4.1 Preparation of sera samples 18

3.4.2 Preparation of 1% CRBC 18

3.4.3 HA and HI test 18

3.5 Statistical analysis 20

Chapter 4 RESULTS AND DISCUSSIONS 21

4.1 Prevalence of antibodies against NDV in commercial chickens 22

4.2 Prevalence of antibodies against NDV in broiler chickens 22

4.3 Prevalence of antibodies against NDV in colour chickens 25

4.4 Prevalence of antibodies against NDV in layers 26

4.5 Prevalence of antibodies against NDV in commercial chickens in relation 29

Chapter 5 CONCLUSIONS AND FURTHER STUDIES 30

5.1 Conclusions 30

5.2 Further studies 30

REFERENCES 31 APPENDIX

LIST OF ABBREVIATIONS

CRBC : Chicken red blood cells

ELISA : Enzyme - linked immunosorbent assay

IBD : Infectious Bursal Disease

ICPI : Intracerebral pathogenicity index

IVPI : Intravenous pathogenicity index

MDA : Maternally derived antibody

MDL : Maternal antibody level

PBS : Phosphate buffered saline

RT-PCR : Reverse - transcription polymerase chain reaction

LIST OF FIGURES, TABLES

Page

Figure 2.1 Diagrammatic representation of Newcastle disease virus 4

Figure 3.1 Diagrammatic presentation of procedure of method of study 17

Figure 3.2 Photograph showing haemagglutination test 21

Figure 3.3 Photograph showing haemagglutination inhibition test 21

Figure 4.1 Percentage of HI titers in different broiler 24

Figure 4.2 Percentage of HI titers in different colour 26

Figure 4.3 Percentage of HI titers in different layers 28

Table 3.1 Protocol of the haemagglutination test 19

Table 3.2 Protocol of the haemagglutination inhibition test 20

Table 4.1 Distribution of NDV antibodies in commercial chickens 22

Table 4.2 Comparison of prevalence of antibody to NDV 23

Table 4.3 Distribution of HI titers against NDV in serum samples of broiler 23

Table 4.4 Comparison of prevalence of antibody to NDV 25

Table 4.5 Distribution of HI titers against NDV in serum samples of colour 25

Table 4.6 Comparison of prevalence of antibody to NDV 27

Table 4.7 Distribution of HI titers against NDV in serum samples of layers 27

Table 4.8 Comparison of prevalence of antibody to NDV in 29

Table 4.9 Comparison of prevalence of antibody to NDV in 29

Chapter 1 INTRODUCTION

1.1 Introduction

Poultry industry is an emerging agri - business and has established its position asfastest growing segment in the agriculture sector of world Poultry industry isemerging in Vietnam from seventies Many small and large scale chicken farms arerapidly growing in Vietnam Vietnam is an agriculture country with 70% of populationliving in rural and 90% of household keep poultry The growth of this profitablesubsector is interrupted by a number of infection diseases Such diseases includeNewcastle disease (ND), Infectious bronchitis (IB) andInfectious bursal disease (IBD) etc

Newcastle disease is a highly contagious and commonly fatal viral poultrydisease Newcastle Disease was discovered in Newcastle upon Tyne, England in 1926

It was widespread throughout the rest of the world Its spread is normally either vianewly introduced birds, selling or giving away sick and carrier birds ND is enzootic inmost countries in Africa, Asia and South America, where it is a major constraintagainst the development of both industrial and village poultry production(Aldous and Alexander, 2001) In Vietnam the ND is the most important causemortality in chicken (Nguyen, 1992) Every year, ND occurs in many localities and isresponsible for economic losses in poultry industry of Vietnam These losses will bedue to losses in productivity and death of poultry Vaccination is practiced widely and

is the recommended method for prevention Farmers or persons in charge ofvaccination are likely to believe that the chicken flocks will be protected aftervaccination But apparent ideal ND vaccination programs do not always guaranteeprotection of chickens flocks against ND due to incautious handling of vaccines and so

on So, seromonitoring of humoral immune response in vaccinated chicken flocks is

necessary for controlling the ND (Rahman et al., 2002) Arising from above, I

conducted a research on “Serological Status of Newcastle Disease in commercialchickens in some provinces of southern Vietnam” with the following objective incommercial poultry

1.2 Objectives

 To determine the serological status of ND in vaccinated chickens

 To investigate the influence of different factors (age, type of chickens, regions)

on NDV antibody prevalence in commercial chickens

1.3 Performance

Collection of samples from different poultry farms in some regions of southernVietnam to perform Haemagglutination test (HA)/ Haemagglutination Inhibition test(HI) to determine antibody titers against NDV

Chapter 2 LITERATURE REVIEWS

2.1 Newcastle disease

Newcastle disease has been one of the most important diseases of poultryworldwide ever since the advent of high - density, confinement husbandry systems

(Murphy et al, 1999) It is a highly contagious, generalized virus disease of domestic

poultry and wild birds characterized by gastro - intestinal, respiratory and nervoussigns

2.1.1 History

ND was the name given to a highly pathogenic disease seen in chickens inEngland in 1926 by Doyle Doyle reported the first outbreak to have occurred, in thespring of 1926, on the farm near Newcastle - upon - Tyne and hence the name Thedisease had also emerged in March 1926 on the island of Java, Indonesia Generally ithas been considered that the presence of the virus in England resulted fromtransportation to the port of Newcastle - upon - Type from South East Asia by ship,either in frozen meat or as a result of the practice of keeping live chickens on board foreggs and meat The disease also appears to have been present in Korea in 1926 Anoutbreak also occurred in Ranikhet in India in July, 1927 What cannot be excluded isthat outbreaks may have been occurred earlier elsewhere but gone unnoticed due tolack of available expertise in recognizing an apparently new disease Wherever thefirst outbreak occurred it is obvious from the literature that a highly virulent disease ofpoultry appeared within a very short time in England, Java, Philippines, India, Ceylon,Korea and Japan and that the disease was sufficiently different from other highlyvirulent disease to be recorded as distinct and recognized as the same disease.Wherever it began and however it was spread, in 1926 a new disease emerged or wasrecognized and within a few years had spread throughout the world (Alexander, 1988)

In Vietnam

In 1956, Nguyen Luong and Tran Quang Nhien had confirmed the presence of

ND in the northern provinces of Viet Nam Since then, outbreaks have been occurredyearly causing heavy losses to poultry production

a haemagglutinin (in most species with neuraminidase activity also) and a fusionprotein Replication takes place in the cytoplasm, and assembly occurs via budding onplasma membranes The viruses have narrow host ranges and have only beenidentified in vertebrates, primarily in mammals and birds Transmission is mainly by

aerosol and droplets (Murphy et al, 1999).

Newcastle disease virus is a member of the family paramyxoviridae in the genusRubulavirus There are nine serotypes of avian paramyxoviruses designated APMV-1

to APMV-9 and ND virus has been designated APMV-1 NDV has also been

categorized into five pathotypes based on clinical signs in infected chickens,designated: a) viscerotropic velogenic, b) neurotropic velogenic, c) mesogenic, d)lentogenic or respiratory and e) subclinical enteric Pathotype groupings are rarelyclear - cut.

The virus is inactivated by 56oC in 3 hours or 60oC in 30 minutes The virus isknown to be destroyed by 1:5000 potassium permanganate, 1:1000 cresol, 3%formalin and 1:5 ethanol It also is inactivated by acid, pH lesser or equal 2 It survivesfor long periods at ambient temperature, especially in faces (Chauha and Roy 2007).

2.1.2.1 Genome of NDV

The genome of NDV codes for six proteins, which have been described by

Samson (cited by Saif et al, 2003) include L protein is the RNA - directed RNA

polymerase associated with the nucleocapsid; HN is responsible for thehaemagglutinin and neuraminidase activities, forming the larger of two types ofprojections seen on surface of paramyxovirus particals; F, fusion protein, forms thesmaller of the surface projections; NP, nucleocapsid protein; P, phosphorylated,nucleocapsid - associated; and M, matrix The order of the genes for these proteins inthe virus genome is 3’N – P – M – F – HN – L 5’

2.1.2.2 Important viral proteins and biologic properties

Two proteins of NDV are inserted in the envelope They are thehaemagglutinin/neuraminidase protein and the fusion protein These two proteins areimportant in determining the virulence of the virus and how the virus infects host cells.Haemagglutination activity: the ability of NDV and other avian paramyxoviruses

to agglutinate red blood cells (RBCs) is due to the binding of thehaemagglutinin - neuraminidase (HN) protein to receptor on the surface of the RBCs.This property and the specific inhibition of agglutination by antisera have proven to bepowerful tools in the diagnosis of the disease Chicken RBCs usually are used in HAtests, but NDV will cause agglutination of all amphibian, reptilian, and avian cells.Neuraminidase activity: the enzyme neuraminidase is also part of the HNmolecule and presented in all members of the Rubulavirus genus An obviousconsequence of the possession of this enzyme is the gradual elution of agglutinatedRBCs The exact functions of the neuraminidase in virus replication is unknown, but it

seems likely that neuraminidase removes virus receptors from the host cell whichprevents the reattachment of released virus particles and virus clumping.

Cell fusion and hemolysis: NDV and other paramyxoviruses may bring abouthemolysis of RBCs or fusion of other cells by essentially the same mechanism.Attachment at the receptor site during replication is followed by fusion of the virusmembrane with the cell membrane, which may result the fusion of two or more cells.The rigid membrane of the RBCs usually results in lysis from the virus membranefusion In order for fusion to occur, the shape of the native fusion protein must bechanged This change happens when a host cell protease cleaves or cuts the protein at aspecific cleavage site After this has happened, the fusion protein is activated and cannow fuse to the membrane of the cell The sequence of the amino acids around thecleavage site determines the range of proteases that can activate cleavage of theprotein This sequence therefore determines the virulence

2.1.3 Epidemiology

2.1.3.1 Hosts

The virulence of NDV strains varies greatly with the host Chickens are highlysusceptible, but ducks and geese may be infected and so few or no clinical signs, evenwith strains lethal for chickens

2.1.3.2 Pathogenesis

In chickens, the pathogenicity of ND is determined chiefly by the strain of virus,dose, route of administration, age of the chicken, and environmental conditions allhave an effect In general, the younger the chicken is, the more acute the disease is.With virulent viruses in the field, young chicken may experience sudden deathswithout major clinical signs; however, in older birds the disease may be moreprotracted and with characteristic clinical signs Breed or genetic stock does not appear

to have a significant effect on the susceptibility of chickens to the disease Naturalroutes of infection (nasal, oral, and ocular) appear to emphasize the respiratory nature

of disease, and intramuscular, intravenous, and intracerebral routes appear to enhancethe neurologic signs

Initially the virus replicates in the mucosal epithelium of the upper respiratoryand intestinal tracts, shortly after infection, virus spreads via the blood to the spleen

and bone marrow, producing a secondary viremia This leads to infection of othertarget organs, lung, intestine, and central nervous system.

2.1.3.3 Sources of NDV

Sources of NDV are respiratory discharge, feces of infected birds and all parts ofthe carcass Virus is shed during the incubation period, during clinical stages and for alimited period during convalescence Wild birds and waterfowl may act as reservoirhosts for lentogenic pathotypes of ND; subsequently, these viruses could becomevirulent following mutation upon establishment in domestic poultry Some psittacinebirds have been demonstrated to shed NDV intermittently for over 1 year and beenassociated with introduction into poultry

2.1.3.4 Spread of NDV

The following virus sources or methods have been implicated in variousepizootics: 1) movement of live birds, racing pigeons, and commercial poultry; 2)contact with other animals; 3) movement of people and equipment; 4) movement ofpoultry products; 5) airborne spread; 6) contaminated poultry feed; 7) contaminatedwater; and 8) vaccines The importance of any of these factors will depend on thesituation in which the epizootic occurs In countries where poultry are kept exclusively

in bird proof housing, the ability of feral birds to invade affected flocks and transferthe disease will be minimal, whereas birds kept an open range are more likely to beinfected with strains carried by feral birds

2.1.4 Clinical signs

The incubation period of ND after natural exposure has been reported to varyfrom 2 - 15 days (average 5 – 6 days) The speed with which signs appear, if at all, isvariable depending on the infecting virus, the host species and its age and immunestatus, infection with other organisms, environmental conditions, the route of exposureand the dose

With extremely virulent viruses, the disease may appear suddenly, with highmortality occurring in the absence of other clinical signs In outbreaks in chickens due

to the virulent NDV pathotype, clinical signs often begin with listlessness, increasedrespiration, weakness, ending with prostration and death Green diarrhea is frequentlyseen in birds that do not die early in infection, and prior to death, muscular tremors,

torticollis, paralysis of legs and wings Mortality frequently reaches 100% in flocks offully susceptible chickens.

The neurotropic velogenic: sudden onset of severe respiratory disease followed aday or two later by neurologic signs Egg production falls dramatically, but diarrhea isusually absent Morbidity may reach 100% Mortality is generally considerably lower,although up to 50% in adult birds and 90% in young chickens have been recorded.Mesogenic strains of NDV usually cause respiratory disease in field infections

In adult birds, there may be a marked drop in egg production that may last for severalweeks Nervous signs may occur but are not common Mortality in fowl is usually low,except in very young and susceptible bird, but may be considerably affected byexacerbating conditions

Lentogenic viruses do not usually cause disease in adults In young, fullysusceptible bird, serious respiratory problems can be seen, often resulting in mortality.Lentogenic strains usually cause mild respiratory disease with coughing, gasping,sneezing and rales It can produce more severe symptoms if the flock is co - infectedwith other pathogens

2.1.5 Lesions

There are no pathognomonic gross lesions, several birds must be examined todetermine a tentative diagnosis and final diagnosis must await virus isolation andidentification Only velogenic strains produce significant gross lesions Lesions thatmay be found include: swelling of periorbital area or entire head, oedema of theinterstitial or peritracheal tissue of the neck Congestion and sometimes haemorrhages

in the caudal pharynx and tracheal mucosa Petechiae and small ecchymoses on themucosa of the proventriculus, concentrated around the orifices of the mucous glands.There have oedema, haemorrhages or degeneration of ovaries Spleen may appear

(http://www.oie.int/fileadmin/Home/eng/Animal_Health_in_the_World/docs/pdf/NEWCASTLE_DISEASE FINAL.pdf)

2.1.6 Immunity

2.1.6.1 Cell - mediated immunity

The initial immune response to infection with NDV is cell mediated and may bedetectable as early as 2 - 3 days after infection with live vaccine strains This has been

thought to explain the early protection against challenge that has been recorded invaccinated birds before a measurable antibody response is seen However, a later studyconcluded that the cell mediated immune response to NDV by itself is not protectiveagainst challenge with virulent NDV The importance of cell mediated immunity inprotection conferred by vaccines is, therefore, not clear, and a strong secondaryresponse to challenge similar to the antibody response does not seem to occur.

2.1.6.2 Humoral immunity

When chickens survive NDV infection long enough, antibodies usually aredetectable in the serum within 6 - 10 days The levels largely depend on the infectingstrain, but generally, peak response is at about 3 - 4 weeks Decline in antibody titervaries with the titer achieved but is much slower than their development.Haemagglutination inhibition antibodies may remain detectable for up to one year inbirds recovered from infection with mesogenic viruses

2.1.6.3 Local immunity

Antibodies appear in secretions of the upper respiratory tract and intestinal tract

of chickens at about the time humoral antibodies can be first detected In the upperrespiratory tract, the immunoglubulins appear to be chiefly IgA with some IgG

2.1.6.4 Passive immunity

Hens with antibodies to NDV will pass these on to their progeny via the eggyolk Levels of antibody in day - old chicks will be directly related to titers in theparent Maternal immunity is protective and, thus, must be taken into account whentiming the primary vaccination of chicks

2.1.6.5 Immunosuppression

Suppression of the immunoresponse has important effects on both thepathogenicity of infecting NDV strains and protection levels achieved by vaccination.Under natural conditions, immunosuppression may occur due to infection with otherviruses such as IBDV The subsequent immune deficiency may result in a more severedisease caused by some NDV strains and a failure to respond adequately tovaccination

2.1.7 Diagnostics

2.1.7.1 Serology

The presence of specific antibody to NDV in the serum of a bird give littleinformation on the infecting strain of NDV and, therefore, has limited diagnosticvalue Nevertheless, in certain circumstances the demonstration that infection hastaken place is sufficient for the needs of the diagnostician Postvaccinal serology can

be used to confirm successful application of vaccine and an adequate immune response

by bird In the absence of vaccination, the presence of specific antibodies against theNDV indicates that the bird has been infected by the virus at some time, but notnecessarily that it was suffering from the disease at the time of sampling In practice, ahigh antibody titer is indicative of a recent infection HI is the most commonly usedserological test Other tests include virus neutralization, ELISA

2.1.7.2 Virus isolation

Newcastle disease can be diagnosed by isolating APMV-1 from affected birds.This virus is usually recovered by inoculating samples into 9 – 11 day oldembryonated chicken eggs Choriolaallantonic fluid from the eggs is tested forhaemagglutinating activity, and any agents that haemagglutinate are examined for HIwith a monospecific antisera to APMV-1

2.1.7.3 Pathogenicity tests

The pathogenicity of the isolate can be quantified by 1) the mean death time(MDT) in chicken embryos, 2) the intracerebral pathogenicity index (ICPI) inone - day chicks, or 3) the intravenous pathogenicity index (IVPI) in six - week oldchickens In the MDT assay, velogenic isolates have an MDV of less than 60 hours,mesogenic strains have an MDV of 60 – 89 hours, and lentogenic viruses have anMDT greater than 90 hours The ICPI and IVPI tests are scoring systems that evaluateillness or death in chickens The values in the ICPI test range from 0 to 2.0, the mostvirulent viruses approach 2.0, while lentogenic strains are usually close to 0.0 Thevalues in the IVPI test are from 0 to 3.0, the velogenic strains approach 3.0, whilelentogenic strains and some mesogenic strains have IVPI values of 0.0 However,some viruses that can produce severe disease have IVPI values of 0.0, the ICPI test isgenerally preferred for this reason

2.1.7.4 Molecular techniques

Reverse - transcription polymerase chain reaction (RT-PCR), gene sequencing,restriction enzyme analysis and other molecular techniques are also used to identifyAPMV-1 in eggs or clinical specimens.

2.1.8 Prevention

2.1.8.1 Vaccination

Inactivated vaccines are produced by growing a NDV in eggs, and then treatingthe infective allantoic fluid with an inactivating agent, such as formalin orbetapropiolactone An adjuvant, such as mineral oil, is usually then added to make theinactivated virus more immunogenic Since the vaccine is no longer capable ofreplication or spread, it has to be injected individually into every bird needingvaccination It is normally injected into the back of the thigh muscle (sometimes thebreast muscle is used), using 0.3 or 0.5 ml per bird Inactivated vaccines produce veryhigh levels of antibodies against NDV, and provide good protection against thevirulent virus

In intensive poultry production, inactivated vaccines are usually applied after aninitial priming vaccination with a live vaccine Although inactivated vaccine givesgood protection, it is relatively expensive to produce It also carries a slight risk to theuser of accidental self - injection While inactivated vaccines are, to some extent, heatsensitive, they are much less so than conventional live vaccines which makestransporting them to villages more feasible

Live vaccines differ from inactivated vaccines in that they can replicate in thehost This is both an advantage and a disadvantage It is an advantage in that it is notnecessary to vaccinate every bird individually; the vaccinal virus can spread on its ownfrom one bird to another It is, however, a disadvantage in that, since an infection with

a live virus is involved, this may result in clinical signs because of the innate virulence

of the vaccine virus or by exacerbating other organisms that may be present, especially

in the respiratory tract The severity of this reaction depends therefore on the particularvaccinal strain used and the presence or otherwise of concurrent infection with otherpathogens Another advantage of live vaccines compared to inactivated vaccines, istheir ease of application as they can be applied to the drinking water or with aneye - dropper

Although NDV has essentially only one serotype, there is a wide difference inthe pathogenicity of different strains, ranging from those that cause virtually no signs

to those that kill within a few days The majority of live vaccines are derived fromasymptomatic enteric or lentogenic strains, although some vaccines derived frommesogenic strains are still in use Live lentogenic vaccines are usually derived fromfield viruses that have been shown to have low pathogenicity for poultry but produce

an adequate immune response Typical vaccine strains are HB1, LaSota and F strainand some viruses from the asymptomatic enteric pathotype, which are usually based

on the V4 or Ulster 2C viruses And mesogenic vaccine such as Roakin, Mukteswarand Komarov However, these viruses have been frequently subjected to selectionpressures by manufacturers in order to improve their immunogenicity or to enable theiruse by a particular method of application

2.1.8.2 Biosecurity and hygiene

On commercial farms, control measures should attempt to prevent viruses frominfecting the flock Biosecurity aimed at preventing disease should begin at theplanning stage of commercial poultry farms Farms and flocks should be wellseparated, hatcheries should be isolated from poultry farms, different species should bereared on different sites, and there should be an adequate fresh water supply,preferably one that does not draw on surface water On the farms the following pointsshould be observed:

• Houses, food stores and water tanks should be bird - proofed

• Movements on and off the farm should be kept to a minimum

• All equipment, especially vehicles, should be disinfected before access to thesite is permitted

• Movements between different farms for egg collection, carcass collection, fooddelivery and the like should be confined to a specified collection and delivery pointaway from the poultry flocks

Visits by personnel such as bleeding or vaccination teams, inseminators andveterinarians are the most likely method of introduction of ND and if such visits areunavoidable, regimens of clothing change, equipment disinfection and other basichygiene controls must be enforced

2.1.9 Zoonosis

NDV is a human pathogen Reported infections have beennon - life - threatening and usually not debilitating for more than a day or two Themost frequently reported and best substantiated clinical signs in human infections havebeen eye infections, usually consisting of unilateral or bilateral reddening, excessivelacrimation, oedema of eyelids, conjunctivitis and subconjunctival haemorrhage.Although the effect on the eye may be quite severe, infections are usually transient andthe cornea is not affected Reports of other clinical symptoms in humans infected withNDV are less well substantiated but suggest that a more generalized infection maysometimes occur resulting in chills, headaches and fever, with or withoutconjunctivitis There is evidence that both live vaccine and virulent (for poultry)strains of NDV may infect and cause clinical signs in human Human infections withNDV have usually resulted from direct contact with the virus, infected birds orcarcasses of diseased birds There have been no reports of human to human spread,although spread by contagion is theoretically possible The types of person known tohave been infected with NDV include: laboratory workers (usually as a result ofaccidental splashing of infective material into the eye), veterinarians in diagnosticlaboratories (presumably as a result of contact with infective material duringpost - mortem examinations), workers in broiler processing plants and vaccinationcrews when live vaccines are given as aerosols or fine dust (Mark Pattison, 2008).

2.2 Hemagglutination and haemagglutination inhibition test

2.2.1 Haemagglutination

The ability of suspensions of virus to agglutinate erythrocytes was first observedwith influenza virus, and the reaction involved has been extensively studied with thisvirus and other members of myxovirus group In the myxoviruses the reaction with theerythrocyte differs from that of some other viruses because most members of the grouppossess the enzyme neuraminidase, and the virion can be eluted, by mean of it, fromthe receptors on the erythrocyte surface Besides being of important from the purelypractical point of view, the phenomenon allows the study of the interaction of viruswith the surface of the cell in which it does not multiply, and can also be used forserological identification of viruses and for titration of antibody by means of thehaemagglutination - inhibition phenomenon The condition for haemagglutination,

e.g., pH and temperature, may be very critical, and the species of the red blood cell isimportant.

After adsorption and elution of the virus, the red blood cells have a reducedelectrophoretic mobility and decreased negative charge on the surface They areinagglutinable by the same myxovirus as before and usually by some others Byheating at 56oC under various conditions, the enzyme can be inactivated withoutgreatly affecting the ability to hemagglutinate Such virus does not elute fromerythrocytes and is known as indicator virus Inhibitors of myxovirushaemagglutination are mostly themselves mucoproteins and behave like themucoprotein on the surface of erythrocytes, they adsorb onto the virus surface, andelute from it by the action of viral neuraminidase There inhibators are nonspecific inthat they can act on a much wider variety of myxoviruses than do antibody, which arehighly strain - specific, even though some cross - reactions occur This specificity isthe basis of haemaglutination - inhibition test (Betts and York, 1967)

2.2.2 The haemagglutination inhibition test

The HI test is based on the principle that the haemagglutinin on the viralenvelope can bring about the agglutination of chicken red blood cells and that this can

be inhibited by specific antibodies U - bottomed microtitration plates are used Theserum samples are diluted in serial twofold dilutions in phosphate buffered saline andthen a fixed quantity of viral antigen is added to each well Following incubation, asuspension of red blood cells is added to each well and the plate is incubated again Inthe absence of any antibody against the virus, haemagglutination occurs, appearing as

a diffuse red colour at the bottom of the well In the wells where the antibody againstthe virus is of a sufficient level, haemagglutination is inhibited and the red blood cellssediment and appear as a small pellet at the bottom of the well The presence orabsence of agglutination is accurately assessed by tilting the plates Only those wells inwhich the RBCs stream at the same rate as the control wells (containing RBCs andPBS only) should be considered to show inhibition The HI titer is the reciprocal of thehighest dilution of serum which completely inhibits haemagglutination and is usuallyand most conveniently expressed as the logarithm to the base 2 Although the test isdifficult to standardize between laboratories, the HI titer gives an indication of theimmune status of the bird A titer of log23 is indicative of protection and a titer of