UNIVERSITI PUTRA MALAYSIA

DEVELOPMENT OF AN RT NESTED PCR-ELISA DIAGNOSTIC TEST FOR THE DETECTION OF NEWCASTLE DISEASE VIRUS

KHO CHIEW LING

FSAS 1999 32

DEVELOPMENT OF AN RT NESTED PCR-ELISA DIAGNOSTIC TEST FOR THE DETECTION OF NEWCASTLE DISEASE VIRUS

By

KHO CHIEW LING

Thesis Submitted in Fulfilment of the Requirements for the Degree of Master of Science in the Faculty of

Science and Environmental Studies, Universiti Putra Malaysia.

June 1999

This thesis is specially dedicated to

my beloved family and

friends ........ .

ACKNOWLEDGEMENTS

I would like to sincerely acknowledge the chairman of the supervIsory

committee, Associate Professor Dr. Khatijah Yusoff for her guidance and continuing

support of this project. I also wish to thank the members of the supervisory

committee, Dr. Siti Suri Arshad and Dr. Mohd. Azmi Mohd. Lila, for providing

prompt and helpful advice when required.

My appreciation goes to Dr. Abdul Rahman for providing some of the tissue

samples, Professor Dr. Aini Ideris for providing the influenza and fowl pox viruses,

Dr. Hair Bejo for providing the infectious bursal disease virus, Dr. Siti Suri Arshad

for providing the infectious bronchitis viruses and Veterinary Research Institute (VRI)

for supplying the NDV isolates. Special thanks go to Mr. Wahid for his excellent

assistance in the plaque assay. I would also like to thank Mr. Adam and Mr.

Kamaruddin for their kindly help. Many thanks are also conveyed to all the members

of the Virology Laboratory: Ainu, Alan, Chui Fung, Filza, Omeima, Pria, Putery,

S ing King, Subha, V. 1. Wei Hong for their kind help, thoughtful comments and

friendship .

My gratitude also goes out to the Universiti of Putra Malaysia for providing

the scholarship (P ASCA), and, as always, much love and appreciation to my parents

for their patience, support and sacrifices.

This study was supported by IRPA Grant No. 0 1 -02-04-0 1 07.

iii

TABLE OF CONTENTS

ACKNOWLEDGEMENTS LIST OF TABLES LIST OF FIGURES LIST OF PLATES LIST OF ABBREVIATIONS ABSTRACT ABSTRAK

CHAPTER

I INTRODUCTION Objective of the Study

II LITERATURE REVIEW General Introduction of Newcastle Disease Occurrence Importance Nature of the Virus

Virus classification Virus morphology Virus pathotype

Pathogenesis ofNDV Mode of Spread Approaches to Virus Diagnosis Molecular Amplification Technique Diagnostic Application of PCR Reaction Specificity Nested PCR Contamination of PCR ReactIOn and Prevention PCR Optimisation PCR Product Detection Method Colorimetric Detection Primer Dimerization

III MATERIALS AND METHODS Virus Isolates Virus Propagation Virus Harvesting and Haemagglutination Test Virus Purification

IV

Page

111 VII

VllI IX X

Xlll XVI

4

5 5 5 6 7 7 7 9

1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 8 20 20 2 1 23

25 25 25 27 28

IV

Titration of Virus Stock Cell culture Plaque assay

Chicken Tissue Specimens Conventional Laboratory Test

Sample processing Egg passage

RNA Extraction Allantoic fluid samples Tissue samples

Primers One-Tube Nested PCR

cDNA synthesis and first amplification reaction Second amplification reaction

Sensitivity Studies Specificity Determination Analysis ofPCR Product

Agarose gel electrophoresis Colorimetric detection (ELISA) ofPCR product

RESULTS Nested PCR Optimisation

Annealing temperature Concentration of the outer primer and . . lOner pnmers MgCh concentration Reaction cycle

Virus Titre Determination by Plaque Assay Specificity Evaluation of the RT Nested PCR Sensitivity Determination of the RT Nested PCR Standardisation of ELISA-based Colorimetric Detection Method

Optimal condition of microtiter plate ( MTP) coating Optimum enzyme conjugate concentration Optimum incubation time of enzyme conjugate-substrate reaction

Determination of Relative Sensitivity of the ELISA Detection Method R T Nested PCR-ELISA for the Detection of NDV in Tissue Samples Comparison of One-Tube and Two-Tube RT Nested PCR

v

29 29 29 30 3 1 3 1 33 33 33 34 35 37 37 38 38 39 3 9 3 9 40

4 1 4 1 4 1

42 47 47 49 52 52

55

57 57

59

62

65

69

V DISCUSSION 74 Rationale in Method Development 74 Strategies Toward Method Establishment 75

Primer design 75 PCR optimisation 76 ELISA standardisation 77

Performance of the Established R T Nested PCR-ELISA 80

Assay specificity and sensitivity 80 Determination of the Cutoff Value 82 Detection ofNDV in Tissue Samples 83 Semiquantitative Assay 86 Turnaround Time Estimation and Cost Analysis 87 Precautions in RT Nested PCR-ELISA Practices 88

VI CONCLUSION 9 1 Application, Limitation and Future Aspect 92

BIBLIOGRAPHY 94

VITA 1 03

VI

Table

1 2

3 4

5

LIST OF TABLES

List of ND Viruses Used in this Study List of Suspected NDV Infected Tissue Samples obtained from Diagnostic Virology Laboratory, Faculty of Veterinary Medicine, UPM List of Primers Used in this Study Absorbance Value (405 nm) for Suspected NDV Infected Tissue Samples Comparisons of Methods for Detection ofNDV in 3 5 Tissue Samples

VII

Page

26

32 3 5

68

71

Figure

2 3

4

5

LIST OF FIGURES

Schematic Diagram of NDV Virion Illustration of the Nested PCR-ELIS A Protocols Result of Streptavidin Titration Against Various Dilutions of Peroxidase Anti-digoxigenin Conjugate Checkerboard Titration between Peroxidase Anti-digoxigenin Conjugate and Dilutions ofPCR Product The Effect of Incubation Time of the Enzyme Conjugate-Substrate Complex on Absorbance Value

VlIl

Page

8 36

58

60

61

LIST OF PLATES

Plate Page

Outer Primers Annealing Temperature Optimisation . . .. . . . . . . 43 2 Effect of Outer Primer (FOP) Concentrations on the

Amplification of the 280 bp PCR Product by RT Nested PCR . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . .. . . . . . . . . . 45

3 Effect of Reducing the Inner Primer (FIP) Concentration . . . . 46 4 Optimisation of the Magnesium Concentration of

RT Nested PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 5 Cycle Effect in the R T Nested Amplification Reaction . . . . .... 50 6 Plaque Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 1 7 Specificity Evaluation of the One-Tube RT Nested PCR . . . 53 8 Sensitivity Determination of One-Tube R T Nested PCR . . . . 54 9 Sensitivity Testing of Non-Nested RT-PCR using Outer

Primers (FOPI, FOP2) or Inner Primers (FIPI, FIP2) only 56 1 0 Detection of Nested PCR Product by the ELISA (A) and

Agarose Gel Electrophoresis (B) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . 63 1 1 Comparison of ELISA and Gel Electrophoresis for the

Detection of Nested PCR Product. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 12 Detection ofNDV in 20 Tissue Samples by RT Nested

PCR-ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 1 3 Detection ofNDV in 1 5 Tissue Samples by RT Nested

PCR-ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 1 4 A Typical Microtiter Plate at the end of the Colorimetric Assay 70 1 5 Detection ofNDV in Tissue Samples by Conventional

Two-Tube RT Nested PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . 73

ix

ABTS

BCP

bp

cDNA

DEPC

DIG

DNA

dNTP

EDTA

ELISA

F

fg

FIP

FOP

xg

h

LIST OF ABBREVIATIONS

2,2' - azinodi-ethylbenzothiazolinesulphonate

acid

I-bromo-3 -chloropropane

base pairs

degree CeJcius

complimentary DNA

diethyl pyrocarbonate

distilled water

digoxigenin

deoxyribonucleic acid

deoxynucleoside triphosphate

ethylenediamine tetraacetic acid

egg infective dose 50%

enzyme linked immunosorbent assay

fusion

fentogram

. . Inner pnmer

outer primer

x gravity

hour

x

HA

HN

HI

kb

KCl

KH2P04

L

M

MgCI2

MTP

�I

mm

mM

Na2HP04

NO

NDV

ng

NP

NTE

P

PBS

haemagglutination

haemagglutinin-neuraminidase

haemagglutination -inhibition

kilobase pairs in duplex nucleic acid, kilobases

in single-stranded nucleic acid

potassium chloride

di-potassium hydrogen phosphate anhydrous

large

molar

magnesium chloride

microtiter plate

microliter

minute

milimolar

di-natrium hydrogen phosphate

Newcastle disease

Newcastle disease virus

nanogram

nucleoprotein

natrium choride-Tris-EDT A buffer

phosphoprotein

phosphate-buffered saline

xi

PCR

PD

pfu

pg

RNA

RNase

RT-PCR

s

SD

TBS

U

UPM

UV

V

VRI

VVNDV

v/v

w/v

polymerase chain reaction

primer-dimer

plaque forming unit

plcogram

ribonucleic acid

ribonuclease

reverse transcriptase-polymerase chain reaction

second

standard deviation

Tris-buffered saline

melting temperature

unit

Universiti Putra Malaysia

ultraviolet

volt

Veterinary Research Institute

viscerotropic velogenic Newcastle disease virus

volume per volume

weight per volume

xii

Abstract of thesis submitted to the Senate of Universiti Putra Malaysia in fulfilment of the requirements for the degree of Master of Science.

DEVELOPMENT OF AN RT NESTED PCR-ELISA DIAGNOSTIC TEST FOR THE DETECTION OF NEWCASTLE DISEASE VIRUS

By

KHO CHIEW LING

June 1999

Chairperson: Associate Professor Khatijah Mohd. Yusoff, Ph.D.

Faculty: Science and Environmental Studies

Newcastle disease virus causes an economically important poultry disease

known as Newcastle disease in Malaysia. The velogenic strain of this virus causes

1 00% mortality in infected chicken. Therefore, a rapid and sensitive diagnostic

method is necessary for the detection of the virus. In this study a sensitive one tube

reverse transcription nested PCR was developed by using two nested pairs of primer

which were designed from the consensus fusion gene sequences. The outer primer

sequences are 5' -T ACACCTCATCCCAGACAGGGTC-3' (FOP I) and 5'-

AGGCAGGGGAAGTGATTTGTGGC-3' (FOP2). The inner primer pair with the

sequence of 5' -T ACTTTGCTCACCCCCCTT -3' (FIP I ) and 5' -

CA TCTTCCCAACTGCCACT -3' (FIP2) were labeled with biotin and digoxigenin at

their 5' ends respectively. The PCR condition for the outer primers is 90°C/30 s,

67°C/3 0 s and 72°C/30 s for 20 cycles in which a 532 bp PCR product was generated.

While for the inner primers, the PCR condition used is 90°C/30 S, 55°C/30 s and

xiii

72°CII 5 s for a total of 30 cycles for the amplification of a labeled 280 bp PCR

product. The primer pairs used are highly specific enabling the identification of all the

three different pathotypes of NDV. No cross-reactions with other avian infectious

agents such as infectious bronchitis virus, infectious bursal disease virus, influenza

virus, and fowl pox virus were observed. The detection limit of this one tube nested

peR technique was 3 pfu/ml of NDV by agarose gel electrophoresis detection

method and was about 1 00 fold more sensitive compared to that of a non-nested R T -

peR. To facilitate the detection of the peR product, the amplified peR product was

then subjected to a colorimetric detection method using ELISA where the labeled

peR product was captured in a streptavidin coated microtiter plate and was detected

by using anti-digoxigenin-peroxidase enzyme conjugate and 2,2'- azinodi­

ethylbenzothiazolinesulfonic acid (ABTS) as the substrate. Comparisons between the

detection methods of agarose gel electrophoresis and ELISA showed that the latter

was 1 0-fold more sensitive than the former. The efficacy of the nested PCR-ELISA

was also compared with the conventional NDV detection method (HA test) and non­

nested RT-PCR by testing against a total of 35 tissue specimens collected from ND­

symptomatic chickens. With the cutoff value of 0. 154 having been calculated from 1 5

known negative samples, 2 1 of 35 (60%) samples were tested to b e NDV positive by

nested peR-ELISA. One of these positive samples, however, was negative by nested

peR and gel detection method. Only 8 of 35 (22.9%) samples were tested positive by

non-nested RT- PCR and 2 of 35 (5.7%) samples were positive by the conventional

HA test. Due to the high sensitivity of nested PCR-ELISA for the detection of NDV

xiv

from tissue specimens, a peR-ELISA based diagnostic test may be a useful screening

test especially in dealing with large number of samples.

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia untuk memenuhi keperluan bagi mendapat Ijazah Master Sains.

PENGEMBANGAN UJlAN DlAGNOSTIK TINDAKBALAS RANT AlAN

POLIMERASE PENY ARANGAN ELISA (NESTED PCR-ELISA) UNTUK MENGESAN VIRUS PENY AKIT NEWCASTLE

Oleh

KHO CHIEW LING

Jun 1999

Pengerusi: Profesor Madya Khatijah Mohd. Yusoff, Ph.D.

Fakulti: Sains dan Pengajian Alam Sekitar

Virus penyakit Newcastle menyebabkan penyakit Newcastle atau sampar ayam

yang amat penting dari segi ekonomi di Malaysia. Strain velogenik virus ini boleh

mengakibatkan 1 00% kematian pada ayam yang dijangkiti. Oleh itu, satu teknik

diagnostik yang cepat lagi sensitif adalah amat diperIukan untuk mengesan virus ini.

Dalam kajian ini, satu teknik yang dikenali sebagai transkripsi berbalik tindakbalas

rantaian polimerase penyarangan (R T nested PCR) yang sensitif telah direka. T eknik

ini menggunakan dua pasang primer (primer luaran dan primer dalaman) yang direka

berasaskan jujukan nukleotida pada gen pertaupan. Jujukan nukleotida pasangan

primer luaran adalah 5' -T ACACCTCA TCCCAGACAGGGTC - 3' (FOP 1) dan 5'-

AGGCAGGGGAAGTGATTTGTGGC - 3' (FOP2) . Jujukan nukleotida pasangan

primer dalaman adalah 5' - T ACTTTGCTCACCCCCCTT - 3' (FIP 1 ) dan 5' -

CA TCTTCCCAACTGCCACT - 3' (FIP2) yang masing-masing telah diiabelkan

dengan biotin dan digoxigenin pada setiap hujung 5' . Keadaan tindakbalas rantaian

xvi

polimerase (PCR) untuk pasangan primer luaran adalah 90°C13 0 s, 6rC13 0 s dan

nOC/3 0 s sebanyak 20 pusingan dimana tindakbalas ini akan menghasilkan produk

PCR yang bersaiz 532 pasangan bes Manakala keadaan PCR untuk pasangan primer

dalaman pula adalah 90°C130 s, 55°C/30 s and 72°C1 1 5 s sebanyak 30 pusingan untuk

mengamplifikasikan produk PCR berlabel yang bersaiz 280 pasangan bes Primer yang

digunakan adalah amat spesifik di mana ia berupaya mengenal pasti ketiga-tiga

patotaip NDV yang diuji Tiada tindakbalas silang diperhatikan apabila teknik ini diuji

terhadap virus-virus "infectious bronchitis", " infectious bursal disease", "avian

influenza" dan "fowl pox" Had pengesanan teknik ini berserta dengan penggunaan

elektroforesis gel agarose ialah 3 pfu/ml NDV Had pengesanan ini adalah lebih

kurang lOOx lebih sensitif berbanding dengan teknik "non-nested RT-PCR" Demi

kemudahan mengesan produk PCR, kaedah pengesanan secara kalorimetri (ELISA)

telah digunakan, di mana ia melibatkan pemerangkapan produk PCR yang berlabel di

dalam piring mikrotiter yang terlebih dahulu diselaputi dengan streptavidin Produk

PCR yang terperangkap itu kemudian dikesan dengan menggunakan enzim

berkonjugat peroksidase anti-digoxigenin dan ABTS sebagai substrat Perbandingan

kaedah pengesanan produk peR di an tara elektroforesis gel agarose dan ELISA

menunjukkan bahawa kaedah ELISA adalah lOx lebih sensitif berbanding dengan

yang terdahulu Keberkesanan teknik "nested PCR-ELISA" ini juga dibandingkan

dengan kaedah mengesan NDV secara konvensional iaitu ujian HA dan "non-nested

PCR" dengan menguji 3 5 sampel tisu yang dikutip daripada ayam yang menunjukkan

simptom ND Dengan nilai "cutoff' sebanyak 0 1 54 yang dikira daripada 1 5 sampel

":Vll

negatif yang dikenali, 2 1 daripada 35 (60%) sampel tisu didapati positif terhadap

NDV melalui teknik "nested PCR-ELISA" . Walau bagaimanapun satu daripada 21

sampel yang positif ini didapati negatif apabila dikesan secara "nested PCR" berserta

elektroforesis gel agarose. Hanya 8 daripada 3 5 sampel (22. 9%) adalah positif

terhadap NDV melalui teknik "non-nested RT-PCR", manakala 2 daripada 3 5 sampel

(5 . 7%) adalah positif dengan ujian HA. Memandangkan sensitiviti tinggi yang

ditunjukkan oleh teknik "nested PCR-ELISA" terhadap pengesanan NDV dalam

sampel tisu, teknik ini berpotensi sebagai ujian penyaringan terutamanya berhubungan

dengan jumlah sam pel yang banyak.

xviii

CHAPTER 1

INTRODUCTION

Since first isolated in 1 926, Newcastle disease virus (NDV) remains a hazard

to the poultry industry of many countries. In Malaysia, Newcastle disease (ND) is still

endemic throughout the country despite the introduction of various vaccination

strategies in commercial poultry farming. It is believed that the backyard type

operation where a vaccination programme is seldom extended to and the migration of

wild birds are the main culprits of the disease recurrence.

The poultry industry forms a major component of the livestock industry in

Malaysia. According to data released from Department of Veterinary Services,

Malaysia, in 1 996 (Livestock / Livestock Products Statistics, 1 996), the ex-farm value

of poultry production in Malaysia is estimated to be RM 2 .7 billion. The poultry

industry contributed more than 60% of the total value of livestock in 1 996. Poultry

meat and eggs are consumed chiefly as the main source of protein for Malaysians.

Currently, Malaysia is self sufficient in poultry eggs and meat production. The total

local consumption of poultry meat has increased from 242. 88 metric tonne in 1 987 to

486,59 metric tonne in 1 996 and per capita consumption also increased sharply from

1 7 . 86 kg in 1 987 to 29.23 kg in 1 996.

2

Due to the expanding population, the demand for poultry products is expected

to expand substantially from year to year. In view of this, the problem of ND which

may cause a great loss to the poultry owners must be taken seriously.

Correct and easy diagnosis of ND surely would pave way for a more effective

control of the disease. Unfortunately, rapid detection of NDV at the onset of the

disease has always been hampered by lack of a sensitive and fast detection method.

Clinical signs ofND vary according to the age of the bird and the pathotypes ofNDV

involved. Symptoms always involve both the respiratory and nervous systems in

addition to the effects in egg production and eggshell quality. These typical

symptoms, however, cannot be assumed as definite proofs that the responsible agent

is NDV. In fact there are other avian diseases which also produce some similar

symptoms to ND; such as avian encephalomyletis, infectious bronchitis, infectious

coryza and laryngotracheitis (North, 1978).

Although vaccination programs and importation quarantine requirements have

provided significant protection against any outbreak, the virus remains a potential

threat to the commercial poultry producers as well as the backyard type operation

owners. Current diagnosis of NDV infection by conventional virus isolation and

serological tests such as hemagglutination inhibition (HI) and serum neutralisation

are time consuming, labour intensive and expensive. To overcome these

disadvantages, an enzyme-linked immunosorbent assay (ELISA) was developed to

detect NDV antibody in the chicken sera (Bidwell et aI. , 1 977; Soula and Moreau,

3

1 98 1 ) However, the reported ELISA tests were of l imited use, mainly because of

non-specific reactions in chicken sera

With the advent in molecular biology based techniques, polymerase chain

reaction (PCR) based assay has become a new approach for detecting many veterinary

important viruses The first paper to describe the detection of NDV in infected

allantoic fluids of embryonated chicken eggs by PCR appeared in the year of 1 991

(Jestin and Jestin, 1 99 1 ), and thereafter there have been several similar publications

attesting to the perceived importance of this new diagnostic tool (Hodder et al ,

1 992, Yusoff et at , 1993 )

The use of reverse transcriptase-PCR (RT-PCR) followed by ethidium

bromide staining gel electrophoresis of the amplified product has indeed provided an

easy means for detection of various infectious viruses The application of this

technique, however, has always been limited on the research bench and is less suitable

for routine laboratory where normally, higher degrees of sensitivity and specificity are

required for analysing large numbers of clinical samples To fulfil these requirements,

nested PCR coupled with a microtiter plate based colorimetric detection system or

ELISA has been developed by several research groups to improve both the sensitivity

and specificity of their respective diagnostic assays (Wilson et at, 1 993 , Lage et at ,

1996, Britten et at, 1997, Shaio et at, 1997)

Objective of the Study

The development of a rapid, specific, sensitive and relatively inexpensive

detection method for the detection of NDV would be advantageous under a variety of

circumstances including clinical disease investigation, molecular epidemiological

studies for understanding the biology of NDV as well as the enhancement of

international trade of poultry. Therefore the objective of the study was to establish a

highly sensitive and specific R T nested PCR and colorimetric detection of the PCR

product for direct detection ofNDV in tissue samples.

This research, therefore, comprised the following steps:

1. establishment of a one tube R T nested PCR;

2. standardisation of the colorimetric detection method or ELISA;

3 . sensitivity and specificity evaluation of the R T nested PCR-ELISA; and

4 . evaluation of the method efficacy by testing on tissue specimens.

CHAPTER II

LITERA TURE REVIEW

General Introduction of Newcastle Disease

Newcastle disease (ND) is a highly contagious and destructive avian disease

caused by Paramyxovirus type 1 or better known as the Newcastle disease virus

(NDV). This disease has been recognised in most species of free living and also

domestic birds throughout the world. Both the exotic and free living nature birds can

act as reservoirs for the virus thus making it possible both recrudescence and rapid

spread of the ND to domestic birds as well as domestic poultry. Generally, chickens

are highly susceptible compared to other avian species. Therefore ND is a serious

threat upon the development of poultry industry.

Occurrence

The outbreaks of ND were first reported for poultry in Batavia (now known

as Jakarta), Indonesia in 1 926. In the same year Doyle observed a similar disease near

5

6

Newcastle-upon-Tyne, England and he named the disease after the locality and

demonstrated it to be distinct from any previously known fowl pest (Brandly and

Hanson, 1984) . Owing to the absence of effective control measures, it spread to many

parts of the world within a decade following the first recognition of this disease. In

Malaysia, it was believed that the ND made its first apparence in the 1 930s'.

However, it was initially thought to be a new disease known as 'diptheritic­

stomatopharyngitis' (Chong et aI., 1 960) until it was proven later as ND by Orr and

John (1 960).

Importance

ND represents a serious economic challenge to all segments of the poultry

industry. In chickens, outbreaks may be so severe with high rates of mortality in the

affected flocks. The loses from non-fatal outbreaks, while less spectacular, may be

equal to or greater than those where the death loss is high. The greatest loss

frequently results from the loss in body weight from the surviving chickens, reduction

of the egg production and impaired eggshell and also albumin quality (North, 1 978).

Besides that, ND also reduces the fertility and hatchability of eggs (Brandly and

Hanson, 1 984). ND however, is not a serious problem to human. To date, no severe

infection has been officially reported in human. Those who had contracted with the

disease might show illnesses like headache, flu-like symptoms and mild conjuntivitis

(Brandly and Hanson, 1 984).