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).
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