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ACKNOWLEDGEMENTS
First of all, I wish to express my sincere gratitude to my advisor Dr. Kibeb Legese and Dr.
Jaleta Shuka for their valuable advice, guidance and constructive comments in directing and
shaping the research idea.
I am also indebted to National Veterinary Institute (NVI) and its management who provided
me this opportunity to attend this program.
I would like to thank staff members of Ada district veterinary clinic Dr. Tsigie G/Michael, Ato
Belay Temesgen and Ato Efirem shimelis, who helped me during clinical examination and
sample collection.
My appreciation also goes to W/ro Berhan Demeke who helped me during the period of
laboratory analysis and for her constructive comments.
Last but not least, my colleagues at NVI helped me a lot; especially Dr. Hundera Sori, Ato
Kasaye Adamu for their help during laboratory works.
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Contents
ACKNOWLEDGEMENTS ... 2
Contents ... 3
LIST OF ABBREVIATION ... 4
LIST OF TABLES ... 5
LIST OF FIGURES ... 6
SUMMARY ... 7
1. INTRODUCTION ... 8
2. METHOD OF CLINICAL DATA COLLECTION ... 9
2.1. Study area ... 9
2.2. Study animal ... 9
2.3. Case report ... 9
2.4. Clinical Examination ... 9
2.5. Sample collection ... 10
3. LABORATORY ANALYSIS ... 10
3.1. Sample processing and storage ... 10
3.2. Virus isolation ... 10
3.2.1. Haemagglutination test (HA) ... 12
3.2.2. Haemagglutination test results ... 12
3.3. Virus identification ... 13
3.3.1. Haemagglutination inhibition test (HI) ... 13
4. RESULT AND DISCUSSION ... 14
4.1. Clinical Diagnosis ... 14
4.2. Virus isolation ... 14
4.3. Virus identification ... 15
5. CONCLUSION AND RECOMMENDATION ... 16
6. REFERENCES ... 17
7.APPENDIXES ... 18
4
LIST OF ABBREVIATION
APMV
AB
APMV
0
c
cRBC
FAO
HA
HAU
HB1
HI
ML
NCD
NCDV
NVI
OIE
PBS
PCV
RBCS
RPM
SPF
l
Avian paramixo virus
Anti body
Avian paramixo virus
Degree celcious
Chicken red blood cell
Food and agricultural organization
Haemagglutination
Haemagglutination unit
Vaccinal strain for Newcastle virus
Haemagglutination inhibition
Mili liter
Newcastle disease
Newcastle disease virus
National Veterinary Institute
Office Des International Epizootics
Phosphate buffered saline
Packed cell volume
Red blood cells
Revolution per minute
Specific pathogenic free
Micro liter
5
LIST OF TABLES
Table 1. Result of history and clinical examination
Table 2. Result of virus isolated using NCD virus antibody free specific embryonated eggs
Table 3. Haemagglutination inhibition test of collected serum
6
LIST OF FIGURES
Figure 1. SPF eggs for inoculation
Figure 2. Incubated SPF eggs
Figure 3. Inoculated and ceiled eggs
Figure 4. Candling eggs
Figure 5. Harvesting of allantoic fluid
Figure 6.RBC and different materials used for test
Figure 7. Micro haemagglutination test in a v- bottomed micro plate
Figure 8. Haemagglutination inhibition test on micro plate
7
SUMMARY
Newcastle disease (NCD) is a contagious bird disease affecting domestic and wild avian
species characterised by respiratory signs (gasping, coughing, nervous signs depression, in
appetence, muscular tremors, and drooping wings, twisting of head and neck, greenish watery
diarrhoea, misshapen, rough-or thin shelled eggs and reduced egg production. This study was
designed for isolation and identification of Newcastle disease virus from sick and suspected
chicken in poultry farm, Bishoftu, Ethiopia, during October 16, 20015- May 30, 2016 which
brought to research and diagnostic laboratory in National Veterinary Institute (NVI), Ethiopia
by owner. Total suspected chicken were 200 and out of these 30 chickens were brought to NVI
and samples were collected. The sample included serum, cloacal and tracheal swab. Serum
sample was collected for confirmatory diagnosis by serological HI test. HI result has showed
that more than 80% of the total samples collected were positive for Newcastle disease virus
(NCDV). Isolation of the virus from cloacal and tracheal swabs was performed by inoculating
each suspected sample in to 10 day old embryonating eggs. Out of 30 NCDV suspected samples
24 (6 layers and 18 broilers) were positive for NCD virus isolation. Out of the 24 HA positive
NCD virus suspected samples 24(6 layers and 18broilers were HI positive.
Keywords:- Newcastle disease virus, isolation,Haemagglutination, Haemagglutination
inhibition.
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1. INTRODUCTION
Newcastle disease (NCD) is a highly contagious and often severe disease found worldwide that
affects birds including domestic poultry. It is caused by a virus in the family of
paramyxoviruses. The disease appears in three forms: lentogenic or mild, mesogenic or
moderate and velogenic or very virulent, also called exotic Newcastle disease. The lentogenic
strains are very widespread, but cause few disease outbreaks. It usually presents as a respiratory
disease, but depression, nervous manifestations, or diarrhea may be the predominant clinical
form. NCD, in its highly pathogenic form, is a disease listed in the World Organization for
Animal Health (OIE) Terrestrial Animal Health Code and must be reported to the OIE (OIE
Terrestrial Animal Health Code. 2010).
The poultry sector of Ethiopia is mainly based on chicken production and concerned with egg
and meat requirements. The total chicken in the country is estimated to be about 58 million of
which 95.87%, 3.6% and 0.53% were reported to be indigenous, hybrid and exotic breeds,
respectively (CSA,2 008; Ashanafi, 2000). The chicken kept under traditional or "back yard"
conditions accounts for 99%, while only 1% birds are rearing under intensive management
system in commercial farms (CSA; Almargot, 1987).
Despite of large number of chicken, the benefit of chicken contribution to the country is far
away from the existing potentials. This is because of inadequate feed supply, improper
production system, and high prevalence of disease, poor animal genetic resources and poor
marketing (Alemu, 2010). Among those disease affects poultry Newcastle disease is one of
poultry disease inflicting heavy loss in Ethiopia. The disease has a local name "Fengel"which
literary mean collapsing (Ashanafi, 2000).
Bio-security and vaccination are two important measures for prevention and control the disease
in the country and have been successful where the measures are properly implemented.
However, there both measures have mostly been used in commercial intensive and semi-
intensive production systems of the country. This gap makes the disease prevalent in backyard
production system. According to work done by Ashenafi (2000) some farmers have given up
rearing poultry because of disease problem.
There have been several cases of the Newcastle disease come to NVI for diagnosis from nearby
areas. Virus isolation, haemaglutination test and Serological diagnosis test are most utilized to
diagnose the disease. However, serological test is necessary to confirm the disease since
haemagglutination has its own drawback.
9
Therefore, the objective of this study is:
To isolate and identify Newcastle disease virus from cases brought to national
veterinary institute diagnostic laboratory
To determine NCD virus strain present in the Bishoftu town
To forward prevention methods to the stoke holders
2. METHOD OF CLINICAL DATA COLLECTION
2.1. Study area
Accordingly, clinical materials (serum, tracheal and cloacl swab) were collected from NCD
suspected chicken reared under semi-intensive poultry farm located at Bishoftu town which was
located at distance of 47km to the south east of Addis Ababa. The area is situated at latitude of
09
0
and longitude of 04
0
and has elevation of 1850m above sea level. The mean minimum and
maximum temperature of the town ranges from 12.3
0
c to 27.7
0
c with an average annual rain
fall of 800 mm and mean relative humidity of 61.3% (CSA, 2001).
2.2. Study animal
From total 200 clinically sick exotic breeds chicken samples were collected from 30 highly
sick chickens (20 broilers and 10 layers) which were in different ages (3weeks-6months). The
chickens were come from one farm found in Bishoftu. According to the information obtained
from the owner, all chickens were reared under semi-intensive farming.
2.3. Case report
Owner was brought his clinically sick chickens after one to two days of the commencement of
the disease. Loss of appetite, depression, diarrhea, inability to stand, swelling of comb and
wattle, nasal and eye discharge and reduction of egg yield were the main symptoms which
were explained by the owner. The owner also indicated the presence of death of some chickens
after they had showed the similar signs. Moreover, the owner responded that his flocks were
not vaccinated against NCD.
2.4. Clinical Examination
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Clinical examination was made based on history the owner of the farm complained. Based on
inspection, the sick chickens had showed depression, weakness paralysis of legs and wings,
dehydration, edema of the head and wattles, difficulty in breathing were the main clinical signs
observed during inspection.
2.5. Sample collection
Samples were collected, transported and stored according to (OIE.2010) protocol. Out of 200
clinically sick chickens, cloacal and tracheal swabs were collected from 30 chickens which
were tentatively diagnosed as highly infected by NCD and selected for sample collection. The
samples were collected by using sterile cotton swab. Each sample was placed in labeled screw
cap test tube having isotonic phosphate buffered saline (PBS), PH 7.2 7.4, containing
antibiotics. The blood serum of bird had also collected for inhibition test. (OIE, 2012)
3. LABORATORY ANALYSIS
3.1. Sample processing and storage
Standard sample storage and processing for NCDV test was employed (OIE, 2012). Each
cloacal and tracheal sample was centrifuged at 2500rpm for 10 minutes to obtain clarified
supernatant suspension of test virus at temperature not exceeding 25
0
c by using labeled test
tube having isotonic phosphate buffered saline (PBS), pH 7.07.4, containing antibiotics and
stored at +4
0
c until viral isolation.
3.2. Virus isolation
Incubated eggs were candled and eggs which have viable embryo selected and marked on air
space for inoculation. Marked eggs were disinfected with 70% ethanol. The supernatant fluid
expected to contain the virus was inoculated in 0.2 ml volumes into the allantoic cavity of 10
day embryonated SPF fowl eggs. After inoculation, it was incubated at 37°C for 7 days.
Candling was carried out every 24 hours during incubation period of the infected embryonated
eggs. Eggs containing dead or dying embryos as they arose, and all eggs remaining at the end
of the incubation period was chilled to 4°C for overnight and then allantoic fluid was harvested.
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The harvested allantioc fluid from each egg was checked for haemagglutination activity (OIE,
2012) and the remaining harvested allantoic fluid was kept at -20
0
c for further study.
Fig 1. SPF eggs
Fig 2. Incubated SPF eggs
Fig3. Inoculated and sealed
Fig 4. Candling of inoculated
eggs
Fig5.Harvesting of allantoic
fluid
Fig 6. Materials needed for
test
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3.2.1. Haemagglutination test (HA)
This test was conducted to confirm the presence of NCD virus in the allantoic fluid of eggs
inoculated with supernatant fluid which got from samples.50 L of allantoic fluid from each
eggs was taken and placed each drop in a separate well of ''V'' shaped microtitre plate.25 L of
1% fowl red blood cell was added to each well that had allantoic fluid and mixed gently. This
kept at room temperature for 45 minutes and read the result.
3.2.2. Haemagglutination test results
Agglutinated red blood cells in suspension have a clumped appearance distinct from
non-agglutinated red blood cells.
The red blood cells mixed with the positive control allantoic fluid will clump within
one minute.
The red blood cells mixed with the PBS and negative control allantoic fluid remain as
an even suspension and do not clump. Judge the results of the test sample by
comparison with the positive and negative controls.
Fig.7. Micro haemagglutination test in a V-bottome microwell plate
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3.3. Virus identification
3.3.1. Haemagglutination inhibition test (HI)
To perform HI test, 4 Haemagglutination units (4HA units) was prepared for working antigen.
This known antigen was obtained from National Veterinary Institute (NVI), Ethiopia. (The
procedure to prepare 4HA is written in Appendix 1.4.). Each blood serum collected from sick
chicken was tested for the presence of antibody of NCD virus. 25 L PBS was dispensed in to
each well of a 96-well V-bottomed microtitre plate. After shaking a test serum, 25 L of serum
added to the first and the last (control) well of each row. By using multichannel pipetter, made
serial two fold dilutions of each serum sample along the row by transferring 25 L of fluid from
one well to the next stopped at the second last well. 25 L of fluid from the second last well of
the row was discarded. The last well of each well did not diluted (control).Then 25 L of 4 HA
units added to each well except control well. Mixed the well gently, covered and kept at room
temperature for 30 minutes. Then after 25 L of 1%suspension of red blood cell added to each
well and tapped gently the side of plate to mix and cover and kept at room temperature for 45
minutes and read the result.
Fig. 8. Haemagglutination inhibition test on micro plate
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4. RESULT AND DISCUSSION
4.1. Clinical Diagnosis
Clinically, 18(90%) birds of 20 layers chickens and 7(70%) of 10 affected broilers were
diagnosed as Newcastle (Table 1) which means 83.33% were suspected. The most clinical signs
observed were depression, edema of the head and wattle, twisted neck and paralysis, loss of
weight and greenish diarrhea.
Table1. Result of history and clinical examination
4.2. Virus isolation
A total of 24(90%) samples for 30 clinically diagnosed Newcastle affected chickens, were
positive for virus isolation in embryonated eggs (table 2). In all positive cases embryo died
within 72 - 168 hours of post inoculation. All of 24 samples showed positive to HA activity
which indicate that the isolate were heamagulitinating virus.
Table2. Result of virus isolated using NCD virus antibody free specific embryonated eggs
Total no, of
hi k
No, of suspected chickens for NCD Prevalence (%)
Broilers
Layers
Broilers Layers
Total
Broilers
Layers
Total
20
10
18
17
60
23.33
83.33
25
No, of samples inoculated
No, of positivechickens for NCD
percentage (%)
Broilers Layers Total
Broilers
Layers
Total
20
10
30
18 6
24
80
15
4.3. Virus identification
All 25 tested serum showed settled red blood cell in the bottom of well ( Figure ) and HI result
of total sample found in range of 16:128 ( Table 3). This amount range showed the chicken is
infected with ND virus.
Table 3. Hemaglutination inhibition test result of collected serum
Sample code sample type HI titer result Interpretation
S1
Serum
1:16
Positive NDV
S2
Serum
1:32
Positive NDV
S3
Serum
1:128
Positive NDV
S4
Serum
1:32
Positive NDV
S5
Serum
1:64
Positive NDV
S6
Serum
1:64
Positive NDV
S7
Serum
1:32
Positive NDV
S8
Serum
1:64
Positive NDV
S9
Serum
1:128
Positive NDV
S10
Serum
1:128
Positive NDV
S11
Serum
1:64
Positive NDV
S12
Serum
1:32
Positive NDV
S13
Serum
1:64
Positive NDV
S14
Serum
1:32
Positive NDV
S15
Serum
1:32
Positive NDV
S16
Serum
1:64
Positive NDV
S17
Serum
1:64
Positive NDV
S18
Serum
1:128
Positive NDV
S19
Serum
1:64
Positive NDV
S20
Serum
1:128
Positive NDV
S21
Serum
1:64
Positive NDV
S22
Serum
1:64
Positive NDV
S23
Serum
1:32
Positive NDV
S24
Serum
1:128
Positive NDV
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5. CONCLUSION AND RECOMMENDATION
In this study the presence of NCD virus in chickens coming from poultry farm was confirmed
based on clinical signs, virus isolation and identification.
The prevalence of ND in poultry farms of the country should be taken as a potential threat for
the poultry industry. As ND is viral disease, no specific treatment is effective after infection.
Therefore, practicing preventive strategies is the only mechanism to control the disease.
Based on the above conclusion the following recommendations are forwarded
The vaccine should be availed through local production by the NVI and vaccination of
all the chicken at risk age group.
Training and awareness creation about the disease should be made to all stockholders'
of the sector including farmers.
Effort must be done to study how to minimize the disease incidence to improve
productivity and to eliminate the disease from the country.
17
6. REFERENCES
Alemu, (2010): Drug Administration and control authority of Ethiopia, (2006): standard
treatment guidelines for veterinary practice, 1
st
edition. Addis Ababa Ethiopia.
Alexander DJ (2003): Newcastle disease, other avian paramyxoviruses, and pneumoviru
Infections. In: Saif Y, Barnes JH
Alexander DJ,(2001):Newcastle disease- the Golden Memorial lecture Britain veterinary
science, 42,5-22.
CSA, 2008; Almargot, 1987: avian pathology of industrial farms in Ethiopia. In IAR proceed
ings, First National livestock improvement conference, Addis Ababa agricultural
research institute, Ethiopia. Pp 114-117.
CSA,2008; Ashenafi ,H. (2000): survey on identifications of major disease of local chickens in
three selected agro climatic zone in central Ethiopia. DVM thesis, Faculty of veterinary
medicine, Addis Ababa University, Debre Zeit, Ethiopia.
CSA, 2001: statics agency report, Addis Ababa Ethiopia
NVI, (1974): Poultry disease and Newcastle disease record book, Debre Zeit, Ethiopia.
OIE (2012): Manual of diagnostic test and vaccine for terrestrial animals, version adopted by
world assembly delegate in may.
OIE (2012): Manual of diagnostic test and vaccine for terrestrial animals, adopted may 2012,
6
th
ed. Office international des epizootics. Paris, France. World assembly delegate in
May.
Tadelle, D. (1996): Studies on village poultry production system in central high lands of
Wilson, Ethiopia. Msc thesis, university of Uppsala, Sweden.
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7.APPENDIXES
Annex.1. Materials and reagent required
Water bath, incubator, and vortex
V-bottomed micro titre plates
Single channel micropipettes
Fowl RBC(ARBC)
Trough
Syringe
PBS buffer at PH 7.2 7.4
Elsevier's solution
NCD virus antigen
Positive and negative control
Bi-distilled water
Arranged test sera sheets of plate lay out
Annex.2. Reagent preparation
1. Phosphate buffered saline (PBS) without calcium or magnesium
Sodium chloride
8.0g
Potassium chloride
0.2g
Potassium dihydrogen orthophosphate
0.2g
Disodium hydrogen orthophosphate
1.44g
Distilled water
1000ml
Sterilized by Autoclave
2. 70% ethanol alcohol
Mix 70ml of absolute ethanol (100%) with 30ml distilled water
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3. Preparing a washed red blood cell suspension
Measure the required volume of anticoagulant into a sterile screw-capped bottle or
centrifuge tube. Use equal volumes of Alsever's solution and blood.
Draw the anticoagulant (Alsever's solution or ACD) into a 10 mL syringe.
Collect blood from one donor chicken. Gently mix the blood and anticoagulant in the
syringe.
Remove the needle and discharge the blood into the bottle or centrifuge tube
containing anticoagulant. Roll gently to mix the blood.
Collect blood from the remaining donor chickens (repeat steps 3 and 4 for each
chicken).
Fill the bottle with PBS and mix gently.
Centrifuge at 1500 rpm for 10 minutes to sediment the red blood cells.
Remove the supernatant using a Pasteur pipette and discard. Do not disturb the red
blood cell layer at the base.
Refill the bottle or centrifuge tube with PBS.
Mix gently to resuspend the cells and centrifuge again for 10 minutes at 1500 rpm.
Rough mixing may cause haemolysis of the red cells.
Remove the supernatant using a Pasteur pipette and discard.
Refill the centrifuge tube with PBS.
Mix gently to resuspend the cells.
Pour the blood into 10ml calibrated centrifuge tubes and centrifuge again for 10
minutes at 1500 rpm.
Remove the supernatant using a Pasteur pipette and discard,
Measure the volume of the red blood cell layer using the graduations on the wall of
the centrifuge tube.
Add PBS to the red blood cell layer to make a final 10% red blood cell suspension.
For example, if the volume of the red blood cell layer is 1ml, add 9 ml PBS to make
a total volume of 10 ml.
Mix gently to resuspend the cells, transfer the red blood cell suspension to a screw -
capped bottle, label and store at 4
0
C.
20
4. Preparation of 4 HA units of ND virus antigen suspension
Using the quantitative HA test, titrate the ND virus antigen suspensions and calculate
the HA titer.
Divide the HA titer by four to calculate the dilution factor.
Calculate the volume of diluted antigen suspension required. Allow 2.5 ml for each
micro-titer plate.
Measure the volume of antigen suspension required and dilute in PBS
5. NCD virus antigen titration
Dispense 50l of PBS in each micro plate.
Add 50l of known NCDV antigen in to each well.
Add 50l of 1% ARBC suspension in all wells & shake the plate &let it stay 30
minutes at room temperature.
The last well where there is agglutination represents the dilution having one
haemagglutinatin unit ( HAU) per 50 l
Adjust your antigen to 4-8HAU 50 l
6. The proper hem agglutination test (OIE, 2012)
Before testing, the sera are treated in a water bath at 58
0
c for 30 minutes.
Make two fold test serum dilution at avolumes of 25 l
Add 4-8 HAU 25 l antigens in each well.
Shake plate and keep for 15minutes at room temperature.
Finally add 50 l of a 1% RBC suspension. Shake it well and let it stay for 30
minutes.
Interpretation:- The last dilution where there is complete inhibition of
haemagglutination represents the titration of the serum. the of point of NCDV is
1:16, so the sample which has the last dilution 1:16 are positive.
21
7.Haemagglutination inhibition test procedure(OIE, 2012)
o
25 l of PBS was dispensed in to each well of a plastic V-bottomed micro titre
plate.
o
25 l of chicken anti-sera was placed in first well of the plate.
o
Two fold dilution of 25 l volumes of the serum made across the plate.
o
4HAU virus/antigen 25 l is added in each well and the plate is left for
minimum of 30minutes at room temperature.
o
25 l of 1%(v/v)chicken RBCs is added to each well and after gentle mixing,
the RBCs are allowed to settle for about 40minutes at a room temperature.
o
The HAI titre is highest dilution of serum causing complete inhibition of
4HAU of antigen. The agglutination is assessed by tilting the plate. Only those
wells in which the RBCs stream at the same rate as control wells (positive
serum, virus/antigen and PBS control) was considered to show inhibition.
o
The validity of result should assess against a negative control serum which
should not give a titre greater than ¼ and positive control serum for which the
titre was within dilution of known titre. HAI regarded as being positive if there
was inhibition at antigen dilution 1/16 or more against 4HAU.
Final del extracto de 21 páginas
- Citar trabajo
- Wakjira Kebede (Autor)Dr. Kibeb Legese (Autor)Dr. Jaleta Shuka (Autor), 2016, Isolation and identification of the Newcastle disease virus, Múnich, GRIN Verlag, https://www.grin.com/document/336144
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