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, 2015 - 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 (6layers and 18broilers) 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: Diseases, Haemagglutination, Inhibition. Isolation, Newcastle, Virus

Excerpt

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.

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

LIST OF ABBREVIATION

APMV

cRBC

FAO

HAU

HB1

NCD

NCDV

NVI

OIE

PBS

PCV

RBCS

RPM

SPF

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

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

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

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.

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.

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

and longitude of 04

and has elevation of 1850m above sea level. The mean minimum and

maximum temperature of the town ranges from 12.3

c to 27.7

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

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

c by using labeled test

tube having isotonic phosphate buffered saline (PBS), pH 7.07.4, containing antibiotics and

stored at +4

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.

The harvested allantioc fluid from each egg was checked for haemagglutination activity (OIE,

2012) and the remaining harvested allantoic fluid was kept at -20

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

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

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

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

23.33

83.33

No, of samples inoculated

No, of positivechickens for NCD

percentage (%)

Broilers Layers Total

Broilers

Layers

Total

18 6

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

Serum

1:16

Positive NDV

Serum

1:32

Positive NDV

Serum

1:128

Positive NDV

Serum