Evaluation of the Serum Haemagglutination Inhibition Antibody Titre Required to Protect Against Drop in Chicken Egg Production by Velogenic Newcastle Disease Virus Infection.


This study investigated the serum  haemagglutination  inhibition (HI)  antibody titres required to protect against drop in chicken egg production in  velogenic  Newcastle  disease  virus (NDV) infection. Isa brown pullets were obtained at 12 weeks of age.

At 18 weeks, they were vaccinated against Newcastle disease (ND) using LaSota vaccine. Also at the peak of lay (27 weeks of age), they were revaccinated with ND LaSota vaccine. Two more booster  vaccinations were also given at one week intervals to achieve  hyperimmunity.

The  HI antibody titres of these birds were monitored weekly. At antibody geometrical mean titres (GMT) titres of 84.4, 42.2 and 21.1, twenty birds were randomly selected, taken to a distant location and challenged intramuscularly with a local velogenic strain of NDV, KUDU 113.

After the third challenge, the remaining layers came  down naturally with ND and  showed  drop in egg production. The groups of birds were designated batches A-D representing layers challenged at HI antibody GMT titres of 84.4, 42.2, 21.1 and natural infection respectively.

After each challenge, the layers were monitored for clinical signs especially drop in egg production. Gross and histopathological examinations were performed  on  two  layers  that were sacrificed on day 3 PC from every challenge and control groups.

Cloacal swabs were collected from three layers from every challenged group on days 3, 6 and 9  post challenge  (PC) for virus isolation, confirmation and quantification. Serum samples were collected from every challenge and control layers on days 0, 7, 14 and 21 PC for antibody assay using haemagglutination inhibition (HI) test.

Results showed reduced water and feed intake, slight cough and drop in egg production in batch D. No death was recorded. The three weeks percentage egg production was significantly lower (p < 0.05) in the  infected  batch  D  than their controls.

Batches A, B and C showed enlargement of the spleen in  their  challenged groups while batch D showed atrophy of the spleen and  oviduct with atretic  ovarian  follicles in the infected group. Histopathological results showed inflammation and oedema of the oviduct with infiltration of mononuclear cells.

Serology showed increased antibody titres two weeks after hyper-immunisation which started declining two weeks after. Increased antibody titres were also observed on days 7- 21 PC.

Virus was isolated from all the cloacal swabs collected on days 3, 6 and 9 PC from all the batches and was confirmed to be NDV using specific antiserum.

From the results, it could be concluded that  higher  antibody  titre  is required to protect layers from clinical disease of ND than that required to protect them from drop in egg production.

In laying chickens, antibody titre of 24 (16) was observed to be the protective titre against drop in egg production in velogenic NDV infection.


Title page i
Declaration ii
Certification iii
Dedication iv
Acknowledgements v
Abstract vi
Table of contents vii
List of Tables and figures viii


1.1 Background of the study 1
1.2 Statement of the problem 2
1.3 Specific objectives 3


2.1 Historical background 4
2.2 Incidence and Distribution 5
2.3 Host range 7
2.4 Age and breed susceptibility 8
2.5 Nature of the virus 9
2.6 Classification of the NDV 11
2.7 Classification of viral pathogenicity 12
2.8 Pathogenesis 14
2.8.1 Mode of transmission 14
2.8.2 Route of infection 15
2.9 Clinical signs 17
2.10 Pathology 23
2.10.1 Gross lesions 24
2.10.2 Histopathological changes 26
2.11 Diagnosis 28
2.11.1 Virus isolation in embryonated chicken eggs and typing 29
2.11.2 Virus isolations in cell cultures 31
2.11.3 Molecular methods of NDV diagnosis 32 Reverse transcription polymerase chain reaction (RT-PCR) 34 Real time PCR era of detection and typing of APMV-1 35
2.12 Differential diagnosis 36
2.13 Prevention and control 38
2.13.1 Quarantine and movement control 38
2.13.2 Tracing 38
2.13.3 Destruction of birds 39
2.13.4 Disposal 39
2.13.5 Decontamination 39
2.13.6 Public awareness 39
2.13.7 Pest and vector control in flocks 39
2.13.8 Vaccination 39


3.1 Experimental chickens 41
3.2 Vaccine 41
3.3 Newcastle disease challenge virus 41
3.4 Experimental design 42
3.5 Vaccine administration 43
3.6 Virus challenge 43
3.7 Clinical signs and gross lesions 43
3.7.1 Histopathological examination 43
3.8 Serology 44
3.8.1 Haemagglutination and Haemagglutination inhibition tests 44 Washing of red blood cells 44 Haemagglutination test 44 Haemagglutination inhibition test 45
3.9 Virus isolation 46
3.9.1 Sample collection and preservation 46
3.9.2 Processing of samples 46 Cloacal samples 46 Spleen tissue 47
3.9.3 Embryonated egg inoculation 47
3.9.4 Harvesting of allantoic fluid 48
3.9.5 Spot testing 49
3.9.6 Microhaemagglutination Test 49
3.9.7 Confirmatory NDV Test 49
3.10 Statitical analysis 50


4.1 Clinical signs 51
4.2 Percentage egg production 51
4.3 Gross and histopathological changes 51
4.4 Serology 52
4.5 Newcastle disease virus isolation 52
4.5.1 Microhaemagglutination test 52
4.5.2 NDV confirmatory HI Test 52


5.1 Discussion 64
5.2 Conclusion 69
5.3 Recommendation 69


1.1 Background of the study

Newcastle disease (ND) is one of the most important viral diseases of poultry (Orsi et al.,  2010). It is an infectious, rapidly spreading, contagious,  nervous and  respiratory viral disease of domestic poultry and other species of birds regardless of sex and age (Okeke and Lamorde, 1988; Alexander, 2003; Haque et al., 2010; Iram et al., 2013).

The aetiological agents of ND  are virulent strains of avian paramyxovirus-1(APMV-1) (Yu etal., 2001; Qin  et  al.,  2008; Choi et al., 2010) that are circulating in poultry flocks (Munir etal., 2012b).

The disease is characterised by respiratory and or nervous system impairment, gastrointestinal tract and reproductive problems (Nanthakumar et al., 2000; Tiwari et al., 2004).

Newcastle  disease  virus (NDV) has a wide host range which include chickens, turkeys, ducks,pigeons, guinea fowls, Japanese quails and many wild and cagedbirds of all ages (Nanthakumar et al., 2000; Zhang et al., 2011).

The most susceptible are the chickens (Razaeianzadeh et al., 2011). In several developing countries, ND is endemic and has greater impact on villages  where  people’s livelihood depends upon poultry farming (Mohamed et al.,  2011;  Razaeianzadeh etal., 2011).

Newcastle disease causes huge economic losses to  the  commercial  poultry farmers round the world. It is highly fatal and a top ranked poultry disease. Annual  losses caused by the disease worldwide are in millions of dollars (Aldous  et  al., 2003;  Waheed  et al., 2013; Wakawa etal., 2014).

Newcastle disease is an economically important disease  and also a major threat to the poultry industry. Depending on the NDV strain, the rate of mortality and morbidity in a flock vary from 90% – 100% along with decrease in egg production(Nanthakumar et al., 2000; Choi et al., 2010; Haque et al., 2010;Narayanan et al., 2010).

The disease has remained a notable problem in Nigeria (Oladele et al., 2002). Due to the severity of ND and the related consequences, it is included in “listed” agents (reportable disease)between 2002 and 2004 by the World Organisation for Animal Health (OIE).

Notification is required by this organisation of any outbreak of ND, when it meets certain criteria of virulence (Aldous and Alexander, 2001; Cattoli et al., 2011; Munir et al., 2012b; Boynukara etal., 2013; Cao et al.,2013).

Vaccines are used to  control and prevent ND  in many countries. Chickens and turkeys are immunised against ND.

The prophylactic vaccination is not used in developed countries because of virus multiplication and shedding  after vaccination but there is a wide use of these vaccines on commercial  flock  s  in developing countries (Munir et al., 2012b;OIE, 2012).


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