Detection of Avian Influenza, Newcastle Disease and Infectious Bronchitis Viruses in Domestic and Captive Migratory Wild Birds Using Nested ...
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Journal of Veterinary Advances
Detection of Avian Influenza, Newcastle Disease and Infectious
Bronchitis Viruses in Domestic and Captive Migratory Wild
Birds Using Nested Polymerase Chain Reaction, Yobe State
Nigeria
Garba J., Nwankwo I. O., Manu I. J. and Faleke O. O.
J Vet Adv 2012, 2(10): 481-487
Online version is available on: www.grjournals.comISSN: 2251-7685 GARBA ET AL.
Original Article
Detection of Avian Influenza, Newcastle Disease and
Infectious Bronchitis Viruses in Domestic and
Captive Migratory Wild Birds Using Nested
Polymerase Chain Reaction, Yobe State Nigeria
1
Garba J., 2Nwankwo I. O., 3Manu I. J. and 2Faleke O. O.
1
Department of Veterinary Services, Ministry of Animal and Fisheries Development, P.M.B. 1155, Damaturu 620001, Yobe State,
Nigeria.
2
Department of Veterinary Public Health and Preventive Medicine, Usmanu Danfodiyo University, P.M.B. 2346, Sokoto, Nigeria.
3
Nigeria Field Epidemiology and Laboratory Training program, Polio Response Unit, Asokoro Abuja, Nigeria.
Abstract
Yobe State is located in Nigeria; it lies between latitudes 11°56ʹ and 13°20ʹE and longitudes 9°38ʹ and
12°25ʹN and shares boundary with Niger Republic. The state is known for rearing of livestock and local poultry
production. Viruses are known to cause most of the important respiratory diseases of poultry, these diseases
results in morbidity, mortality and decline in production. We conducted a survey to detect Avian Influenza (AI),
Newcastle Disease (ND) and Infectious Bronchitis (IB) viruses in domestic and captive migratory wild birds
using Nested Polymerase Chain Reaction (nPCR), we sampled 159 domestic and 34 wild migratory birds from
poultry slaughter slabs located in four Local Governments Areas of Yobe state. Tissue samples were collected
processed and tested using nPCR. The prevalence of ND virus was 30.8% and of IB virus was 2.5% in
domestic birds. The prevalence of ND and IB viruses was highest (33.1% and 2.9% respectively) among local
chickens when compared to other domestic birds. ND, IB, and AI viruses were not detected in wild migratory
birds. Some of these viruses are zoonotic and of economic importance in poultry production, an effective
control and prevention strategy should be established.
Keywords: Domestic bird, wild migratory birds, avian influenza virus, infectious bronchitis virus, Newcastle
disease virus, Yobe state, Nigeria
Corresponding author: Department of Veterinary Public Health and Preventive Medicine, Usmanu Danfodiyo University, P.M.B. 2346, Sokoto, Nigeria.
Received on: 23 Aug 2012
Revised on: 25 Sep 2012
Accepted on: 02 Oct 2012
Online Published on: 30 Oct 2012
481 J. Vet. Adv., 2012, 2(10):481-487DETECTION OF AVIAN INFLUENZA, NEWCASTLE DISEASE AND …
Introduction Avian Influenza and Newcastle Disease viruses
are of public health concerns with clinical
Viruses cause most of the important respiratory manifestations ranging from inflammations to
diseases of poultry and these diseases generally systemic infections among humans (Alexander D.J.
results in morbidity, mortality, decline in production 2003). Infectious Bronchitis viruses however, have
functions, decrease in body weight and poor feed not been reported to be a public health threat to
conversion. Viral diseases are characterised by little humans yet. However, the virus continues to be an
or no response to conventional antibiotic therapy, economically significant problem by causing severe
however, a number of these diseases can be decline in egg production (Cavanagh D. and Naqi
prevented by vaccination and good management S.A. 2003; Chousalkar K.K et al., 2009).
practices (Abdu P.A. 2007; FAO 2008). Live bird markets provides avenue for mixing
The potential ways of spread of viral infections of birds of different species. These markets exist in
include movement of contaminated fomites, feed or different locations within and outside Yobe state.
water and introduction of carrier bird to susceptible Biosecurity level in rural poultry production and
flocks. Migratory wild birds, use of vaccines and live bird market systems is minimal or in some
mix poultry populations can equally help in spread cases non-existing, this may lead to spread of
of viral infections (Adene D.F. 2007; CIDRAP multiple infections (Permin A. 1997).
2008). Despite serological evidence of AI and ND
Direct economic consequences of outbreaks of viruses in domestic birds in some parts of Nigeria
respiratory viral infections on the poultry industry (Owoade A.A et al., 2002; Saidu L. et al., 2004;
generally include the loss of birds as a result of Ocholi R.A et al., 2006; Owoade A.A. et al., 2006;
death, disruption of production and trade in poultry Abdu P.A. 2007; Abubakar M.B et al., 2008) there
and poultry products from affected areas, increase is dearth of information on these respiratory tract
of price of retail of poultry and poultry products as viral infectious agents in local poultry in north-
well as cost of logistics on the part of authorities to eastern Nigeria. There is no documented evidence
put control measures in place (Owoade A.A 2006; describing the presence of IB virus in both domestic
CIDRAP 2009). The World Bank estimated in and wild birds in the area under study.
October 2006 that about 1.5 billion was needed to This study investigated the presence of AI, ND
combat Highly Pathogenic Avian Influenza (H5N1) and IB viruses in domestic and wild birds using
globally. However, by October 2008, $1.5 billion nested polymerase chain reaction (nPCR).
has been disbursed; yet, the infection was far from
being contained in many nations of the world. In Materials and Methods
Nigeria, over 1.3 million birds have been culled and
over $5.4 million has been paid as compensation to Study area
farmers due to Highly Pathogenic Avian Influenza Yobe State is located in north-eastern Nigeria.
H5N1 between 2006 and 2008 (Adene D.F. It is located between latitudes 11°56ʹ and 13°20ʹE
2007;CIDRAP 2008; CIDRAP 2009). and longitudes 9°38ʹ and 12°25ʹN of the Northern
The mutagenic tendencies of some respiratory hemisphere. It shares international boundary with
viral agents of avian species make them a potential Niger Republic in the extreme north and interstate
threat to human health especially in some boundaries with Borno State in the east, Bauchi and
developing countries where there is intimate Jigawa States in the west and Gombe State in the
interaction between birds and humans. Some of south.
these viral agents are known to remain in Hadejia-Nguru and Dogona Wetlands is located
evolutionary stasis in their avian hosts. Such in Yobe state, it lies between Sudanian Savanna to
evolutionary stasis is often indicated by low genetic the south and the drier Sahel to the north. It is part
mutation rates. When disease determinants supports of the Chad Basin National Park which covers
cycles of infection, the viral agents may mutate and 938 km2; some of the land is permanently flooded,
adapt to a new hosts (Grain 2006; Greger M 2006). while other parts are flooded only in the wet season
482 J. Vet. Adv., 2012, 2(10):481-487GARBA ET AL.
(August and September). The annual rainfall ranges 2008). AIV oligonucleotide primers: ChenF(M52C)
between 200–600 mm, during the period May– and ChenR(M253R) were used as forward and
September. The wetlands are protected by five reverse primers respectively for AIV matrix assay
Forest Reserves, and a Wildlife Sanctuary (Ramsar, (Fouchier and Wallensten, A, 2005). NDV forward
2008). The wetland supports over 25 bird species, a primer F0P1 and F0P2 reverse primer first round
wide range of fish species and is an important while forward primer F1P1and reverse primer F1P2
source of drinking water for local cattle (Ramsar, were used for nested NDV PCR assay (Kho C.L et
2008). al .,2000). Infectious bronchitis virus (IBV) was
assayed using forward primer N784 and reverse
Sampling primer N1145 for first round PCR while forward
Gashua, Nangere, Nguru and Potiskum Local primer N791 and reverse primer N1129 were used
Government Areas (LGAs) out of the seventeen for nested PCR (Akin A et al ., 2001). A 1kb plus
LGAs of the State were selected at random by DNA marker was used to determine the DNA
balloting, four out of six Live bird markets and five segments size of each virus.
out of ten slaughter slabs in the selected LGAs were
identified and selected by balloting (Snoeck C.J et Results and Discussion
al., 2009).
Thirty five captive wild birds were sampled All the birds examined were about 22 weeks of
from Nguru LGA by convenient sampling technique age and apparently healthy at ante mortem, there
(Singh K et al., 2005). Cloacal and tracheal swabs was no record of vaccination and general health
were collected aseptically using sterile cotton management. The live bird markets in the study
swabs, with double ends (one for each bird). Each areas served as feeder for the attached slaughter
of the cotton ends were cut and deposited into slabs. The overall sanitary conditions in the live
Eppendorf tubes containing 1.5 ml of Hanks viral bird markets and the slaughter slabs were observed
transport medium. to be associated with inadequate water supply, lack
Samples were placed in a sterile transparent of proper drainage system and minimal control of
plastic bag and stored in a customized cool box movement of people on and around the slaughter
containing ice packs. Samples were preserved at slabs.
−20°C for less than 48 h before transportation to A total of 159 domestic birds were sampled
Biotechnology Research Laboratory, Faculty of from four LGAs, 87.4% were local chickens, and
Veterinary Medicine, and University of Ibadan, 10.1% were turkeys and 2.5% Muscovy ducks.
Nigeria for analysis as recommended (Thrusfield The Average number of birds slaughtered per
M, 1997). day in the study area was 401, average number of
birds slaughtered in the various LGAs are, Potiskum
Sample processing 199 (49.6%), Nguru 95 (23.7%), Gashua 59(14.7%)
The samples were prepared for viral genome and Nangere 48(12%) (See table 1).
extraction. Using QIAGEN kits manual (Hilden, The prevalence of NDV in domestic birds
Germany), 140 µl of each field sample was pulse slaughtered in the study area was 30.8%, a local
vortexed, centrifuged and dispensed into a set of chicken was 33.1% and in turkeys was 18.8%. NDV
new sterile 1.5 ml Eppendorf.. was not detected in ducks (N=4) slaughtered in the
Total RNA was extracted directly using study area.
QIAamp viral RNA mini spin protocol (Hilden, The prevalence of IBV in local chickens
Germany). The extracted RNA was subjected to slaughtered in the study area was 2.9%. IBV was
reverse transcription polymerase chain reaction not detected in Turkeys and ducks slaughtered in
using superscript III reverse transcriptase (Life the study area. AIV was not detected in domestic
Technologies, California, US). birds slaughtered in the study area (See table 2).
PCR amplification was carried out for each of A total of 34 wild birds, consisting of seven
the viruses as described by (WHO, 2002; OIE, species were captured. The highest was Goose
483 J. Vet. Adv., 2012, 2(10):481-487DETECTION OF AVIAN INFLUENZA, NEWCASTLE DISEASE AND …
(Anser anser) 32.4% and the least was peacock
(Afropavo congensis) 2.9%. NDV, AIV and IBV
were not detected in wild birds (See table 3).
Table 1: Distribution Of Domestic Birds Sampled From Slaughter Slabs By Local Government Areas
Average Number of birds sampled No (%)
Location
laughter/day Chicken Duck Turkey
Gashua 59 26 (44.1) 0 0
Nangere 48 20 (41.7) 0 0
Nguru 95 40 (37.9) 0 4 (4.2)
Potiskum 199 73 (28.6) 4 (2.0) 12 (6.0)
Total 401 (34.7) 4 (0.9) 16 (3.9)
Table 2: Prevalence Of NDV, AIV And IBV In Domestic Chickens Slaughtered In The Study Area
No. Positive (%)
Domestic Bird No. Sampled
AIV NDV IBV
Local chickens 139 0 (0.0) 46 (33.1) 4 (2.9)
Turkey 16 0 (0.0) 3 (18.8) 0 (0.0)
Duck (Muscovy) 4 0 (0.0) 0 (0.0) 0 (0.0)
Total 159 0 49 (30.8) 4 (2.5)
AIV= Avian Influenza Virus, NDV =Newcastle Disease Virus, IBV =Infectious Bronchitis Virus
Table 3: Distribution of captured wild birds in Yobe State Nigeria and the prevalence of NDV, AIV and IBV
among the various species
Number Number positive (%)
Wild bird
sampled AIV NDV IBV
Goose (Anser anser) 11 0 (0.0) 0 (0.0) 0 (0.0)
Common scoter (Melanitta nigra) 4 0 (0.0) 0 (0.0) 0 (0.0)
Little bittern (Ixobrychus minutus) 1 0 (0.0) 0 (0.0) 0 (0.0)
Peacock (Afropavo congensis) 7 0 (0.0) 0 (0.0) 0 (0.0)
Purple gallinule (Porphyrio porphyrio) 3 0 (0.0) 0 (0.0) 0 (0.0)
Speckled pigeon(Columba guinea) 6 0 (0.0) 0 (0.0) 0 (0.0)
White stork(Ciconia ciconia) 0 (0.0) 0 (0.0) 0 (0.0)
Total 34
Domestic birds in Yobe state are commonly across the international borders of Chad and Niger
seen to be raised in semi-intensive settings mainly Republics. It is common to see birds in the study
in the rural communities where there was little or no area hanged on handles of bicycles and motorcycles
veterinary cares and birds scavenge for feed most as they are transported to live bird markets. These
part of the day. This type of management practice is practices not only put stress on the birds but may
common in most African and Asian countries. This encourage the spread of infectious agents through
practice is known to encourage easy acquisition and aerosol or contact from infected to susceptible birds
spread of infectious agents (Permin A, 1997; Adene on transit. Similarly, zoonotic infections can be
D.F, 2007; Gueye E.F. 2007). transmitted from infected individual(s) to
The birds presented for sale at the poultry susceptible humans or birds (CIDRAP, 2008).
markets and the slaughter slabs for slaughter were Furthermore, infected birds from one locality can
sourced from different locations both within and serve as source of infection to susceptible bird’s
outside the state; occasionally the birds are sourced population in another locality (CIDRAP, 2008;
484 J. Vet. Adv., 2012, 2(10):481-487GARBA ET AL.
Saidu L. et al., 2004). Research have shown that the findings of this study. This may be related to
most birds found in live bird markets in Nigeria the less number of sample investigated when to the
harbours one or another disease (Saidu L. et al., previous studies. This may also mean that AI
2004). response and control measures put in place by
Newcastle disease virus had the highest Nigerian Government during the outbreak of AI in
prevalence in local chickens and turkeys (Table 2). year 2006 (Ocholi R.A et al., 2006) might have
This may be supported by the findings of Saidu et aided the containment of the disease in the area.
al., (2004) that local chickens are more susceptible None of the three viruses was detected in
to NDV infection than other domesticated birds. Muscovy ducks and wild birds. This is contrary to
NDV has been described as the most important the report by Mai H.M et al., 2004) who obtained
virus in poultry causing huge economic losses in 6.7% infection rate of Newcastle disease virus
poultry farms in developed and developing among local ducks in a North-central Nigeria. The
countries (Snoeck et al., 2009). The prevalence of absence may be attributed to the fewer number of
NDV in this study was low when compared to 92% ducks examined in this study. HPAI (H5N1) virus is
reported by Saidu et al., (2004). Although serology known to circulate among ducks without serious
is less sensitive when compared with PCR effect but the ducks may serve as source of
techniques (Singh et al., 2005), the wide difference infection for other birds species and human
in prevalence may be as a result of the variations in (Henning et al., 2010).
geographical locations, usage of varied ND Generally, the viral agents detected in this
vaccines and the study design. Saidu et al., (2004) study are known to infect birds and cause
worked in north central area of Nigeria which is respiratory diseases or diseases associated with
popular for intensive commercial poultry rearing. respiratory system in infected flock(s) (Owoade
Indiscriminate usage and mishandling of vaccines A.A et al., 2006). Infections with either of these
on commercial farms may lead to contamination of viruses have been reported to lead to reduced feed
equipment and the immediate environment, this intake and utilization and diverse pathological
increase possibility of transmission to neighbouring conditions resulting ultimately in reduction of
farms production functions and death of the infected birds;
IBV was detected in local chicken only. The consequent to these are economic loses to the
prevalence of 2.9% found in this study is low when farmer in particular and to the poultry industry at
compared to 26% reported in commercial poultry in large (Alexander D.J. 2003; Van Gils J.A et al.,
south western Nigeria (Owoade A.A et al., 2006). 2007).
However this study confirms Ducatez et al (2009) Great losses have been experienced from avian
findings that IBV infections seemed to be less epizootics such as AI outbreaks in different parts of
common in live bird markets in northern Nigerian the world in recent times (CIDRAP, 2008). The cost
and in backyard poultry in Niger Republic,. Sero- of control measures including vaccination
prevalence evidence in Nigeria suggests increase in represents a continuing loss to the industry and
new variant serotypes of IBV currently circulating increase cost to the consumer. In many developing
in Nigeria and Niger Republic (Ducatez M.F et al., countries NDV is endemic and pose an important
2009). This may portend danger as IBV is known to limiting factor in the development of commercial
cause significant economic losses because of poultry production and the establishment of trade
reduced productivity other than bird mortality links (Sonaiya E.B. and Swan 2008; Iroegbu C.U
(Cavanagh D. and Naqi S.A, 2003). and Nchinda, 1999.). Infection with IBV in small
Although Ocholi et al., (2006) listed the study and medium scale poultry farms has led to severe
area among the sites in Nigeria that had AI (H5N1) declines in quality and quantity of egg thereby,
outbreak in 2006, and Gaidet et al., (2007) reported leading to losses from production inefficiencies
the presence of LPAI in wild migratory birds (Cavanagh D. and Naqi S.A. 2003).
sampled from wetlands areas of Nigeria, which There is a growing prospect for large scale
includes Yobe State, these reports are contrary to poultry farming in Yobe State to complement the
485 J. Vet. Adv., 2012, 2(10):481-487DETECTION OF AVIAN INFLUENZA, NEWCASTLE DISEASE AND …
existing wide spread backyard production of local coronavirus and avian infectious bronchitis virus. Acta
chickens. Thus, measures have to be taken to Virol., 45, 31-38. DOI:10.1099/vir0.01247-0
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