Avian Influenza Survellance In Wild Migratory, Resident, Domestic Birds And Poultry In Maharashtra And Manipur, India During Migratory Season 2006 2007
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Avian Influenza Survellance In Wild Migratory, Resident, Domestic Birds And Poultry In Maharashtra And Manipur, India During Migratory Season 2006 2007
1. RESEARCH COMMUNICATIONS
Avian influenza surveillance in wild ZOONOTIC diseases like Avian Influenza (AI), Newcastle
Disease (ND) and West Nile (WN) are some of the
migratory, resident, domestic birds emerging viral diseases in water birds1. Due to large out-
and in poultry in Maharashtra and breaks in recent years and in some cases virus transmis-
Manipur, India, during avian sion from poultry to human with a high fatality rate,
Avian Influenza A virus has currently aroused concern
migratory season 2006â07 and received serious attention2. There may be an associa-
tion between Highly Pathogenic Avian Influenza (HPAI)
Shailesh Pawar1,*, Satish Pande2, outbreaks and the presence of rapidly increasing poultry
Aniruddha Jamgaonkar1, Santosh Koratkar1, farms in several parts of the world3. It has been postu-
Bishwajoy Pal1, Satish Raut1, Madhuri Nanaware1, lated that H5N1/97 virus for humans principally came
Koninika Ray1, Alok Chakrabarti1, from retail and live poultry markets in Hong Kong in
Sadhana Kode1, Vishal Thite1, Madhukar Khude1, 1997 and subsequently spread to Cambodia and South
Korea4. It is believed that poor bio-security and poor
Satish Randive1, Atanu Basu1, Amit Pawashe2,
hygiene were responsible for the spread of the virus and
Aditya Ponkshe2, Pranav Pandit2 and it is more likely that wild birds had no role in the spread5.
Pramod Deshpande2 Long-term screening and surveillance of migratory
1
National Institute of Virology, Microbial Containment Complex, birds for the presence of AI virus is necessary as a part of
131/1, Sus Road, Pashan, Pune 411 021, India wider range of preparedness to avert the future appear-
2
Ela Foundation, C-9 Bhosale Park, Sahakarnagar, Pune 411 009, India
ance of the virus in a pandemic form in humans6. Since
2003, HPAI H5N1 virus has spread to Europe and Africa
India reported outbreaks of Highly Pathogenic Avian
Influenza (HPAI) H5N1 in poultry in the states of and virus from birds in West Siberia, Europe and Africa
Maharashtra, Gujarat and Madhya Pradesh (Febru- is similar to that from Qinghai lake, China7,8. Importantly,
aryâApril 2006); Manipur (July 2007); West Bengal East and Central Asian Flyways of migratory birds,
(January 2008) and Tripura (April 2008). The role of which include India in their path, overlap extensively in
migratory birds in the transmission of the HPAI H5N1 West China (around Qinghai Lake), Mongolia and Central
remains a subject of debate. Avian Influenza (AI) sur- Siberia allowing interchange of diseases between these
veillance in wild migratory, wild resident, domestic areas and particularly with India9,10. India reported AI
birds and poultry was undertaken by National Insti- H5N1 outbreaks in poultry8,11â13. The role of migratory
tute of Virology (NIV) jointly with Ela Foundation, birds in the movement of the HPAI H5N1 remains a sub-
Pune, India during 2006â07. A total of 1968 faecal ject of debate14. Therefore, in view of these recent AI
specimens (1369 droppings from wild migratory and H5N1 outbreaks in poultry in India, screening of wild
wild resident birds; 474 droppings from poultry and migratory, wild resident and domestic birds as well as
125 cloacal swabs from chickens and ducks) were col- poultry was undertaken by National Institute of Virology
lected. These samples representing 10 avian families of
(NIV) jointly with Ela Foundation to study the role of
wild migratory birds, four families of wild resident
birds totalling 36 species, were from eight districts of these birds in transmission of AI viruses. Migratory birds
Maharashtra covering 20 water bodies and two districts visit India during winter season (OctoberâApril) every
of Manipur. The samples were screened for AI viruses year. There are no reports of AI surveillance in migra-
by reverse transcriptase polymerase chain reaction tory/wild resident birds from India. This report presents
(RT-PCR), real-time PCR and were processed for the findings of AI surveillance during avian migratory
virus isolation in embryonated chicken eggs and cell season 2006â07.
culture. Two samples from wild ducks were positive Faecal samples (FS) of migratory birds were collected
for viruses other than AI, newcastle disease virus from several sites in Maharashtra, which are known for
(NDV) and infectious bursal disease virus (IBDV). the arrival of migratory birds, during the avian winter
During the study period no sample was positive for migratory season 2006â07 (Table 1). The samples were
Influenza A viruses, Influenza A (H5N1) or any other collected and transported in viral transport medium
strain of HPAI by RT-PCR and virus isolation. In (VTM) (Hankâs balanced salt solution) with antibiotics
view of the recent HPAI H5N1 outbreaks in poultry in
(Penicillin, Streptomycin, Gentamycin, Amphotericin B)
India, continued and more widespread AI surveillance
on wet ice/ice packs15.
is necessary to elucidate the role of wild migratory,
resident, domestic birds and poultry in the transmis- Samples of local birds were also collected during the
sion of AI viruses. same period. Poultry was sampled by site visits to com-
mercial and backyard poultries. The samples consisted of
Keywords: Avian influenza surveillance, faecal sam- faecal droppings in all birds, oral pellets and faecal drop-
ples, migratory birds. pings in case of gulls. Only fresh and wet samples were
collected. When mixed flocks were encountered, names
of all the species composing such flocks were entered for
*For correspondence. (e-mail: pawarshailesh@hotmail.com) such samples.
550 CURRENT SCIENCE, VOL. 97, NO. 4, 25 AUGUST 2009
2. RESEARCH COMMUNICATIONS
Table 1. District-wise samples collected from December 2006 to April 2007
No. of faecal samples
Wild migratory/ Poultry
Location resident birds and ducks Total
Maharashtra
Nandurbar 248 59 307
Raigad 58 â 58
Ahmednagar â 20 20
Pune 706 365 1071
Nanded 3 30 33
Nagpur 51 â 51
Ratnagiri 167 â 167
Satara 136 â 136
Manipur
West and East Imphal districts â 125 125
Total 1369 599 1968
The various sites/water-bodies/dams visited were Vir, inoculated in Madin Darby Canine Kidney (MDCK) cell
Ujani, Bhor, Naryangoan, Yedgoan, Kavdi, Khadakvasla, line.
Panshet, Pashan Lake, Lonawala, Vadaj, Chaskaman, All the 1968 samples were screened for the presence of
Revas, Akshi, Guhagar, MakarâDhokla, Rangavali, Bor- influenza A and H5N1 viruses using standard one step
pada, Bhaura and Khekada. These study sites included RT-PCR method. Viral RNA was extracted using QIAamp
water-bodies from Navapur, where outbreaks of HPAI viral RNA mini kit (Qiagen Inc, Germany). Qiagen One
H5N1 have been previously reported in poultry. step RT-PCR kit (Qiagen Inc, Germany) was used to
All the avian species were correctly identified follow- detect influenza-specific amplification of different genes
ing standard field guides; FS were collected with sterile according to manufacturerâs instructions. WHO recom-
swabs or spoons in VTM. Sample tubes were immediately mended influenza A-specific primer sets were used15.
sealed with parafilm and stored in icebox. Aseptic pre- PCR protocols standardized at AI laboratory at NIV, us-
cautions like wearing latex gloves, facemasks and correct ing primers for detection of matrix (M) gene were also
disposal of used equipment were meticulously carried used for verification.
out. FS were characterized species-wise by performing All 125 samples from Manipur and 300 representative
measurements of liquid splash (urinary tract contribution) samples collected from and around the outbreak locations
and solid pellet (digestive tract contribution) components, were tested by real-time RT-PCR using the TaqMan
as well as noting their consistency, colour and pH. As far influenza A/H5 detection kit cv1.0 (Applied Biosystems,
as possible, five droppings from single species from the USA). Analyses were carried out on Applied Biosystems
same flock were pooled to make one sample. If pooling 7300 real-time platform.
was not possible, single dropping was considered as one Ten-day-old SPF embryonated chicken eggs were used
sample16â19. for inoculation. Each sample was inoculated in two eggs
AI H5N1 outbreak occurred in poultry in Manipur12 in by allantoic route. These eggs were incubated at 37°C for
July 2007. A total of 125 cloacal swabs from chickens 72 h, chilled at +4°C overnight, and allantoic fluid was
and ducks were received from in and around 5 and 10 km harvested. The allantoic fluids were screened by haemag-
distances from the H5N1-affected area, West and East glutination (HA) test using 0.5% fowl and 1% horse
Imphal districts, Manipur (received from the Director of erythrocytes (RBCs)15. Representative allantoic fluids
Veterinary and Animal Husbandry Services, Government were tested by RT-PCR for confirmation.
of Manipur, Manipur; Table 1)12. All the samples were Each T-25 flask with confluent monolayer was infected
processed for virus isolation in embryonated chicken eggs with 500 ÎŒl of inoculum, allowed to adsorb for 30 min at
and tested by reverse transcriptase polymerase chain 37°C followed by washing of monolayers with medium to
reaction (RT-PCR). remove un-adsorbed virus particles. Flasks containing
The contents of each collection vial were stirred and 5 ml of Dulbeccoâs Modified Eagle Medium (DMEM)
each vial was centrifuged at 2000 rpm for 5 min to remove containing 2 ÎŒg/ml of TPCK trypsin without calf serum
debris. The supernatant was used for molecular diagnosis were then incubated at 37°C for 4â6 days. The flasks
and for inoculation in specific pathogen free (SPF) embry- were observed daily for cytopathic effect (CPE). MDCK
onated chicken eggs obtained from Venkateshwara cell line infected with influenza viruses shows degenera-
Hatcheries, Pune. Representative samples were also tion of cells which come out from the surface in super-
CURRENT SCIENCE, VOL. 97, NO. 4, 25 AUGUST 2009 551