Surveillance of Avian Influenza Viruses in Wild Birds in Areas Adjacent to Epicenter of an out Break in Federal Capital Territory of Pakistan

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1 Internatinal Jurnal f Pultry Science 4 (1): 39-43, 2005 Asian Netwrk fr Scientific Infrmatin, 2005 Surveillance f Avian Influenza Viruses in Wild Birds in Areas Adjacent t Epicenter f an ut Break in Federal Capital Territry f Pakistan J.Z. Khawaja, K. Naeem, Z. Ahmed and S. Ahmad 1 Natinal Reference Lab Fr Pultry Diseases, Animal Health Prgram, Natinal Agricultural Research Center, Islamabad, Pakistan Department f Bilgical Sciences, Quaid- I- Azam University, Islamabad, Pakistan ajkhawaja@htmail.cm 2 Abstract: Influenza is a highly cntagius acute respiratry disease f avian rigin that has caused epidemics and pandemics in humans fr centuries. Amng these avian influenza viruses (AIV), nly sertypes H5 and H7 are cnsidered highly pathgenic in pultry. Hwever, sertype H9N2 has als been fund t prduce severe respiratry and reprductive tract infectins in chickens. The previus studies have suggested that mvement f pultry and wild birds play a majr rle in the spread f influenza viruses t distant areas in a cuntry. The present study was carried ut t mnitr the prevalence f AIV in wild birds in certain areas f the Pakistan, which were free frm infectin during the utbreak f Nvember In this regard, 7 wild bird species were selected and their bld, clacal swabs and tissue samples were cllected bth fr serlgical evaluatin and virus islatin. The results indicated that antibdies t AIV sertype H9N2 were present in 10% f wild birds, whereas the virus was itself islated nly frm 6.72% f the samples. These data prvide the evidence regarding the wild birds as ne f the majr carrier f the AIV infectin. Key wrds: Avian influenza, sertype H9N2, wild birds Intrductin Avian influenza (AI) is caused by type A Orthmyxviruses. Only type A influenza viruses are knwn t cause natural infectin bth in birds and human. On the basis f different cmbinatin f 15 haemagglutinin and 9 neuraminidase antigens present n the surface f the avian influenza viruses, 256 cmbinatins f Avian Influenza Virus (AIV) may exist in nature. Influenza A viruses infecting pultry can be divided int tw distinct grups n the basis f their ability t cause disease. The very virulent viruses, named as highly pathgenic avian influenza viruses (HPAI), can cause heavy mrtality and drastic decline in prductin. This grup f viruses includes subtypes H5 and H7, althugh nt all viruses f these subtypes cause HPAI-assciated disease. All ther viruses cause a milder, primarily respiratry disease, which may be exacerbated by ther infectins r envirnmental cnditins. HPAI viruses are rarely islated frm wild birds. Hwever, extremely high islatin rates f viruses f lw virulence fr pultry have been reprted in surveillance studies in which 15% fr ducks and geese and abut 2% f all ther species have been shwn t be the carriers f AIV. Influenza viruses have been shwn t affect all types f dmestic r captive birds in all areas f the wrld, but the frequency with which primary infectins ccur in any type f bird depends n the degree f cntact with feral birds. Secndary spread is usually assciated with human invlvement, prbably by transferring infective feces frm infected t susceptible birds (Alexander, 2003). Avian influenza f HPAI type was first reprted in Pakistan in 1995 (Naeem and Hussain, 1995). The disease caused by sertype H7N3 prduced high mrtality amng the affected flcks especially in the briler breeder rearing areas f the cuntry. Anther influenza utbreak in nrthern areas f Pakistan was reprted in 1999, which resulted in 10-20% mrtality with decrease egg prductin frm 10 t 75%. It was fund t be H9N2 subtype and was named as A/Chicken/Pakistan/3/99(H9N2) (Naeem et al., 1999). Since then the disease has been repeatedly reprted frm varius pultry rearing areas at different lcatins thrughut the cuntry. In Nvember f 2003 a new utbreak f AIV invlving sertype H7N3 and H9N2 was reprted frm the castal areas f Karachi, nly affecting cmmercial layer flcks and als in briler breeder rearing regin in the nrth. The disease caused high mrtality in these regins. This disease cnfined t the affected areas but there was a pssibility f its spread t ther pultry raising area in adjacent twns. Althugh very strict bisecurity measures were intrduced in the affected areas, there was a pssibility f AIV transmissin t the nrthern part f the cuntry thrugh wild bird ppulatin, as there is heavy wild bird ppulatin in this area. Sme earlier studies f avian influenza A viruses carried ut in Eastern Germany during shwed virus islatin directly frm feral ducks and ther wild birds (Suss et al., 1994). The wild ducks are als knwn t be a majr reservir fr avian influenza viruses but there are few recent published reprts f surveillance directed at 39

2 Khawaja et al.: Surveillance f Avian Influenza Viruses in Wild Birds Table 1: Serprevalence f avian influenza virus kept at -20 C till further prcessing. The tissues were sertype H9N2 in wild birds ppulatins as determined by Haemagglutinatin Inhibitin assay minced, freeze-thawed thrice, and centrifuged at 800xg fr 10 mins and the supernatant was cllected fr in v inculatin. Bird Type Number GMT Titre Range f Serum Samples Eagles Myna Crws Ducks Gse Water Fwls Dves this grup. Predminant AIV hemagglutinin (HA) subtypes reprted in ne f such studies f ducks in Nrth America included H3, H4, H6, H5, H7, and H9 subtypes (Hansn et al., 2003). The present study was undertaken t assess the rle f different wild birds in harbring bth H7 and H9 sertypes f avian influenza virus in the federal capital territry f Pakistan that is clse t sme majr brilerbreeder and layer rearing areas in the cuntry. Materials and Methds Specimen cllectin: A number f wild birds were either caught, r sht at each f the three designated lcatin frm December 2003 t May These lcatins were Lake f Bhera, Chakshehzad, Islamabad (which is a thickly ppulated pultry area); banks f River Sindh near Attck and Wild Life Park f Lhi Bher. All these areas are lcated within 100 km f the epicenter f AI utbreak. The types f specimens cllected during this study are described belw: a. Bld: Bld was taken frm the fllwing species f wild birds: Crws, Cmmn Myna, Mallard ducks, Dves, Cmmn Gse, Fwls and Eagles. Frm each bird 1-2ml f bld was drawn and allwed t clt. The serum was remved after centrifugatin at 800xg fr ten minutes. Sera were subsequently stred at -20 C until used. Virus islatin: Each tube-cntaining swab in virus transprt medium was vrtexed, and the swab carefully remved after squeezing. It was centrifuged and supernatant was filtered thrugh 0.2µm filter fr in v inculatin. Bth the tissues hmgenates and clacal swab preparatins were inculated in 9-day-ld embrynated eggs, via allantic rute. After fur days, the eggs were chilled ver night. Under aseptic cnditins, the allantic fluid (AF) was cllected. The embrys were als examined fr lesins. The AF frm each egg was tested fr haemagglutinating activity as previusly described (Naeem et al., 1999). The negative samples were repassaged in fresh embrynated eggs. Haemagglutinatin and Haemagglutinatin inhibitin (HI) tests: In rder t mnitr the antibdy titres against AI viruses in wild birds, HA and HI tests were perfrmed accrding t the prtcls described earlier (Olsen et al., 2003). Briefly, 2- fld dilutin f the AF was made in PBS (ph 7.2) in a 96-well micr titratin plate. Chicken RBC s were added t each well at 0.5% cncentratin. The plates were incubated fr 30 mins at 37 C befre recrding the haemagglutinating activity. Haemagglutinatin inhibitin (HI) titre f each serum sample was als determined. Briefly, 25 µl f the test sera were serially diluted in PBS (ph7.2) using a 96-well titratin plate. T this 25 µl f the knwn sertype f the AIV (4HA) was added in each well. The plates were incubated fr 30mins at 37 C. Nw 50ml f 0.5% f the chicken RBC s were added t each well and the plates were again incubated fr 30 mins. at 37 C. The results were recrded and subjected t gemetric mean titres (GMT) analysis. Virus Neutralizatin (VN) test: The cnstant-serum, diluted-virus methd was used fr the identificatin f the islated sertypes using the methd as previusly described by Beard, (1980). b. Clacal swabs: Clacal swabs were taken frm the wild birds and cllected aseptically with sft Dacrn tipped applicatrs in 1.5 ml vial cntaining virus transprt medium (BHI brth with Penicillin, Streptmycin, Gentamycin). The samples were prmptly transprted in ice t the labratry where they were stred at -20 C. c. Clinical specimens: Trachea and lung specimen were cllected frm each bird fr virus islatin. The samples were placed in transprt media and then Results Out f 84 serum samples cllected frm the wild birds, 12 samples representing every bird type shwed HI antibdy titres against H9N2 AIV sertype. Table 1 shws the results f HI against AIV amng different species f wild birds. As shwn, the eagles had the highest GMT value f 16. All ther birds had a GMT value within a range f 1-4 (myna 1, crws 3, ducks 2.3, cmmn gse 3.5, waterfwls 4 and the dves 2). All the samples tested fr the presence f antibdies against H7N3 were fund negative. 40

3 Khawaja et al.: Surveillance f Avian Influenza Viruses in Wild Birds Table 2: Distributin f avian Influenza virus islatin Table 3: Virus Neutralizatin Test fr sertype frm tissues f serpsitive samples identificatin f avian influenza islates Bird Type Virus Detectin at Passage (P) Level Islate designatin Virus neutralizatin activity against P1 P2 P Fwl # H9N2 H7N3 Eagle# Fwl#1 + - Crw# Fwl#2 - - Crw# Dve# Crw# Cmmn Myna# Crw# Mallard Duck#1 - - Duck# Crw#1 + - Gse# Crw#2 + - Gse# Crw#3 + - Gse# Crw#4 + - Gse#1 - - Gse# Gse# = Negative + = Psitive. NOTE: All the samples tested fr the presence f H7N3 sertype was fund negative. Gse#3 + - Gse#4 + - Table 2 shws HA activity f the islates recvered frm the tissue samples f wild birds. All the samples were prcessed and passaged twice in eggs. Out f 84 samples, 8 samples shwed haemagglutinating activity. All samples were further tested thrugh Agar Gel Precipitatin Test (AGPT), using AIV plyvalent antisera and 4 samples were fund psitive n AGPT (data nt shwn). Fig. 1 shws the HA activity f islates recvered frm the clacal swabs and tissues f these wild birds. The crws shwed the highest activity i.e.128, 32 and 2, while fwls shwed the HA activity f 32, 8, and 4; ducks shwed the HA activity f 2, 2, and 2; eagles shwed the HA activity f 4, 2, and 2; while the gse shwed the HA activity f 32, 4, and 2. Table 3 shws the results f virus neutralizatin test (VNT) perfrmed fr the sertype identificatin f AIV. Eight islates were psitively identified as H9N2 sertypes using the reference antisera. N psitive islates f H7N3 were fund. Fig. 2 depicts the distributin f AIV psitive cases amng varius species f wild birds, tested by HI. It was seen that 50% eagles, 17% crws, 10% ducks, 30% gse and 33% fwls were the carriers f AIV (H9N2 sertypes). It was further seen that all the samples tested fr the presence f antibdies against H7N3 were fund negative. Discussin There have been a number f AIV ut-breaks in pultry during the past decade in different parts f the wrld. A recent utbreak invlving H7N3 and H9N2 ccurred in Pakistan frm Nvember 2003 t May The study under reprt was carried ut t analyze the rle f wild birds in the spread f avian influenza viruses in this utbreak. Fr this purpse mallard ducks, geese, crws, cmmn myna, Indian fwl, dves and eagles were examined. Bld samples, clacal swabs and the tissue samples, where available, were cllected. The antibdy titres against AIV sub types H9N2 and H7N3 were tested by HI. 10 % f the mallard ducks shwed the high antibdy titres against influenza virus sub type H9N2, which migrated in summer t Lake Kalar Kahar (Chakwal). N titres were fund against H7N3 in these birds. Presence f high titres f HA antibdy supprted the idea that mallard ducks may be the natural carrier f AIV. Similarly the titres against AIV were als checked in ther wild birds. The percentage f serpsitive birds against H9 antibdies was 30% geese, 17% crws, 50% eagles and 33% Indian fwls. This shws that the wild birds are the natural carriers f AIV sertype H9N2. The titre range (GMT) in mallard ducks was 2-8, in geese 2-256, in crws 2-64, in eagles and Indian fwls The high antibdy titres against subtype H9N2, is the indicatin fr the presence f AIV (H9) in these birds persistently. High antibdy titres against H9N2 were fund in 17% f these samples. This shws the rle f crws in the spread f avian influenza virus in pultry ppulatin. As the crws eat pultry waste and are carnivrus animal, this might be the reasn f transfer f virus t crws. On the ther hand cmmn myna shwed n titres against the influenza virus and seems nt t be participating in influenza spread. The dves were als examined in this study. It was fund that there was n antibdy titre against AIV (H9 and H7) in these birds. Mrever n virus was islated frm the tissues f these dves. As dves are nt invlved in flesh eating, they remained free frm virus expsure. It further indicates that the water reservirs in the vicinity frm where sampling was dne are free frm 41

4 Khawaja et al.: Surveillance f Avian Influenza Viruses in Wild Birds Fig. 1: Haemagglutinain (HA) titres f AIV islates recvered frm clacal swabs and tissues Fig 2: Distributin f aiv psitive cases amng different species f wild birds as tested by HI AIV as there is great pssibility f sharing f water reservir amng all types f birds which culd have transmitted the virus t myna and dves as well. The AIV sertype H9N2 was als detected frm the clacal swabs f the birds examined abve. Here it was fund t be 50 % (2/4) in geese, 50% (½) in fwls, 50% (½) in eagles and 75% (3/4) in crws. The AIV subtype H9N2 was recvered frm the clacal swabs f these birds by in v inculatin. The prpagatin f influenza virus in embrynated eggs cnfirms the previus knwledge abut the reliability f this system. It was als fund that mst f the psitive islates in this study were detected after 2nd passage thrugh the embrynated eggs. Rle f wild birds in maintaining and spreading the avian influenza virus has als been reprted by sme ther wrkers. Lee et al. (2000) reprted the islatin f an H9N2 influenza virus (A/duck/Nrth Carlina/91347/01) (Dk/Nc) frm wild ducks in the United States. Genetic analysis shwed that this duck virus had the same human /classical swine/avian reassrtment gentype as the H9N2 viruses that have been islated frm pigs and turkeys in the US since In anther study, serum samples frm 163 slaughter age striches in Ohi and Indiana were tested fr antibdies t avian influenza virus. Sme strich had antibdies t AIV H5N9. This is the first reprt f antibdies t avian influenza in striches in the United States. In anther reprt AIV sertype H7N3 was recvered frm a falcn in Syria in Upn the mlecular analysis f these islates, the sequencing data revealed its hmlgy t the islates frm Pakistan in It was eventually fund that this falcn was brught frm Pakistan (Banks et al., 2000). Such studies f wild bird mnitring have been helpful in predicting the new sertypes f AIV in an area. Such intensive surveillance study f aquatic birds especially in Siberia has earlier prvided infrmatin n the future pandemics f influenza viruses and fr vaccine preparatin (Okazaki et al., 2000). Significance f such studies is als reflected frm many previus studies indicating the inter species transmissin f influenza A viruses circulating in the wild aquatic birds ccasinally results in influenza ut breaks in mammals, including humans (Matrsvich et al., 2000). This happens due t genetic reassrtment amng different types f AIV infecting wild birds. The rle f AIV sertype H9N2 in causing infectin in humans in Hng Kng and China (Peires et al.,1999) als signifies the prevalence f H9N2 sertype in pultry. In this part f the wrld, it is highly likely that this virus is spreading amng humans, wild birds and pultry and may result in causing new utbreaks after its mutatin during interspecies transfer and replicatin. Therefre the wild birds can be cnsidered as majr reservirs f H9N2 in this area. In the abve scenari, the detectin f AIV H9N2 in wild birds in this cuntry pses a cntinuus threat fr the emergence f mre pathgenic strains f influenza virus in pultry, alng with the pssibility f further spread f these viruses t ther parts f the Pakistan. The persistence f this AIV sertype frm the wild birds, in this part f the wrld signifies the existence f virus as a pssible candidate fr future ut-breaks in pultry in Asia. Based n the significance f wild birds as the carrier f AIVs, it is recmmended t keep an eye n the migratry patterns f the wild birds in a regin. As the 42

5 Khawaja et al.: Surveillance f Avian Influenza Viruses in Wild Birds glbal climate is changing rapidly, the temperature f the Lee, C.W., C.S. Sng, Y.J. Lee, I.P. M, M. Garcia, D.L. earth is increasing and the glaciers are melting, the Suarez and S.J. Kim, Sequence analysis f patterns f the migratin f these birds are fluctuating as the hemagglutinin gene f H9N2 Krean avian well. S it is necessary t watch the patterns f migratin influenza virus and assessment f the pathgenic and sermnitr these birds n regular basis. It will help ptential f islate MS96. Avian Dis., 44: t implement the prper precautinary measures in Asia Matrsvich, M., A. Tuzikv, N. Bvin, A. Gambaryan, A. and Sub-cntinent t cntrl the frequent relapse f AIV Klimv, M.R. Castrucci, I. Dnatelli and Y. Kawaka, in pultry ppulatin in Pakistan Early alteratins f the receptr-binding Serlgical analysis f varius species als indicates prperties f H1, H2, and H3 avian influenza virus the activity f this subtype in several types f wild birds. hemagglutinins after their intrductin int It wuld, therefre, be apprpriate t sermnitr the mammals. J. Virl., 74: wild birds n regular basis t evaluate the rapidly Naeem, K. and M. Hussain, An utbreak f avian changing status f this virus. It is further suggested that influenza in pultry in Pakistan. Vet. Rec., 137: 439. imprvement in bisecurity at the farms shuld be given Naeem, K., A. Ullah, R.J. Manvell and D.J. Alexander, tp pririty t stp the spread f AIV sertypes thrugh Avian influenza A subtype H9N2 in pultry in cntacts between wild birds and cmmercial pultry. Pakistan. Vet. Rec., 145: 560. Okazaki, K., A. Takada, M. Imai, H. Takakuwa, M. Hatta, References Alexander, D.J., Reprt n avian influenza in the Eastern Hemisphere during Avian Dis., 47: Banks, J., E.C. Speidel, J.W. McCauley and D.J. Alexander, Phylgenetic analysis f H7 haemagglutinin subtype influenza A viruses. Arch Virl.,145: Beard, C.W., Serlgic prcedures. In: Islatin and identificatin f avian pathgens, 2nd ed. S.B. Hitchner, C.H. Dmermuth, H.G. Purchase, and J.E. Williams, eds. Amer. Assc. Avian Pathl., Cllege Statin, Texas, pp: Hansn, B.A., D.E. Stallknecht, D.E. Swayne, L.A. Lewis H. Ozaki, T. Tanizaki, T, Nagan, A. Ninmiya and V.A. Demenev, Precursr genes f future pandemic influenza viruses are perpetuated in ducks nesting in Siberia. Arch. Virl., Olsen, C.W., A. Karasin and G. Ericksn, Characterizatin f a swine-like reassrtant H1N2 influenza virus islated frm a wild duck in the United States Virus Res., 93: Peires, M., K.Y. Yuen, C.W. Leung, K.H. Chan, R.W. Lai, W.K. Orr and K.F. Shrtridge, Human infectin with influenza H9N2. Lancet, 354: Suss, J., J. Schafer, H. Sinnecker and R.G. Webster, Influenza virus subtypes in aquatic birds f eastern Germany. Arch Virl., 135: and D.A. Senne, Avian influenza viruses in Minnesta ducks during Avian Dis., 47:

The estimator, X, is unbiased and, if one assumes that the variance of X7 is constant from week to week, then the variance of X7 is given by

The estimator, X, is unbiased and, if one assumes that the variance of X7 is constant from week to week, then the variance of X7 is given by ESTIMATION PROCEDURES USED TO PRODUCE WEEKLY FLU STATISTICS FROM THE HEALTH INTERVIEW SURVEY James T. Massey, Gail S. Pe, Walt R. Simmns Natinal Center fr Health Statistics. INTRODUCTION In April 97, the