A focal outbreak of Japanese encephalitis among horses...

A focal outbreak of Japanese encephalitis among horses...
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  . 1119 2003- 40 is A Focal Outbreak of Japanese Encephalitis among Horses in.Pune District, India.(Received or publication: ian. 2002,Focal outbreak of Japllne5'e Encephalitisoccuued among horses n stlld t'alms situatedabout 50 kms west of district Pune,Maharashtra state, India, The stud farmshoused around 150 horses and are surrounded by well-cultivated paddy fields. These horses\~:ere of thorough breeds ofboth genders and up to two years ofage. Duling October 1999,tour horses de\ eloped symptoms of pyrexia,anol-exia and abnormal behaviow" with central nen'ous system involvement. The affectedhorses were treated symptomatically, Ofthese, wo severely affected horses died while the remaining two reco\'ered subsequently.Paired and single serum samples werecollected from two affected and 59unaffected (contact) hol-ses respectively.Serum samples were also collected from thefour farm workers, who had close contactwith the diseased horses. To detemline thepossible viral aetiology, an autopsy sample ofbrain was collected from a dead animal. Allthe samples were.transported to NIV, Puneon wet ice, horse serum was further inoculatedintrathoracically in adult Cl/le.\:quinquefasciatll.5 and .4edes ae'5.1.primosquitoes \\,ith approximately 0.2 ~ll/mosquito, as described by Rosen and Gubler.'Head squashes of these n1osquitoes \\ereprepared on the 12th post inoculatiol1 ( PI )day and screened for the presence offlavivirus antigen using polyclonal antiselllmagainst Japanese encephalili.5 virus (.JEV )by indirect immunofluorescence antibody(IF A) technique.2 Two out of 48 headsquashes of C.~. quinqu~tascialIl5.mosquitoes gave weak positive reaction inthe IFA for the JEV antigen and.4e. ae6':1Plihead squashes were found negative 10 481.Bodies of these Cule.\" mosquitoes \\.erepassaged once more in a fresh batch of C.\:.quinquefasciatus mosquitoes andimmunofluorescence was detected \\.ithpolyclonal antisel"Um to lEV in 5136 squashes.To confirm the lEY aetiology the suspensionof the bodies of these positive mosquitoeswere inoculated by ic route in suckling mice.On the third PI da)', the mice came downwith characteristic sickness. The sick micebrains \\'ere further passaged in themosquitoes and mice. The brain suspensionof the dead horse, acute phase serum. 4thpassage suspension of mosquito and mousebrain were found to be positive in the .JE\;antigen capture ELISA, employIng JEVspecific mouse monoclonal antibodi~s.jHOweveT, the brain suspension of the 4thpassage in mice \\'as found to be negativeA 10% suspension of the post-mortem brain sample in Bovine albuminphosphate saline and acute serum of the sameanimal diluted I: lOin PBS were inoculatedintracerebrally (ic) in S\viss albino sucklingmice, Vero, PS IPorcine stable cell), andC6/36 (Aede .' albopictus) cell lines,Characteristic c)1opathological changes orsickness was not observed in cell culture andsuckling mice respecti\ ely, Blind passagesalso failed to yield any viral agent, The acute  '(II Outbre(lk (~/~/apa/1e.\,e ¬£ncephalilis (lmo/1g IIvr Figure-: Lane-M is marker (100bp ladder), PCR products using Japaneseen~phalitis virus specific primers: Lane-l, Normal mouse brain; Lane-2, Fieldmaterial inoculated mouse brain; Lane-3, JE prototype virus inoculated mousebrain; I~ane-4, Original horse brain. M 1 2 34 against lEV polyclonal and West Nile virus(",rNV) specific monoclonal antibodies in thequick complement fixation test.4 Two pairedsl:ra of the horses sho\\ed a significantscroconversion of hat:magglutinationinhibition (HAl) antibodies (inonotypic)against .JE antigen. The antibody tin"es rangedfi"om 10- 80. Twelve out of59 single horsesera sho\\:ed the presence ofantibodies (titreslal1ged 1 20 to 1: ] 60) against .JE antigenin HAl test. 011 the other hand, all the contacthuman sera \Vere found to be r,egati\'e. Thesamples w~re also al1aj~,s~d by re, ersetI-al1scliptase polYInerase chain reaction (RT -PCR) using primers that are specific for JEVil-US" TotarR.NA was extI"acted using Trizol(Life Technologies); RT PCRwas pertoImedas described earlier by Paranjape andBaneIjee.5 Figure shows that mouse braininfected with prototype vil"Us as well as withfield material show the expected 488 hpfragments, confiffi1ing the presence of .JE\ irus in the field material. Results of thesevirolog1cal investigations confilmed the JEVaetiology in this focal outbreak.This is the first report of JE\'isolation from hol"Ses n India. However. thereare similar repoI1s trom other countries. Inthe recent past serologically confirmed humancases have been repol1ed Pune dis.tlict(I\TIV, unpublished data). Similar results in the  42 Rout et 01 horses have been repol1ed rom Java.6 n thepresent repOl1, t was interesting to note thatthese sera were negati\'e for other twocommonly found fla\'iviruses in this countryi.e., ~'N and Dengue viruses. The stud faffi1sare sulTounded by paddy fields and presenceof important JE vector species n this areahas been documented in the past.7 The factthat the human sera were negati\'e for lEV,could be attributed to the zoophilic nature ofthe. lectors. ACK.~OWLEDGEMENT .The authors are thankful to the Director. NI V,Pune, for all the help extended during thestudy. Thanks are extended to Dr. M. M.Gore & Mrs. R.G. Damle for providingmonoclonal antibodies and Dr. B.L. Rao forher valuable suggestion. They are alsothankful to Mr. Anand Singh, Mr. C.B. LandeandT.L.G. Rao for their technical support. *I\ational Institute of \'irology,20-A. Dr. Ambedkar Road. Pune 411001c.(;. Raul.*J.P. Thakare.*V .S. Padhidri.*G.". Sapkal.**A.C. 'lishra.*.R. Paramasivan.*.M.D. Gokhale.**D. T. 'Iourya.**V.S. Shouche.***P.C. Jayal,.omar*.. **Microbial Containment Complex.Sus Road. Pashan. Pune 411021. ***Natio"nal Centre for Cell Science.Pune University Campus. Ganeshkhind. Pune 411 007" 5. 2.6. 3.REFEREl\"CESRosen L and Gubler D. The use ofmosquitoes to detect and propagatedengue viruses. .4m J Trop Med Hyg1974:23:1153-1160.Mourya DT and ~ishra AC. Antigendistribution pattern of Japaneseencephalitis virus in Cule.Ttritaeniorhynchus. C. vishnui and C.pseudo\"ishnui mosquitoes. lIlt/ion JMed Res 2000: 111: 157-161.Gore MM, Gupta AK, Ayachit VM,Athawale SS, Ghosh SN and Banerjee,K. Selection of a neutralization-escapevariant strain of Jupanese encephalitisvirus using monoclonal antibodies.llIt/ian J Med Re.'i 1990; 91: 231-233.Pavri K~, Gokhale TB and Shah KV.Serological response to I~ussian springsun1l11er encephalitis virus vaccIne asmeasured with Kyasanur forest diseasevirus. Indian J Med Res 1962: 50: 153.Paranjape S and Banerjee K.Phylogenetic analysis of the envelopegene of Japanese encephalifis virus.~ intsRes 1996;42: 107-117.Widja.ia S. Soekotjo W. Hartati S.Jennings GB and Corwin AL. Prevalenceof haemgglutination inhIbition andneutralizing antibodies to arboviruses inhorses of Java. A.\ill J TrapMetfPubHlth.1995;26: 109-113.Rao TR and Rajagopalan PK.Observations on mosquitoes of Poonadistrict. Inctia, with special relerence totheir distribution. seasonal prevalenceand the biology of adults. IfIdiaJI Jf\lalarioI1957: II: 1-54. 7. 4
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