Amoroso, Mg

Aprea, G

Fusco, G

Galiero, G

Guarino, A

VENEZIANO, VINCENZO

Viscardi, M.

Archivio della ricerca - Università degli studi di Napoli Federico II

Short CommunicationFirst report of naturalBoHV-1 infection inwater buffaloG. Fusco, M. G. Amoroso, G. Aprea,V. Veneziano, A. Guarino, G. Galiero,M. ViscardiBovine herpesvirus 1 (BoHV-1) is a double-stranded envelopedDNA virus belonging to the family Alphaherpesvirinae, genusVaricellovirus. It is the causative agent of infectious bovine rhino-tracheitis/infectious pustular vulvovaginitis (IBR/IPV), a world-wide disease that affects ruminants. BoHV-1 causes respiratoryand reproductive symptoms, abortion, vulvovaginitis, encephal-itis and fetal death (Rana and others 2011, Amoroso and others2013). The virus is not always restricted to its natural hostspecies. Animals such as goat, sheep, red deer and reindeer weresuccessfully infected with BoHV-1 under experimental condi-tions (Thiry and others 2007). In addition, Mediterraneanbuffalo have been shown to be sensitive to BoHV-1 by experi-mental infection (Thiry and others 2007, Scicluna and others2010), and seropositivity to BoHV-1 in water buffalo has beenwell documented (Peshev and Christova 2000). Nonetheless, thevirus has never been identiﬁed in naturally infected buffaloes. Tothe authors’ knowledge, this study reports the ﬁrst isolation ofBoHV-1 from water buffaloes.Over the period September 2011 to December 2012, theauthors received the samples of 65 buffaloes with pathologiespossibly ascribable to herpesvirus: in detail, samples from 35animals with rhinotracheitis (nasal swabs and organs), samplesfrom 27 cases of abortion (which occurred during the third/fourth month of gestation), organs from one calf born with ahind limb deformity and euthanased when three weeks old, andorgans from two calves that had died a few days (4–7) afterbirth. With respect to abortion, in 14 cases (which we deﬁnedcomplete cases) both mothers’ samples (cervical–vaginal swaband blood) and the organs of the fetus were received, in one caseonly the aborted fetus, and in the remaining 12 cases only cer-vical–vaginal swab and blood samples of the aborted buffaloes.All the samples came from animals belonging to three herds inSouthern Italy.All the blood samples were screened for the presence of anti-bodies against BoHV-1 glycoprotein B (gB) and glycoprotein E(gE) by ELISA (respectively IBR gB IDEXX and IBR gE IDEXXELISA antibody test kits; Herd Check Laboratories). Serumsamples from female buffaloes that had had abortions and fromthe euthanased calf were positive for both gB and gE antibodies.Ruminant α-herpesviruses related to BoHV-1 (Thiry andothers 2006) were detected by real-time PCR by amplifying aregion of a highly conserved gene of herpesviruses (gB gene)(Wang and others 2007). DNA was extracted from cervical–vaginal swabs and organs using the Nucleic Acid Puriﬁcation Kit(Macherey-Nagel) following the manufacturer ’s instructions.Real-time PCR was carried out as described in the OIE TerrestrialManual (2010) using a RotorGene Q PCR ampliﬁcation system(Qiagen). Results showed a total of 14 animals positive toruminant α-herpesvirus. In detail, samples collected from heart,lung, liver and spleen tissues from the two dead newborn calveswere all positive for α-herpesvirus. From these samples, it wasalso possible to isolate the virus by cell culture. Viral isolationfollowed the procedures described in the OIE Terrestrial Manual(2010). α-Herpesvirus was also detected by real-time PCR inliver and brain samples from the euthanased buffalo calf, eventhough isolation of the virus by tissue culture gave negativeresults. As to abortion, we registered positivity to α-herpesvirusfor 11 cases out of the 27 analysed: ﬁve positive cases among the14 complete cases, one positivity for the fetus alone, and sixpositive results among the cases for which we could onlyanalyse the vaginal swab. As to virus isolation, cell culture waspositive for only two cervical–vaginal swabs. Samples from casesof rhinotracheitis were all negative to ruminant α-herpesvirusand were sent to other laboratories for further investigations(data not shown). All the described results are summarised inTable 1. A sequencing assay based on pyrosequencing technologywas developed to classify the α-herpesviruses identiﬁed by bothreal-time PCR and cell culture (Fusco and others 2015). Bysequencing a small polymorphic segment of the US8 gene (gE),we could distinguish, on the basis of the nucleotide sequencedifferences (Table 2), between Bovine herpesvirus 1.1, Bovineherpesvirus 1.2, Bovine herpesvirus 5, Bubaline herpesvirus 1 andCaprine herpesvirus. The US8 gene was ﬁrst ampliﬁed in aTABLE 1: Detection of BoHV-1.1 by real-time PCR, sequencingand tissue cultureCaseAnimalsanalysed(n)Real-time PCR and sequencingVirusisolationresultsPresence ofBoHV-1.1(number ofanimals)Type of samplewith positivityDead newborncalf2 2 Heart, lung,liver, spleenIsolationfrom all thesamplesCalf with hindlegmalformation1 1 Liver, brain NoisolationAbortion (fetus+vaginal swab)14 5 Organs of thefetus (heart,lung, liver,spleen) vaginalswabNoisolationAbortion (fetus) 1 1 Heart, lung,liver, spleenNoisolationAbortion(cervical–vaginalswab)12 5 Vaginal swab Isolationfrom 2samplesRhinotracheitis 35 0 – –Veterinary Record (2015) doi: 10.1136/vr.103139G. Fusco,M. G. Amoroso, PhD,A. Guarino,G. Galiero,M. Viscardi, PhD,Department of Animal Health,Experimental Zooprophylactic Instituteof Southern Italy, Via Salute, 2, PorticiNA 80055, ItalyG. Aprea,Department of Food Hygiene andTechnology, ExperimentalZooprophylactic Institute of Abruzzoand Molise ‘G. Caporale’, via CampoBoario, Teramo 64100, ItalyV. Veneziano,Department of Pathology and AnimalHealth, University of Naples ‘FedericoII’, Via Delpino, 1, Naples 80137, ItalyE-mail for correspondence:giovanna.fusco@cert.izsmportici.itProvenance: not commissioned;externally peer reviewedAccepted June 26, 201510.1136/vr.103139 | Veterinary Record | 1 of 3Short Communicationgroup.bmj.com on July 26, 2015 - Published by http://veterinaryrecord.bmj.com/Downloaded from RotorGene Q Real Time PCR ampliﬁcation system (Qiagen)using the PyroMark PCR kit (Qiagen) as indicated by the manu-facturer. Samples were pyrosequenced by a PyroMark Q24instrument (Qiagen) programmed with cyclic dispensation ofdeoxynucleoside triphosphates (8× (ACGT)). The results wereelaborated by Geneious v7.1 software (Biomatters). The pyrose-quencing technique allowed us to classify the α-herpesvirus fromthe speciﬁc polymorphic site sequenced (Fig 1 and Table 2). In allanalysed samples, we identiﬁed the nucleotide polymorphicsequence speciﬁc for BoHV-1.1 (GAGGCCG). Pyrosequencingresults were conﬁrmed by sequencing as previously reported(Amoroso and others 2013). Sequences showed 100 per centidentity with the BoHV-1 gE (GenBank accession numberEF624466.1).The assay created represents a valid alternative to the con-ventional Sanger sequencing method because it requires muchless time for processing of the samples but offers the same speci-ﬁcity. PyroMark can efﬁciently detect subtle differences insequences, allowing identiﬁcation of very closely related organ-isms. This technique has already been successfully employed forthe detection of herpes simplex virus types 1 and 2 (Adelson andothers 2005) and for the determination of Equine herpesvirusnucleotide polymorphism (Tewari and others 2013). In allsamples analysed by pyrosequencing, we identiﬁed and classiﬁedthe isolated α-herpesvirus as BoHV-1 and not as BuHV-1.Buffalo sensitivity to BoHV-1 has been widely demonstrated byexperimental infection and serological surveys (Peshev andChristova 2000, Scicluna and others 2010). However, the virushas never been isolated from naturally infected buffaloes. Thepresent results show that the samples collected from both deadnewborn and euthanased buffalo calves were positive forBoHV-1 and negative for all the other pathogens investigated(Bovine viral diarrhoea virus, Bovine coronavirus, Schmallemberg virus,Bluetongue virus, Border disease virus, Chlamydophila species,Coxiella burnetii, Leptospira species, Neospora caninum, Toxoplasmagondii, Ovine herpesvirus-2). It has been largely reported that neo-natal bovine calves infected with BoHV-1, either before or earlyafter birth, usually die within a few days (Muylkens and others2007). The present ﬁndings seem to indicate that BoHV-1 exhi-bits the same level of pathogenicity, including death, in newbornbuffaloes, as both aborted fetal tissue samples, and cervicalswabs analysed were positive for the virus. Fetal abortion as aresult of BoHV-1 infection has been broadly described for cattle(Thiry and others 2006, Muylkens and others 2007, Nandi andothers 2009). The present results suggest that BoHV-1 may beresponsible for the abortions observed in buffaloes. As to the calfwith hind limb deformity, viruses other than BoHV-1 have beendescribed as possible causes of malformation (Roger 2015), andBoHV-1 has never been associated with cases of limb deformity.Since liver and brain of the euthanased calf tested positive forBoHV-1 in real-time PCR, we could ascribe responsibility for themalformation to the virus, speculating that the infection hadoccurred during pregnancy at the time of limb genesis, probablyengendering a teratogenic effect on the fetus. However, thisassumption needs to be investigated further for corroboration.Through experimental infection with BoHV-1 in buffaloes,Scicluna and others (2010) reported no clinical symptoms char-acteristic of a BoHV-1 infection and therefore concluded theTABLE 2: Strains of α-herpesviruses identifiable by thedeveloped pyrosequencing assayVirus strainnameHostspeciesGenBank glycoprotein Ereference strain sequencePolymorphicsiteBovineherpesvirus 1.1Bovine Bovine herpesvirus 1 strain Lam(1.1) glycoprotein E (US8) gene(EF624467.1)GAGGCCGBovineherpesvirus 1.2Bovine Bovine herpesvirus 1 strain K22(1.2) glycoprotein E (US8) gene(EF624466.1)GAGGCCTBovineherpesvirus 5Bovine Bovine herpesvirus 5 strainN569 glycoprotein E (US8) gene(EF624468.1)GCGGCCGCCBubalineherpesvirus 1Bubaline Bubaline herpesvirus 1 strainB6 glycoprotein E (US8) gene(EF624469.1)GAGGCCGCGCaprineherpesvirus 1Caprine Caprine herpesvirus 1 strainBa-1 glycoprotein E (US8) gene(EF624470.1)GAGGCCGCGGGSequencing of the polymorphic site allows unambiguous classification of theherpesvirus identified.ConsensusConsensusIdentityIdentityConsensusIdentityConsensusIdentity1. BoHV1.2|EF624466.12. BoHV1.1 EF624467.13. BoHV5 EF624468.14. BoHV1 EF624469.15. CaHV1 EF624470.11. BoHV1.2|EF624466.12. BoHV1.1 EF624467.13. BoHV5 EF624468.14. BoHV1 EF624469.15. CaHV1 EF624470.11. BoHV1.2|EF624466.12. BoHV1.1 EF624467.13. BoHV5 EF624468.14. BoHV1 EF624469.15. CaHV1 EF624470.11. BoHV1.2|EF624466.12. BoHV1.1 EF624467.13. BoHV5 EF624468.14. BoHV1 EF624469.15. CaHV1 EF624470.1FIG 1: Geneious alignment of the ﬁve herpesvirus glycoprotein E sequences used to create the PyroMark assay. Yellow arrows indicate thesequences of the primers used: (1) forward primer, (IBRpyro-for) 50-TGCTGCAGTACAACGGCCACGT-30; (2) biotinylated reverse primer,(IBRpyro-rev) 50-GCCCGAAGGGGTTGAGGA-30; (3) sequencing primer, 50-ACCGACCACACGCGCCCC-30. The blue arrow indicates thepolymorphic site where the sequence allows unambiguous identiﬁcation of the herpesvirus.2 of 3 | Veterinary Record | 10.1136/vr.103139Short Communicationgroup.bmj.com on July 26, 2015 - Published by http://veterinaryrecord.bmj.com/Downloaded from infection to be subclinical. The present results are in contrastwith those of Scicluna and others. Naturally, there are manyfactors that can inﬂuence the severity of BoHV-1 infection,including virulence of the virus strain, host resistance factors,animal age and potential concurrent bacterial infections(Muylkens and others 2007). The present study suggests thatBoHV-1, similarly to other ruminant α-herpesviruses, can adaptto other related species (Thiry and others 2006) and cause thesame symptomatology present usually in the species of origin.One of the most important features of BoHV-1 is that it has thecapacity to persist in infected animals long term, becominglatent, periodically being re-activated, and readily transmittedbetween animals (Raaperi and others 2014). BoHV-1-infectedbuffaloes could therefore become permanent carriers of BoHV-1and a potential source of infection for the rest of the herd.Considering that in southern Italy it is common to ﬁnd farmswith cattle and buffaloes living together (Scicluna and others2010), any systematic IBR eradication plan to be applied to thesefarms should involve not only the cattle but also the water buf-faloes. At the same time, in the authors’ opinion, any nationalsurveillance/eradication programme implemented for cattleshould mandatorily also include buffalo herds.ReferencesADELSON, M. E., FEOLA, M., JASON, T., TILTON, R. C. & MORDECHAI, E.(2005) Simultaneous detection of herpes simplex virus types 1 and 2 byReal-time PCR and Pyrosequencing. Journal of Clinical Virology 33, 25–34AMOROSO, M. G., CORRADO, F., DE CARLO, E., LUCIBELLI, M. G.,MARTUCCIELLO, A., GUARINO, A. & GALIERO, G. (2013) Bubaline herpesvirus1 associated with abortion in a Mediterranean water buffalo. Research inVeterinary Science 943, 813–816FUSCO, G., AMOROSO, M. G., MONTESANO, N. G. & VISCARDI, M. (2015)Development of a pyrosequencing assay for the typing of alphaherpesviruses.MethodsX 2, 47–52MUYLKENS, B., THIRY, J., KIRTEN, P., SCHYNTS, F. & THIRY, E. (2007) Bovineherpesvirus 1 infection and infectious bovine rhinotracheitis. Veterinary Research38, 181–209NANDI, S., KUMAR, M., MANOHAR, M. & CHAUHAN, R. S. (2009) Bovineherpesvirus infections in cattle. Animal Health Research Reviews 10, 85–98OIE TERRESTRIAL MANUAL (2010) Infectious Bovine Rhinotracheitis/InfectiousPustular Vulvovaginitis. Chapter 2.4.13, 6–7.PESHEV, R. & CHRISTOVA, L. (2000) Study of bovine herpes virus 1 spreadingamong buffalo herds in Bulgaria. Acta Virologica 44, 229–230RAAPERI, K., ORRO, T. & VILTROP, A. (2014) Epidemiology and control of bovineherpesvirus 1 infection in Europe. The Veterinary Journal 201, 249–256RANA, S. K., KOTA, S. N. L. S., SAMAYAM, P. N. R., SRINIVASAN, R. &SRINIVASAN, V. A. (2011) Use of Real-time polymerase chain reaction to detectbovine herpesvirus 1 in frozen cattle and buffalo semen in India. Veterinaria Italiana47, 313–322ROGER, P. (2015) Schmallenberg virus: an update. In Practice 37, 33–37SCICLUNA, M. T., CAPRIOLI, A., SARALLI, G., MANNA, G., BARONE, A.,CERSINI, A., CARDETI, G., CONDOLEO, R. U. & AUTORINO, G. L. (2010)Should the domestic buffalo Bubalus bubalis be considered in the epidemiology ofBovine herpesvirus 1 infection? Veterinary Microbiology 143, 81–88TEWARI, D., DEL PIERO, F., CIEPLY, S., FERIA, W. & ACLAND, H. (2013) Equineherpesvirus1 (EHV-1) nucleotide polymorphism determination using formalinﬁxed tissues in EHV-1 induced abortions and myelopathies with Real-time PCRand pyrosequencing. Journal of Virological Methods 193, 371–373THIRY, J., KEUSER, V., MUYLKENS, B., MEURENS, F., GOGIEV, S.,VANDERPLASSCHEN, A. & THIERY, E. (2006) Ruminant alphaherpesvirusesrelated to Bovine herpesvirus 1. Veterinary Research 37, 169–190THIRY, J., WIDEN, F., GREGOIRE, F., LINDEN, A., BELAK, S. & THIRY, E. (2007)Isolation and characterization of a ruminant alphaherpesvirus closely related toBovine herpesvirus 1 in a free-ranging red deer. BMC Veterinary Research 28, 3–26WANG, J., O’KEEFE, J., ORR, D., LOTH, L., BANKS, M., WAKELEY, P., WEST, D.,CARD, R., IBATA, G., VAN MAANEN, K., THOREN, P., ISAKSSON, M. &KERKHOFS, P. (2007) Validation of a real-time PCR assay for the detection ofbovine herpesvirus 1 in bovine semen. Journal of Virological Methods 144, 103–10810.1136/vr.103139 | Veterinary Record | 3 of 3Short Communicationgroup.bmj.com on July 26, 2015 - Published by http://veterinaryrecord.bmj.com/Downloaded from water buffaloFirst report of natural BoHV-1 infection inGaliero and M. ViscardiG. Fusco, M. G. Amoroso, G. Aprea, V. Veneziano, A. Guarino, G. published online July 21, 2015Veterinary Record  http://veterinaryrecord.bmj.com/content/early/2015/07/21/vr.103139Updated information and services can be found at: These include:References #BIBLhttp://veterinaryrecord.bmj.com/content/early/2015/07/21/vr.103139This article cites 14 articles, 1 of which you can access for free at: serviceEmail alertingbox at the top right corner of the online article. Receive free email alerts when new articles cite this article. Sign up in theNoteshttp://group.bmj.com/group/rights-licensing/permissionsTo request permissions go to:http://journals.bmj.com/cgi/reprintformTo order reprints go to:http://group.bmj.com/subscribe/To subscribe to BMJ go to:group.bmj.com on July 26, 2015 - Published by http://veterinaryrecord.bmj.com/Downloaded from 

First report of natural BoHV-1 infection in water buffalo

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Archivio della ricerca - Università degli studi di Napoli Federico II