Pesq. Vet. Bras. 35(6):557-561, junho 2015 DOI: 10.1590/S0100-736X2015000600012
Outbreaks of canid herpesvirus 1 disease in puppies in southern Brazil1
2 3 4, Rudi Weiblen2 2* Juliana F. Cargnelutti , Eduardo K. Masuda , Mariana G. Neuls ABSTRACT.- and Eduardo F. Flores Out- breaks of canid herpesvirus 1 disease in puppies in southern Brazil Pesquisa Vete- rinária Brasileira Cargnelutti 35(6):557-561. J.F., Masuda E.K., Neuls M.G., Weiblen R. & Flores E.F. 2015. - . Setor de Virologia, Departamento de Medicina Veteriná ria CanidPreventiva, herpesvirus Universidade 1 (CHV-1) Federal is a widespread de Santa Maria, pathogen Av. Roraimaof dogs and 1000, produces Santa infertility,Maria, RS 97105-900, Brazil. E-mail: [email protected] -
abortions and severe systemic disease in young puppies. Clinical data indicate the circula tion of CHV-1 among Brazilian dogs yet definitive diagnosis has rarely been accomplished.- This article describes the clinicopathological findings of four independent cases/outbreaks of neonatal disease by CHV-1 in Bulldog puppies followed by virus identification and gene tic characterization. Three events occurred in a kennel holding dogs of different breeds at- reproductive age (March 2013, October 2013 and April 2014). Puppies from three French or English Bulldog litters, aging 9 to 30 days were affected, presenting dyspnea, agonic bre- athing, pale mucous, abdominal pain and tension, evolving to death within about 24 hours. At necropsy, the puppies presented necrohemorrhagic hepatitis, multifocal and modera te necrohemorrhagic nephritis and fibrinonecrotic interstitial pneumonia. Virus isolation was positive in clinical specimens from one litter and CHV-1 DNA was detected by PCR in glycoproteintissues from allB, Cfour and cases. D genes Virus-neutralizing revealed 99-100% assays of identity with samples among of the the viruses affected and kennel with revealed 9/12 adult animals with high antibody titers to CHV-1. Nucleotide sequencing of - gsCHV-1 support sequences CHV-1 infectionavailable as in the GenBank. cause of Phylogenetic disease and deathanalyses in these of gC dog sequences litters, reinforcing showed a thesegregation need for of correct the samples, etiologic even diagnosis, among three prevention isolates and from immunization the same kennel. against These CHV-1 findin in
dogs from Southern Brazil. INDEX-TERMS: Canid herpesvirus 1, CHV-1, diagnosis, dogs, glycoproteins and pathological RESUMO.- [Surtosfindings. de doença por herpesvírus canino 1 em filhotes de cães no sul do Brasil.] O herpesvírus indicado a circulação do CHV-1 em cães no Brasil, embora canino (CHV-1) é um patógeno de cães que possui distri- sistêmica severa em filhotes de cães. Achados clínicos tem buição mundial e que causa infertilidade, abortos e doença artigo descreve os achados clinicopatológicos de quatro ca- o diagnóstico definitivo seja raramente determinado. Este
1 sos/surtos independentes de morte neonatal de filhotes de- 2 Received on October 31, 2014. ramcães noda mesmoraça Bulldog canil que causados abriga peloanimais CHV-1, de diferentes a identificação raças Accepted for publication on March 16, 2015. - eme a caracterizaçãoidade reprodutiva genética (março do de vírus. 2013, Três outubro eventos de ocorre2013 e Setor de Virologia, Departamento de Medicina Veterinária Preventiva, - Universidade Federal de Santa Maria (UFSM), Av. Roraima 1000, Santa Ma ria,3 RS 97105-900, Brazil. *Corresponding author: eduardofurtadoflores@ cês e/ou Inglês, com idade de 9 a 30 dias, foram afetados e gmail.com apresentaramabril de 2014). dispneia, Filhotes derespiração três ninhadas agônica, de mucosasBulldog Fran páli- 4 Axys Análises Diagnóstico Veterinário e Consultoria, Av. Salvador Leão- das, dor e tensão abdominal, que evoluíram para morte dos 147, Salas 101 and 202, Porto Alegre, RS 91130-700, Brazil. - Fertivida - Clínica Veterinária, Rua Lucas de Oliveira 2689, Porto Ale gre, RS 90460-001. cães dentro de, aproximadamente, 24 horas. Na necrop 557 Juliana F. Cargnelutti et al.
558 sia foram observados hepatite necro-hemorrágica, nefrite necro-hemorrágica multifocal e moderada e pneumonia a pathological description of neonatal death of two litters vance. To date, only few reports were described in Brazil:- - intersticial fibrinonecrótica. O isolamento viral foi positivo (Oliveira et al. 2009); a case of virus isolation and molecu- em amostras clínicas de um filhote e DNA de CHV-1 foi de tionlar identification of neonatal deathof CHV-1 in dogs in a puppywith 10 with days-old neonatal (Ávila death et provenientestectado por PCR do emcanil tecidos afetado de revelaramfilhotes de quetodos nove os surtos.de 12 (Kurissio et al. 2012) and a clinicopathological descrip- animaisTeste de adultossoroneutralização possuíam altos com títulosamostras de anticorposde soro de paracães zil, CHV-1 infection of bitches and neonate puppies seem - toal. occur2011). endemically As no vaccine and, is ascommercially such, have acquiredavailable relative in Bra coproteínas B, C e D revelaram 99-100% de identidade en- importance for dog breeders and domestic dog owners in treo CHV-1. as amostras Sequenciamento e com as sequências de nucleotídeos de CHV-1 do gene disponíveis das gli CHV-1 infection in Bulldog puppies and molecular iden- do gene da glicoproteína C mostrou uma segregação das past years. This report describes four cases/outbreaks of amostras,no GenBank. mesmo A análise entre osfilogenética três isolados baseada de vírus na provenien sequência- the understanding of CHV-1 biology and, most importan- tly,tification to call of attention the agent. to Weand hope help ourveterinarians report contribute in recogni for- ates necessidade do mesmo canil.do correto Esses achadosdiagnóstico demonstram etiológico que e a oimple CHV-- mentação1 é a causa de da medidas doença dee da prevenção morte dos e imunização filhotes, reforçando contra o zing such cases.MATERIALS AND METHODS Outbreak description CHV-1 em cães no sul do Brasil. TERMOS DE INDEXAÇÃO: Herpesvírus canino tipo 1, CHV- The four cases of CHV-1 infection occurred in Bulldog litters 1, diagnóstico, cães, INTRODUCTIONglicoproteínas e lesões. in Porto Alegre, Rio Grande do Sul. Three occurred from March- to October 2013 (fall to spring) and the later occurred in April Canid herpesvirus 1 (CHV-1) belongs to family Herpesvi- 2014.Case The 1 main epidemiological and clinical findings are descri ridae, subfamily Alphaherpesvirinae, genus Varicellovirus bed below: abdominal (SV822/13).pain and tension, Two 9 withto 15-day-old a fast evolution puppies to out death of a litter (ap- manifestations in dogs including neonatal deaths, respira- of eight English Bulldogs presented severe dyspnea, pale mucous, (ICTV, 2015) and it is associated with a variety of clinical prox.Case 24 h).2 After birth, the bitch developed a severe mastitis that latenttory, ocular, infections and in reproductive neural and non disorders neural (Evermanntissues after et acu al.- puppieshampered developed breast-feeding. respiratory agony and pulmonary edema that te2011). infection Like and other can alphaherpesviruses, be reactivated in stressful CHV-1 establishesand immu- (SV 823/13). Four 22-day-old litter of English Bulldog Case 3 - Bulldogsevolved to presented death in anasal few hours.discharge, abdominal pustules and neu- nosuppressive situations (Miyoshi et al. 1999). - (SV1110/13). A 30-day-old puppy of a litter of French The outcome and clinical manifestations of CHV-1 infec tion in dogs are associated with several risk factors, inclu- rologicalCase 4 signs, characterized by ataxia and incoordination. The ding age, breeding and kennel cough (Ronsse et al. 2004). clinical course evolved to death in approximately 24 h. - (SV242/14). Two 23-day-old puppies of a litter of six virusAdult anddogs infected with CHV-1 usually develop a mild res - Bordetella bronchiseptica English Bulldogs developed abdominal pain and showed pale mu piratory disease, frequently associated with Parainfluenza cosae, with fast evolution to death (24 h). The bitch was seronega associated with CHV-1 infection, however,infection generally (Decaro occur et al. tive to CHV-1 when introduced in the kennel, immediately before- 2008, Evermann et al. 2011). The most important disorders proximatelythe pregnancy. one hundred male and female dogs at reproductive in pregnant females may result in transplacentary CHV-1 Case 1, 3 and 4 occurred in the same kennel, which holds ap transmissionin pregnant bitches and fetal and ininfection, young puppies. leading Systemic to fetal viremiadeaths, age, but in different occasions (SV822/13 occurred in October Pathologic2013; SV1110/13 analyses in March 2013, and SV242/14 in April 2014). abortion, mummification, infertility and the birth ofin weakutero orpuppies during (Hashimoto the birth, the et mortality al. 1983, usuallyAnvik 1991, approaches Decaro 100% et al. - due2008, to Evermann a multisystemic et al. 2011). necrohemorrhagic In neonates infecteddisease (Ronsse Animals from all reported cases were submitted to necropsy and tissue samples (kidney, lung and liver) were collected for his age of the puppies at the time of primary infection is deter- topathology and virology diagnosis. For histopathology, samples- minantet al. 2005, to the Decaro outcome et al. of 2008, the disease Evermann and theet al. clinical 2011). signs The were fixed in 10% buffered formalin and processed for routine histopathological examination. The slides were stained using he matoxylin and eosin (H & E) standard protocol. Hence, CHV-1 should be considered an important agent of Virus isolation are usually more severe in neonates (Decaro et al. 2008). were grinded, homogenized and resuspended to 10% (weight/ Cases of disease clinically compatible with CHV-1 in- For virus isolation, tissue fragments (kidney, lung and liver) fectiondisease haveand death been infrequently neonates reported and young by dogs. veterinarians in supernatants were inoculated onto monolayers of primary cani- - volume) in culture medium. After low speed centrifugation, the 2 and monitored for - notBrazil been yet conducteda definitive to diagnosis date, is spiteis rarely of thisreached. clinical Likewi rele- ne kidney cells, stored at 32°C with 5% of CO se, serological studies and/or virus identification have cytopathic effect (cpe) during three passages of five days each be fore considering negative for virus. Pesq. Vet. Bras. 35(6):557-561, junho 2015 Outbreaks of canid herpesvirus 1 disease in puppies in southern Brazil 559
Virus-neutralization Table 1. Primers and PCR conditions used to amplified - fragments of glycoproteins C and D of Canid Herpesvirus-1
Thirteen serum samples collected from dogs and bitches of di - positions size (bp) fferent breeds at reproductive age from the kennel (cases 1, 3 and Gene Primer sequence (5’-3’) Nucleotide Amplicon PCR conditions 4) were submitted to a standard virus-neutralization (VN). VN as 50/well) and says were performed in 96-well plates, testing two-fold dilutions gC F: CGGCATCTACATCCAAAC Based on gC - 768 95°C - 5 min, 30x of sera against a fixed dose of virus (100-200 TCID AF361074 (95°C - 1min, MDCK cells as indicators. The CHV-1 used in VN assays was kindly R: GACCGTCAATCCATACAC 65-82 50°C - 1 min, provided by Dr. João Pessoa Araújo Júnior (Unesp, Pirassununga, 832-815 72°C - 1min), dilutionSP). VN readingsof sera that were prevented performed the after production five days of of cpe incubation. Sera from at 72°C - 7min a32 dog °C naturallyand VN titers infected were with considered CHV-1 and as thefrom reciprocal a seronegative of highest dog gD F: CTTCTACTGACCCATGTG Based on gD – 647 95°C – 5 min, 30x AF361076 (95°C - 1min, R: TGGCCTCGATGGTAATTC 3614-3631 47.5 °C - 1 min, were used as positive and negative controls, respectively. 4260-4243 72°C – 1min), Molecular analysis 72°C – 7 min
a and submitted to PCR for (Hall 1999) and compared with other CHV-1 sequences deposited CHV-1Total glycoprotein DNA was extracted B gene (gB), from using tissue the samples primers (pool and conditions of kidney, Edit Sequence Alignment Editor Software suite, version 7.0.5.3- liver and lung) using DNAzol Reagent in GenBank. To building the phylogenetic tree, based on sequen previously described (Ronsse et al. 2005). DNA extracted from- ces of gC gene, it was used the Neighbor-Joining method with the liver of a dog infected with CHV-1 (SV435/08) (Oliveira et al.- 2000 bootstrap replicates implemented by MEGA5.0 (Tamura et 2009) was used as positive control. Additionally,a PCRaccording to glycopro to the al. 2011), based on Tamura 3- parameters model. manufacturer’steins C and D (gC instructions and gD; Table and submitted1) were performed. to nucleotide The sequen ampli- RESULTS AND DISCUSSION cons were purified using the PureLink PCR kit - - sease, characterized by multifocal to aleatory petechia in cing in an automatic sequencer ABI-PRISM 3100 Genetic Analyzer At necropsy, all puppies had severe necrohemorrhagic di- armed with 50 cm capillaries and POP6 polymer (Applied Biosys- - tems, Carlsbad, CA, USA). The obtained sequences were analyzed liver and kidneys. In the lungs had severe edema and he by the Staden package (Staden 1996) and aligned using the Bio morrhage. At histopathologic examination, there were ne
A B C) - Fig.1. ( D) Pulmonary lobes with severe edema and hemorrhage. ( ) Aleatory foci of hemorrhage (petechia)C, D over the renal cortex. ( Multiple alveoli are necrotic and filled by fibrinous plaques and mild to moderate neutrophilic and histiocytic inflammatory infiltra te. ( ) Severe necrosis of hepatocytes with Councilmann-like bodies and moderate hemorrhage. ( ) Hematoxylin and eosin. Bar = 100.00µm. Pesq. Vet. Bras. 35(6):557-561, junho 2015 Juliana F. Cargnelutti et al.
560
sequences that resulted in amino acid changes when com- cleotide changes in SV822/13, SV823/13 and SV1110/13
pared with a standard CHV-1 sequence (GenBank accession: 242/14AF361074): - amino SV822/13 acid position - amino 199 acid (valine positions to leucine) 35 (proline and to alanine) and 199 (valine to leucine); SVs 823/13 and - Phylogenetic tree based on the nucleotide sequences of larSV1110/13 chemical -group amino and acid structure, position the 36 substitution(threonine to of serine).proline Although most mutations in gC involved amino acid of simi- Fig.2. - glycoprotein C gene of Canid Herpesvirus 1. The tree was constructed using the Neigbohr-Joining method with 2,000 to alanine (SV822/13) rarely occurs and it might affect pro bootstrap replicates based on Tamura-3 parameters model tein folding and/or function. The sequences were deposi and implemented by MEGA 5.0. Values>70% are shown. CHV- ted in GenBank and named), SV1110/13SV822/13 (gB (gB - - KJ676503, gC 1 samples recovered of the four reported cases (SV822/13,- - KJ676504, gD - KJ676505), SV823/12 (gB - KJ676506, gC SV823/13, SV1110/13 and SV242/14) are identified with a - KJ676507, gD - KJ676508 KJ676509, gC- black diamond. Brazilian CHV-1 sample from other case (Oli ber- KJ676510, of sequences gD - KJ676511 available) inand databases SV242/14 precludes (gB - KJ946357, further crohemorrhagicveira et al. 2009) hepatitis, is identified multifocal with a white and diamond.moderate necro- gC - KJ946358, gD – KJ946359). Unfortunately, the low num genetic and/or functional analysis of these glycoproteins. herpesviralhemorrhagic viral nephritis corpuscles (Fig.1A), were not and observed fibrinonecrotic in all cases, and The obtained gC sequences were then submitted to hemorrhagic interstitial pneumonia (Fig.1B). Intranuclear phylogenetic analysis using the Neighbor-Joining method- - with 2000 bootstrap replicates implemented by MEGA5.0 yet the findings in all target organs were characteristic of (Tamura et al. 2011), based on Tamura 3- parameters mo- CHV-1 infection in neonates (Love & Huxtable 1976, Olivei del (Figure 2). The phylogenetic three was constructed- ra etCells al. 2009). inoculated No distemper with tissue virus-associated homogenates lesions of werecase using CHV-1 sequences obtained from GenBank and Brazi observed in any case (differential diagnosis). lian samples, such as SV453/08 - identified in a puppy in incubation, characterized by cell rounding and detaching fected with CHV-1 in Southern Brazil in 2008 (Oliveira et al. SV822/13 developed typical herpesvirus cpe after 48 h of 2009) (Genbank accession - KJ676501). - The presumptive diagnosis of the four events was based from the monolayer. The other samples remained negative neonatalon the clinical CHV-1 signs, infection, high mortality characterized and pathological by a multisystemic findin atfor different cpe after times,three passages.we tested Since serum three samples cases obtainedoccurred in gs. At necropsy, the macroscopic features were typical of the same kennel (SV822/13, SV1110/13 and SV242/14) collected from dogs and bitches of different breeds at re- infectionnecrohemorrhagic and disease disease in Brazil (Decaro - there et al.are 2008, few exceptionsOliveira et productivethis kennel agefor CHV-1were submittedantibodies. to Thirteen a standard serum virus-neu samples- al. 2009). In spite of the lack of published reports of CHV-1-
such as Oliveira et al. (2009), Ávila et al. (2011) and Ku- previoustralization exposure (VN). Nine to dogsthe viruspresented since antibodiesthese dogs to had CHV-1 no rissio et al. (2012) - veterinarians frequently report cases- historyin titers of ranging CHV-1 vaccinationfrom 16 to 128.and, inThese addition, data demonstrateindicate that compatible with CHV-1-induced disease. Virtually all sus - particularpected cases, cases, however, however, remain the support without of a a definitivevirology diag etio- tive serology for CHV-1- and for other alphaherpesviruses logical (virology, serology, molecular) diagnosis. In these asCHV-1 well infection - indicate is the probably condition endemic of latent in the carrier, kennel. the As serolo posi- Positive virus isolation in primary canine cells, PCR and nostic laboratory allowed a definitive etiological diagnosis.- - gical findings undercovered an important epidemiological nucleotide sequencing confirmed the presumptive diagno situation in this kennel since seropositive animals may oc- sis. Subsequently, serology confirmed the circulation of the casionally reactivate and transmit the virus. Thus, the virus duringvirus in autumn the affected and kennel.winter, when the environmental tem- is probably perpetuated in the kennel through periodic epi Neonatal mortality in puppies is frequently reported- lung,sodes were of reactivation submitted and to PCR transmission. to gB gene and a band of ap- ronmental temperatures, housing conditions and the body proximatelyTotal DNA 443bp was extracted - corresponding from pools to the of kidney,expected liver ampli and- temperatureperatures fluctuate exert afrom strong 0 to effect +10-15°C. on CHV-1 In addition infection to envi sin- ce optimal virus replication occurs at temperature below con for CHV-1 - was detected in DNA extracted from tissue- severe and frequently fatal in young puppies in which the samples of all investigated cases (SV822/13, SV823/13,- 35°C (Huxsoll & Hemelt 1970). The disease is particularly- SV1110/13 and SV242/14). Additionally, PCR to glycopro tion may be subclinical or mild in older dogs due to the abi- teins C and D (gC and gD; Table 1) were performed to con litybody of temperaturesthese animals areto stabilize usually theirbelow body 35-36°C. temperatures The infec at firm the etiologic agent and to analyze gene sequences. fromThe 99 nucleotideto 100%, and identity the lower of genesidentities gB, were gC, gD observed from the in samples and CHV-1 sequences available in GenBank ranged- maternal38°C (Jenson immunity & Ederstrom may worsen 1955). neonatal CHV-1 disease In addition to the low body temperature, the lack of gB and gC sequences. The gC sequence analysis showed nu Pesq. Vet. Bras. 35(6):557-561, junho 2015 Outbreaks of canid herpesvirus 1 disease in puppies in southern Brazil
561 Acknowledgements.- protect puppies from fatal CHV-1 infection even though the de Desenvolvimento Cient This study was supported by Conselho Nacional (Evermann et al. 2011). Maternal antibodies apparently ífico e Tecnológico (CNPq). J.F. Cargnelutti, E.F. Flores and R. Weiblen are CNPq research fellows. whichanimals the develop infection subclinical is endemic infection or can (Decarobe frequently et al. intro2008,- REFERENCES duced,Huxsoll vaccination & Hemelt 1970). of females Thus, at in heat a population and/or during of dogs preg in- nancy should be highly recommended to minimize the con- Anvik J.O. 1991. Clinical considerations of canine herpesvirus-infection. Vet. Med-Us 86:394-395. - In the present report, the affected puppies presented Ávila V.P.F., Esmeraldino A.T., Fallavena L.C.B., Césaro C., Rodrigues N.C., severesequences respiratory of CHV-1 signs infection that ofevolved puppies. to death in a day or Braga A.C. & Cerva C. 2011. Herpesvirus canino em filhotes da raça Gol Case den Retriever - relato de caso. Clín. Vet. 92:52-56. 3) also presented neurological signs, which are rarely ob- Decaro N., Martella V. & Buonavoglia C. 2008. Canine adenoviruses and- so. Interestingly, a puppy from a French Bulldog litter ( herpesvirus. Vet. Clin. North Am., Small. Anim. Pract. 38:799-814. reason, differential diagnosis for canine distemper virus Evermann J.F., Ledbetter E.C. & Maes R.K. 2011. Canine reproductive, res piratory, and ocular diseases due to canine herpesvirus. Vet. Clin. North.- (CDV),served anin agentpuppies involved naturally in neurologic infected with disease CHV-1. in dogs For wasthis Am., Small. Anim. Pract. 41:1097-1120. Hall T.A. 1999. BioEdit: a user-friendly biological sequence alignment edi tor and analysis program for Windows 95/98/NT. Nucleic Acids Symp.- performed (Tipold et al. 1992). Again, the pathological and placental41:95-98. transmission of canine herpesvirus in pregnant bitches during virologicalSequence findings analysis were of CHV-1 conclusive gC sequences of CHV-1 (including infection thoand- Hashimoto A., Hirai K., Suzuki Y. & Fujimoto Y. 1983. Experimental trans histopathological analyses dismissed CDV infection. - the 2nd trimester of gestation. Am. J. Vet. Res. 44:610-614. se from the reported cases and two obtained in GenBank) Huxsoll D.L. & Hemelt I.E. 1970. Clinical observations of canine herpesvi revealed a high level of nucleotide identity. Nonetheless, rus. J. Am. Vet. Med. Assoc. 156:1706-1713. SV822/13, SV242/14 and SV1110/13 isolates – all coming ICTV. International Committee on Taxonomy of Viruses. Virus Taxonomy: 2013 Release.
Pesq. Vet. Bras. 35(6):557-561, junho 2015