See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/234034744 An atypical clinical presentation for the first isolation of Canid herpesvirus 1 in Argentina Article in Arquivo Brasileiro de Medicina Veterinária e Zootecnia · September 2010 DOI: 10.1590/S0102-09352010000500034 CITATIONS READS 6 74 5 authors , including: Miguel Ayala Cristina Gobello Universidad Nacional de La Plata Universidad Nacional de La Plata 22 PUBLICATIONS 110 CITATIONS 106 PUBLICATIONS 1,093 CITATIONS SEE PROFILE SEE PROFILE Maria Gabriela Echeverria Cecilia M Galosi Universidad Nacional de La Plata Universidad Nacional de La Plata 138 PUBLICATIONS 417 CITATIONS 145 PUBLICATIONS 528 CITATIONS SEE PROFILE SEE PROFILE Some of the authors of this publication are also working on these related projects: Indígena View project Platform for the recombinant proteins production of interest in veterinary medicine View project All content following this page was uploaded by Cecilia M Galosi on 28 May 2014. The user has requested enhancement of the downloaded file.
Arq. Bras. Med. Vet. Zootec. , v.62, n.5, p.1267-1270, 2010 Communication [ Comunicação ] An atypical clinical presentation for the first isolation of Canid herpesvirus 1 in Argentina [ Uma apresentação clínica atípica para o primeiro isolamento do herpesvirus canino 1 na Argentina ] V.E. De Palma 1 , M.A. Ayala 1 , C. Gobello 1,2 , M.G. Echeverria 1,2 , C.M. Galosi 1,3* 1 Facultad de Ciencias Veterinarias - UNLP 60 y 118, CP 1900 CC296 – La Plata, Buenos Aires, Argentina 2 Consejo Nacional de Investigaciones Científicas y Técnicas – Buenos Aires, Argentina 3 Comisión de Investigaciones Científicas de la Provincia de Bs As – La Plata, Buenos Aires, Argentina 1 (CaHV-1) is a dog population. The disease was reported in Canid herpesvirus Varicellovirus of the subfamily numerous countries and the prevalence of Alphaherpesvirinae , family Herpesviridae, and antibodies (Abs) against CaHV-1 varies from 6% order Herpesvirales , with a host range restricted in some countries to higher than 90% in other to domestic and wild canids (Remond et al., (Ronsse et al., 2005; Nöthling et al., 2008). 1996). CaHV-1 was first recognized as the agent responsible for causing a highly fatal A first preliminary serological study performed hemorrhagic viral disease in newborn puppies in in Argentina by a previously standardized 1965. Apart from being an important disease in enzyme linked immunosorbent analysis (ELISA) newborn puppies, CaHV-1 also affects indicated a 23% of Abs prevalence (De Palma et reproduction of dogs in other ways: the virus al., 2006) In addition, typical clinical signs and may cause vesicular lesions in the vestibulum neonatal mortality, suggesting viral activity, have and vagina of the bitch, as well as on the penis been observed by Argentine breeders. This study and the preputial mucosa of dogs and may cause reports an atypical clinical manifestation for embryonic resorption, abortion, and fetal death CaHV-1 and the first viral isolation of this virus (Carmichael, 1970). In addition, the virus is in Argentina. associated with respiratory (kennel cough syndrome) and ocular disease in dogs (Erles and A four-year-old privately owned female Brownlie, 2005; Ledbetter et al., 2009). Oronasal Labrador Retriever, which had been and venereal transmission are the common routes mastectomized two weeks before, presented of infection but transplacental infection has also vesicular lesions preceded by erythema in the been described. Lesions in the vestibulum and internal part of the right thigh (Figure 1). vagina of bitches may recur when bitches come Vesicles were surrounded by suppurative crusts into pro-estrus and regress when they go into and regional lymphoid nodes were enlarged. The anestrus (Carmichael, 1970; Hashimoto et al., lesions were first treated with local gentamicine 1982). Affected animals may remain latent (Kualcoderm, Kualcos – Buenos Aires, carriers and the virus was identified in the Argentina) for two days without improvement. lumbosacral ganglia, tonsils, parotid salivary Vesicular fluid was aspirated by a sterile syringe glands, and liver of dogs that showed no sign of and transported in a refrigerated box to the herpesvirus infection (Burr et al., 1996). Several laboratory of virology. In addition, blood studies suggest that CaHV-1 is enzootic in the samples for antibodies (Abs) detection were also Recebido em 16 de fevereiro de 2010 Aceito em 28 de setembro de 2010 *Autor para correspondência ( corresponding author ) E-mail: cmgalosi@fcv.unlp.edu.ar
De Palma et al. drawn. Then, the lesions were treated with 5% standardized indirect ELISA test, using a soluble acyclovir (ACV - Aciclovir Lafedar – Entre antigen produced with CaHV-1 YP-11 strain Rios, Argentina) cream three times daily for five infected MDCK cells (De Palma et al., 2006). days. A first rapid diagnosis was done by polymerase chain reaction (PCR) using specific The DNA was specifically amplified by PCR, oligonucleotide primers derived from the generating a product that yielded a sharp visible glycoprotein B gene of CaHV-1 (P1: 5’ CAG band of 120bp on an ethidium bromide gel. GAC TAT TGG ACT ATA GT 3’, P2: 5’ TTG Identical bands were obtained from positive CAA TGC CCC TCA TAA TT 3’) (Burr et al., controls. A focus of CPE consisting of spherical 1996). The reaction was performed from a cells and lysis was detected on day 3 pi and then portion of boiled vesicular fluid and the DNA of increased after successive passages (Figure 2). the YP-11 Japanese strain (Dr. T. Mikami – The IFA showed specific nuclear fluorescence Tokyo, Japan) was used as positive control. The (Figure 3). The isolated virus named LPJ (10 4.5 CCID 50% /50 μ L) was confirmed as CaHV-1 conditions for PCR amplification were: a) 35 cycles of 94°C for 30s, 57.5°C for 1min, 72°C by the VN test. The ELISA test was also positive for 1min; b) final extension at 72°C for 5min. and indicated that a CaHV-1 infection was The PCR products were examined on 3% produced. agarose gel in TBE buffer (50mM Tris pH 8.0, 50mM boric acid, and 1mM EDTA). The gels ACV is an antiviral agent with activity against a were examined under UV light following variety of viruses that has been found to be ethidium bromide staining. The molecular sizes efficacious in the management of human herpes of fragments were compared with those of a labialis in several formulations and routes of 100bp (base pairs) ladder (Promega Lab – administration. In addition, ACV has been shown Madison, USA). A visible band of weight equal to have an effect on veterinary herpesvirus to 120bp was considered a positive result. As a (Asano, 1995; Garre et al., 2007). In the case second step, the sample was diluted 1:10 in reported in this work, it was observed that the phosphate-buffered saline (PBS), centrifuged lesions decreased rapidly from day 2 of ACV (3000g for 15min at 4°C) and inoculated over treatment. The exact explanation of this unusual confluent monolayers cultures of Madin-Darby CaHV-1 clinical presentation is not known. In Canine Kidney (MDCK) cells grown in twenty- this particular case, no other previous clinical four-well plates. Plates were incubated at 35°C in signs were observed. As in other an atmosphere of 5% CO 2 and examined daily alphaherpesviruses, the CaHV-1 latency in for the appearance of viral cytopathic effects sensory ganglia has been reported (Miyoshi et (CPE). When CPE was extensive, the al., 1999). It is also known that latent viruses in supernatant was separated by low speed the lumbosacral nodes have an important role in centrifugation (3000g for 15min at 4°C) and venereal infections (Burr et al., 1996). A possible titrated in MDCK cells using the Reed and explanation is that the virus had replicated in the Muench method. The titer was expressed at 96h epithelial cells of the vagina (self-limited post infection (pi) as CCID 50 (cell culture primary infection), established latency, and then infectious dose 50%)/50 μ L. In addition, infected was reactivated by the post surgical immune and non-infected MDCK cells grown on depression, producing the skin lesions described. coverslips were fixed for 30min in cold acetone The PCR, VI, and IFA confirmed that the lesions for routine immunofluorescence analysis (IFA). were produced by CaHV-1. To confirm the isolates as CaHV-1, serial two- fold dilutions of dog anti-CaHV-1 reference To the authors' knowledge, the occurrence of serum were mixed with 100CCID 50 of the vesicular lesions produced by CaHV in the area viruses isolated and after 60min of incubation in described was never reported. This is not only a 5% CO 2 atmosphere, 100 μ L of MDCK cells the first report of an atypical localization for to (3×10 5 cells/mL) were added. For standard IFA, consider in the future, but also the first isolation CaHV-1 monoclonal antibodies and anti mouse of this virus in Argentina. conjugated to fluorescein isothiocyanate (Zymed Laboratories – San Francisco, USA) were used Keywords: dog, Canid herpesvirus 1 , atypical as primary and secondary Abs, respectively. The clinical lesions, viral isolation Abs were determined by previously conventional 1268 Arq. Bras. Med. Vet. Zootec. , v.62, n.5, p.1267-1270, 2010
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