Detection of bovine herpesvirus types 1 (BoHV-1) and 5 (BoHV-5) in the central nervous system of cattle

RENSETTI D; MARIN M; MORÁN P; VERNA A; ODEÓN A; PÉREZ S

San Miguel de Tucumán

Otro; XX REUNIÓN CIENTÍFICO TÉCNICA ASOCIACIÓN ARGENTINA DE VETERINARIOS DE LABORATORIOS DE DIAGNÓSTICO; 2014

Introduction Bovine herpesvirus type 1 (BoHV-1) is an alpha-herpesvirus responsible for several syndromes in cattle, including respiratory disease, abortions and genital disorders. Bovine herpesvirus type 5 (BoHV-5), a closely related alpha-herpesvirus and formerly classified as BoHV-1.3, is the causative agent of non suppurative meningoencephalitis in calves, a condition which is highly prevalent in South America. Unlike BoHV-5, BoHV-1 is only occasionally reported as responsible for cases of bovine encephalitis. However, recent reports identified BoHV-1 as a frequent cause of encephalitis in cattle and demonstrated that BoHV-1 participation in neurological disease is not a rare event. The aim of this study was to determine the presence and the distribution of both alpha-herpesviruses in the central nervous system of experimentally inoculated cattle. Comparisons are made between the detection of both viruses by cell culture isolation and nested PCR. Materials and methods Fourteen crossbred, 1 year-old calves were used in this study. All animals were free of detectable antibodies to BoHV-1 or BoHV-5. Calves were intranasally inoculated by aerosolization with 10 ml BoHV-1 Cooper or BoHV-5 97-613 strains. The inoculum was evenly distributed in each nostril. Animals were grouped as follows: Group 1 (n = 4) for study of primary (acute) infection, Group 2 (n = 4) for the study latency, Group 3 (n = 4) for the study of reactivation from latency and Group 4 consisted of mock-infected calves (n = 2). Calves in Group 1 were inoculated with 106.3 tissue culture infective doses (TCID50) of Cooper (2 calves) or 97-613 (2 calves) strains to induce a primary, acute infection. They were euthanized at 6 days post-inoculation (dpi). Calves in Group 2 were inoculated with 103 TCID50 of Cooper or 97-613 strain to induce a latent infection. Calves in Group 3 received the same inoculum than Group 2. At 21 dpi they were administered dexamethasone to induce reactivation. Euthanasia was performed at 25 dpi. Mock-infected calves were inoculated with MEM and they were euthanized at 6 dpi and at 2 days post-reactivation. At necropsy, brain samples were collected for virus isolation and PCR. The following areas were evaluated: anterior cerebral cortex, including the olfactory cortex (sample 1), frontal cortex (samples 2 and 3) and dorso-lateral cortex (sample 4); posterior cerebral cortex, including the marginal groove area (sample 5) and the ectomarginal groove area (sample 6); cerebellum (sample 7); cervical medulla (sample 8); medulla oblongata (sample 9), pons (sample 10) and diencephalon (sample 11). Furthermore, trigeminal ganglia (TG) were also collected. Virus isolation was performed on MDBK cells. Nested PCR techniques previously described by Wang et al. (2001) (gD BoHV-1) and Mayer et al. (2006) (gB BoHV-5) were used for virus DNA detection in neural samples. Results and Discussion During acute infection (6 dpi) BoHV-1 was isolated from both inoculated calves. The highest titers for BoHV-1-infected tissues were observed in the olfactory cortex, the ectomarginal groove area in the posterior cortex, pons and diencephalon. In the case of BoHV-5 infected calves, the virus could not be isolated from any SNC sample. Infectious virus was only detected in the TG of one BoHV-5-infected calf. As expected, BoHV-1 or BoHV-5 were not isolated in samples from latently-infected cattle. BoHV-1 was not isolated in tissue samples from animals that received DEX administration. However, BoHV-5 was isolated from frontal, dorso-lateral and posterior brain cortex after DEX-induced reactivation. The highest titers were detected in frontal cortex. BoHV-1 and BoHV-5 DNA was consistently detected in several areas of the SNC, including cervical medulla and medulla oblongata of acutely-infected calves. Unlike BoHV-1, the presence of BoHV-5 DNA was detected in several brain regions and TG from latently-infected calves and from calves that received DEX treatment. The results from this study demonstrate that BoHV-1 is able to acutely replicate in the bovine brain and that virus recovery from this tissue appears to be easier than isolation of BoHV-5. Difficultness in the isolation of BoHV-5 from neural tissue has been previously suggested (Belknap et al., 1994, Pérez et al., 2003). Despite these findings it is interesting to note that BoHV-1encephalitis is not as frequently reported as that caused by BoHV-5. During latency and reactivation, the distribution of BoHV-5 DNA in the nervous system is broader than that of BoHV-1 and it is consistent with the mild clinical reactivation observed in cattle at this stage of BoHV-5 infection. Furthermore, the findings of this work highlight the importance of using molecular techniques for the diagnosis of herpesvirus encephalitis in cattle. As expected infectious virus or viral DNA were not detected in mock-infected calves.