Identification of equine herpesvirus type 1 as cause of abortion in mares in Southern Brazil

1 Programa de pós-graduação  Faculdade de Veterinária  Universidade Federal de Pelotas  (UFPel) Pelotas, RS 2 Instituto Nacional de Investigación Agropecuaria (INIA)  Estación Experimental La Estanzuela Colonia del Sacramento, Colonia, Uruguay 3 Faculdade de Veterinária  Centro Universitário do Espírito Santo (UNESC)  Colatina, ES 4 Faculdade de Veterinária (FV)  Universidade Federal de Pelotas (UFPel) – Pelotas, RS 5 South Dakota State University  Brookings, USA


South Dakota State University  Brookings, USA
Equine herpesvirus type-1 (EHV-1) is classified into the family Herpesviridae, subfamily Alphaherpesvirinae, gênero Varicellovirus (International…, 2018). This virus is a ubiquitous pathogen that affects the horse population worldwide causing epidemic abortion, neonatal death, respiratory disorders and myeloencephalopathy (Schulmann et al., 2015). Infection with this virus may cause a serious economic impact in the horse industry, especially for breeding farms (Gilkerson et al., 1999). In Brazil, serological studies have demonstrated the circulation of EHV-1 in horses (Lara et al., 2010;Diaz et al., 2015). Latency has been demonstrated in both lymphoid and neural tissues and allows the virus to circulate silently in the horse populations (Patel et al., 2005).  Equine herpesvirus-1 abortion is diagnosed by detailed examination of the aborted fetus using a combination of gross findings, histopathology, virus isolation, polymerase chain reaction (PCR), and immunostaining (Reed et al., 2004). Conventionally, these tests have been applied to selected target organs, particularly the liver, lung, thymus and spleen (Easton et al., 2009). The main purpose of this study was to describe and confirm EHV-1 as cause of abortion in southern Brazil.
Four EHV-1 infection outbreaks in thoroughbred mares were identified in the necropsy protocols of the LRD/UFPel. These cases occurred between 1994 and 2012. In the first outbreak, eight out of 16 pregnant mares aborted. Seven abortions occurred between 10 and 11 months of gestation, and one abortion occurred in a 9month-pregnant mare. The mares of this stud farm were not vaccinated against equine rhinopneumonitis. In another outbreak, there were two abortions and one stillbirth out of 35 pregnant mares. In the third outbreak, one abortion of a 9-month old fetus occurred in one mare out of 50 pregnant mares. In the fourth outbreak, one abortion of a 10 months old fetus was recorded in a herd of 50 pregnant mares, which have been vaccinated with a commercial vaccine against rhinopneumonitis annually. No serologic status of the vaccinated animal was available.
In all outbreaks, only formalin-fixed organ samples were received for evaluation at the laboratory. The clinicians described the findings grossly as a diffuse yellow tinge to all tissues and abundant amber fluid in body cavities.
Microscopic evaluations in all cases showed multiple random foci of degeneration and necrosis ( Figure 1A) with moderate infiltration by lymphocytes and macrophages in the liver. Several acidophilic nuclear inclusion bodies were also observed in the hepatocytes and Kupffer cells ( Figure 1A). Multifocal necrosis was also evident in the spleen along with multiple acidophilic intranuclear inclusion bodies. The lungs exhibited multifocal necrotic foci randomly distributed in the parenchyma and acidophilic intranuclear inclusion bodies were also observed in the epithelia of the bronchioles. Extramedullary hematopoiesis was observed was evident in some less affected areas of the liver. EHV-1 antigen was detected using IHC staining within the nucleus and cytoplasm of several hepatic cells and leukocytes of the tissue samples from all the cases. Positive IHC staining was associated with necrotic foci in the liver ( Figure  1B), lung and in the spleen in all analyzed samples. The amplification of a specific 188bp amplicon from DNA extracted from two paraffinembedded tissues confirmed the presence of EHV-1 in tissue samples from two aborted fetuses. Additionally, the sequencing of the PCR products and BLAST results obtained with the consensus sequence (100% identity with multiple gB sequences available in the GenBank) confirmed the presence of EHV-1 DNA in the tissues analyzed.

EHV-1 abortion was first diagnosed in Southern
Brazil in 1994 by isolation of virus associated with the high prevalence of EHV-1 antibodies in the equine population (Weiblen et al., 1994). These authors suggested that abortions due to this virus are more frequent than reported as a consequence of absence of laboratory diagnosis. Several other reports have demonstrated the presence of specific antibodies to EHV-1 in Brazil (Vargas e Weiblen, 1991). However, it was demonstrated in the present study that the prevalence of viral abortion was less frequent in Southern Brazil compared with other causes of equine abortion in the same region. EHV-1 abortion accounted for 3.8% out of 104 abortion outbreaks diagnosed in this region between 1978and 2016(Schild et al., 2017. The low prevalence observed in these cases could be explained, at least partially, by the systematic vaccination of the animals in stud farms. Abortion outbreaks are described in vaccinated mares from a reproductive center that specializes in the impregnation of mares using artificial insemination and embryo transfer. The authors suggest that broodmare management and improvement of the vaccine efficacy could prevent abortion outbreaks (Barrandeguy et al., 2002). Foote et al. (2006) demonstrate that EHV-1 and EHV-4 circulate in vaccinated populations of mares and their unweaned foals. This could explain the occurrence of sporadic abortion in vaccinated animals, which was observed in the present report. The annual vaccination without serological monitoring of mares may also have contributed to the occurrence of the abortion.
Histological lesions observed in the present study were characteristic of abortions by EHV (Schlafer e Miller, 2007). IHC analysis showed the presence of EHV-1 antigen in tissues of aborted fetus. IHC analysis is an important diagnostic tool especially in cases suspected of EHV infection in which specific histopathological changes are not found (Szeredi et al., 2003). In the present study the viral etiology of the abortions outbreaks was confirmed based on the histological lesions along with the detection of antigens of EHV-1 by immunohistochemical analysis. Furthermore, specific EHV-1 DNA was detected (by the multiplex nested-PCR) and confirmed (by nucleotide sequencing and BLAST analysis) on tissue samples of two cases, corroborating the findings of IHC and histological analysis. Taken together, these findings indicated that EHV-1 should be considered in the differential diagnosis of equine abortion in southern Brazil.