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EQUINE VETERINARY EDUCATION / AE / SEPTEMBER 2018


the colostrum. VN antibodies appear in the serum of newborn foals a few hours after colostrum intake, levels peak at age of 1 week, and titres decline to extinction at the age of 2–6 or, rarely, 7 months. The mean half-life of EAV maternal antibodies in serum from foals is 32 days (McCollum 1976; Hullinger et al. 1998). Even though Castillo-Olivares et al. (2003a) demonstrated


that EAV induces specific cytotoxic CD8+ T lymphocytes and that their precursors may last for at least 1 year following experimental infection, there are no further comprehensive studies that describe the cell-mediated immune response (CMI) to EAV.


Diagnosis


Confirmatory diagnosis of EVA cannot be achieved solely based on clinical presentation, since clinical signs frequently resemble other infectious and noninfectious diseases of equids. Differential diagnosis should include other infectious agents such as equine herpesviruses-1 and -4, equine influenza virus, equine rhinitis A and B viruses, equine adenoviruses, equine infectious anaemia virus, Leptospira interrogans, and several transboundary diseases to the Americas including Getah virus, African horse sickness virus and Hendra virus. In regards to noninfectious diseases, purpura haemorrhagica, urticaria and toxicosis due to hoary alyssum (Berteroa incana) should be taken into consideration. Hence, it is imperative to confirm a provisional clinical diagnosis by laboratory means before implementing appropriate preventive and control measures (Balasuriya et al. 2013, 2014). Laboratory diagnosis of EVA is based on classical virus


isolation (VI); detection of viral nucleic acid by standard reverse transcription–polymerase chain reaction (RT-PCR), real-time RT-PCR (RT-qPCR) or reverse transcription–insulated isothermal PCR (RT-iiPCR); detection of viral antigens by immunohistochemistry; or serological assays that demonstrate a specific antibody response elicited by EAV as an indirect method to identify EAV infection (Hyllseth 1969; Senne et al. 1985; St-Laurent et al. 1994; Lopez et al. 1996; Del Piero et al. 1997; Gilbert et al. 1997; Starick 1998; Ramina et al. 1999; Del Piero 2000b; Fukunaga et al. 2000; Balasuriya et al. 2002b; Westcott et al. 2003; Mankoc et al. 2007; Lu et al. 2008; Miszczak et al. 2011; OIE 2013; Hans et al. 2015; Carossino et al. 2016a,b). Clinical samples for laboratory diagnosis should be ideally


collected from suspected cases in the acute phase of infection to confirm the aetiology of the disease. All clinical samples should be stored and submitted under conditions of refrigeration via overnight delivery for laboratory testing. Freezing whole blood samples is discouraged as it could potentially hamper viral recovery. Tissue samples from foals and aborted fetuses could also be submitted in 10% neutral buffered formalin for histopathological and immunohistochemical evaluation. However, in such cases it is important to use an appropriate tissue: fixative volume ratio (at least 1:10) to ensure proper fixation. Suitable clinical samples to attempt VI or detection of


viral nucleic acid include nasopharyngeal swabs or nasal washings, conjunctival swabs, and whole blood (in EDTA or citrate). The use of heparin as anticoagulant is not suitable for laboratory diagnosis of viral infections; thus, its use is discouraged. During outbreaks of abortion, tissue samples


© 2016 EVJ Ltd


from aborted fetuses, fetal membranes, and amniotic or allantoic fluid should be submitted for laboratory testing. Most frequently, a pool of samples including primarily fetal placental membranes (allantochorion and/or amnion), lung, thymus, spleen, liver, and other lymphoid tissues (if available) is recommended to perform VI and detection of viral nucleic acid. In cases of neonatal foals that die as a consequence of developing interstitial pneumonia or the pneumoenteric syndrome, appropriate tissue samples should be submitted for VI and/or detection of viral nucleic acid. These samples should include lung, spleen, and other lymphoid tissues such as thymus, mesenteric and bronchial lymph nodes, and kidney.


Serological diagnosis of EVA is considered an indirect


method for identification of infected animals, and it is based on the World Organisation for Animal Health (OIE)-prescribed virus neutralisation test (VNT). Thus, EAV infection can be demonstrated by a rise in neutralising antibody titres (4-fold or greater) in paired sera collected 21–28 days apart as indicated previously. A competitive enzyme linked immunosorbent assay was recently developed and validated for the detection of EAV specific antibodies, and could be considered as an alternative assay for the serological diagnosis of EVA (Chung et al. 2013a,b, 2015; Pfahl et al. 2016).


The approach for the diagnosis of EAV carrier stallions is


currently dependent on the demonstration of neutralising antibodies in serum and detection of virus in their semen (Timoney et al. 1987b; USDA-APHIS 2004; OIE 2013, 2015). Initial identification of carrier stallions is achieved by the detection of neutralising antibodies in their serum (seropositive, titres ≥1:4), and persistent infection is confirmed either by isolation of the virus from semen, test-breeding using two seronegative mares (where seroconversion within 28 days after test-breeding indicates that the tested semen is infective) or detection of viral nucleic acid in semen. Semen samples submitted for virological assessment should contain the sperm-rich fraction of the ejaculate (Timoney et al. 1986, 1987a). Pre-ejaculatory fluids are inappropriate since presence of virus in this fluid is inconsistent. Currently, the VI and VNT are the OIE prescribed tests for international trade (OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals; OIE 2013). Even though VI is the gold-standard for the detection of EAV in semen and the OIE-prescribed test for international trade, the performance of certain RT-qPCR and a RT-iiPCR described in the literature have an equal or higher sensitivity when compared to VI in semen samples (Balasuriya et al. 2002b; Lu et al. 2008; Miszczak et al. 2011; Carossino et al. 2016b). In addition, the newly developed RT- iiPCR assay has been demonstrated to have a high sensitivity and accuracy when compared to VI in tissue samples derived from aborted fetuses (Carossino et al. 2016b). Therefore, molecular assays could be used as an alternative method for EAV diagnosis (St-Laurent et al. 1994; Gilbert et al. 1997; Starick 1998; Ramina et al. 1999; Fukunaga et al. 2000; Balasuriya et al. 2002b; Westcott et al. 2003; Mankoc et al. 2007; Lu et al. 2008; Miszczak et al. 2011; Hans et al. 2015; Carossino et al. 2016a,b).


Treatment


Even though antiviral compounds have been evaluated (van den Born et al. 2005; Zhang et al. 2010b), there is still no


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