Isolation and Identification of Equine Herpes Viruses Ali, W.F. Animal Health Research Institute Virology Dept. Dokki, Giza, Egypt

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1 Animal Health Research Journal Vol. 5, No. 3, September 2017 pp Isolation and Identification of Equine Herpes Viruses Ali, W.F. Animal Health Research Institute Virology Dept. Dokki, Giza, Egypt ISSN: Received in 11/7/2017 Accepted in 15/8/2017 Abstract In this study using Indirect fluorescent antibody technique (IFAT) for leukocytes of suspected equines samples for herpesviruses in addition to paired serum samples collected for seroconversion using enzyme linked immunosorbent assay (ELISA). Positive staining electron microscopy (EM) and agar gel precipitation test (AGPT) were performed for identification. On conclusion, Out of samples (leukocytes), 13 samples were positive with IFAT and out of 41 samples of 41 animals, 23 samples were positive for isolation in BHK -21 cell culuture and 23 samples were positive in isolation on CAM, positive staining EM detected herpesviral particles Also, All isolates were detected positive with demonstrate considerable seroconversio SPF-ECE (CAM route) used for isolation, IFAT, AGPT and positive staining EM in addition to serology are accurate for EHVs diagnosis and next generation sequencing is recommended. Key Words: Equine herpesvirus- electron microscopy- ELISA. Introduction The wide distribution of equine herpesviruses (EHVs) all over the world resulted in great losses in the horse industry (Patel and Heldens, 2005) the respiratory and neurogical symptoms, venereal disease, keratoconjunctivitis in addition to early death of foals (Hong et al., 1993) were also recorded Latent carrier state exists as a result of infection with herpesviruses (Anthony and Werner, 1992). Different serotypes of herpesviruses were identified in equidae of alphaherpesvirinae and gammaherpesvirinae subfamilies (Davison et al., 2009) Equine herpesvirus 1 (EHV1) and Equine herpesvirus 2 (EHV 2) are the most endemic types among equines all over the world (Patel and Heldens, 2005). Infection with these types occurs among horses through direct contact and inhalation, fomites, water and food sources (Neubauer and Osterrieder, 2004). Equid herpesvirus 3 (EHV 3) causing equine coital exanthema were reported to result in ulceration in mouth and upper respiratory tract. however, EHV 3 do not cause abortion but the urogenital lesions (ulceration of vaginal mucosa) caused by EHV 3 may resemble lesions caused by EHV 1 So differential diagnosis is essential (Anthony and Werner, 1992). Virus isolation could be done in baby hamester kidney (BHK -21 ) cell cultures and specific pathogen free, embryonted chicken eggs (SPF- ECE), was attempted for nasal, nasopharyngeal and ocular swabs, internal organs liver, lung, spleen or thymus gland and lymph nodes of dead animals, aborted faeti and placentae of aborted mares and blood sample on anticoagulant specially if they are taken early during the infection course (Anthony and Werner, 1992 and OIE, 2008). Identification could be achieved rapidly by immunoflourescence especially if type-specific monoclonal antibodies 47

2 Animal Health Research Journal Vol. 5, No. 3, September 2017 Ali, W.E. are available (OIE, 2008). Polymerase chain reaction (PCR) could be used as a sensitive and reliable method (Varrasso et al., 2001). However PCR could not identify empty virions which are produced late in the infection. Also, if mutation occurs in the primer target region, negative PCR may result (Green et al., 2002) Serology could be essential when testing acute and convalescent sera (Thomson et al., 1976) The aim of the current study is the isolation and identification of equine herpesviruses (EHV) from suspected cases. Isolation was attempted in baby hamseter kidney (BHK -21 ) cell culture and specific pathogen free- embryonatedchicken eggs (SPF- ECE) via chorioallantoic route. Identification was achieved by using indirect fluorescent antibody technique (IFAT), agar gel precipitation test (AGPT) and positive staining electron microscopy (EM). Materials and Methods Samples: All the examined samples are of equines from Cairo Governorate. Blood samples: 5 ml of heparinzed blood samples were collected from 15 clinically diseased animals during the febrile phase Samples were layered over Ficoll- Histopaque (Sigma) and centrifuged at 3000 rpm for 20 minutes. Leukocytes at the interphase were collected and washed three times with RPMI 1640 medium (Gibco) Serum samples: Two serum samples were taken from the same (15) animals one in acute phase and the other 2 weeks later. serum samples were stored at - 20 c until examination. Nasal and Ocular swabs: Fifteen nasal and ocular swabs were collected from the same animals suffering from symptoms resemble those of EHVs. Swabs were soaked immediately in 2 ml of transport media and kept on ice and transported as early as possible to the laboratory to be prepared for virological examination. Examination: Tissue samples: (26) nternal organs (spleen, heart, lung, kidney, liver and lymph nodes of dead animals or the aborted fetuses in addition to placenta of the mares showing abortion. Those samples were transported to the laboratory without delay. Control sera: Monospecific monoclonal positive and negative sera for equid herpesvirus, prepared in equines and supplied by Animal and Plant Health Inspection Services, National Veterinary Service laboratory, Ames, IA, USA. Conjugate: Rabbit anti-equine antibody conjugated with fluorescinisothiocyanate (FITC) obtained from Animal and Plant Health Inspection Services, National Veterinary Service Laboratory, Ames, IA, USA. Cell cultures: Baby hamster kidney (BHK -21 ) cell cultures were provided by Virology Department, Animal Health Research Institute (AHRI), Dokki, Giza, Egypt. Specific pathogen free- embryonated chicken eggs (SPF- ECE): SPF- ECE were obtained from NIL SPF farm, KomOsheim, Fayoum. Diagnostic Methods: Isolation: Isolation in BHK -21 cell culture: Suspensions of tested nasal and ocular swabs in addition to tissue samples were subjected for inoculation onto BHK -21 cell cultures according to OIE (2008). The cell cultures were mentioned in Eagle's essential medium containing 2% fetal calf serum and antibiotics The inoculated cultures were incubated at 37 c and ex- 48

3 Animal Health Research Journal Vol. 5, No. 3, September 2017 pp amined for cytopathic effect (CPE) daily and up to one weak. If no CPE is detected, cultures inoculated should be frozen and thawed 3 times then used for inoculation up to 3 blind passages. Isolation in SPF- ECE : Suspensions of the tested swabs and tissue samples were subjected for inoculation of SPF - ECE days old embryo via chorioallantoic membrane (CAM) route according to Versteeg (1990) and eggs were examined daily for bock lesions from 3 to 7 days. Identification: Positive staining EM: Blood leukocytes and CAM with pock lesions were harvested and processed for positive staining EM and ultra thin sections are examined with EM according to method described by Payment and Trudel (1993). Indirect fluorescent antibody technique (IFAT): The harvested leukocytes were fixed on slides with chilled acetone and subjected for IFAT according to method described by OIE(2008). Agar gel precipitation test (AGPT): This test is carried out according to method described by Payment and Trudel(1993) using suspensions of the inoculated BHK -21 cell cultures showing CPE and SPF- ECE isolate suspensions showing pock lesions in CAM. Detection antibodies of Herpesviruses using indirect enzyme linked immunosorbent assay (Ingezim, Spain): The antigen on a solid support (Polystyreme plate). When a serum sample contains specific antibodies against the virus, they will bind the antigen adsorbed on plate. After washing to eliminate non-fixed material from the seum sample, we can put conjugate which is anti equine immunoglobulins conjugated with peroxidase. After the addition of substrate, a colorimetric reaction will appear which could be measured by an ELISA reader. In this way the presence of colour mean the presence of antibodies against the virus in the horse sera and the absence of colour mean the absence of specific antibodies. Depending on need, the kit can be used to differentiate animals which present antibodies or to know the titer of antibodies when different dilutions of sera are performed. Results Detection of Equid Herpes virus antigen with (IFAT): Out of 15 samples (Leukocytes), 13 samples were positive where fluorescent cells were detected this indications presence of EHV antigen (Fig. 1 and Table2 ). Isolation: Isolation of Equid Herpes virus in BHK -21 cell cultures: Out of 41 samples (of 41 animals), 23 samples were positive (table 1) where CPE were detected which is characterized by rounding and detachment of cells (Fig. 2). Isolation in SPF-ECE (CAM route): Out of 41 samples (of 41 animals), 27 samples were positive (Table 1) where bock lesions were detected which are characterized by being white and circumscribed (Fig. 3) Positive staining EM : Two leukocyte samples of the examined animals that were positive with IFAT and their swabs were negative in isolation along with 4 CAM which are positive in isolation in SPE- ECE and their samples were negative in isolation in BHK -21 cell cultures, they were subjected for positive staining EM where herpes viral particles were detected (Fig. 4, 5 and 6). Identification of equid Herpes virus with Agar Gel Precipitation test (AGPT): All isolates of BHK -21 cell culture and SPE- ECE were detected positive (Table 2) where a clear precipitation lines appear between antigens and control positive antiserum but no for the negative control serum. Detection of equine Herpes virus antibodies with Indirect ELISA: paired serum samples (acute and convalescent) were illustrated in table (3) which denotes considerable seroconversion. 49

4 Animal Health Research Journal Vol. 5, No. 3, September 2017 Ali, W.E. Fig (1). Infected leukocytes presented fluorescence (fluorescent cells) when examined with IFAT indicating presence of EHV antigen. (x 40) Fig (2). Right : uninoculated BHK -21 cells (control) showing confluent sheet of spindle shape cell. (x 40). Left: BHK -21 cell. Inoculated with suspected sample showing CPE which is charathier detachment (x 40). Fig.(3): pock lesions were detected in CAM when suspected samples were inoculated in SPF-ECE. Bock lesions are white and circumscribed and they become larger on prolonged incubation 50

5 Animal Health Research Journal Vol. 5, No. 3, September 2017 pp Fig (4). Positive staining EM of thin section of blood leukocytes ( ) after their harvesting with ficoll histopaque showing cross section of herpesviral particles ( ) which are of different sizes presenting tegument between nucleocapsid and envelope ( x) Fig (5). Positive staining EM of thin section for inoculated CAM demonstrating multiple herpesviral particles which are characterized by nucleocapsid ranging from 89.8 to 147 nm surrounded by tegument and envelope. 51

6 Animal Health Research Journal Vol. 5, No. 3, September 2017 Ali, W.E. Table (1). Results of isolation of Equid Herpes virus in BHK -21 cell cultures and SPF-ECE (CAM route). Animals Number of samples Type of samples Results of isolation BHK -21 cell SPF-ECE (CAM) Showing abortions 8 Placenta 4 5 Aborted feti 8 Organs 5 7 Dead animals 10 Showing clinical symptoms Oragns and lymph nodes 15 swabs Table (2). Results of identification of Equid Herpes virus from blood leukocytes and isolates of BHK -21 cell cultures and SPF-ECE (CAM). Type of samples Number of samples Test Number of positive Blood leukocytes 15 IFAT 13 Suspensions of CAM 27 AGPT 27 Suspensions of BHK - 21 cells 23 AGPT 23 Table (3). Results of serum samples (acute and convalescent) examined by commercial ELISA Number of serum samples Titer of acute serum samples 1/100 1/100 1/100 Titer of convalescent serum samples 1/800 1/1600 1/800 Discussion Herpesviruses biology has a silent character which able them for production of latency where herpesviruses could persist with the host life (Foote et al., 2003 and abdelgawad et al., 2016). This character makes its control very difficult. Accurate diagnosis is essential for controlling these viruses. Because virus isolation is considered a gold standard for diagnosis of EHVs as mentioned by Lunn et al., (2009), the present study used BHK -21 cell cultures and SPF-ECE to compare between them where the current study illustrated that SPF-ECE (CAM route) is more sensitive. This result may be due to BHK - 21 cells as any cell culture may have viral latency specially for herpesviruses where hamster is very susceptible to infection with herpesviruses (Blood et al., 1983) but ECE is SPF so it is more sensitive for isolation. Result obtained by Hassanien et al., (2002) for isolation of EHVsin CAM of SPF-ECE are in agreement with the result of this study IFAT is used for rapid diagnosis of EHVs as a group specific test on using polyclonal antisera 52

7 Animal Health Research Journal Vol. 5, No. 3, September 2017 pp so it could mainly identify herpesvirus without typing it if monoclonal antibodies are not available as stated by OIE (2008). Electron microscopy (EM) could detect different pathogens present in the sample or isolate leads to differential diagnosis so other suspected diseases are excluded (Hzelton and Gelderblom, 2003). That is why EM must be the method of choice and as a frontline as stated by Green et al., (2002). However, result of the current study illustrated that positive staining EM could help for confirmation of herpesvirus infection with its characteristic shape (envelope, tugument and nucleocapsid) and characteristic size ( nm) as mentioned by Brooks et al., (1998). The current study denoted that AGPT could be used for identification of herpesviruses in equines on using the specific antiserum and it could be used as alternative for EM if it is not available because it is group specific (Anthony and Werner. 1992). Serological diagnosis could be used for diagnosis if 2 samples (acute and convalescent) of the same animal and considered positive for recent infection if there is increase in titer of antibodies as four fold dilution due to equines usually had level of antibodies to EHVs as stated by OIE (2008). However, serology has no significance in case of abortion due to at time of abortion, animals not seroconvert because antibody titer reached its maximum as detected by James and Potgieter (1985). On conclusion, isolation specially in SPF- ECE (CAM route), IFAT, AGPT and positive staining EM in addition to serology are suitable methods for diagnosis of EHVs although they are all group specific so next generation sequencing for EHVs isolates is recommended to avoid cross reactions which may occurs frequently between tests conducted for diagnosis and due to it is very important to certain and confirm the type or types of herpesviruses concerned with a lot of problems among equines. References Abdelgawad, Assa; Damiani, Armando; Ho, Y.W. Simon; Strauss, Gunter; Szentiks, A. Claudia; East, L. Marion; Osterrieder, Nikolaus and Greenwood, D. Alex (2016). Zebra Alphaherpesviruses (EHV_9): Genetic Diversity, Latency and Co-Infections. Viruses, 8(262). WWW. mdpi.com/ journal/ viruses. Anthony, E.C. and P.H.E. Werner (1992). Veterinary Diagnostic Virology a practitioner's guide, Mosby year book. Blood, D.C.; O.M. Radostits and J.A. Henderson (1983). Veterinary Medicine, sixth edition. Brooks, G.F.; Bute, J.S. and Morse, S.A. (1998). Medical Microbiology. Twenty First Edition. Clapham, P. (1976). Serological detection of equid herpesvirus 1 infections of the respiratory tract. Equine Veterinary Journal, 8: Davison, A.J.; Eberle, R., Ehlers, B.; Hayward, G.S.; McGeoch, D.J.; Minson, A.C.; Pellett, P.E.; Roizman, B.; Studdert, M.J. and Thiry, E. (2009). The order Herpesvirales. Archives of Virology, 154: Foote, C.E.; Gilkerson, J.R.; Whalley, J.M. and love, D.N. (2003). Seroprevalence of equine herpesvirus 1 in mares and foals on a large Hunter Valley stud farm in years pre and post vaccination. Australian Veterinary Journal, 81: Green, K.Y.; G. Bolliot; J.L. Taylor; J. Valdeuso; J.F. Lew and A.Z. Kapikian (2002). A predominant role for nor walk_ like viruses as agents of epidemic gastroenteritis in malignant nursing homes for the eldery. J. Infect. Dis., 185:

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