EQUINE HERPES VIRUSES PREVALENCE IN HORSE POPULATION IN LITHUANIA

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1 EQUINE HERPES VIRUSES PREVALENCE IN HORSE POPULATION IN LITHUANIA V. Liutkevičien 1, M. Stankevicien 1, V. Mockeliunien 2, R. Mockeliunas 1 Lithuanian Veterinary Academy, Kaunas, Lithuania 1 Lithuanian Veterinary Academy, Veterinary Institute, Kaisiadorys, Lithuania 2 ABSTRACT Equine herpes viruses are outspread in most countries. The most prevalent among equine viral diseases is equine herpes virus type 1 (EHV-1) (1). The ultimate epidemiological reservoir for EHV-1 is the large and globally distributed pool of latently infected (7). Mares abort, herd s reproduction is affected and disordered so too many funds are used to eliminate the disease and to restore herd s reproduction. Equine herpes case noticed first time in USA and investigated by W.W. Dimock (1932) and P.R. Edwards (1936) (22) while equine herpes research started in Lithuania in from 9 stud farms, riding clubs and agriculture farms in different districts of Lithuania were in connection with herpes viruses. The research has shown that herpes virus does affect the cells (CPE) and also EHV-1 antigen was found. EHV-1 viruses latently infected were investigated almost in all herds of. Results show that herpes viruses circulate in population of Lithuanian. Introduction Equine herpes viruses are populated in most countries and cause large economical damages because of abortions of mares, disordered reproduction of herds, losses of fund to eliminate the disease. Disease noticed first time in USA and investigated by W.W. Dimock (1932) and P.R. Edwards (1936) (22). Viruses belong to DNA virus group, Herpesviridae family. DNA of virus is doublechaine, nucleocapside cubic symmetry, virion size nm. Some viruses have supercapside. Optimal media ph for virus survival is Viruses inactivate at ph below 4 and above 10. Virulence is kept 10 minutes at 56 ºC temperature, 7-8 month at 4 ºC, and month at ºC in cultures solution. Viruses are sensitive to ether and chloroform (4, 18). The structure of virus antigens is still ambiguous except S (soluble) and V (viral) antigens which have been segregated. Replication of viruses takes place in the cell s nucleus and results in forming eosinophylic bodies. 20 polipeptides of molecular mass D mol are identified using electrophoresis. 14 polipeptides determined in non supercapside virions and 3 polipeptides in. Viruses cytopathogenic effect (CPE) (12, 15, 21). EHV- 1 and EHV-4 are main types of this family (2, 11, 13). These viruses cause complex of different symptoms: 1. Rhinopneumonia (ehv-4, rather ehv-1); 2. Mare viral abortion (especially EHV-1); 3. Myeloencephalopatia (especially EHV-1). Besides the mentioned types of equine herpes viruses other types are exist in the nature, but not classified yet to any genus, pathogens of Alphaherpesvirinae EHV 3, EHV 6, EHV 8 and Betaherpesvirinae EHV -2, EHV 5, EHV 7 subfamilies (20, 22). Decrease of tale tonus, paralysis of urine bladder, back limbs and sphincters paresis, atacsia is noticed in case of myelophatia. Viruses are identified in CNS cells, there are no lesion of respiratory and fever in 111

2 typical case of myelophatia (6, 10). EHV-1 and EHV-4 are pathogenic for different age of. It is possible to infect experimentally guinea pigs and hamsters, which usually die. Sick and recovered spread viruses with nasal, respiratory and genital secretion. Respiratory lesions usually occur in spring and winter and genital in different year time (8, 9, 19). Virus pass into organism trough respiratory path, multiply in respiratory cells, pass into leucocytes and into placenta. Viruses toxins, tissue metabolites and transudate mechanically separate fetal involucres and mares abort at 7 11 foal month (5, 14, 17). EHV-1 is the main cause of mares abort. Though sometimes mare succeeds to born foal, however it is weak. EHV can cause such pathology as paralysis and respiratory disease. EHV-1/ EHV-4 caused infection for foal pass as acute, contagious respiratory disease with fever and ends % lethal. As prevention is used vaccination of mares with live or inactivated vaccines (3, 8, 16, 17). Aim of research: Estimate the outspread of viruses in population of Lithuanian. Materials and Methods Blood for analysis was taken from 9 herds of (in stud farms, agriculture farm, riding clubs). Specific antibodies were investigated using Indirect fluorescent antibody (IFA) method and reaction of virus neutralization (VN). Elimination of viruses was performed by classic method of virusology. Blood for serological analysis was taken from Jugular vein to 10 ml vacuum tubes without coagulant. Samples were centrifuged at 1500 rpm for 15 min. to separate serum. Numbered blood samples were kept at -20 ºC temperature pending analysis. Commercial diagnostic kit made and standardized in Sweden (Svanovir) was used for equine herpes viruses analysis IFA method. 140 randomly selected were in point of herpes viruses. Prevalence of EHV 1 infection in groups of of various ages and genders was investigated using retrospective method analyzing results from four horse farms and classifying 114 to the groups. Comparable EHV-1 and EHV-4 infections occurrence was analyzed in five herds (26 ). Determination of viruses: Fetus internal organs of 7 mares which had abort were collected and made as 10 % suspension in media 199. Equine derma cells (ED 39.P.7.6.) were used for determination of viruses. Cells were grown in MEM media with 5 % calves embryo serum in 50 ml plastic containers in 37 C temperature. 0.5 ml 10 % suspension was poured to all containers when cells monolayer was grown. Incubation was carried out for 1 hour in 37 C temperature with careful 10 min. horizontal shake. Following incubation 5 ml of MEM media with 5% EVS was poured on infective material and kept in thermostat doing daily revision. One container without infection was incubated also. EHV-1 and EHV-4 standards were used for control. Cells were colored with acridin orange for analysis of citopathogenic effect (CPE) and proximate immunefluorescence reaction with Gamakon diagnostic (Slovenia) was used to determine herpes virus antigen. Results and Discussion Blood samples for analysis were taken from 9 herds of in different regions of country. Specific antibodies were determined and compared results of IFA and VN methods. 114 were analyzed for herpes virus infection in four riding clubs. Results are shown in Table 1. Data presented in Table 1 shows that EHV-1 antigens were determined in every stud farm, though level of pervasion was different. The smallest part of seropositive 112

3 Teigiamų mginių skaičius procentais :5 1:10 1:20 1:40 1:80 1:160 1:320 Antikūnų titrai Figure. Repartition of specific antibodies titers for EHV-1. A žirgynas ( 90 vnt.) B žirgynas (6 vnt.) C žirgynas (8 vnt.) D žirgynas (10 vnt.) Results of serological analysis TABLE 1 number % positives Stud farm A B C D Total: was determined in stud farm B during investigation. While almost all were infected with EHV-1 in stud farms A and D. Different repartition of specific antibodies titers for EHV-1 was determined analyzing with standard neutralization reaction. Results are shown in Figure. The Figure above shows results in farm stud B differs significantly. Titers of antibodies were 1:20 in stud farm B, 1:20 1:60 in stud farm C. While titers of antibodies were 1:5 1:160 in stud farm A and D. Results show that EHV-1 circulation is active in stud farm of Lithuania. In point of epizootical situation it is important to determine main factors which influence infection agent prevalence. Influence of sex for EHV-1 prevalence is shown in Table 2. TABLE 2 Analysis of blood serum depending on gender Animal gender Number positives % Stallion Mare Total: % seropositive animals were determined in mare group what is 16.2% more comparing with stallions group. While essaying repartition of specific antibodies in groups were determined lower titers of specific antibodies (1:5 1:40) in mares group comparing with stallions group, where titers were 1:10 1:80 for majority of animals. Age of animal is important parameter. Age and environment factors influence physiological and immune status of horse as well as metabolism. 114 were assessing influence of age for EHV-1 latently infected animals. Results are given in Table 3. Analyzing results given in Table 3 the smallest part (33.3%) of seropositive animals is in less then 2 years old group. Results of other groups show there is much bigger part of infected animals even %. Thus general tendencies are increase of horse age increases 113

4 TABLE 3 Repartition of EHV-1 latently infected animals depending on age Group Animal age Nr. (years) positives number % > TABLE 4 EHV-1 and EHV-4 analysis by IFA method Herds of EHV-1 positives EHV-4 positives I II III IV V Total number of EHV-1 infected animals. Assessing of specific antibodies repartition in different age animal groups was determined that titers of antibodies are 1:10 1:80 in all groups. Analyzing population of EHV-1 and EHV-4 in herds was determined that both types of viruses (60% cases) circulate in (Table 4). Analysis of seropositive repartition showed that in 38.5% were determined EHV-4 monoinfection. Animals contacts with both types of viruses were determined in 26.9% of the. Determination of specific antibodies using serological methods (IFA, VN) in herds shows circulation of herpes viruses inside. Fetus internal organs of 7 mares which had abortion were analyzed. Results of EHV determination are given in Table 5. Microscoping six samples of pathological TABLE 5 Analysis of cytopathogenic effect in cells culture Samples of pathologic material after 12 hours Changes in cells culture after After after hours hours hours after 72 hours Immunefluorescence material colored with acridin orange did not inhibit growth and make cytopathogenic effect of cells. Cytopathogenic effect (CPE) was seen in cells culture after 18 hours after infection with suspension made from fetus internal organs of mare which had abortion. Form of cells was varied in small areas of monolayer. Most cells form changed after 48 hours: vacuoles were seen in cytoplasm and capsule got rough edge. Infected cells started to move from surface and large empty spaces were seen in samples after 72 hours. Comparing to standard viruses, CPE and cytoplasm cells change accord with EHV-1 standard. (Table 5) Complex antigen antibody was formed after proximate immunofluorescence reaction and was glittering by luminescence microscope. That shows herpes virus antigen was located in the infected cells. EHV-1 is widely spread in most horse populations. The herpes virus has evolved to occupy a unique ecological niche within the horse that allows viral persistence over lifetime of the individual animal. The ultimate epidemiological reservoir for EHV-1 is the large and globally distributed pool of latently infected (7). 140 from 9 stud farms, riding clubs and agriculture farms in different districts of Lithuania were in connection with herpes viruses. The research has shown that EHV-1 latently in- 114

5 fected were investigated almost in all herds of. The smallest part (33.3%) seropositive were evaluated in stud farm B during investigation while most or all (75-100%) were infected EHV 1 in other herds. Determined EHV-1 antigen and inspired CPE in cells typical for herpes viruses. It shows that herpes viruses circulate in population of Lithuanian. For fully identification of abstracted virus is needed to make neutralization reaction with EHV- 1 immune serum and PCR. Integrated analysis is needed to clarify herpes viruses pervasion in horse population in Lithuania and to prepare elimination and control program, which allows stud farms reach healthy herd status and enables owners to breed healthy pedigree herds. Presented data are statistically insignificant. We used SPSS statistical package (SPSS Inc., ) for statistical evaluation of data. For the data analysis we applied the ANOVA method (23). For reliability of difference between groups (p criterion) we used the multiple comparison method described by Sheffe. We took the difference as statistically significant if p<0.05. For graphic presentation of the data we used Microsoft Excel 7.0 program. Conclusions 1. Herpes viruses circulate in population of Lithuanian. Determined that from 33.3 till 100 % animals had contact with EHV-1 and/or EHV-4 in horse herds. 2. Determined cytopathogenic effect for derma cells and EHV-1 antigen is typical for herpes viruses. REFERENCES 1. Allen G.P., Kydd J.H., Slater J.D., Smith K.C. (2002) Equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4) infection. In: Infectious Diseases of Livestock. (J.A.W. Coetzer, G.R. Thomson, R.C. Tustin, Eds.), Oxford University Press, Cape Town. (In Press). 2. Borchers K., Slater J. (1993). Journal of Virological Methods, 45, Breathnach C.C., Yeargan M.R., Sheoran A.S., Allen G.P. (2001) Equine Veterinary Journal, 33 (7), Bryans J.T., Prickett M.E.A. (1970) Infection Diseases, II, Carvalho R., Passos L.M.F., Gouvea A.M.G., Resende M., Martins A.S., Franco G.C. (2000) Arquivo Brasileiro de Medicina Veterinaria e Zootechnia, 52 (3), Dutta S.K., Talbot N.C., Myrup A.C. (1983) American Journal of Veterinary Research., 44, Edington N., Welch H.M., Griffiths L. (1994) Equine vet. Journal, 26, Foote C.E., Gilkerson J.R., Whalley J.M., Love D.N. (2003) Australian Veterinary Journal, 81(5), Gilkerson J.R., Love D.N., Whalley J.M. (2000) Australian Veterinary Journal, 78 (4), Huleihel M., Salman A., Talyshinsky M., Erukhimovitch V. (2002) Antiviral Research, 53, Kirasawa R., Endo A., Iwai H., Kawakami Y. (1993) Veterinary. Microbioogy, 36, Lawrence G.L., Gilcerson J., Love D.N., Sabine M, Whalley J.M. (1994) Journal of Virological Methods, 47, Maanen C. (2002) Veterinary Quarterly, 24 (2), Mair T.S. (1996) Equine Veterinary Education, 8(6), Mizukoshi F., Maeda K., Hamano M., Iwata H., Matsumura T., Kondo T., Sugiur T. (2002) Veterinary Immunology and Immunopathology, 88(1 2), Patel J.R., Bateman H., Williams J., Didlick S. (2003) Veterinary Microbiology, 91 (1), Schroer U., Lange A., Glatzel P., Ludwig H., Borchers K. (2000) Berliner Und Munchener Tierarztliche Wochenschrift, 113 (2), Singh B.K., Yadav M.P., Tewari S.C. (2001) Veterinary Research Communications, 25 (8), ). 19. Smith K.C., Blunden A.S., Whitwell K.E., Dunn K.A., Wales A.D. (2003) Equine Veterinary Journal, 35 (5, Szeredi L., Palfi V., Molnar T. (2003) Acta Veterinaria Hungarica, 51 (2), Tearle J.P., Smith K.C., Platt A.J., Hannant D., Davis-Poynter N.J., Mumford J.A. (2003) Research in Veterinary Science, 75 (1), Thein P. (2000) Pferdeheilkunde, 16 (1), Glantz C. (1999) Medico-biologicheskaja statistica, Practica, Moskow, p