SEROSURVEILLANCE OF EQUINE INFECTIOUS ANAEMIA IN A REGION OF VOJVODINA

Equine infectious anemia is a consequence of a persistant infection of the horses with Lentivirus. Pathogenesis of the disease is very variable, what can bee seen through a wide range of clinical forms of the disease – from inaparrent infection to death. Diagnostics of EIA is based on clinical symptoms, detection of antibodies and virus. Antibodies can be identifi ed with Hi, VN, CFIT, cELISA, SA-ELISA and AGID test. RT-PCR technique enables the detection of and/or quantifi cation of viral RNA level in blood of infected animal. First reliable serological test for EIA was AGID test. Modifi ed AGID test is considered today as aknowledged, international standard for the detection of antibodies against EIA virus and it enables detection of more then 95% of ll positive animals. Horses with positive fi ndings with this test are considered infected and should be euthanized or placed in strict isolation. Further measures to control the spread of this disease are insectvector control and disinfection of surgical and other equipment in use on successive animals. Th e results of a study during a twenty year period, in the region of AP Vojvodina show that from the total of 11.972 horses blood samples, with te use of AGID test, positive results were found in 21 or 0,17%


INTRODUCTION
Equine infectious anaemia (EIA) is a persistent viral infection of equidas.Th e causative agent, EIA virus (EIAV) belongs to Lentiviruses from family Retroviridae, subfamily Orthoretrovirinae.Equine infectious anaemia occurs in cases of persistent Lentivirus infection of horses.Th e disease is spread out in the whole world and occurs in horses, ponies, mules and donkeys.Clinical cases of the disease was described for the fi rst time in France in the middle of 19 th century and viral etiology was identifi ed in 1904 (Timoney et al.,1988).During the following years there was no progress in the introduction of pathogenesis of the disease, only in epidemiology, clinical manifestation of the disease and pathology.All attempts for the virus to be transfered to diff erent experimental animals, except horses, were unsuccsesfull (Cook et al. 1996).A reliable diagnostic method was not introduced until 1970 (Coggins and Norcross, 1970).On the contrary to that, during the last few years a break through was done in analysing biological and biochemical characteristics of EIA virus and its pathogenesis.

EPIZOOTIOLOGY
EIA virs can be found in many countries in the world, at all continents.Th e disease can be found in horses in Europe and the level of incidence depends of the density of the equine population, presence of the vectors and also diff erent programs for control of this disease (Sellon, 1993;Toma, 1980).Equine infectious anaemia is found also in USA, Canada and Latin America, where a high level of incidence was found (Cook et al. 1996;Hall et al.,1988;Timoney et al., 1988).Th e most important way of infection with EIA in nature is with contaminated blood, mostly transfered by blood sucking artropods (Kemen and Coggins, 1972).Th e virus is spread via interrupted feeding of bloodsucking horsefl ies on a clinically ill horse and then on susceptible horses.Transmission can also occur by the iatrogenic transfer of blood through the use of contaminated needles, because the virus can remain vital up to 4 days (Cook et al. 1996).
Epidemiological proof, with a fact that EIA virus cannot replicate in mamals other then equides, (Kemen and Coggins, 1972) shows that persistently infected horses, ponies, donkies and mules represent the only reservoir of the virus in the nature.Besides, according to the known data so far, wild type of EIA virus will not replicate in any kind of insect or insect cell lines (Foil and Issel, 1991).Th at it why it is considered that artropod transimission of the virus is only mechanical (Kemen i Coggins, 1972).Succsessful machanica transmission of the virus depends on everal factors such as level of the viru in blood or tissues of the host (titar), characteristics of the vector and its behaviour, behaviour of the host, climate, close presence of the woods, shelters (most of the Tabanides do not enter indoor spaces).

PATHOGENESIS AND CLINICAL SYMPTOMS
Pathogenesis of EIA virus is very variable, caused by the characteristics of the host and the virus, which refl ects in a wide range of clinical forms if the disease, from inaparen infection to death.Two important parametrs in pathogenesis of EIA are a lifetime persistence of EIA virus in infected host with a spo-radic occurence of the disease.Life time persistence of the virus comes from a capability of the virus to intergrete itself into a hromosome DNA of the host.Proviral DNA in integrated and non intergrated state can easily be detected during the period of acute infection.Yet, specifi c sequences of the virus cannot be detected in asymptomatic horses which indictes that the number of cells which contain inergreted DNA viruses is accually low (Rice et al., 1989).In special conditions such as immunosupression (Dreguss and Lombard, 1954), the reapperiance of clinical symptoms is conditioned with the characteristics of EIA virus -it can mutate and make new demands for the immune system of the host.
Th e disease is characterised by recurrent febrile episodes, thrombocytopenia, anaemia, rapid loss of weight and oedema of the lower parts of the body.If death does not result from one of the acute clinical attacks, a chronic stage develops and the infection tends to become inapparent.Th e incubation period is normally 1-3 weeks, but may be as long as 3 months.Th e severity of the disease varies and it can be in a asymptomatic form or in a form with a high morbidity rate, even with fatal ending.Factors of the host and virus which infl uence this variability are still not completely clear.
Clinical manifestation of EIA disease can be acute or chronic and there is also the inapparent state of the carrier.Acute form of the disease is oft en connected with the primary infection and clinical symptoms include pyrexia, anorexia, depression and petechial bleeding of mucosa.Anaemia is not characteristic in acute infections, except in very severe cases when there can also be seen epistaxys and ventral oedema.In chronic infections cycles of healing and return of the disease can be seen with classic symptoms of anemia, edema and weight loss.Death of the animal can occur 4 week later at the earliest aft er the infection.If the animal lives through the acute phase, the frequency and the severity of the clinical episodes progressively drops (90% can be seen during the fi rst zear aft er the infection), until the animal becomes an inaparent reservoir of pathogens (Timoney et al., 1988.)Nevertheless, some animals serologicaly positive to EIA virus, never had any clinical symptoms, or they were in uch a mild form that they could not b noticed by the owner (Issel and Coggins, 1979).

DIAGNOSTICS
Diagnostics of EIA is based on clinical symptoms , analysis of fagocytic blood cells which contain ingested erythrocites (sideroleucocytes / which are not pathognomonic for EIA, but oft en are present in acute infection), detecti-on of antibodies against viral components and detection of the virus.Diagnostics based on clinical symptoms is complex because of the variability of the symptoms and the exsistence of inaparent carriers.For fi eld strains of EIA virus which replicte only in monocitic/macrofage cells, detection of the virus can be done with a transfusion of 250ml of whole blood (collected in acid-cytrate solution of dextrosis) from a horse suspected for EIA to a seronegative horse as a receiver.RT-PCR technique enables detection and/or quantifi cation of the viral RNA level in blood of infected animal.
Antibodies can be detected by the following tests: Hi, VN, CFIT, cELISA, SA-ELISA and AGID test.Th e fi rst reliable serological test for EIA was the AGID test (Coggins and Norcross, 1970).However, there are reports that in infected horses can be gained negative fi ndings and what is even more important, some of them can spread the virus (Toma, 1980).Detection in these animals requires more sensitive serological tests.Noumerous modifi cations of ELISA method have been described so far with a p26 as antigen.Competitive ELISA method (cELISA) uses mAb for p26 and ELISA with synthetic antigen (SA-ELISA)can also be used.When using ELISA methods, false positive fi ndings can be expected.Although cELISA and SA-ELISA will detect antibodies somewhat earlier and at lower concentrations than the AGID test, positive ELISAs have to be confi rmed using the AGID test ( Lew et al.,1993) Th e AGID test also has the advantage of distinguishing between EIA and non-EIA antigen-antibody reactions by lines of identity.Agar gel immunodiff usion (AGID) tests (Coggins et al., 1972) and enzyme-linked immunosorbent assays (ELISAs) (Suzuki et al., 1982) are accurate, reliable tests for the detection of EIA in horses, except for animals in the early stages of infection and foals of infected dams.In rare circumstances, misleading results may occur when the level of virus circulating in the blood during an acute episode of the disease is suffi cient to bind available antibody, and if initial antibody levels never rise high enough to be detectable (Toma, 1980).
More advanced dignostics is possible with the use of RT-PCR technique, which enables detection and/or quantifi cation of the viral RNA level in blod (Nagarajan and Simard 2001).

OUR INVESTIGATIO
During a period of twenty year, blood serum semples of horses were analysed, from two diff erent epizootical regions -from southern Backa and Srem region.Horses originated from two stables, several horse clubs, private owners and one collection point where horses were in qurantine before tran-sportation for exportation.
Agar-gel immunodifussion test was used (Coggins test-VMRD Inc.) and in total 11.972 horse serum samples were analysed.
Findings aft er the analysis of blood serum samples from horses, for equine infecious anemia are shown in Table 1.In the perid of study from the total of 11.972 horse serum samples, positive results were found in 21 animal, which is 0,17%.

DISCUSSION
Equine infectious anaemia can cause damage and it can be a deadly disease.Until today, treatment for horses that have EIA is not known.Also, there is no vaccine on the market against EIA virus for the protection of horses.But with control measures and management techniques that are strict and appropriate for implementation in horse breeding, the chances of infection with EIA virus can be pretty much reduced.
Horses infected with EIA virus represent a danger and threat to the community.Th e consequences can be of diff erent levels and risk from EIA is still to be estimated.In order to prevent the spreading of EIA virus, strict control measures should be applied (Cook et al. 1996).In some rural areas horses still participate in everyday life, taking part in agriculture and transportation.A decrease of the potential risk of great economical losses resulting from infectious diseases occurring in horses is very important.
A signifi cantly higher number of infected animals has been found in Greece.During the period 2001-2008, a total of 7.872 horse serum samples were tested at the Centre of Veterinary Institutes of Athens.Antibodies against equine infectious anaemia (EIA), were found in 4.5% of the samples and seropositivity for EIA was determined ( Mangana-Vougiouka et.al.2013).
To the contrary of this, there are data from several regions in Turkey.A study was done where the material consisted of 8.947 horse serum samples, including 8.769 horses and 178 donkeys, from Ardahan, province in north east part of Turkey (Albayrak and Ozan, 2010).Blood was collected from all horses and donkeys and the sera were analysed for the presence of antibodies for equine infectious anaemia virus (EIAV) using an enzyme-linked immunosorbent assay.All animals were negative for antibodies against EIA virus.EIA infections are also reported in diff erent countries (Pearson and Knowles 1984;Lew et al. 1993;Nagarajan and Simard 2001).In Armenia and Georgia EIA has not been reported so far, but the presence of haematophagous vectors as important risk factors of EIA for equines has been found (Erdem, 2007).Since 1981, in Serbia there is a program for prevention and, eradication of EIA in horses ( Vidić et. al. 1998.).EIA is a disease that is mandatory for reporting to the OIE and disease which is under annual program of monitoring, administered by the Ministry of Agriculture and environment protection of Serbia.
Gained results show that EIA is present in horses from the region included in the study and there is a very low prevalence.Th ere is no tendency of the virus to spread and we can assume that the gained results are a consequence of a horse trade market, because EIA was found in horses from other regions of Serbia, intended for slaughter.

PROPHYLAXIS AND CONTROL
An attenuated live vaccine, developed in the early 1970s, was extensively used in China (OIE Terr. Manuel, 2008) between 1975 and 1990.Until today, there is no available vaccine against EIA virus, except in China, because of the complex development of the vaccine which is conditioned by the life cycle and antigen performances of Lentivirus.Control measures for EIA have the aim to reduce the probability of the appearance of infection.Th is is achieved by the good management on the farm, strict laws and regulations and the capability of detecting and separation of infected animals.Good practice on farms involves separation of infected animals, grazing animals far from the edge of the woods, shelter from the tabanides attack, usage of spray and repellents for reduction of the vectors and also the usage of the proper procedures of vaccination or blood sampling can help in the prevention of the spreading of EIA.Besides all this, most of the countries have their own regulations for EIA with the measures "test and remove", which limit the breeding and trade of the infected animals.All of the horses with positive fi nding are considered infected and should be euthanized or placed in strict isolation.Further measures to control spread of this disease are insect-vector control and disinfection of surgical and other equipment between use on successive animals.Th ese regulations include the obligation of permanent identifi kation of infected animals (lip tatoo, stamps, electronic implants).

Aknowledgments
Th is investigation was supported by a grant from the Ministry of Education and Science of the Republic of Serbia, Project No. 31071.

Table 1 .
Finding of blood serum samples from horses nalysed for equine infectious anemia during the period 1994-2013