PREVALENCE OF SMALL RUMINANT LENTIVIRUS INFECTIONS IN SHEEP AND GOATS IN SOME REGIONS OF VOJVODINA PROVINCE

Small Ruminant Lentivirus causes a chronic lifelong, multisystemic diseases in sheep and goats with or without clinical manifestation. Maedivisna virus (MVV) and Caprine arthritis encephalitis virus (CAEV) are often considered together as small ruminants lentivirus (SRLV) because of their phylogenetic correlation and the interspecies transmission between


INTRODUCTION
Maedi-visna virus (MVV) and Caprine arthritis encephalitis virus (CAEV) are oft en considered together as small ruminants lentivirus (SRLV) because of their phylogenetic correlation and the interspecies transmission between sheep and goats. SRLV belongs to genus Lentivirus, subfamily Orthoretrovirinae and family Retroviridae. SRLV can cause a chronic lifelong, multisystemic diseases in sheep and goats with clinical manifestation in adults. Due to a slow progress of the disease, the possibility of infection within a fl ock is high. Most of infected animals never develop clinical symptoms. In sheep, name "visna" stands for the disease that aff ects central nervous system, a neurological form of the disease and predominantly causes meningoencephalitis in sheep. Th e term "maedi" stands for infection of the lungs. Besides this, the virus can also aff ect other organs such as udder.
Small ruminant lentiviruses include viruses with diverse genotypes that frequently cross the species barrier between sheep and goats that display a great genetic variability. Th ere are fi ve genotypes (A-E) of SRLV (Minguijón et al., 2015). Genetic variability is a key feature of small ruminant lentivirus genome and is essential for accurate diagnosis. Th ere is a slight variation of the strains of SRLV and specifi c symptoms can be more dominant depending on the differences in genetic susceptibility patterns. Depending on the cell type, visna/ maedi virus infection may lead to a cytopathic eff ect, or remain unnoticed.
Caprine arthritis and encephalitis virus (CAEV) can aff ect joints, central nervous system and udder in goats (Marcom et al., 1991). Arthritis can be found in infected animals which are 1 to 6 years old, with swollen carpal joints, causing limp and weight loss in sick animals (Blacklaws, 2004 Domesticated host animals of the virus are sheep (Ovis aries) and goats (Capra hircus). European moufl on (Ovis aries musimon) is also recognized as a host in the wilderness (Straub, 2004). Th e earliest reports on the disease come from South Africa and the USA (Straub, 2004, Randall et al. 1988), but nowadays the disease can be found in all the countries where sheep are bred. Th is virus is distributed all over Europe and in many other countries worldwide. According to International Animal Health organization (OIE), back in 2005, the disease was either reported as present or known to be present in most of European countries (Austria, Belgium, Bulgaria, Czech Republic, Denmark, Finland, France, Germany, Hungary, Iceland, Ireland, Luxembourg, Netherlands, Norway, Poland, Portugal, Sweden, Switzerland and UK) (OIE, Handistatus, 2005). One of the most important factors that aff ected the spreading of this disease in diff erent countries (such as Denmark, Norway, Finland and the United Kingdom) is the trade of animals without control (Gjerset et al 2007). In some other countries there was serological evidence and/or isolation of the agent (Latvia, Slovakia) and in some countries the virus was not found at that time. Later on, the evidence of the disease appeared in those countries too, like in Serbia and Romania (Vidić et al., 2008;Mihai et al., 2018). So far, SRLV-free countries are New Zealand and Australia. Th e fi rst survey in Serbia was conducted in 2008, when a prevalence of 16.24% was found in the whole country by ELISA test (Vidić et al., 2008). Th e attention was brought to this disease at that time and then in 2012 a seroprevalence of 21% was found in sheep (varying from 14-30% in diff erent regions) to 6.81% in rams (Savić et al., 2012). It is very important to identify the presence of Maedi-visna infection because of the long incubation period, and control of this disease is complicated since there is no treatment, no vaccine and the options for prevention are limited. Th is is why a monitoring program for breeding animals was introduced in 2016. Th e purpose of this study was to analyse the seroprevalence in sheep, rams, goats and bucks aft er the 3 years of active monitoring program in breeding animals for SRLV and to show if and how seroprevalence has changed over the study period.
Diff erent studies have shown that all productivity parameter measures appeared to be reduced in the seropositive groups for both goats and sheep, even though the diff erences were not statistically signifi cant (Leginagoikoa et

Material/sampling and sample distribution
During a fi ve-year period, from 2014-2018, an annual monitoring on male animals (rams and bucks) used for breeding was conducted. Th e purpose of this program was determination of antibodies against SRLV with ELISA serological method. Th is program was proposed and coordinated by the Veterinary Directorate of the Ministry of Agriculture, Forestry and Water Management. Th e monitoring program is planned and announced every year and it applies to the whole country, including all regions. Th e analyses in this study were done according to that mandatory annual program introduced by the government of Serbia and so they included all male animals (rams and bucks) used for breeding. Besides male animals, female animals were also analysed when there was a suspicion that the disease is present in a fl ock. Th e region in which the sampling was performed was South Backa and Srem District -a region which is controlled by the Scientifi c Veterinary Institute "Novi Sad". Th is means that during the whole study period, each year the animals from the same regions were analysed. In total, serum samples from 5,039 animals were included in the study. Out of the total, 1,316 samples were from sheep, 2,587 samples were from rams, 994 samples were from goats and 142 samples were from bucks. Th ere were 2,729 samples from male and 2,310 samples from female animals analysed for the presence of antibodies against SRLV.

Th e ELISA methods
Th e kits used were ELISA diagnostic kits of two types. First, the screening diagnostic kit was used in order to identify samples positive for antibodies against SRLV. Positive samples identifi ed with screening ELISA kit (ID Screen MVV-CAEV Indirect Screening test, IDvet, France) were then analysed with a conformation ELISA kit (INgezim MAEDI Confi rmation, Ingenaza, Spain) in order to obtain the conformation of positive samples. Screening kit is designed to detect antibodies against SRLV in sheep and goat serum samples, based on an indirect ELISA technique using a purifi ed pool of SRLV antigenic peptides. Conformation assay was designed to confi rm the positive samples obtained in the screening test by detection of specifi c antibodies against SRLV through the use of specifi c peptides of genotypes A and B. Th e sensitivity and specifi city of the kits were 95%.

RESULTS
During the fi ve-year period, the results were categorized by year and by animal species and categories into tables and pictures. From the total number of 5,039 analysed animal samples, 282 were positive for SRLV, which is 5.59% of anlysed animals. Th e seroprevalence for the whole period was 5.15% only in male animals, while and in female animals it was 6.15%.
Seroprevalence for SRLV for the whole 2014-2018 period was 4.52% in rams, and 17.61% in bucks. In sheep, seroprevalence for the whole period was 1.14% and in goats it was 12.57%. Th e number of positive samples for antibodies against SRLV and seroprevalence in sheep, rams, goats and bucks is shown in Table 1 and Figures 1 and 2. Th e number of positive animals in the years 2014 and 2015 was 0 and 2 respectively, leading to no seroprevalence and a very low seroprevalence, respectively. Th is was most probably due to a very low number of total samples taken during those two years, and also due to a low number of samples taken from goats/bucks. Th e monitoring program was in preparation and the actual numbers were gained during the next three years, over the period from 2016 to 2018. During this period, the seroprevalence did not change much in sheep (0.85 -1.93%), but in rams it signifi cantly decreased from 9.96 to 3.06%. Th e seroprevalence in goats was 15.05% in 2018, which is rather high compared to other categories, but in the previous years it was not looked into. Th e seroprevalence in bucks was mostly inconsistent, but it was decreasing signifi cantly from 47.62% to 8.64%. Seroprevalence in sheep and rams (3.38%) is signifi cantly lower than in goats and bucks (13.20%).
Th e overall seroprevalence for the whole study period (2014-2018) in sheep, rams, goats and bucks is shown in Figure 2.

DISCUSSION
During the 2014-2018 period, seroprevalence for SRLV decreased from 9.96% to 3.06% in rams and from 47.62% to 8.64% in bucks. In sheep, the seroprevalence did not change much over the years (0.85-1.93%), but in goats it appeared to be very high during the last year of the study (15.06%). Compared to previous studies, it can be observed that the overall seroprevalence decreased to 5.59%, and it is very interesting regarding the sex and species categories.
Th e fi rst survey in Serbia was conducted in 2008 and seroprevalence of 16.24% was found in the whole country (Vidić et al., 2008). A study from 2012 showed a seroprevalence of 21% in sheep in Vojvodina region, while in rams it was 6.81% (Savić et al., 2012). Th en, in the period from 2013 to 2015 seroprevalence seemed to be very low, but it was only because a very small number of samples (or even none) were analysed. When the monitoring program coordinated by the Veterinary Directorate started in 2016, a signifi cantly higher number of samples were analysed, but still not from all the categories. Only in 2018, a representative number of samples were gathered from sheep, rams, goats and bucks. What is also important is that seroprevalence in goats and bucks together (13.20%) was signifi cantly higher than seroprevalence in sheep and rams (3.38%), even though the number of analysed samples was significantly lower in goats and bucks.
Th e characteristic of SRLV antibodies is that they are slow in appearance aft er the infection and this has to be taken into consideration when fi ndings are discussed. Passively obtained antibodies can last for 6 months and when serological examination of sheep is performed before this period, this fact needs to be taken into account as well. Positive serology fi ndings do not mean that symptoms will appear. It means that the animals were in contact with the virus, but the animals will not necessarily be sick. Prevalence can be high in a fl ock, especially in older animals meaning that there was a contact between the fl ock and the virus (Gomez-Lucia et al., 2018).
Th e prevalence of SRLV seems to be much higher in farms where lambs and kids are fed with a pool of colostrum or milk from the tank, a practice that favours transmission to the management system (Leginagoikoa et al., 2010;Barquero et al., 2013). Besides a type of management system (higher SRLV seroprevalence in intensively reared sheep than in semi-intensively and extensively reared sheep), fl ock size also can be a factor. According to Junkuszew et al. (2016) 70 dams are the limit above which there is an increased risk for lentiviral infection. Th e route of transmission is related to body fl uids, mainly respiratory exudates and milk or colostrum and also airborne, favoured by overcrowding (Barquero et al., 2015).
Th e region in the study area is very developed in agriculture with plant production and soil exploitation. Th us, sheep and goats are mostly kept in stables with a lot of animals in a small area. Traditionally, the size of the fl ock is from one hundred to a few hundred animals. Only in the places where the soil is not suitable enough for plant production, animals can be kept on pastures where they can easily get in contact with other fl ocks. Also, traditionally, male animals are traded between diff erent fl ocks. Sheep are most commonly used only for meat production and lambs are not separated from mothers that nurse them, which carries a great risk of getting infected with SRLV through milk or colostrum. Th ere is also a great risk of other ways of infection due to the direct contact with older animals in fl ock which are all kept together. Goat breeding is oriented exclusively towards milk production and lambs and kids are separated from dams at early stages of life, so it is more appropriate to take measures in order to diminish the risk of infection with SRLV.
Th e main economic losses resulting from SRLV infection come from the eff ect of subclinical infection on the productivity of infected fl ock. Th e economic impact of this disease is a direct damage from increased mortality in a fl ock. But besides that, there is a decreased production of animals, a higher level of illness incidence, etc. Seropositive animals have lower level of milk production, shorter lactation period and reduced reproduction abilities. Th e presence of the disease in a fl ock signifi cantly aff ects the value of the animals on the market.

CONCLUSION
Th e number of positive animals is a signifi cant fi nding especially with regards to a longer screening period. It is important to keep the disease under control, because the losses resulting from this disease are signifi cant, which means that the annual monitoring program is crucial. What has not been accomplished so far is including a larger number of goats and bucks in the program in order to gain much more precise results, and to identify the infected fl ocks.
In the observed region there are seropositive animals within the fl ock which need to be under control. Th ere is uneven distribution of seropositive animals within the region. However, all the municipalities are aff ected by SRLV virus.
Annual monitoring program organized by the Veterinary Directorate is an ongoing and continuing program. Th e obtained results show that this program is very important and needs to be maintained in the future in order to keep the disease under control. Th e program helps in raising the awareness and familiarizing the owners with SRLV and the importance of breeding seronegative animals. A monitoring program that includes more bucks and goats is required in the future as well as a program for seropositive animals. Also, a plan on what to do with them while the seroprevalence in the country is still relatively low is required.

ACKNOWLEDGEMENT
Th is paper is part of the research done in the project TR31071 granted by the Serbian Ministry of Education and Science.

Author's contributions:
Individual contributions of authors to the manuscript, specifi ed by initials in this section are the following: SS made contributions to the concept and design of the study, organisation of work, writing the manuscript and data analysis and prepared the fi nal draft of the manuscript. MŽS completed laboratory analysis of all the samples, drew the maps and worked on technical preparation of the manuscript. DB participated in writing of the manuscript and contributed mostly to data analysis process. DM collected all the epizootical data needed for map drawings and participated in the writing of the manuscript. AM participated in writing of the manuscript and preparation of the fi nal draft of the manuscript, mostly contributing to data analysis. AP participated in writing of the manuscript and preparation of the fi nal draft of the manuscript.