BLUETONGUE DISEASE - EPIZOOTIOLOGY SITUATION IN SERBIA IN 2015, DIAGNOSIS AND DIFFERENTIAL DIAGNOSIS

Bluetongue disease is non-contagious, vector borne, viral disease mainly of sheep but also of other domestic and wild ruminants. Bluetongue virus (BTV) belongs to the family Reoviridae , genus Orbivirus and is characterized by segmented double-stranded RNA. Virus is transmitted from one to another susceptible animal by hematophagous insects of the genus Culicoides . According to offi cial data, between 2002 and 2014, Serbia has belonged to BTV free countries. Aft er that, the fi rst outbreak occurred in August 2014. Th e last case was reported in December of the same year. During 2015, 74 samples were examined for exclusion of bluetongue disease: 8 in cattle, 65 in sheep and one in goat. In order to detect viral genome, 73 blood samples and one tissue sample were examined by reverse transcription - polymerase chain reaction (RT-PCR). None of tested samples was confi rmed to be BTV positive. Following the Instruction of the Ministry of Agriculture and Environmental Protection - Veterinary Directorate, monitoring program for Bluetongue disease in Serbia started from October 2015. Th e program consists of insect identifi cation and detection of viral genome in Culicoides spp. by RT-PCR assay. Of the 80 samples that were received during the program realization in 2015, only four, which were collected during late autumn, have contained insects of Culicoides spp . In none of them, BTV was detected. For diff


INTRODUCTION
Bluetongue disease is non-contagious, vector borne, viral disease that infects mainly sheep but also other domestic and wild ruminants. Bluetongue virus (BTV) belongs to the family Reoviridae, genus Orbivirus. Th e genome of bluetongue virus is segmented, double-stranded RNA. Up to date, 27 serotypes of bluetongue virus have been discovered (Maan et al., 2012, Jenckel et al., 2015. Th e virus is transmitted between susceptible animals by hematophagous insects of the genus Culicoides. Only females can transmit the virus. Th ey live around 70 days, and suck blood every 3-4 days (Radojičić et al., 2011). Being the vector borne disease, the presence of vectors is crucial for occurrence of the infection in animals. Seasons with low temperatures, which are free of vectors, infl uence the epizooty of disease in such way that the disease incidence decreases to zero.
According to offi cial data, Serbia was considered BTV free country during the period 2002 -2014 2 . Th is period ended with an outbreak of the disease in August 2014, in the south of the country. Later on, the virus has extended over almost complete territory of Serbia. It was confi rmed that the virus that caused the disease belonged to serotype 4. Th e last case was reported in December of the same year. During this period, 644 outbreaks have been reported, all of them caused by BTV serotype 4 (BTV4) (Anonymous, 2015, Veljović et al., 2015). Laboratory diagnostic and confi rmation were carried out in National Reference Laboratory (NRL) for Bluetongue Disease -Institute of Veterinary Medicine of Serbia (IVMS).
Following the Instruction of the Ministry of Agriculture and Environmental Protection -Veterinary Directorate (Anonymous, 2015), Monitoring program of bluetongue disease has been implemented in October 2015. Th e program included insect identifi cation and detection of viral genome in Culicoi-des spp. by Reverse Transcription -Polymerase Chain Reaction (RT-PCR) and was aimed to determine the vector free periods.
Although clinical signs in sheep are quite typical, there are many other viral diseases with similar manifestation. Clinical signs in cattle are rather rare but can easily be misinterpreted. Th erefore, all ill domestic ruminants with facial oedema, erosions of the nasolabial plate, congestion and haemorrhage of mucous membranes, infl ammation and necrosis of the skin should be diff erentially tested for contagious ectyme of sheep, bovine viral diarrhoea, poxvirus infections, viral stomatitis etc.
Th e aim of this study was to present epizootiological situation of bluetongue disease during 2015 and establishment of diff erential diagnosis aft er BTV has been excluded in samples originating from clinically ill ruminants.

MATERIAL AND METHODS
Laboratory diagnostics of the bluetongue disease was performed at virology department of Th e Institute of Veterinary Medicine of Serbia, which is a NRL for bluetongue. During 2015, 74 samples originating from sheep, cattle and goat with symptoms of bluetongue were tested by RT-PCR. Th e majority of tested samples included unclotted blood from ill animals (73 samples). Only one sample originated from a dead goat and was composed of parts of morphologically altered organ (spleen).
Monitoring program for bluetongue disease, issued by the Ministry, prescribes entomology and virology examination of insects. During twelve months

Culicoides, which can transmit the virus. Until now, four species of Culicoides in which BTV can propagate have been identifi ed in the territory of Serbia:
Culicoides pulicaris, C. nubeculosus, C. obsoletus and C. parroti (Pavlović Ivan, 2015). Besides the necessity of the presence of competent vector species, the vectors during their lifetime have to be fed on viremic animal and survive extrinsic incubation period (which depends on outside temperature, humidity, etc), whilst virus multiplies in the gut of the insect. To spread the infection, infected midges have to bite susceptible, naïve animal and to excrete suffi cient amount of the virus into its blood. Many physiological barriers may act to limit or constrain dissemination of the virus throughout the insect's organs and thus prevent transmission. It is important to emphasize that only Culicoides females suckle blood, and that they live probably 10-20 days, exceptionally longer (between 44 and 90days) (Mellor et al., 2000).
With the arrival of wintertime, conditions for reproduction of insects become unfavorable. Among other weather conditions that can infl uence vectorial capacity of Culicoides spp., the temperature is the most important one. It aff ects the number of generations that can arise during one season and the population size. Temperature infl uences the virus infection/replication in the bodies of adult midges. For few vector species, it was proved that the complete inhibition of virus replication occurs when ambient temperature decreases below 15ºC (Mullens et al., 1995). Contrary to that, infection rate and speed of virus replication grows at higher temperatures, but the life of midges shortens (Mullens et al., 1995). Increase of precipitation during summer season favors their reproduction.
Beside the competent vector, viremic animal is another essential factor in this equation with many variables. Th e duration of viremia in bluetongue disease is variable. It lasts from 3 to 300 days (Radojičić et al., 2011). Longer duration of viremia implies higher possibility for the occurrence of new disease outbreaks aft er winter. It is well known that among susceptible species the longest duration of viremia is recorded in cattle -maximum 300 days (Radojičić et al., 2011). Th erefore, this species is considered most responsible for overwintering of the virus and new outbreaks aft er long period with unfavorable climatic conditions.
Having in mind the aforementioned facts, it is clear that the process of transmission and overwintering of the virus can fail in many points.
Although the predictions from the beginning of 2015 suggested that new outbreaks of bluetongue will appear in our country again (Veljović et al., 2015), it became obvious that climatic factors were not favorable for the development of Culicoides spp. Most probably, cold wave with temperatures below -10°C during December 2014 and January 2015 6 caused disappearance of adult competent vectors and termination of reproductive cycle. On the other hand, long and very hot summer in 2015, with six hot waves and average precipitation 6 , reduced the number of newly borne Culicoides spp. Moreover, animal population that is partially seroconverted makes unfavorable circumstances for new epizooty of BTV serotype 4.
Many surrounding countries encountered outbreaks of bluetongue during 2015 (Hungary, Romania, Macedonia, Albania, Bosnia and Herzegovina and Croatia) 7 . Only Hungary and Croatia faced the outbreaks that happened close to the border with Serbia, but transmission across the border has not been confi rmed.
Monitoring program that has begun in October 2015 is aimed at examining and establishing local presence of Culicoides spp. and defi ning seasons throughout the year that are free of vectors. To the end of 2015, 80 pooled samples of insects have been collected and delivered to the IVMS. Among them, only four, which were collected during November, contained insects of Culicoides spp. (C.obsoletus). As RT-PCR and nested PCR for detection of NS1 gene 8 gave negative results it could be concluded that midges had not been infected with bluetongue virus.
Diff erential diagnostic assays were performed on all 73 animal blood samples. Sixty-fi ve ovine samples have been examined for viruses of contagious ectyme, and sheep and goat pox. All samples gave negative results for the presence of genome segment that encodes synthesis of viral growth factor of sheep and goat poxviruses. According to the offi cial data, the virus circulated in neighboring countries between 2013 and 2015 (Bulgaria -2013 and Greece 2013-2015) 9 , but not in close proximity to the Serbian border. As this is highly contagious disease that cause high morbidity (around 75%) and mortality (around 50%) in naive populations (Radojičić et al., 2011), it was expected that these analyses would give negative result.
Contagious ectyme is widespread across our territory. During 2014, six positive samples (animals) were detected at virology department of Th e Institute of Veterinary Medicine of Serbia. Out of 65 blood samples tested during 2015, not even one gave positive reaction. Genome of parapoxvirus has not been detected in blood, though it was expected because of its presence in ovine population during 2014. For a more accurate diagnosis, the sampling and sam-ple submission for laboratory diagnostics should be done during the clinical phase of the disease. As soon as the fi rst symptoms with skin changes appear, the sample of choice is a fl uid from the vesicles, and later on a scab. Although unclotted blood is the best sample for BTV detection (because of quite long viremia), in case of poxvirus infections, virus is present in blood for short period of time and therefore this type of sample is not reliable particularly if taken aft er viremic stage of the disease.
Aft er primary and secondary viremia, the highest amount of the virus is present in skin pustules and in scabs that arise aft er eruption of pustules (Baxby D., 1996). Th us, these are the preferable samples for laboratory testing when speaking of animals in this phase of the disease. Negative results in our testing were probably due to the fact that blood is not enough suitable sample for poxvirus PCR analysis.
Bovine samples collected for the detection of bluetongue virus were diff erentially examined for viruses of bovine viral diarrhea, infective bovine rinotracheitis / pustular vulvovaginitis and malignant catarrhal fever. Among these viruses, only viral genome of bovine viral diarrhea virus was detected. Two samples positive for bovine viral diarrhea virus originated from cattle from the settlements of Gaj, municipality Kovin, and Dobrica, municipality of Alibunar. Such result was expected having in mind unfavorable epizootiological situation in whole country.

CONCLUSION
Opposite to predictions, bluetongue disease was not confi rmed in NRL for bluetongue during 2015. Which combination of factors drive up to the preferable epizootiology situation is still an open question. Th ank to it, epizootiological situation is better now than in the last year (2014). Th e monitoring program has been carried out until the autumn 2016, and its results should help us to deal with new potential epizooties. In addition, a program of vaccination of ruminants in regions where outbreaks occurred and in regions where combination of abiotic, biotic and climatic factors is favorable for bluetongue disease was started at the end of 2015. Killed vaccine should prevent infection of ruminants with BTV serotype 4 and decrease the probability of new outbreaks. Diff erential diagnostic assays served for exclusion and confi rmation of other viral diseases with similar symptoms. In order to obtain better and more reliable results, appropriate samples (beside blood) from ill animals should be collected and used for laboratory examination.