First detection of bluetongue virus serotype 14 in Poland

Here, we present the first detected cases of bluetongue virus (BTV) in native cattle from Poland. The virus was found in animals located near the Polish-Belarusian and Polish-Lithuanian borders. The positive animals were detected through an official epidemiological surveillance program. A combination of type-specific real-time RT-PCR and phylogenetic tests revealed the presence of BTV serotype 14 (BTV-14). This serotype is highly homologous to the vaccine strain and BTV-14 present in Russia, Lithuania, and Spain (from an animal imported from Lithuania). The most probable route of virus introduction to Poland was transmission through midges. All of the cases were subclinical.

Bluetongue virus (BTV) causes direct economic losses due to overt clinical disease in animals. The presence of BTV in ruminants restricts the international trade of animals [9]. BTV spread depends on circulation among ruminants, and it is transmitted by vectors that include several species of midges of the genus Culicoides [14]. BTV was previously considered an exotic disease in Europe, with only a few sporadic cases prior to 1998, e.g., a BTV-3 outbreak in Cyprus, BTV-4 in Turkey and Greece, and BTV-10 in the Iberian Peninsula [7,11]. However, since 1998, different BTV serotypes including 1, 2, 4, 6, 8, 9, 11 and 16 have been continuously recorded in several Mediterranean regions, including European Union (EU) member states [16]. In August 2006, the first BTV cases were identified in northwestern Europe in domestic ruminants infected with BTV serotype 8 [4,15]. Subsequently, BTV-6, BTV-11 and BTV-14 isolates that are genetically similar to vaccine strains have been found in northern Europe [1,2,8]. However, the route of their introduction is unclear. Poland remained free from BTV infections until 2012.
The  [13]. No positive results were found in a total of 494 samples. A calf born on September 13, 2011 on a farm in Podlaskie voivodeship, Sokolski region, Miedzianowo village ( Fig. 1) was sold in Spain on October 17, 2011. The calf was subsequently tested for BTV in Spain in December and had a positive BTV result. Before reaching its final destination, the calf was transferred three times between various holdings in Spain during the season when the ambient temperature was still highly suitable for midge activity. Finally, the calf was placed in a group of 127 calves also originating from Poland. However, this was the only calf among the 128 animals that tested positive (both ELISA and rtRT-PCR). Further molecular investigation including serotype-specific RT-PCR testing revealed negative results for BTV 1, 2, 4, 6, 8, 9, 11, 16, 24 and 26. Sequence analysis of the fragment of 194 nt from virus segment 5 revealed 100 % sequence identity to strain 600572 BTV-14, available in the GenBank database (accession no. FJ713346) [12].
In the same northeastern region of Poland (Podlaskie voievodeship, Sokolski region, Łowczyki village) in September 2012, BTV was diagnosed in cattle without clinical signs. Although the cattle lacked symptoms, the cases were identified due to the BTV surveillance program. In total, 19 animals were found seropositive, and four animals were rtRT-PCR BTV positive [13]. Furthermore, BTV genetic material was identified in cattle in the neighbouring administrative units (Suwalki region, Szypliszki village and Augustowski region, Bargłów Kościelny village) ( Table 1). All of the animals were immediately slaughtered. The CVO requested further identification and virus typing, and blood samples were sent to the European Union Reference Laboratory for Bluetongue, Pirbright Institute, UK. According to the EU-RL protocol [8], the samples were tested twice by groupspecific rtRT-PCR assays with the positive results of 24-33 Ct values. The serotyping results were negative for BTV 1, 2, 4, 6, 8, 11, and 16. The samples were also tested with a type-specific rtRT-PCR assay for BTV-14 (targeting segment 2, Seg-2), and the results were positive, with Ct values of 16-25. The rtRT-PCR typing results were confirmed by direct sequencing of the amplified products and The high degree of sequence similarity between the Polish, Russian, and Spanish/Lithuanian strains and the vaccine strain may indicate the use of attenuated BTV-14 vaccine in the field. BT cases caused by vaccine strains (BTV-6 and BTV-11) were previously recorded in Europe. However, a live vaccine containing BTV-14 is not available on the EU market. Furthermore, vaccination against BTV is strictly forbidden by law in Poland. Additionally, the nucleotide sequences of the different strains collected from geographically distinct places are similar. Thus, the strains were derived from a common source and spread in the field. Phylogenetic comparison of whole genome sequences of BTV-14 isolates from different countries of Europe and isolated around the world revealed sequence similarities between BTV-14 European strains and existing vaccine and field BTV strains. Moreover, BTV-14 strains that have spread in Europe are reassortants containing genome segments derived from different reference vaccine strains originating from South Africa [6]. Because the virus is transmitted by midges, controlling the spread of BTV is difficult during periods of high Culicoides activity. We assume that BTV-14 appeared in Eastern Europe (possibly by illegal use of vaccine) and was spread to the West. The BTV-14 serotype appeared in 2011 in Smolensk at first.
The following year, the virus was identified in Lithuania, Poland (but only the northeastern part of the country near Belarusian and Lithuanian borders, Fig. 1), and Spain, but only in an animal imported from Lithuania. BTV-14 infections in cattle have also been recorded in Latvia and Estonia. Animal movement as a route of virus transmission was excluded, as all of the animals imported into Poland were tested for the presence of BTV, and all rtRT-PCRpositive animals were slaughtered immediately. Thus, the introduction of BTV-14 by live animals was unlikely.
Another possibility could be vehicles transporting live animals travelling through Poland (west-east and eastwest). In the northeastern region of Poland, drivers stop to rest in a parking lot for lorries prior to crossing the Polish-Belarusian border. During this rest stop, midges could come into contact with BTV-positive animals transported from the east and transmit the virus to Polish animals located near the parking area.
Regarding the active monitoring programme for BTV, 11,405 blood serum samples were tested in Poland in 2012.  Fig. 1). All of the infections were subclinical and were detected only under the obligatory monitoring program by routine commercial diagnostic tests for moving animals using the rtRT-PCR method [12]. Positive animals were slaughtered according to the decision of CVO.
From November 2014 to the end of December 2015, no BTV-14 cases were detected in Poland.

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