Epizootic Hemorrhagic Disease Virus Serotype 6 Infection in Cattle, Japan, 2015

During October–December 2015, an epizootic hemorrhagic disease outbreak occurred in cattle in Japan. Forty-six animals displayed fever, anorexia, cessation of rumination, salivation, and dysphagia. Virologic, serologic, and pathologic investigations revealed the causative agent was epizootic hemorrhagic disease virus serotype 6. Further virus characterization is needed to determine virus pathogenicity.

In October 2015, an outbreak of febrile illness occurred in cattle in Hyogo Prefecture, Japan, and lasted 3 months. We summarize the features of this outbreak, which affected 46 cattle at 38 farms.

The Study
Of the 46 affected cattle, 40 were beef cattle (Japanese Black) and 6 were dairy cattle (Holstein heifers). The average age of all affected animals was 114.7 (range 11-187) months. The clinical signs observed in beef cattle were fever, anorexia, cessation of rumination, swollen eyelids, salivation, paralysis of the tongue, difficulty swallowing, nasal and ocular discharge, and abortion (Table 1). Seven beef cattle died, and 2 were euthanized because they did not recover despite treatment of symptoms. The dairy cattle showed fever, anorexia, coughing, conjunctivitis, cessation of rumination, salivation, difficulty swallowing, and reduced milk production.
Early in the outbreak, Ibaraki disease was suspected, so blood samples were collected. We washed the blood cells and used them for virus isolation and reverse transcription PCR (RT-PCR) for group-specific and serotypespecific EHDV detection (9,10). Paired serum samples were also collected from 20 of the 46 affected animals at early onset of the outbreak and after the outbreak at 1-or 2-month intervals. We used the paired serum samples for neutralization tests and conducted necropsies on 2 euthanized animals.
At necropsy, we observed edema of the esophagus, pharynx, and tongue in an affected cow with dysphagia. The lumen of the esophagus was also dilated (Figure 1 We further characterized the causative agent by performing sequence analysis of genome segment 2, which correlates with serotype, and segment 3, which correlates with geographic genetic type, of outbreak isolate HG-1/E/15. We designed RT-PCR primers to amplify segments 2 and 3 from the full-length cDNA on the basis of sequence data of EHDV strains from Japan and Australia available in GenBank. We performed RT-PCR using RNA obtained from an affected cow (no. 9; Table 2) as template; we then purified the RT-PCR products and subjected them to direct sequencing. We used the sequence data to analyze the phylogenetic relationships between EHDV HG-1/E/15 and other EHDV isolates.

Conclusions
We determined this febrile illness affecting 46 cattle in Japan in 2015 was an epizootic hemorrhagic disease outbreak caused by EHDV-6 infection on the basis of clinical findings, RT-PCR results, neutralization tests, and sequence analyses. Clinical EHDV-6 cases in cattle have been reported in Turkey, Bahrain, Israel, Morocco, Tunisia, Algeria, and Réunion Island (3,5,13,14), but the EHDV-6    The neutralization tests showed significant increases of antibody titers in 5 cattle, suggesting recent EHDV-6 infection. At the same time, no significant increase of antibody titers was observed with the other 15 cattle. These results suggest that the onset of the epizootic hemorrhagic disease outbreak might have occurred >3-5 weeks after the EHDV-6 infection in the 15 seronegative cattle, considering that neutralizing antibody titer increases have been observed to occur in cattle 11-14 to 21-37 days after experimental EHDV-6 infection (15).
The clinical and pathologic findings of this outbreak are similar to those observed for Ibaraki disease in cattle. Thus, EHDV-6 HG-1/E/15 possibly has pathogenic characteristics similar to those of Ibaraki strain EHDV-2 JPN1959/01 in cattle. Further investigations are needed to clarify the genetic characteristics of EHDV-6 HG-1/E/15 to determine why this outbreak occurred.