In September 2012, several cows and a calf showed decreased activity, anorexia and fever on Ishigaki Island, Okinawa Prefecture, Japan, and the cases were diagnosed as bovine ephemeral fever (BEF). We isolated BEF virus (BEFV) from one of the affected cows and then determined the complete genome sequence of the G gene, which encodes a class I transmembrane glycoprotein of BEFV. The BEFV isolate in this case, ON-3/E/12, was sorted into the same cluster as other BEFV isolates in Japan, Taiwan and China obtained in 1996−2004 and was most closely related to a 2002 Chinese isolate, JT02L, according to the phylogenetic analysis of the complete G gene. Since inactivated vaccines for BEF available in Japan are considered effective against the ON-3/E/12 isolate as well as other isolates in East Asia from 1996−2004, annual vaccination should be conducted to prevent BEF in Okinawa. Additionally, in this study, we developed an RT-PCR assay to detect the BEFV gene in Japan and neighboring countries. Our assay was able to amplify target sequences in all of the tested BEFV isolates, including 18 isolates in Japan and another isolate in Australia. The assay was found to be useful also for testing RNA samples extracted from bovine peripheral blood mononuclear cells, and the detection limit of the assay was 10 copies per tube. We believe that our assay would be an important tool for the screening of BEFV infection and the diagnosis of BEF.

BackgroundBovine ephemeral fever (BEF) is a febrile disease of cattle that is transmitted by arthropod vectors such as mosquitoes and Culicoides biting midges. An outbreak of BEF recently occurred in Ishigaki Island and surrounding islands that are located southwest of Japan. In this study, an epidemiological analysis was conducted to understand the temporal and spatial characteristics of the outbreak. Factors associated with the disease spread within Ishigaki Island were investigated by hierarchical Bayesian models. The possibility of between-island transmission by windborne vectors and transmission by long-distance migration of infected vectors were examined using atmospheric dispersion models.ResultsIn September 2012, the first case of the disease was detected in the western part of Ishigaki Island. In 1 month, it had rapidly spread to the southern part of the island and to surrounding islands, and led to 225 suspected cases of BEF during the outbreak. The dispersion model demonstrated the high possibility of between-island transmission by wind. Spatial analysis showed that paddy fields, farmlands, and slope gradients had a significant impact on the 1-km cell-level incidence risk. These factors may have influenced the habitats and movements of the vectors with regard to the spread of BEF. A plausible incursion event of infected vectors from Southeast Asia to Ishigaki Island was estimated to have occurred at the end of August.ConclusionThis study revealed that the condition of a terrain and land use significantly influenced disease transmission. These factors are important in assessing favorable environments for related vectors. The results of the dispersion model indicated the likely transmission of the infected vectors by wind on the local scale and on the long-distance scale. These findings would be helpful for developing a surveillance program and developing preventive measures against BEF.

ABSTRACT. CD56+ cells in canine blood leukocytes were characterized by flow-cytometric analysis of peripheral blood of 30 healthy adult beagle-dogs (15 males and 15 non-pregnant females). In 19 of the 30 dogs, anti human CD56 antibody, reacted CD56, which is a neural cell adhesion molecule (N-CAM), is a membrane glycoprotein belonging to the immunoglobulin superfamily [3,8]. CD56 is expressed mainly on natural killer (NK) cell in human peripheral blood and is used as a surface marker of human NK cell [2]. Although the precise functional role of CD56 molecules on human NK cells is not fully understood, CD56 is thought to be involved in cell-attachment of NK cells to the target cells in their cytokilling process [10].In rhesus monkeys, however, peripheral blood NK cells predominantly show CD56 -phenotype [1]. Furthermore, CD56 is expressed on almost all peripheral blood monocytes in cynomolgus monkeys [9,12] and in rhesus monkeys [1]. Therefore, CD56 is not always a specific marker for NK cells in mammals other than humans.An anti human CD56 antibody, Leu-19 (Becton Dickinson, San Jose, California, U.S.A.), cross-reacts with canine leukocytes [14]. However, detailed characteristics of canine CD56 + cells remain unclear. In this study, we clarified the phenotypic characteristics of canine CD56 + cells by flow-cytometric analysis on canine peripheral blood leukocytes.Thirty clinically healthy beagle-dogs (15 males and 15 non-pregnant females) 1.4 to 7.3 years old were used in this study. By vaccination, all dogs were free from rabies virus, canine distemper virus, canine herpesvirus, canine parvovirus, canine parainfluenza virus, and canine adenovirus. Blood was collected from each dog's jugular vein, using sodium citrate as an anti-coagulant. We confirmed that the complete blood counts and the leukocyte differential counts of all blood samples were within normal ranges (data not shown).Direct staining of peripheral blood leukocytes was done as follows. The following pairs of antibodies were reacted with 50 µl samples of blood for 15 min at room temperature: anti human CD56 phycoerythrin conjugated (-PE) antibody (Leu-19; Becton-Dickinson) and anti canine CD3 fluorescein isocyanate conjugated (-FITC) antibody (CA17.2A12; Serotec, Oxford), anti human CD21-PE antibody (B-ly4; Becton-Dickinson) and anti canine CD3-FITC antibody, or isotypic control-FITC antibody (Beckman-Coulter, Fullerton, California, U.S.A.) and isotypic control-PE antibody (Beckman-Coulter). To each of these samples, we added 1.5 ml of Erythrocytes Lysing Buffer (0.83% ammonium chloride, 0.1% potassium hydrocarbonate, and 0.004% EDTA disodium salt in distilled water), and then left the sample for 7 min at room temperature. After centrifugation, the supernatant was removed by aspiration. The pellet was washed twice with wash buffer (phosphate buffered saline containing 0.5% bovine serum albumin, 2 mM EDTA disodium salt, and 0.03% sodium azide) at 4°C. The samples were fixed with 1% paraformaldehyde-phosphate buffered saline, and then subjected to ...

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