Molecular identification, phylogenetic status, and geographic distribution of Culicoides oxystoma (Diptera: Ceratopogonidae) in Israel.

Culicoides oxystoma (Diptera: Ceratopogonidae) is an important vector species, reported mainly from Asia, with high potential to transmit viral diseases affecting livestock. In Japan, many arboviruses have been isolated from C. oxystoma, suggesting it as a key player in the epidemiology of several Culicoides-borne diseases. Over the years, C. oxystoma has also been reported in the Middle East region, including Israel. In this region, however, C. oxystoma cannot be easily distinguished morphologically from its sibling species included in the Culicoides schultzei complex. We therefore used genomic data for species identification and phylogeny resolution. Phylogenetic analyses based on internal transcribed spacer 1 (ITS-1) of ribosomal DNA and the mitochondrial gene encoding cytochrome oxidase subunit I (COI) showed that C. oxystoma from Israel is closely related to C. oxystoma from Japan. Using differential probing PCR, we showed that C. oxystoma is distributed all over the country, especially in Mediterranean climate regions. Culicoides oxystoma is less common or even absent in arid regions, while the other genetic cluster of C. schultzei complex was found only in the east of the country (mostly arid and semiarid regions). The molecular finding of C. oxystoma in wide geographical regions, together with its high proportion in the general Culicoides population and its vectoring potential, imply that it may be an important vector species in the Middle East.

Epidemiological investigation of bovine ephemeral Fever outbreaks in Israel.

Outbreaks of bovine ephemeral fever (BEF) occurred in Israel in 1990, 1999, and 2004. The main patterns of BEF spread were similar in the 1990 and in 1999 epidemics, and the BEF virus was probably carried in vectors transported by air streams across the Rift Valley and the Red Sea. In the 2004 outbreak, the primary focus of the disease was the southern Mediterranean coastal plain and the disease agent was apparently brought by infected mosquitoes carried from their breeding site in the Nile Delta by the south-western winds. The disease broke out under optimal ecological conditions, among a vulnerable cattle population and spread rapidly; it showed essentially a spring-summer herd incidence and terminated soon after the night average ambient temperature fell below 16 degrees C in late autumn. The herd incidence of the disease reached 78.4%, 97.7%, and 100% in 1990, 1999, and 2004, respectively. The highest herd incidence, morbidity, and case fatality rates were noted in dairy cattle herds in the Jordan Valley, with morbidity of 20%, 38.6%, and 22.2%, and case fatality rate among affected animals of 2%, 8.6%, and 5.4% in 1990, 1999, and 2004, respectively. The average sero-positivity to BEF in 1999 was 39.5%, which matched the morbidity rate. Comparison among the various age groups showed that the lowest morbidity rates were observed in the youngest age group, that is, heifers up to 1 year, with 3.2%, 3.6%, and 4.2% in 1990, 1999, and 2004, respectively. In heifers from 1 year to calving, the morbidity rates were 13.8%, 14.9%, and 28%, respectively, in first calvers 30.8%, 31.6%, and 28.3%, respectively, and in cows 34.3%, 35.7%, and 27.2%, respectively. All affected cattle were over the age of 3 months. It is hypothesized that mosquitoes and not Culicoides spp. are the vectors of the BEF virus in Israel.

The association of winds with the spread of EHDV in dairy cattle in Israel during an outbreak in 2006.

Winds may play a major role in spread of arthropod-borne viruses (arboviruses). Arboviruses like epizootic hemorrhagic disease virus (EHDV), bluetongue virus and bovine ephemeral fever virus (BEFV) frequently cause major outbreaks in Israel with a unique pattern of spread. Most of these outbreaks begin in the Jordan valley, near the Sea of Galilee and then spread to the north, south and west through the major valleys of Israel. The aim of this study was to describe the spread pattern in such an outbreak and to find if this pattern can be explained by winds. Herein, we compared the spread rate to each direction and used Cox proportional hazards model to test factors associated with the spread of EHDV, which emerged in diary cattle in Israel during the summer of 2006. Documented, clinical and serological data on spread of the outbreak were then compared with wind data collected by meteorological stations along the trail of virus spread and with modeled winds at high altitude (>500 m). The analysis revealed that both the hazard and the rate of outbreak spread to the south and to the north were significantly higher than to the west. Average rate of outbreak spread during periods in which at least 3 h of winds to spread direction were recorded was 20,880 m/week (SD=13,230) vs. 7486 m/week (SD=4936) in periods during which no such winds were recorded. Serological evidence demonstrated exposure to the virus up to 166 km away from the location of the initial outbreak center. Modeled wind data showed that this spread may be explained by winds at high altitudes. Animal movements due to shipments of feedlot calves and slaughters could not explain the spread pattern observed during the outbreak. This study therefore shows that winds are probably a major contributory factor for long and medium distance spread of Culicoides borne viruses in this region.

Newly emerged nulliparous Culicoides imicola Kieffer (Diptera: Ceratopogonidae) with pigmented abdomen.

The method of segregating nulliparous and parous females of Culicoides spp. based on the presence of burgundy-red pigment inside the abdominal wall of parous Culicoides midges, is used worldwide. Out of 320 females of Culicoides imicola trapped by emergence traps, set over an artificial breeding site for 10 and 24 days, 73 (22.8%) showed a red-pigmentation despite the fact that they were nulliparous. This finding indicated that 23% of the "parous" females that are examined for the presence of arboviruses and other pathogens or for age-grading purposes, are actually old nulliparous females, which had no chance of acquiring pathogens. This bias in parous rate distorts upward the calculation of vectorial capacity.

Insect contribution to horizontal transmission of Reticuloendotheliosis virus.

The involvement of insects in Reticuloendotheliosis virus (REV) transmission was examined by testing insects trapped at commercial farms and by controlled feeding experiments using mosquitoes, Culex pipiens L. and house flies, Musca domestica L. We established sensitive methods of REV detection, including reverse transcription-polymerase chain reaction (PCR) for REV-LTR and REV-gag genes, REV antigenemia measurements by enzyme-linked immunosorbent assay, and virus isolation in tissue cultures. A variety of blood-sucking species of insects were trapped at farms with infected poultry and tested, but none were positive. To rule out the possibility of PCR inhibition by insect RNA, spiking experiments were conducted and no interference was observed. Because Cx. pipiens mosquitoes were trapped frequently at farms, we performed feeding experiments with mosquito females fed on a REV-containing tissue culture medium and chicken blood mixture. Virus was detected in the mosquitoes up to 5 h postfeeding, compared with 96 h in the feeding mixture, indicating that Cx. pipiens can only harbor REV for a short period. House flies were suspected to be involved in the virus transmission because they frequently were trapped on positive farms. In contrast to mosquitoes, REV was harbored within the house fly digestive tract for up to 72 h and could infect chickens, as demonstrated by seroconversion and by detection of viral gag-sequence in the cloaca. The current study is supportive for the role of house flies as a mechanical vector of REV among poultry.

Akabane virus in Israel: a new virus lineage.

This report describes the first molecular characterization of Akabane virus (AKAV) in Israel. The virus was recognized by real-time RT-PCR in extracts from Culicoides imicola insects trapped at the Volcani Center located in the center of Israel. This is also the first report on the use of real-time RT-PCR to identify the virus. The quantitative capability of this technique was applied, and it was calculated that the insect extract contains 1.5 x 10(5) copies of the genome segment S. Following amplification of the small (S) genome segment, its nucleotide sequence was determined to have 93.4% identity or greater with the S segment of other AKAV isolates. The deduced amino acid (aa) sequence of the combined nucleocapsid and the non-structural protein showed more than 96.6% identity. Phylogentic trees constructed using the combined deduced nucleocapsid and the non-structural protein aa sequences showed that the Israeli isolate forms a fourth cluster of AKAV, indicating a separate virus lineage. Attempts to isolate the virus by inoculation to Vero cells and by intracerebral inoculation to mice were unsuccessful.