Characterizing the Epidemiology of Bluetongue Virus Serotype One in South Louisiana

NON-TECHNICAL SUMMARY: In November, 2004, a sick deer was harvested in St. Mary Parish, La.; and the deer was positive for a strain of bluetongue virus (BTV-1) that had not previously been reported in the United States. BTV-1 occurs in the Caribbean and is transmitted by the biting midge, Culicoides insignis, and we need to know what species of ceratopogonids are competent vectors of BTV-1 in La. The purpose of the study is to determine the species of Culicoides present in the area where a BTV-1 infected deer was harvested and also at nearby farms where BTV-1 seropositive livestock have been found. We also propose to prepare pools of specimens of the different Culicoides species and then to determine if the pools contain BTV RNA.

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APPROACH: In November, 2004, a sick deer was harvested in St. Mary Parish, La.; and the deer was positive for a strain of bluetongue virus (BTV-1) that had not previously been reported in the United States. BTV-1 occurs in the Caribbean and is transmitted by the biting midge, Culicoides insignis. The purpose of the study is to determine the species of Culicoides present in the area where the deer was harvested and also at nearby farms where BTV-1 seropositive livestock have been found (3 of 72 head on one farm and 2 of 21 head on another farm). Given the prevalence of BTV-1 antibody, we anticipate that there are competent vectors for BTV-1 in the area. We propose to conduct periodic light trap collections which will allow us to determine the seasonal patterns of the different Culicoides species. This information alone can provide associations between suspected vectors and disease outbreaks. Miniature CDC black light traps baited with dry ice as a source of carbon dioxide, will be used to collect ceratopogonids. Collections will be made at three livestock farms (two traps per farm) in the first week and the third week of each month from October 2005 to October 2007. Collections in the area where the infected deer was harvested will require transportation by boat, and we will trap with the help of the La. Department of Wildlife and Fisheries at 4 sites in this area once per month. We also propose to prepare pools of specimens of the different Culicoides species and then to determine if the pools contain BTV RNA. The Culicoides specimens will be sorted according to species and combined in pools of no greater than 50 individuals. The vials will be stored at -80 C, and then transported to the ABADRL on dry ice. At Laramie, the pools will be homogenized in a cell culture compatible buffer and 10% of the homogenate will be held for cell culture if necessary. RNA extraction will be performed according to the standard protocol with modifications to allow for use of cell culture buffer for homogenization using the Qiagen RNeasy 96 Kit. Pools will be assayed for BTV using a newly developed Infra Red Reverse Transcriptase PCR (IR RT-PCR) method. The assay has been developed to process 96 sample pools in plate format from RNA extraction to the single step RT-PCR assay, and gel electrophoresis. The method is fast and sensitive compared to the BTV PCR methods previously described. IR RT-PCR assay sensitivity detects 0.5 plaque forming units (pfu) of purified RNA. Therefore, the sensitivity of the assay is well below the threshold for an individual insect infected with BTV. Positive pools will undergo further PCR and sequencing analysis to determine BTV serotype. The results of the proposed studies should allow us to identify the species of Culicoides that is a competent vector of BTV-1. The geographic range of the vector species will allow us to predict if there will be barriers for the spread of BTV-1 in the United States. The impact of the spread of a new BTV serotype across the nation relative to disease in domestic and wild ruminants is difficult to estimate but could be significant.
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PROGRESS: 2005/11 TO 2007/11 <BR>
OUTPUTS: In November, 2004, a sick deer was harvested in St. Mary Parish, La. The deer was positive for a strain of bluetongue virus (BTV-1) that had not previously been reported in the United States. Subsequently a serum survey was conducted on farms nearby the area where the BTV-1 positive deer was found. Blood samples were collected from March to July in 2005 and 2006 from cattle, goats, and sheep grazing within a 20 miles radius from the site of the BTV-1 infected deer in St. Mary Parish. In 2005, a total of 460 cattle, 47 sheep and 42 goat sera were screened for the presence of antibodies to bluetongue. In 2005, 61 (11.11%) animals tested positive for BTV. Of these, 5 cattle and 1 sheep were considered to be BTV-1 positive animals. In 2006, 68 (12.98 %) animals tested positive for BTV, but none of these animals were BTV-1 positive. Another objective of our study was to determine the species of Culicoides present in the area where the deer was harvested. During 2006, a total of 3160 ceratopogonids (members of the genus Atricopogon, Forcipomyia, and five species of Culicoides) were captured in the Atchafalaya Delta area where the first deer was harvested. We also collected ceratopogonids on three livestock farms that are immediately inland from the Atchafalaya Delta area. In 2006, a total of 1136 ceratapogonids (8 species of the genus Culicoides) were captured on the farms. All of the pools of the 3160 ceratopogonids that were collected in 2006 were tested for the presence of orbiviruses (BTV and EHDV) by infrared-RT-PCR. Two pools of specimens of C. debilipalpis and one pool of specimens of C. crepuscularis were positive for BTV. The positive results were confirmed by DNA sequencing and the sequences were identical to either BTV 17 or BTV 13. During 2007, a total of 932 ceratapogonids were captured at the three livestock farms between March and August, and 78.5% of them were in the genus Culicoides representing 9 species. Pools of all specimens collected between March and August 2007 were tested for the presence of orbiviruses (BTV and EHDV). An individual specimen of C. furens was found positive for BTV by IR-RT-PCR. The sequence for this BTV was identical to those identified in 2006. These results have been presented by Mike Becker in a presentation entitled "Characterizing the Epidemiology of Bluetongue Virus Serotype One in south Louisiana" at the 82nd Annual Meeting of the Southeastern Branch of the Entomological Society of America in Jacksonville, Fl March 2-5, 2008 and published in the thesis of Mike Becker (2007) Characterizing the Epidemiology of Bluetongue Virus Serotype One in South Louisiana. Master's Thesis. Louisiana State University, Baton Rouge, La. <BR> PARTICIPANTS: Scott DeJean, USDA, APHIS, VS, Baton Rouge, LA E.N. Ostlund, APHIS, National Veterinary Services Laboratories, Ames, IA M.P. Emery, APHIS, National Veterinary Services Laboratories, Ames, IA D. J. Johnson, U.S. Department of Agriculture, APHIS, Veterinary Services, National Veterinary Services Laboratories, Ames, IA Cassidy Lejeune, Louisiana Department of Wildlife and Fisheries, Coastal and Nongame Resources, New Iberia, LA Dr. Will Reeves, Research Entomologist, APHIS, ABADRL, Laramie, WY <BR> <BR> IMPACT: 2005/11 TO 2007/11<BR>
BTV-1 occurs in the Caribbean and is transmitted by the biting midge, Culicoides insignis. During 2005 in Louisiana, 31% of serum samples from hunter killed deer were positive for BTV. There were six samples that provided possible evidence of BTV-1 exposure. This information indicates that the BTV infection in the deer harvested in 2004 was not an isolated case. In 2005, we found 11.1% of the livestock tested in St. Mary Parish, La. were seropositive for BTV and approximately 10% of those animals were positive for BTV-1. In 2006, 13% of the animals from the same area tested positive for BTV, but none of them were positive for BTV-1. These data indicate that BTV-1 virus transmission was not sustainable on the livestock farms. We did not find the primary vector of BTV-1 in the Caribbean, Culicoides insignis, or the primary vector of BTV in the United States, Culicoides sonorensis, but there was evidence of autochthonous transmission of BTV-1 in south Louisiana. Therefore, there is likely a native species of Culicoides that is a competent vector of BTV-1. The results of our studies should allow us to identify which species of Culicoides that is a competent vector of BTV-1. The geographic range of the vector species will allow us to predict if there will be barriers for the spread of BTV-1 in the United States. The impact of the spread of a new BTV serotype across the nation relative to disease in domestic and wild ruminants is difficult to estimate but could be significant.