Lane Foil, Mike Becker and Claudia Husseneder

Two viruses that can lead to massive disease outbreaks with death of white-tailed deer are bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV). These viruses are primarily transmitted between ruminants by the biting midges of the genus Culicoides. In Louisiana, these flies are commonly called gnats and are nasty pests that can ruin a good day in environments like coastal marshes, particularly under calm conditions because these flies are weak flyers. Approximately 22 species of Culicoides are found in Louisiana, and some are associated with health issues in horses and cattle. The bluetongue and epizootic hemorrhagic disease viruses, however, cause acute hemorrhagic disease of both wild and captive deer with mortality rates exceeding 50% in infected animals.

The LSU AgCenter Bob R. Jones-Idlewild Research Station near Clinton, Louisiana, hosts wildlife research projects for many AgCenter researchers and is the center for the Bob R. Jones Wildlife Research Institute. Herds of cattle, red deer, and both captive and wild white-tailed deer at the station make it the ideal location for the study of BTV and EHDV transmission.

In 2011, the researchers initiated a prospective study on the transmission of these viruses. A prospective study is one where all of the elements of a potential event are considered and measured prior to the event, such as virus transmission to deer. The primary elements were:

— Measure what proportion of the animals have antibodies and thus had previous exposure to BTV and EHDV.

— Initiate periodic collections of insects suspected to transmit virus (vectors) and test them for the presence of viral genetic material.

— Identify animals that become clinically ill and test for the presence of virus genetic material as well as culture virus from the animals that died.

One of the largest outbreaks of hemorrhagic disease in recorded history for deer occurred in the U.S. in 2012. Wildlife agencies from many states each reported more than 10,000 deer deaths considered to be from hemorrhagic disease, while Louisiana reported approximately 600 deaths. However, the insect vectors were not studied, and the cause of death was rarely confirmed because fresh tissues were not available in carcasses found in the wild. At Idlewild, approximately one-third of the 80 adult deer died due to BTV or EHDV infections confirmed by detection of virus genetic material and culture techniques. An additional one-third were exposed to one of the viruses and developed immunity. Researchers collected specimens of 14 different Culicoides species and found three species containing genetic material of BTV, which identified them as potential vectors.

Publication of the early studies led to a four-year study funded by the U.S. Department of Agriculture National Institute for Food and Agriculture beginning in in 2017. The objectives of that study are to:

— Continue the long-term prospective study on the transmission and maintenance of BTV and EHDV and the fate of captive deer during exposure.

— Increase the knowledge of the life cycles of the Culicoides species present on the research station.

— Develop a diagnostic test for confirming virus presence in the carcasses of deer found in the wild.

An important question for the long-term prospective study is whether and how herd immunity for deer is established. Herd immunity is achieved when a sufficiently high proportion of the deer population has built up immunity after prior virus exposure, preventing spread of disease. Three types of EHDV and four to six types of BTV have been isolated from deer at the research station. Immunity to one group of closely related microorganisms does not appear to offer immunity to others. Therefore, the studies on herd immunity for captive deer are complex. In 2012 and since, the researchers have tested the serum of wild deer at several managed hunts and found about 80% of the deer have antibodies for EHDV or BTV. Researchers are finding that half of the deer exposed to one of the viruses survive the infection and, thus, develop immunity, and females can pass on immunity in the milk to nursing fawns.

If a limited number of strains of the two viruses are in certain geographic ranges, the wild deer herd immunity can be high enough to explain the lower number of deaths recorded annually in Southern states with long summers and midge activity. In the Idlewild herds, the researchers have collected an annual blood sample from each animal from 2011-2019 along with their clinical histories. Over the same period, researchers have tested over 25,000 Culicoides specimens and have found virus-positive samples from five species of Culicoides collected when deer were contracting the viruses, making them top candidates as vectors.

Describing the life cycles of the different Culicoides species present in an area where virus transmission is key to identifying important vectors, and confidence in identification methods for the life stages of the different species is critical. Adults are sorted into species groups according to their wing patterns. Species are then confirmed by clearing the bodies, mounting them on microscope slides, and comparing internal and external structures. This is not practical for sorting thousands of specimens, and the mounting process is prohibitive for testing for viruses. The identification of immature stages is even more difficult, and most species have limited descriptions.

To overcome this limitation, the researchers use DNA barcoding, which is a tool for characterizing species according to species-specific DNA marker sequences. The researchers confirmed four species-specific DNA markers from adult females of 12 related species. DNA barcoding of collected specimen at the research station also indicated the presence of two undescribed species. Although larval habitats are poorly described for most species, tree holes are a larval habitat for certain species. The researchers collected tree-hole samples at the research station and collected both adults that developed and larvae for DNA barcoding. After comparing the DNA of adults and larvae, the researchers have found that tree holes are developmental habitats for at least six species. These studies will facilitate in describing key vector species with certainty, allowing comparisons to other regional studies and aiding in identifying larval habitats for suspected vectors of BTV and EHDV.

When deer carcasses are found in the wild, nothing but bones exist within a few days of death. Both BTV and EHDV are relatively stable viruses, and bone marrow from recent victims can be used to detect virus particles. Researchers tested bone marrow from deer that died at the research station and stored the bones of animals infected with virus in scavenger-proof cages. Researchers found that the genetic material of both viruses can be detected three to four months after death and that they can determine their specific types (although the viruses were not viable after a day). This important detection tool has already been adopted by wildlife personnel.

Lane Foil is the Pennington Regents Chair for Wildlife Research, Mike Becker is a postdoctoral research associate, and Claudia Husseneder is the Paul K. Adams Professor of Urban Entomology, all in the Department of Entomology.

(This article appears in the spring 2020 issue of Louisiana Agriculture, which focuses on entomology.)

Some of the white-tailed deer breeding herd maintained at the Bob R. Jones-Idlewild Research Station in Clinton, Louisiana. Photo by Claudia Husseneder

An example of the methods for collecting bone marrow from the long bones of deer carcasses. Photo by Claudia Husseneder