Serologic detection of adenoviral hemorrhagic disease in black-tailed deer in California

An enzyme-linked immunosorbent assay (ELISA) and a serum neutralization (SN) test were developed to measure serum antibodies against the adenovirus causing hemorrhagic disease in free-ranging and captive experimentally-infected black-tailed deer (Odocoilenus hemionus columbianus) in California (USA). There was a strong (rho = 0.874) and significant (P < 0.0001) correlation between ELISA and SN titers, although the SN assay was more sensitive than the ELISA.     

Antibodies to bluetongue and epizootic hemorrhagic disease viruses in a barrier island white-tailed deer population.

From 1981 through 1989, serum samples from 855 white-tailed deer (Odocoileus virginianus) from Ossabaw Island, Georgia (USA), were tested for antibodies to bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV). During this period, prevalence of precipitating antibodies to BTV and EHDV as determined by agar gel immunodiffusion (AGID) tests decreased from 74% to 3% and from 34% to 1%, respectively. Antibodies were detected in serum samples from 0.5-yr-old deer only during 1981, 1982, and 1983, and with few exceptions, positive serological results after 1983 were restricted to older age classes. A decrease in prevalence of precipitating antibodies to BTV and EHDV in age classes exposed during 1981 indicates that AGID results from white-tailed deer populations underestimate the extent of previous exposure to these viruses. Serum neutralization test results from AGID-positive deer indicated that BTV 11 was the principal serotype responsible for infections during 1981. Since 1983, this serotype has been replaced by BTV 13; however, there has been a low level of transmission within the herd. Infection with EHDV 2 appeared most prevalent during 1982; as with BTV 13, there has been limited transmission in this high density deer population since 1983.     

Precipitating antibodies to epizootic hemorrhagic disease and bluetongue viruses in white-tailed deer in the southeastern United States.

From 1981 to 1989, sera were collected from 3,077 white-tailed deer (Odocoileus virginianus) in Georgia and from 1,749 deer from 12 additional states in the southeastern United States. In Georgia, prevalence of precipitating antibodies to epizootic hemorrhagic disease virus (EHDV) and bluetongue virus (BTV), as determined by agar gel immunodiffusion tests, was dependent on physiographic region, age, and year. Overall prevalence of antibodies to EHDV and/or BTV was 11, 33, 48, and 14% for the Mountain, Piedmont, Coastal Plain, and Barrier Island regions, respectively. Results suggested varying patterns of EHDV and BTV activity throughout the state. Serologic results from other southeastern states were consistent with the Georgia sample; prevalence estimates (EHDV and/or BTV) for corresponding physiographic regions deviated by less than 10%. Over this larger geographical area, antibody prevalence in deer appeared to increase with decreasing latitude.     

Antibodies to bluetongue and epizootic hemorrhagic disease viruses from white-tailed deer blood samples dried on paper strips.

The feasibility of using dried blood samples for serologic testing of white-tailed deer (Odocoileus virginianus) for antibodies to bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) was tested with matched samples of serum and eluted dried whole blood. Results from matched serum virus neutralization (SN) tests indicated that a 1-ml elution from a 1- x 2-cm section of filter paper strip containing dried blood approximated a 1:10 serum dilution. Neutralizing antibody titers detected from 34 matched titrations of serum and dried blood samples were equivalent in 25 (74%) titrations and were within a single dilution in the remaining nine (26%) titrations. Eluted blood samples from SN-positive deer, however, did not produce detectable precipitin lines on agar gel immunodiffusion tests for antibodies to either BTV or EHDV. In a trial using serum and dried blood samples from 108 hunter-killed deer from five locations in Georgia (USA), antibody prevalence and serotype distribution results were similar. Use of dried blood samples for serologic testing for antibodies to BTV and EHDV provides a reliable alternative to serum but should be considered only when serum collection is not feasible.     

Cross-protection between epizootic hemorrhagic disease virus serotypes 1 and 2 in white-tailed deer

Viruses in the epizootic hemorrhagic disease (EHD) serogroup are the most frequent cause of hemorrhagic disease in the southeastern United States, but nothing is known about cross-protection between the two EHD serotypes (EHDV-1 and EHDV-2) present in this region. We experimentally tested whether deer surviving EHDV-2 infection would be protected against subsequent infection with EHDV-1, and used field data to examine the possibility of reciprocal cross-protection. Eleven white-tailed deer fawns (Odocoileus virginianus) were experimentally infected with EHDV-2 and later challenged with EHDV-1. Two EHDV-2-na?ve fawns also were infected with EHDV-1. Deer were monitored via physical examination, complete blood counts, clotting profiles, viral isolation, and serology, and each animal was assigned a quantitative clinical disease severity score based on presence of certain physical and clinical parameters. Infection of na?ve controls with EHDV-1 caused severe clinical disease and death of both fawns, whereas deer previously infected with EHDV-2 exhibited no or minimal signs of disease. Thus, infection with EHDV-2 conferred protection against disease caused by subsequent EHDV-1 infection. Although prior EHDV-2 exposure protected deer from severe clinical disease, it did not prevent infection nor viremia indicating they could still act as virus amplifying hosts. These experimental infections suggest that EHDV-1 and 2 may exist in a state of mutual permissiveness.     

Experimental bluetongue disease in white-tailed deer

Nine white-tailed deer and six sheep were experimentally exposed to the California BTV-8 strain of bluetongue virus. The infections were fatal for seven of the nine deer. An additional deer died from exposure to an isolate of bluetongue virus from bighorn sheep. Clinical signs and lesions of bluetongue in deer were described. The incubation period, signs and lesions of bluetongue and epizootic hemorrhagic disease of deer appear to be similar. Virus isolations were made from the blood and a variety of tissues of exposed deer and identified as bluetongue virus. Neutralizing antibodies were detected in all of the convalescent sera.     

Innate resistance to epizootic hemorrhagic disease in white-tailed deer

Differences in innate disease resistance at the sub-species level have major implications for wildlife management. Two subspecies of white-tailed deer, Odocoileus virginianus borealis and O. virginianus texanus were infected with epizootic hemorrhagic disease (EHD) viruses. These viruses are highly virulent pathogens of white-tailed deer and are endemic within the range of O. virginianus texanus but not within the range of O. virginianus borealis. Two experimental infections were performed. Five O. virginianus texanus fawns and five O. virginianus borealis fawns were infected with 10(7.1) median tissue culture infective doses (TCID50) of EHD virus, serotype 1 and five of each subspecies were infected with 10(7.1) TCID50 of EHD virus, serotype 2. Infections with both EHD virus serotypes caused severe clinical disease and mortality in O. virginianus borealis fawns, whereas disease was mild or nondetectable in O. virginianus texanus fawns. Virus titers and humoral immune response were similar in both subspecies suggesting that differences in innate disease resistance explain the differences seen in clinical disease severity. In white-tailed deer, innate disease resistance may vary at the subspecies level. Should this phenomenon occur in other species, these findings have major implications for managing wildlife populations, both endangered and non-endangered, using tools such as translocation and captive propagation.     

Testicular atrophy in Columbian black-tailed deer in California.

During an 18-year period, 4.1% (34/831) of male deer (Odocoileus hemionus columbianus) killed on a field station during the autumn hunting season had velvet-covered, often misshapen antlers, and at least two deer had testicular atrophy (gonads from most deer were not available for examination). Testes from six similarly affected deer and several normal deer were compared histologically. Lesions ranged from hypocellularity of the semeniferous tubules and relative hyperplasia or degeneration of interstitial cells to complete connective tissue replacement of the testicular parencyma. Chronic vascular changes were present in several testes. The etiology and pathogenesis of the lesions were not determined.     

An epizootic of hemorrhagic disease in white-tailed deer in Missouri

As part of a white-tailed deer (Odocoileus virginianus) survival study in Missouri (USA) we were actively monitoring 97 radio-collared deer when 8 (8%) died. This mortality, which occurred from 20 August to 23 September 1996, consisted of five adult females, two yearling females and one yearling male. Based on the seasonality of this mortality and the isolation of epizootic hemorrhagic disease virus (EHDV) serotype 2 from one of these animals, we believe that these losses resulted from an epizootic of hemorrhagic disease. The remains of five unmarked deer that may have died from HD also were found on the study area during this same period. During the fall following this mortality, we tested serum from 96 deer taken by hunters in the immediate area. Fifteen (16%) were positive for EHDV or bluetongue virus (BTV) antibodies as determined by agar gel immunodiffusion tests. Serum neutralization test results indicated that previous infections were caused by EHDV virus serotype 2. Based on these data, and assuming that there was no prior exposure to EHDV serotype 2 in this population, the exposure rate for this epizootic was 24% of which 8% died. We noted hoof interruptions in only two of the 96 deer sampled. During this mortality event, the Missouri Department of Conservation received no reports of dead deer, and without the radio-monitored animals the event would have been undetected.     

Susceptibility of white-tailed deer (Odocoileus virginianus) to experimental infection with epizootic hemorrhagic disease virus serotype 7

During the fall of 2006, in Israel, epizootic hemorrhagic disease virus (EHDV) serotype 7 caused an intense and widespread epizootic in domestic cattle that resulted in significant economic losses for the dairy industry. The susceptibility of potential North American vector and ruminant hosts to infection with EHDV-7 is not known but is essential to understanding the potential for establishment of this exotic orbivirus in North America if it were introduced. Our primary objective was to determine whether white-tailed deer (WTD; Odocoileus virginianus) are susceptible to infection with EHDV-7. Six, 8-mo-old WTD were experimentally infected with EHDV-7, and all became infected and exhibited varying degrees of clinical disease. Clinical signs, clinicopathologic abnormalities, and postmortem findings were consistent with previous reports of orbiviral hemorrhagic disease (HD) in this species. Four of six animals died or were euthanized because of the severity of disease, one on postinoculation day (PID) 5 and the remaining WTD on PID 7. All deer had detectable viremia on PID 3, which peaked on PID 5 or 6 and persisted for as long as PID 46 in one animal. Deer surviving the acute phase of the disease seroconverted by PID 10. Based on the 67% mortality rate we observed, this strain of EHDV-7 is virulent in WTD, reaffirming their role as a sentinel species for the detection of endemic and nonendemic EHDV. Further, the observed disease was indistinguishable from previous reports of disease caused by North American EHDV and bluetongue virus serotypes, highlighting the importance of serotype-specific diagnostics during suspected HD outbreaks.     

Molecular characterization of epizootic hemorrhagic disease virus serotype 1 associated with a 1999 epizootic in white-tailed deer in the eastern United States

During the autumn of 1999 (mid-August-late September), an outbreak of hemorrhagic disease in white-tailed deer (Odocoileus virginianus) caused by epizootic hemorrhagic disease virus serotype 1 (EHDV-1) occurred along the east coast of the United States from Georgia to New Jersey. An EHDV-1 epizootic of such magnitude had not been described in this region since 1975. To determine the genetic relatedness among the 1999 viruses, as well as among additional EHDV-1 isolates from the eastern and western United States, portions of the S10 and L2 gene segments were sequenced and compared utilizing phylogenetic analyses. Nearly all of the 1999 eastern isolates were identical in nucleotide sequence at one or both loci. Additionally, confirmed cases of EHDV-1 in white-tailed deer occurred in a south (Georgia)-to-north (New Jersey/Virginia) progression over a short period of approximately six weeks. Taken together, these results indicate that this outbreak resulted from the spread of a single viral strain. The phylograms derived from analysis of the entire sample set displayed eastern and western region-specific clusterings (topotypes), as well as an eastern versus western difference in branch lengths, which may reflect the influence of epizootic versus enzootic transmission patterns on viral genetic diversity.     

Oral and fecal shedding of epizootic hemorrhagic disease virus, serotype 1 from experimentally infected white-tailed deer

Epizootic hemorrhagic disease (EHD), one of the most important infectious diseases of white-tailed deer (Odocoileus virginianus), is vectored by species of midges in the genus Culicoides. Although vector borne, fecal shedding of EHD virus, serotype 2 has been reported from infected deer in a previous study. To evaluate the potential for fecal and oral shedding, oral and rectal swabs were obtained on day 8 post-inoculation from white-tailed deer fawns experimentally infected with EHD virus, serotype 1 (EHDV-1). Eight deer were viremic for EHDV-1; virus was detected in oral swabs from three (38%) and in rectal swabs from four (50%). The ability to isolate EHDV-1 in oral secretions or feces was not dependent on being able to detect clinical disease. These results indicate that in a relatively large proportion of EHDV-1 infected deer, virus can be detected in feces and oral secretions. Although more work is necessary, such shedding may be important in experimental studies or pen situations where deer-to-deer contact is prevalent and intense.     

Culicoides, the vector of epizootic hemorrhagic disease in white-tailed deer in Kentucky in 1971.

The biting gnat, Culicoides variipennis (Coquillett), was shown to be a vector of epizootic hemorrhagic disease (EHD) in white-tailed deer, Odocoileus virginianus, in Kentucky because of virus isolations from parous females. Epidemiological evidence showed a close relationship of this vector to the animal host during an outbreak of EHD in penned deer. Larval breeding sites of C. variipennis were found and C. variipennis was the most abundant biting fly present during the outbreak. Females of C. variipennis were commonly observed biting deer, swine, cattle and, occasionally, man.     

Synthesis and characterization of chimeric particles between epizootic hemorrhagic disease virus and bluetongue virus: functional domains are conserved on the VP3 protein.

A functional assay has been developed to determine the conservative nature of the interacting sites of various structural proteins of orbiviruses by using baculovirus expression vectors. For this investigation, proteins of two serologically related orbiviruses, bluetongue virus (BTV) and the less studied epizootic hemorrhagic disease virus (EHDV), were used to synthesize chimeric particles. The results demonstrate that the inner capsid protein VP3 of EHDV-1 can replace VP3 protein of BTV in formation of the single-shelled corelike particles and the double-shelled viruslike particles. Moreover, we have demonstrated that all three minor core proteins (VP1, VP4, and VP6) can be incorporated into the homologous and chimeric corelike and viruslike particles, indicating that the functional epitopes of the VP3 protein are conserved for the morphological events of the virus. This is the first evidence of assembly of seven structural proteins of the virus by a baculovirus expression system. Confirmation at the molecular level was obtained by determining the EHDV-1 L3 gene nucleic sequence and by comparing it with sequences available for BTV. The analysis revealed a high degree homology between the two proteins: 20% difference, 50% of which is conservative. The consequences for Orbivirus phylogeny and the possibility of gene reassortments are discussed.     

Fluorosis in black-tailed deer.

Marked dental disfigurement and abnormal tooth wear patterns were observed in black-tailed deer (Odocoileus hemionus columbianus) taken from an area near an industrial fluoride source in northwestern Washington. Fluoride levels in the bones of these deer were from 10 to 35 times higher than levels in the bones of normal animals. These levels are similar to those associated with fluorosis of cattle.     

Experimental adenovirus hemorrhagic disease in white-tailed deer fawns

Infection with a newly described endotheliotropic adenovirus was the cause of a 1993 epizootic reminiscent of hemorrhagic disease in California mule deer (Odocoileus hemionus columbianus and O. hemionus hemionus). Pulmonary edema and intestinal luminal hemorrhage, or necrotizing stomatitis associated with systemic or localized vasculitis, respectively, were common lesions seen in animals that died during the epizootic. In order to determine if white-tailed deer (Odocoileus virginianus) also are susceptible to infection and fatal disease with the deer adenovirus, eight white-tailed deer fawns (4- to 6-mo-old) were inoculated with purified deer adenovirus. Four were inoculated intravenously and four were inoculated through the mucous membranes. Seven days post-inoculation, one of the fawns inoculated intravenously died. Pulmonary edema and hemorrhagic enteropathy were associated with pulmonary and intestinal vasculitis with systemic multiorgan distribution of endotheliotropic adenovirus as demonstrated by transmission electron microscopy and immunohistochemistry. Adenovirus was reisolated from lung homogenates of the fawn that died of adenovirus hemorrhagic disease.     

Experimental infection of white-tailed deer with rinderpest virus.

White-tailed deer (Odocoileus virginianus) succumbed to experimental infection with virulent rinderpest (RP) virus that was also lethal to cattle and goats. The deer developed clinical signs typical of RP and died 5 and 6 days post-inoculation. Infection was confirmed by recovery of virus from blood before death, from lymph node tissue after necropsy, and demonstration of specific complement fixing antigen in those tissues. Electron micrographs of infected Vero cell cultures revealed extracellular virions and intracytoplasmic and intranuclear inclusions made of randomly distributed fibrillar strands.