Epizootic Hemorrhagic Disease Research Articles

New species of the genus Culicoides (Diptera Ceratopogonidae) for Tunisia, with detection of Bluetongue viruses in vectors.

Bluetongue virus (BTV) and Epizootic haemorrhagic disease virus (EHDV) are double-stranded RNA orbiviruses of the Reoviridae family. Bluetongue virus and EHDV infect domestic and wild ruminants and they are transmitted by biting midges of the genus Culicoides (Diptera: Ceratopogonidae). Since 1999, BTV outbreaks have occurred in Tunisia and 4 serotypes, BTV2, BTV1, BTV4 and BTV3, were involved in 2000, 2006, 2009, and 2016, respectively. Epizootic haemorrhagic disease was detected for the rst time in Tunisia and in other Northern African countries in 2006. These incursions have caused considerable economic losses. Our study had the goal to describe diversity, distribution, and seasonal dynamics of Culicoides. Fourteen sampling sites were chosen throughout the country and 2-night trapping of midges was performed monthly from June 2006 to July 2008. A total of 11,582 Culicoides specimens were collected from 336 light traps, comprising 25 species, of which 7 were identi ed for the rst time in Tunisia, increasing to 35 the total number of Culicoides species now reported in this country. Twenty-three pools of parous females belonging to the Culicoides imicola and Culicoides kingi were tested for detection of BTV and EHDV by molecular assays. Both BTV1 and BTV4 were detected in C. imicola.

Bluetongue in white-tailed deer (Odocoileus virginianus) in Northeastern Mexico

Abstract Bluetongue (BT) and epizootic hemorrhagic disease of deer (EHD) are two distinct viral hemorrhagic diseases of domestic and wild ruminants caused by members of the family Reoviridae and transmitted by Culicoides midges. These conditions have been recognized in Canada and the United States for many years, but not in Mexico. Although in Mexico there is serologic evidence of EHD virus (EHDV) and bluetongue virus (BTV) in domestic and wild ruminants, to our knowledge, there have never been reports of clinical illness or fatalities attributed to either of these viruses. Two free ranging white-tailed deer (Odocoileus virginianus) in two licensed hunting ranches in Northern Mexico near the Texas border died unexpectedly. Postmortem and microscopic examinations revealed hemorrhagic lesions compatible with viral hemorrhagic disease (Reoviridae: Orbivirus). Tissues from one animal tested positive by RT-PCR for BTV but negative for EHDV. To our knowledge, this is the first time in Mexico where deer dying with hemorrhagic lesions consistent with Bluetongue tested positive for BTV by PCR. Key words: hemorrhagic disease, white-tailed deer, Orbivirus, Mexico.

Dynamics of epizootic hemorrhagic disease virus infection within the vector, Culicoides sonorensis (Diptera: Ceratopogonidae).

Culicoides sonorensis biting midges are confirmed vectors of epizootic hemorrhagic disease virus (EHDV), which causes mortality in white-tailed deer and ruminant populations. Currently, of the seven EHDV serotypes, only 1, 2, and 6 are detected in the USA, and very few studies have focused on the infection time course of these serotypes within the midge. The objective of this current research was to characterize EHDV-2 infection within the midge by measuring infection prevalence, virus dissemination, and viral load over the course of infection. Midges were fed a blood meal containing 106.9 PFU/ml EHDV-2, collected every 12 h from 0–2 days post feeding (dpf) and daily from 3–10 dpf, and cohorts of 20 C. sonorensis were processed using techniques that assessed EHDV infection and dissemination. Cytopathic effect assays and quantitative (q)PCR were used to determine infection prevalence, revealing a 50% infection rate by 10 dpf using both methods. Using immunohistochemistry, EHDV-2 infection was detectable at 5 dpf, and shown to disseminate from the midgut to other tissues, including fat body, eyes, and salivary glands by 5 dpf. Stain intensity increased from 5–8 dpf, indicating replication of EHDV-2 in secondary infection sites after dissemination. This finding is also supported by trends in viral load over time as determined by plaque assays and qPCR. An increase in titer between 4–5 dpf correlated with viral replication in the midgut as seen with staining at day 5, while the subsequent gradual increase in viral load from 8–10 dpf suggested viral replication in midges with disseminated infection. Overall, the data presented herein suggest that EHDV-2 disseminates via the hemolymph to secondary infection sites throughout the midge and demonstrate a high potential for transmission at five days at 25°C after an infective blood-meal.

Survey for selected pathogens in Philippine deer (Rusa marianna) from Guam, Marianna Islands, USA.

Philippine deer (Rusa marianna), native to the Philippine Islands, were introduced to Guam in the late 1700's. Dense populations have become established throughout the island where they cause damage to native plant communities resulting in habitat degradation. In addition, cervids can serve as reservoirs for important pathogens of livestock and people. From February-March 2015, blood, tissue and ectoparasite samples were collected from 132 free-ranging Philippine deer on Guam. Data from 10 deer sampled in 1997 were also analyzed. Deer were negative for antibodies to many of the pathogens assessed including epizootic hemorrhagic disease virus, parainfluenza 3 virus, bovine viral diarrhea virus, bovine herpesvirus 1, and Brucella spp.; however, two (2%) and nine (7%) deer were seropositive for bluetongue virus and Toxoplasma gondii, respectively. Five (4%) deer had low titers (1:100) to Leptospira interrogans serovars Bratislava (n=4), Canicola (n=2), and Icterohaemorrhagiae (n=1). None of the kidney samples from Leptospira-seropositive deer were immunohistochemically positive for leptospires. No nematodes or trematodes were detected in lungs, abdomen, abomasum or liver. A few deer had 1-4 Cooperia spp. in the small intestine, although very small nematodes may have not been captured by the #100 mesh used for screening. Of the 105 deer evaluated for ectoparasites, 90.5% were infested with Rhipicephalus microplus. Tick burdens were generally high and classified as low (<500 ticks) (59% of infested deer), medium (500-1000 ticks) (22%), and high (>1000 ticks) (19%). Molecular testing of blood samples for Babesia spp. was negative, but 11 (8%) deer were positive for Anaplasma spp. Sequence analysis revealed that deer were infected with three species of Anaplasma including A. marginale, A. phagocytophilum, and an Anaplasma sp. similar to A. platys. Finding A. marginale, T. gondii, Leptospira and heavy burdens of ticks in Philippine deer is of economic and public health importance.

A serosurvey of bluetongue and epizootic haemorrhagic disease in a convenience sample of sheep and cattle herds in Zimbabwe.

A convenience sample of sheep and cattle herds around the cities of Harare, Kwekwe and Bulawayo, located in the Highveld region of Zimbabwe, was used to estimate the sero-prevalence and sero-incidence of bluetongue virus (BTV) and epizootic haemorrhagic disease virus (EHDV) antibodies. A competitive enzyme-linked immunosorbent assay was used to identify serum antibodies against BTV and EHDV across three rainy seasons. The median sero-prevalence of BTV and EHDV antibodies in cattle was 62% (interquartile range [IQR]: 30–89) and 56% (IQR: 5–77), respectively. In sheep, the median sero-prevalence of BTV and EHDV was 41% (IQR: 19–63) and 0% (IQR: 0–21), respectively. Median sero-incidences of BTV and EHDV antibodies in cattle of 43% (IQR: 22–67) and 27% (IQR: 9–57) respectively were recorded. The median sero-incidence of BTV in sheep was 14% (IQR: 6–23). Based on these preliminary findings, animal health workers in Zimbabwe should continue to monitor the exposure rates of cattle and sheep to BTV and consider the possibility of strains emerging with increased pathogenicity. There are no previous published reports of antibodies against EHDV in Zimbabwe so the possibility of epizootic haemorrhagic disease existing in domestic livestock should now be considered by Zimbabwean animal health officials. Seroconversions to BTV and EHDV occurred predominantly at the end of each rainy season (March and April), which generally corresponds to high numbers of the Culicoides vectors. BTV isolations were made from three individual cows in two of the sentinel herds and all three were identified as serotype 3. This is the first time BTV serotype 3 has been recorded in Zimbabwe, although its presence in neighbouring South Africa is well documented.

Larval Development of Culicoides sonorensis (Diptera: Ceratopogonidae) in Mud Supplemented With Manure of Various Farm Animals.

Understanding factors that affect Culicoides larval development is critical in suppressing adult midge populations that transmit economically important pathogens to ruminants such as bluetongue virus and epizootic hemorrhagic disease virus. In this study, development of Culicoides sonorensis Wirth and Jones (Diptera: Ceratopogonidae) was investigated in sterilized mud mixed with varying concentrations (3.2, 6.4, 12.6, 25.0, 50.0, and 100.0%) of dairy cattle and white tailed deer manures. In addition, C. sonorensis development was also evaluated in manure (25.0% concentration) of six other farm animals (beef cattle, sheep, goat, pig, horse, and chicken). First instar larvae (~100/treatment) were added to each substrate, and adult emergence and development times were monitored for 90 d. In substrates with dairy cattle manure, significantly more adults emerged (≥76.7%) and development time was shorter (≤25.5 d) from 25.0% manure substrate than from lower or higher manure concentrations (≤41.3% emerged; ≥31.2 d). Comparatively, white-tailed deer and chicken manures supported C. sonorensis development poorly with low emergence rates (deer ≤ 13.0%; chicken = 0%) and longer development time (deer ≥ 29.0 d). Mud enriched with manure of beef cattle, sheep, goat, pig, and horse generally supported C. sonorensis development, although adult emergence and development times varied widely between species. These results suggest that manure of several farm animals, except for white-tailed deer and chicken, can contribute substantially to C. sonorensis development in the field. Therefore, the potential of animals other than cattle in sustaining local populations of C. sonorensis cannot be overlooked when designing management strategies.

Genomic Sequences of Epizootic Hemorrhagic Disease Viruses Isolated from Florida White-Tailed Deer.

ABSTRACTEpizootic hemorrhagic disease virus (EHDV) serotypes 1 and 2 were isolated from Florida white-tailed deer in 2015 and 2016, respectively, and their genomes were completely sequenced. To our knowledge, these are the first full genome sequences for EHDV-1 and -2 from Florida.

Epizootic hemorrhagic disease virus serotype 6 outbreak in Israeli cattle in 2015.

In September 2015, a large outbreak caused by epizootic hemorrhagic disease virus (EHDV) was identified in Israeli dairy and beef farms. The main clinical signs were reduced milk production, weakness, drooling, lameness and recumbency, fever, slight erythema of nasal and oral mucosae, weight loss, and abortion. Dyspnea, cachexia, and death were observed less frequently. The clinical diagnosis was confirmed by ELISAs and EHDV-specific real-time reverse transcription PCR (RT-rtPCR), followed by conventional RT-PCR of the VP2 gene and sequence analysis. According to the sequence and phylogenetic analysis of theVP2 gene, the 2015 Israeli EHD outbreak was caused by EHDV-6, which was found not only in clinically ill cattle, but also in aborted fetuses.

Competitive enzyme-linked immunosorbent assay using baculovirus-expressed VP7 for detection of epizootic haemorrhagic disease virus (EHDV) antibodies

Epizootic haemorrhagic disease (EHD) is a vector-borne infectious viral disease of domestic and wild ruminants. EHD could spread from infected northern African countries in free territories like the EU; therefore, the availability of diagnostic assays would represent key components for adequate surveillance and control programs. In this study, the gene encoding the VP7 protein of EHD virus (EHDV) was expressed into a baculovirus-infected insect cell system. With this unpurified protein we developed a home-made competitive ELISA (cELISA) and a total number of 275 serum samples, originating from domestic and wild ruminants, were tested. 74/275 were previously shown to be positive for EHDV antibodies by a commercially available ELISA kit. A “very good” agreement was demonstrated when compared to a commercial ELISA kit (Cohen's kappa value=0.832). Samples which caused disagreement between the two assays originated from wildlife which highlights the need for further validation by using serum samples from wild animals.

The First 10 Years (2006–15) of Epizootic Hemorrhagic Disease Virus Serotype 6 in the USA

Epizootic hemorrhagic disease virus (EHDV) is a Culicoides biting midge-transmitted orbivirus (family Reoviridae) of wild and domestic ruminants and is an important pathogen of white-tailed deer (Odocoileus virginianus). Historically, only two serotypes, EHDV-1 and EHDV-2, have been known to be endemic in the US. However, in 2006, an exotic serotype (EHDV-6) was first detected in the US by a long-term passive surveillance system for EHDV and bluetongue viruses. Here we report EHDV-6 detections made through these passive surveillance efforts by the Southeastern Cooperative Wildlife Disease Study (University of Georgia, Athens, Georgia, USA) and the National Veterinary Services Laboratories (US Department of Agriculture, Ames, Iowa, USA) over a 10-yr period (2006-15). The results demonstrated that EHDV-6 was detected from ruminants every year since 2006 and was widespread in the central and eastern US, providing evidence that EHDV-6 is likely now established in the US.

Characterization of genome segments 2, 3 and 6 of epizootic hemorrhagic disease virus strains isolated in Japan in 1985–2013: Identification of their serotypes and geographical genetic types

We characterized genome segments 2, 3 and 6 (Seg-2, Seg-3 and Seg-6) of 11 Japanese strains of epizootic hemorrhagic disease (EHD) virus (EHDV) isolated in 1985–2013. The Japanese strains were divisible into two groups based on phylogenetic analyses of the nucleotide sequences of Seg-2 and Seg-6. In both of the phylogenetic trees based on Seg-2 and Seg-6, seven of the 11 Japanese strains were grouped together with EHDV-2 and EHDV-7 strains, and the other four Japanese strains were grouped with EHDV-1 strains. The phylogenetic analysis of Seg-2 among EHDV strains identified 10 of the 11 Japanese strains as EHDV-1, EHDV-2 or EHDV-7. The other Japanese strain, ON-4/B/98, isolated from an asymptomatic cow in 1998 was in the same group as the EHDV-2 and EHDV-7 strains in the phylogenetic trees based on Seg-2 and Seg-6, but the results suggested that the strain belongs to another serotype. We thus conducted a serum neutralization test to identify that serotype by using anti-EHDV-2 and anti-EHDV-7 rabbit sera. We observed that the ON-4/B/98 strain was not sufficiently neutralized by any of the antisera, which suggests that the strain could be assigned into a new serotype, tentatively named ‘EHDV-10.’ Sequences of Seg-3 were also determined, and all of the Japanese strains were grouped together with Australian strains, suggesting that the Japanese strains are a part of EHDV distributed in the Asia-Pacific region. The data obtained herein would be beneficial for the diagnosis and prevention of EHD in Japan and neighboring countries.

Emerging vector-borne diseases in dromedaries in Tunisia: West Nile, bluetongue, epizootic haemorrhagic disease and Rift Valley fever.

A total of 118 sera were collected during 2016 from two groups of dromedaries from Kebili and Medenine governorates in the south of Tunisia. The aim of this study was to provide the first serological investigation of four emerging vector-borne diseases in two groups of dromedaries in Tunisia. Sera were tested by ELISA and serum neutralisation test to identify West Nile virus (WNV), bluetongue virus (BTV), epizootic haemorrhagic disease virus (EHDV) and Rift Valley fever virus (RVFV). In the first group, the seroprevalence for BTV was 4.6%, while in the second group, it was 25.8% for WNV and 9.7% for BTV. Only serotype 1 was detected for BTV in the two groups. No evidence for circulation of RVF and EHD viruses was revealed. Results indicated that dromedaries can be infected with BTV and WNV, suggesting that this species might play a significant role in the epizootiology of these viral diseases in Tunisia and neighbouring countries.

The development of a real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay using TaqMan technology for the pan detection of bluetongue virus (BTV)

Bluetongue virus (BTV) is an infectious, non-contagious viral disease of domestic and wild ruminants that is transmitted by adult females of certain Culicoides species. Since 2006, several serotypes including BTV-1, 2, 4, 6, 8, 9 and 16, have spread from the Mediterranean basin into Northern Europe for the first time. BTV-8 in particular, caused a major epidemic in northern Europe. As a result, it is evident that most European countries are at risk of BTV infection. The objective of this study was to develop and validate a real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) assay based on TaqMan technology for the detection of representative strains of all BTV serotypes. Primers and probes were based on genome segment 10 of the virus, the NS3 gene. The assay was tested for sensitivity, and specificity. The analytical sensitivity of the rRT-PCR assay was 200 copies of RNA per reaction. The assay did not amplify the closely related orbivirus epizootic hemorrhagic disease virus (EHDV) but successfully detected all BTV reference strains including clinical samples from animals experimentally infected with BTV-8. This real time RT-PCR assay offers a sensitive, specific and rapid alternative assay for the pan detection of BTV that could be used as part of a panel of diagnostic assays for the detection of all serotypes of BTV.

Seroprevalence and risk factor analysis for exposure to equine encephalosis virus in Israel, Palestine and Jordan.

Equine encephalosis virus (EEV) is an orbivirus transmitted by Culicoides species. Most infected horses show mild clinical signs and mortality is usually very low. EEV is closely related and similarly transmitted to other, more pathogenic and economically important, orbiviruses such as African horse sickness virus (AHSV), bluetongue virus (BTV) and epizootic haemorrhagic disease viruses (EHDV), and may serve as an indicator for possible transmission of the latter. Israel has been reported to be endemic for EEV since 2001. This study was initiated to re‐evaluate the current seroprevalence and risk factors for EEV exposure in Israel, and to assess, for the first time, the seroprevalence of EEV in Palestine and Jordan. Three hundred and sixteen serum samples were collected from apparently healthy horses at 21 farms in Israel, 66 horses at nine farms in Palestine and 100 horses at three farms in Jordan. The presence of EEV antibodies was detected by a serum neutralization assay. Seroprevalence of EEV was 58.2% (184/316 horses) in Israel, 48.5% (32/66 horses) in Palestine and 2% (2/100 horses) in Jordan. Seroprevalence in Jordan was significantly lower than in Israel and Palestine (P < 0.001). The farm (P < 0.001) and horse age (P = 0.003) were found as significant risk factors for EEV exposure in Israel in multivariable statistical analysis. The results of this study further demonstrate that EEV is no longer limited to South Africa and is endemic in both Israel and Palestine and horses in Jordan were also exposed to this virus emphasizing the potential of pathogens to invade new ecological niches.

Novel serotype of bluetongue virus in South America and first report of epizootic haemorrhagic disease virus in Ecuador.

Bluetongue virus (BTV) and Epizootic haemorrhagic disease virus (EHDV) are closely related Orbiviruses that affect domestic and wild ruminants. In Ecuador previous serological studies reported the presence of BTV; however, no data are available about the presence of EHDV. In this study, 295 cattle without symptoms of infection were sampled from two farms located in Andean and Amazonian regions and from a slaughterhouse in the coastal region. ELISA analyses showed high prevalence of BTV (98.9%) and EHDV (81.3%) antibodies, and RT-qPCRs revealed the presence of EHDV (24.1%) and BTV (10.2%) genomes in cattle blood samples. Viral isolation allowed to identify EHDV serotype 1 (EHDV1) and BTV serotypes 9 (BTV9), 13 and 18. These findings suggest that BTV and EHDV are enzootic diseases in Ecuador.

Functional Validation of Apoptosis Genes IAP1 and DRONC in Midgut Tissue of the Biting Midge Culicoides sonorensis (Diptera: Ceratopogonidae) by RNAi.

Culicoides biting midges transmit multiple ruminant viruses, including bluetongue virus and epizootic hemorrhagic disease virus, causing significant economic burden worldwide. To further enhance current control techniques, understanding vector-virus interactions within the midge is critical. We developed previously a double-stranded RNA (dsRNA) delivery method to induce RNA interference (RNAi) for targeted gene knockdown in adult Culicoides sonorensis Wirth & Jones. Here, we confirm the C. sonorensis inhibitor of apoptosis 1 (CsIAP1) as an anti-apoptotic functional ortholog of IAP1 in Drosophila, identify the ortholog of the Drosophila initiator caspase DRONC (CsDRONC), and demonstrate that injection of dsRNA into the hemocoel can be used for targeted knockdown in the midgut in C. sonorensis. We observed CsIAP1 transcript reduction in whole midges, with highest transcript reduction in midgut tissues. IAP1knockdown (kd) resulted in pro-apoptotic caspase activation in midgut tissues. In IAP1kd midges, midgut tissue integrity and size were severely compromised. This phenotype, as well as reduced longevity, was partially reverted by co-RNAi suppression of CsDRONC and CsIAP1. Therefore, RNAi can be directed to the midgut of C. sonorensis, the initial site of virus infection, using dsRNA injection into the hemocoel. In addition, we provide evidence that the core apoptosis pathway is conserved in C. sonorensis and can be experimentally activated in the midgut to reduce longevity in C. sonorensis. This study thus paves the way for future reverse genetic analyses of midgut-virus interactions in C. sonorensis, including the putative antiviral properties of RNAi and apoptosis pathways.

Structural Protein VP2 of African Horse Sickness Virus Is Not Essential for Virus Replication In Vitro.

Members of the Reoviridae family cause major health problems worldwide, ranging from lethal diarrhea caused by rotavirus in humans to economic losses in livestock production caused by different orbiviruses. The Orbivirus genus contains many virus species, of which bluetongue virus, epizootic hemorrhagic disease virus, and African horse sickness virus (AHSV) cause notifiable diseases according to the World Organization of Animal Health. Recently, it has been shown that nonstructural proteins NS3/NS3a and NS4 are not essential for virus replication in vitro, whereas it is generally assumed that structural proteins VP1 to -7 of these nonenveloped, architecturally complex virus particles are essential. Here we demonstrate for the first time that structural protein VP2 of AHSV is not essential for virus replication in vitro Our findings are very important for virologists working in the field of nonenveloped viruses, in particular reoviruses.

Experimental infection of Holstein cows and calves with EHDV‑7 and preliminary evaluation of different inoculation methods.

Infection of cattle with epizootic haemorrhagic disease (EHD) virus (EHDV) is frequently subclinical, yet reports of disease have increased in recent years. In 2006, a widespread EHDV‑7 epidemic caused disease and economic loss in the Israeli dairy industry. In this study, the main objective was to infect cattle with EHDV‑7 and replicate disease observed in Israel during 2006. Two infection studies were performed. Experiment 1, 4 cows inoculated with intradermal (ID) and subcutaneous (SC) injections with an EHDV‑7 blood inoculum. Experiment 2, 6 calves inoculated using 1 of the following 3 methods (2 calves/method): (1) mammalian cell culture supernatant by ID and SC injection; (2) culture supernatant by ID, SC, and intravenous injection; and (3) bite transmission from Culicoides sonorensis. Further, during experiment 2, C. sonorensis were fed on 4 infected calves (18 days post-inoculation) and processed for virus isolation 10 days later in order to evaluate infectivity of low‑titer viraemia. Three cows had detectable viraemia and all 4 seroconverted. No clinical signs were observed. All 6 calves developed viraemia, peaking 7‑10 dpi and all calves seroconverted. No differences in virus kinetics were observed between the inoculation groups. Calves in group 2 had transiently elevated rectal temperatures but no other clinical abnormalities were observed. The 124 midge pools processed after feeding on calves with low‑titer viraemia were virus isolation negative. Detectable viraemia was more consistent in calves than adult cows. This study demonstrates US‑origin cattle are susceptible to EHDV‑7 infection by multiple inoculation methods; however, as reported in other studies, the disease was not replicated experimentally.

Overwintering of epizootic hemorrhagic disease virus in white-tailed deer in Florida, USA: Unanticipated seroconversion and the case for alternative vectors

Overwintering of epizootic hemorrhagic disease virus in white-tailed deer in Florida, USA: Unanticipated seroconversion and the case for alternative vectors

Autogenous vaccine field trial for epizootic hemorrhagic disease virus and bluetongue virus does not result in high titer to homologous virus serotypes

Autogenous vaccine field trial for epizootic hemorrhagic disease virus and bluetongue virus does not result in high titer to homologous virus serotypes