Seven-year prospective study on yearly incidence of Orbivirus infection of captive white-tailed deer and potential Culicoides vectors.

Bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) are arthropod-borne viruses that are transmitted by biting midges in the genus Culicoides (Diptera: Ceratopogonidae) and can cause hemorrhagic disease in certain ruminants. The objectives of this study were to measure the incidence of BTV and EHDV infections in captive white-tailed deer herd as well as tissues and corresponding presence of Culicoides midges at a location near Clinton, LA. During a 7-yr study with yearly outbreaks of hemorrhagic disease in the deer herd, 15 species of Culicoides were captured using Centers for Disease Control (CDC) black light traps. Reverse transcriptase quantitative polymerase chain reaction (PCR) was performed to screen for BTV and EHDV in pools of midges and tissues of deer. From 2012 to 2018, 1,711 pools of midges representing 24,859 specimens were tested, and specimens from 5 of the 15 collected species (Culicoides debilipalpis, Culicoides stellifer, Culicoides venustus, Culicoides haematopotus, and Culicoides crepuscularis) were found to be PCR positive for BTV and EHDV. Most of the BTV-positive pools of biting midges were from specimens of C. debilipalpis and C. stellifer, and most of the EHDV-positive pools were from specimens of C. venustus and C. stellifer. During the 7-yr period, 112 white-tailed deer that died at the study location were PCR positive for BTV or EHDV: detected BTV serotypes were 10 and 12 and EHDV serotypes were 1, 2, and 6. There was a significant increase in BTV/EHDV antibody prevalence in white-tailed deer during the study; antibody-positive rates increased from 15% to 78% in the deer herd of approximately 100 animals.

Comparison of trapping methods for use in surveys for potential Culicoides vectors of orbiviruses.

BACKGROUND: Bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) are orbiviruses that can cause fatal vector-borne diseases in white-tailed deer (Odocoileus virginianus). Trapping methods for collecting potential Culicoides vectors of orbiviruses were compared to optimize surveillance studies. METHODS: The number of captured midges and the virus infection rates of midge pools were compared for dry ice-baited Centers for Disease Control and Prevention (CDC) traps with or without black light. The number of individual midges of different Culicoides species captured at different crepuscular and nocturnal periods using rotator traps also was determined. The number of species/specimens of Culicoides was measured using five different trap methods including three animal-baited methods, a CDC trap with black light, and a CDC trap with no light. RESULTS: In trial one, there was no significant difference (P = 0.37) in the proportion of BTV-infected flies caught in traps with light compared to traps without light. However, there was a significant difference (P = 0.026) for EHDV-infected flies, and 89% were captured in traps with light. In trial two, more specimens of C. debilipalpis were captured in the morning hours (06:00-08:00) than in the evening hours (18:00-20:00). For trial three, the animal-baited traps did not capture any species of Culicoides that were not captured in the CDC light traps. There was no significant difference (P = 0.22) in total specimens captured among all five trap types. CONCLUSIONS: Specimens of Culicoides infected with BTV were not repelled by light traps in the first trial, while the majority of the specimens positive for EHDV were caught in traps with light. For the second trial, specimens of C. debilipalpis were most abundant during early morning hours, and thus spray applications of insecticides for control of that species may be more effective at sunrise rather than sunset. For objective three, no animal-baited trapping method collected different species of midges when compared to the CDC traps with light, which is unlike certain studies conducted in other geographical regions.

Prospective Study of Epizootic Hemorrhagic Disease Virus and Bluetongue Virus Transmission in Captive Ruminants.

Bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) cause hemorrhagic disease (HD) in wild ruminants and bluetongue disease (BT) and epizootic hemorrhagic disease (EHD) in livestock. These viruses are transmitted by biting midges in the genus Culicoides (family Ceratopogonidae). Mortality from this disease can reach 90% in certain breeds of sheep and in white-tailed deer (Odocoileus virginianus). From January until December of 2012, we conducted a prospective study to determine the origin and routes of transmission of BTV and EHDV in captive deer and cattle. The objective was to determine the abundance of Culicoides spp. and BTV/EHDV infection prevalence in midges, cattle, and deer in an area experiencing an outbreak of BT and EHD. Agar gel immunodiffusion (AGID) tests to detect for EHDV and BTV antibodies were conducted on serum collected from cattle and deer, quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) was utilized for BTV/EHDV RNA detection in tissues from dead deer, and CDC miniature black light traps baited with dry ice were deployed to capture insects. The AGID results showed 19 out of 29 cattle and 18 out of 58 white-tailed deer seroconverted for these viruses during the vector season. Tradition gel-based reverse transcriptase polymerase chain reaction was utilized to determine serotype. Sixteen cows were positive for EHDV-2, EHDV-6, or BTV-12 and 15 deer positive for EHDV-1, EHDV-6, or BTV-12. Specimens from 14 species of Culicoides (Dptera: Ceratopogonidae) (Culicoides arboricola Root and Hoffman, Culicoides biguttatus Coquillett, Culicoides crepuscularis Malloch, Culicoides debilipalpis Lutz, Culicoides furens Poey, Culicoides haematopotus Malloch, Culicoides hinmani Khalaf, Culicoides nanus Root and Hoffman, Culicoides neopulicaris Wirth, Culicoides paraensis Goeldi, Culicoides stellifer Coquillet, Culicoides variipennis Coquillet, Culicoides villosipennis Root and Hoffman, and Culicoides venustus Hoffman) were captured and tested for BTV and EHDV using RT-qPCR assays. BTV viral nucleic acid was detected in three pools from three different species of midges: C. crepuscularis, C. debilipalpis, and C. stellifer.

Pregnancy Rates Following Low-Temperature Storage of Large Equine Embryos Before Vitrification.

Satisfactory pregnancy rates can now be achieved following the cryopreservation of large equine embryos. Nonetheless, its wide application might be limited by the fact that the cryopreservation of large equine embryos requires a specialized micromanipulation equipment and micromanipulation/vitrification skills. Alternatives should be developed to increase its utilization and widespread application in the commercial equine industry. To determine if large equine embryos are able to remain viable during transport from farms to specialized centers for embryo cryopreservation, we evaluated pregnancy rates following the low-temperature storage of large equine embryos before vitrification. Grade 1 embryos (n = 37) were randomly assigned to six treatments consisting of day of collection (Day 7 or 8 after ovulation) and cooling for 0, 12, or 24 hours before vitrification in a factorial design. Pregnancy rates of Day 7 embryos cooled for 12 and 24 hours were 55.5% and 75%, respectively. Pregnancy rates of Day 8 embryos cooled for 12 and 24 hours were 0 and 16.6%, respectively. Day 7 cooled embryos resulted in higher pregnancy rate compared with Day 8 cooled embryos (64.7% and 7.7%, respectively; P < .05). Pregnancy rate comparison of cooled embryos grouped by diameter showed that embryos <550 µm resulted in a higher pregnancy rate compared with embryos >550 µm (71.4% and 12.5% respectively; P < .05). In conclusion, Day 7 equine embryos up to 550 µm can be cooled to temperatures of 9-12°C for 12 or 24 hours before vitrification and result in satisfactory pregnancy rates.

Cryopreservation of Day 8 equine embryos after blastocyst micromanipulation and vitrification.

Pregnancy rates after cryopreservation of large equine blastocyst stage embryos have remained lower than other domesticated livestock species. It is generally accepted that the embryonic capsule is the primary barrier to cryoprotectant entry into the embryo proper and techniques need to be developed to circumvent this obstacle. Therefore, the objective of this study was to develop an efficient Day 8 equine embryo cryopreservation protocol through blastocyst micromanipulation and vitrification. Grade 1 and 2 embryos recovered from mares (n = 15) 8 days after ovulation were used in these experiments. In experiment 1, the effect of either one- or two-puncture treatments before aspiration of blastocoel fluid and exposure to vitrification solutions was evaluated. No difference was detected in mean embryo volume across treatment groups after exposure to vitrification solutions or after 1, 24, 48, and 72 hours of culture. Percent of embryos re-expanding at 24 hours and percent of embryos showing diameter increase at 48 and 72 hours during in vitro culture were 100%, 83%, and 75% compared with 93%, 67%, and 50% for one- and two-puncture treatment groups, respectively. Capsule loss was 25% for one-puncture and 50% for two-puncture treatment groups. In experiment 2, no difference was detected in mean embryo volume for indirect introduction (aspiration of blastocoel fluid + equilibration) and direct introduction (injection of cryoprotectant into blastocoel cavity) treatment groups, after exposure to dilution solution or to culture medium. There was no difference in mean embryo volume for the indirect and direct introduction treatment groups after 1, 24, 48, and 72 hours of culture. Percent of embryos re-expanding at 24 hours and percent of embryos showing diameter increases at 48 and 72 hours during in vitro culture were 100%, 76.9%, and 69.2%, respectively, for both treatment groups. Those embryos subjected to the direct introduction treatment had a higher (P = 0.05) percent capsule loss (70%) compared with the indirect introduction treatment group (31%). The pregnancy rate after transfer of vitrified expanded Grade 1 blastocysts using the indirect introduction method was 83% (5/6). Three pregnancies were allowed to continue to term and resulted in the birth of three healthy foals. The vitrification protocol used in this study has the potential to become a key tool for the successful cryopreservation of equine expanded blastocysts.

Sex ratio of bovine embryos and calves originating from the left and right ovaries.

An asymmetric distribution of the sexes within the left and right uterine horns has been described in multiple species. A series of experiments were conducted to evaluate the sex ratio (% male) of calves gestated in the left and right uterine horns, as well as the sex ratio of embryos originating from the left and right ovaries of cattle. The sex ratio of calves gestated in the right uterine horn of naturally mated cows was significantly higher compared with the sex ratio of calves gestated in the left uterine horn. In addition, the sex ratio of the left and right uterine horns differed significantly from parity. The sex ratio of embryo transfer calves born following transfer to the left and right uterine horns was not significantly different. Additionally, the proportion of male embryos collected from the right uterine horns was significantly greater than from the left uterine horns of superovulated cows. The sex ratio of embryos collected from the left and right uterine horns of unilaterally ovariectomized cows was not significantly different. However, more female than male embryos were produced when left ovary oocytes fertilized in vitro. In conclusion, the results of these experiments demonstrate that a significantly greater proportion of males are gestated in the right uterine horn of cattle and a greater proportion of females in the left. Additionally, the data indicate that sex-specific selection pressure may be applied to embryos by ovarian factors rather than by the uterine environment.