Disease Phenotype of Virus-Infected Helminthosporium victoriae Is Independent of Overexpression of the Cellular Alcohol Oxidase/RNA-Binding Protein Hv-p68

ABSTRACT The cellular protein Hv-p68 is a novel alcohol oxidase/RNA-binding protein that is overexpressed in virus-infected isolates of the plant-pathogenic fungus Helminthosporium victoriae (teleomorph: Cochliobolus victoriae). Overproduction of Hv-p68 has been hypothesized to lead to the accumulation of toxic aldehydes and to induce the disease phenotype associated with the virus-infected isolates. We overexpressed the Hv-p68 gene in virus-free isolates and evaluated the morphology of the resulting colonies. We cloned and sequenced the Hv-p68 genomic DNA, which contains five introns and the complete Hv-p68 coding sequence. Vectors for overexpression of the Hv-p68 gene were constructed with either Hv-p68 cDNA or the intron-containing Hv-p68 genomic DNA. Expression of Hv-p68 was significantly higher if the genomic sequence was used for transformation than if the cDNA sequence was used. The virus-free fungal transformants that overexpressed Hv-p68 gene did not exhibit the disease phenotype. In contrast, these transformants showed enhanced growth rates when compared with the nontransformed and empty vector controls. Interestingly, overexpression of Hv-p68 in a fungal isolate infected with both the totivirus Helminthosporium victoriae 190S virus (Hv190SV) and the chrysovirus Helminthosporium victoriae 145S virus (Hv145S) showed enhanced accumulation of the Hv145SV double-stranded (ds)RNA, but not of the Hv190SV. These results are consistent with an earlier report that Hv-p68 co-purified with viral dsRNA, mainly that of the Hv145SV. Elucidation of the role of Hv-p68 in disease induction is important for an understanding of host-virus interactions in this fungus-virus system.     

Impact of Probing the Reproductive Tract During Early Pregnancy on Fertility of Beef Cows

This short communication reports the impact of endometrial biopsies, uterine flushings and follicular fluid aspiration procedures at day 6 post artificial insemination (AI) on pregnancy rates. In Experiment 1, cows were timed AI (TAI) and assigned to the following treatment groups: control (n = 37), uterine flushing (n = 35) and endometrial biopsy (n = 38). On day 30 post AI, pregnancy rates were 40.5%, 33% and 28.5%, respectively (p > 0.1). Pregnancy rate on day 60 was lower (p < 0.004) in flushed cows than in the controls. In Experiment 2, oestrus was detected and cows were assigned to flushing (n = 32) or biopsy (n = 33) treatments 6 days after AI, which resulted in pregnancy rates of 31% and 36%, respectively (p > 0.1). In Experiment 3, cows were, 6 days after TAI, randomly assigned to the following treatments: control (n = 84) or aspiration of the largest follicle (n = 73). Pregnancy rates on day 30 post AI were 63.5% for the control group and 53% for the aspirated group (p > 0.1). In conclusion, uterine flushing and endometrial biopsy negatively affect pregnancy rates, but neither procedure can be considered to be incompatible with pregnancy maintenance. Follicular aspiration during pregnancy does not interact with pregnancy success. The amount and quality of samples obtained are compatible with the use of cellular and molecular analysis of uterine variables from cows that failed or succeeded on maintaining pregnancy.     

Effect of Overnight Staining on the Quality of Flow Cytometric Sorted Stallion Sperm: Comparison with Tradtitional Protocols

Flow cytometry is considered the only reliable method for the separation of X and Y chromosome bearing spermatozoa in equines. The MoFlo SX DP sorter is highly efficient, allowing the production of foals of the desired sex. However, to achieve acceptable pregnancy rates the currently used protocol requires working with fresh semen obtained close to, or at, the sorting facility. An alternative protocol was tested during two consecutive breeding seasons. Fresh stallion semen was cooled for 20 h, during which staining with Hoechst 33342 took place. On the following day, this sample was flow sorted and compared with spermatozoa from the same ejaculate that had been sexed on the previous day. All sperm parameters evaluated remained unchanged when fresh sorted and refrigerated sorted semen were compared. Pre‐sorting storage at 5°C did not alter sperm velocities nor kinetics, viability or membrane permeability, production of reactive oxygen species, mitochondrial membrane potential or DNA fragmentation index of the sorted sample. The findings open for the possibility of using semen from stallions housed far from the sorting facilities. Processed and stained sperm could be shipped refrigerated on the previous day, sorted and inseminated on the next day.     

An ethogram of post-anesthetic recovery behaviors in horses: comparison of pre- and post-anesthetic behaviors

Pain management is an important post-operative concern. Pain scales may rely on the observer's subjective assessment of the level of discomfort and may not correlate with physiologic or pharmacologic measures of pain. The purpose of this study was to develop an objective measure of behavior in healthy pain-free horses recovering from anesthesia that could be used for comparison with the behavior of horses recovering from a surgical procedure.
Focal sampling with videotape and observation was done on five healthy horses before anesthesia to establish baselines. Behavioral measures included head turns, tail swishes, eyelid aperture/size, ear position, angle of neck, weight shifts, and ambulation. Physiologic measures included heart rate, respiratory rate, and temperature. The horses were anesthetized for 2 hours with isoflurane, and in the recovery stall, data were collected continuously on videotape from the time of extubation to standing. Focal sampling of 15 minutes was repeated at 1, 2, 4, and 24 hours after the horses returned to their stalls. Video data were analyzed using the Noldus Observer Video Analysis System.
A wide variation in behavior was observed between horses in the recovery stall. Observations at 1, 2, and 4 hours revealed change from the up/forward ear position to the down ear position, and a decrease in weight bearing of the hind limb from a baseline of 11–12 minutes out of 15 minutes to 7–9 minutes with an increase in toe pointing. Time spent standing was similar to baseline of 93.8%, and the neck angle was not changed from baseline of 173.5°. Values had returned to baseline at 24 hours.
Changes in behavior were induced by anesthesia alone and must be taken into consideration when evaluating analgesic treatments.     

Quantification of transmission of foot-and-mouth disease virus caused by an environment contaminated with secretions and excretions from infected calves

Foot-and-mouth disease virus (FMDV) infected animals can contaminate the environment with their secretions and excretions. To quantify the contribution of a contaminated environment to the transmission of FMDV, this study used calves that were not vaccinated and calves that were vaccinated 1 week prior to inoculation with the virus in direct and indirect contact experiments. In direct contact experiments, contact calves were exposed to inoculated calves in the same room. In indirect contact experiments, contact calves were housed in rooms that previously had held inoculated calves for three days (either from 0 to 3 or from 3 to 6 days post inoculation). Secretions and excretions from all calves were tested for the presence of FMDV by virus isolation; the results were used to quantify FMDV transmission. This was done using a generalized linear model based on a 2 route (2R, i.e. direct contact and environment) SIR model that included information on FMDV survival in the environment. The study shows that roughly 44% of transmission occurs via the environment, as indicated by the reproduction ratio R^02Renvironment that equalled 2.0, whereas the sum of R^02Rcontact and R^02Renvironment equalled 4.6. Because vaccination 1 week prior to inoculation of the calves conferred protective immunity against FMDV infection, no transmission rate parameters could be estimated from the experiments with vaccinated calves. We conclude that a contaminated environment contributes considerably to the transmission of FMDV therefore that hygiene measures can play a crucial role in FMD control.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-015-0156-5) contains supplementary material, which is available to authorized users.     

Cholelithiasis and hyperthyroidism in a cat

A 14-year-old domestic short-hair cat presented with a history of intermittent malaise and increased drinking. A diagnosis of hyperthyroidism and cholelithiasis was made by a combination of blood testing, radiography and ultrasonography. After medical management of hyperthyroidism, thyroidectomy and cholecystectomy were successfully performed. Removed choleliths were comprised of calcium carbonate and bilirubinate. Histopathological analysis of tissue suggested low grade pancreatic and hepatobiliary disease, as well as hyperthyroidism, might have contributed to stone formation.