Development of an Isavirus minigenome system to study the function of the pocket RNA-binding domain of the viral nucleoprotein (NP) in salmon cells

The Isavirus is an orthomyxovirus with a genome composed of eight segments of negative single-strand RNA (−ssRNA). It has been proposed that the eight genomic segments of the Isavirus are organized as a ribonucleoprotein (RNP) complex called a minigenome, which contains all the viral RNA segments, a viral heterotrimeric polymerase and multiple copies of the viral nucleoprotein (NP). Here, we develop an Isavirus minigenome system and show the importance of the formation of active RNPs and the role of viral NP R189, R194, R302 and K325 residues in the NP RNA-binding domain in the context of RNPs. The results indicate it is possible to generate a minigenome in salmon cells, a composite ISAV RNPs with EGFP-based chimeric vRNA with heterotrimeric polymerase (PB1, PB2, PA) and NP protein using CMV-based auxiliary plasmids. It was also shown that NP R189, R194, R302 and K325 residues are important to generate viral mRNA from the constituted RNPs and a detectable reporter protein. This work is the first salmon cell-based minigenome assay for the Isavirus, which was evaluated by a bioinformatic and functional study of the NP protein in viral RNPs, which showed that correct NP-vRNA interaction is key to the functioning of RNPs.     

Use of different geometric morphometrics tools for the discrimination of phenotypic stocks of the striped clam Ameghinomya antiqua (Veneridae) in north Patagonia,Argentina

The striped clam (Ameghinomya antiqua (King, 1832)) metapopulation of the San José Gulf is a good model for phenotypic stocks studies based on the shell shape variation in different fishing grounds. These sites show differences in circulation, tidal currents, coastal topography, and bathymetry. Furthermore, it is known that the diverse environments produce differences in the growth rate of this species. In the current work, we study the shell shape changes in the striped clam from different fishing grounds of the San José Gulf through geometric morphometrics methods. Outlines and landmarks analyses were successful to determinate the provenance of the individuals, with almost the 90% of correct assignations for the most of the fishing grounds. The different methodologies showed variations in diverse morphological traits, and as a result different patterns of the shell shape among localities were evidenced. Moreover, each site presented a typical shell shape. Our results showed that the geometric morphometrics methods are powerful tools to identify and separate intra-specific groups. This approach is applicable to other exploited species with subtle shape variations.     

Ocular and periocular manifestations of leishmaniasis in dogs: 105 cases (1993-1998)

The purpose of this retrospective study was to determine the prevalence, type, and prognosis of ocular lesions associated with leishmaniasis in dogs. One hundred and five dogs (24.4% of all cases of leishmaniasis diagnosed during the study period) had ocular or periocular leishmaniasis, and 16 dogs (15.2% of ocular cases) had only ocular lesions and systemic signs were not apparent. Anterior uveitis was the most common manifestation and other prevalent findings included blepharitis and keratoconjunctivitis. Several distinct variations of eyelid lesions were seen including a dry dermatitis with alopecia, diffuse blepharedema, cutaneous ulceration, and discrete nodular granuloma formation. In some cases with keratoconjunctivitis, corneal lesions clinically resembled nodular granulomatous episclerokeratitis. Twenty-seven of the 34 cases with ocular lesions had improvement in signs following systemic antiprotozoal and topical anti-inflammatory therapy, although many cases with anterior uveitis required long-term topical therapy. Response of ocular signs correlated highly with overall, systemic response to therapy. Ophthalmic manifestations of systemic leishmaniasis are common in the dog, and this disease should be considered in the differential diagnosis of most adnexal and anterior segment ocular inflammatory lesions in dogs in endemic areas.     

Attempts to control haemonchosis in grazing ewes by vaccination with gut membrane proteins of the parasite

A vaccination trial was conducted to evaluate the potential benefit of Haemonchus contortus gut membrane proteins as vaccine antigens under field conditions in Louisiana. The trial was conducted in the summer of 1996 in a flock of ewes grazing pasture naturally infected with H. contortus. Ewes were randomly assigned to three treatment groups (vaccine, adjuvant only, and saline) and fecal egg counts (FEC, measured as eggs per gram of feces), packed cell volumes (PCV), and antibody levels were monitored fortnightly for 12 weeks. It was shown by FEC that there were large individual variations in susceptibility to H. contortus in both vaccinated and non-vaccinated sheep, a finding which could have masked differences between treatments when analyzed by conventional statistical methods. Based on their egg counts before the period when the vaccination could have had an effect, all ewes were categorized as 'susceptible' or 'relatively resistant'. The significance of differences between FEC, PCV and antibody responses of vaccinated and control sheep were tested separately for the 'susceptible' and 'relatively resistant' category. The 'susceptible' vaccinates shed 65% fewer worm eggs during the period when the vaccine could have had an effect, but the difference was only significant on Week 6 post-vaccination. In these experiments, it was difficult to completely exclude the confounding effect of having 'relatively resistant' sheep in the control group. More studies are needed to further evaluate H11 and H-gal-GP antigens under field conditions.     

Effects of Ethylene diurea as a protective antiozonant on beans (Phaseolus vulgaris cv Lit) exposed to different tropospheric ozone doses in Catalonia (NE Spain)

A study to evaluate the effects of ethylene diurea (EDU) as a protective antiozonant for beans (Phaseolus vulgaris) submitted to different ambient ozone concentrations was performed in three stations of the rural Network for Air Quality Monitoring (Catalonia, NE Spain). The highest ozone concentrations were accompanied by significant reductions in fruit yield, number of fruits and shoot biomass. These reductions were lower in plants treated with EDU. The results showed toxic ozone effects on sensitive species such as beans and a protective antiozonant effect of EDU. The intensity of the EDU protective effect differed among the different stations characterised by different meteorological conditions and O₃ concentrations.     

Identification of Apoptotic Bodies in Equine Semen

Apoptosis in the testis is required to ensure an efficient spermatogenesis. However, sometimes, defective germ cells that are marked for elimination during this process escape elimination in the testes, giving rise to ejaculates with increased percentages of abnormal and apoptotic spermatozoa and a high percentage of apoptotic bodies. Apoptosis markers in the ejaculate have been associated with low fertility, either in animals or humans. Therefore, the goal of this study was to investigate whether fresh equine semen contains apoptotic bodies [initially named Merocyanine 540 (M540) bodies] and to study the relationship between the quantity of these bodies and cell concentration, the volume of ejaculate, viability and motility. Moreover, we also studied whether the presence apoptotic bodies in fresh semen was related to the resistance of the stallion spermatozoa to being incubated at 37°C or being frozen and thawed. Fresh equine semen was stained with fluorescent dyes such as M540 and Annexin‐V. Active Caspase 3 was studied in fresh semen through Western blotting and immunofluorescence with a specific antibody. Sperm kinematics was assessed in fresh, incubated and thawed samples using computer‐assisted semen analysis, and viability was evaluated with the LIVE/DEAD Sperm Viability Kit. Overall, our results demonstrate for the first time the presence of apoptotic bodies in equine semen. The quantity of apoptotic bodies was highly variable among stallions and was positively correlated with Caspase 3 activity in fresh samples and negatively correlated with the viability and motility of stallion spermatozoa after the cryopreservation process.     

The Effects of Isoflavones on Androgens and Glucocorticoids During Puberty on Male Wistar Rats

Isoflavones are the most common phytoestrogens found in human diets. However, it is still not clear whether isoflavones have effects on the reproductive and the endocrine systems under normal dietary intake and overdose. The aim of this study was to determine how the most important isoflavones, genistein and daidzein, affect androgen and glucorticoid levels on male prepuberal rats. A hundred and seventy‐five 30‐day‐old male Wistar rats were dosed orally by stomach tube every day for 35 days, with saline solution, low and high doses of genistein, daidzein and a mixture of both. Serum samples were analysed by an enzyme immunoassay for hormone determinations. In control group, there was a peak of testosterone (T) and dihydrotestosterone levels associated to the onset of puberty, at the third week. However, in low‐dose groups, the same peak was found at the fourth week (p < 0.05), indicating a delay in the onset of puberty in these groups. Moreover, high doses groups serum androgen levels were significantly lower (p < 0.05) than the control group from the first week until fifth week. This fact was supported by a epididymal histological analysis that indicate in low doses there were several content of spermatozoa at fourth week and in high doses there were few content of spermatozoa. Besides, corticosterone levels followed the same pattern of androgens in all groups. We can conclude that oral administration of isoflavones in male rats decreased the secretion of androgens and glucocorticoids causing a delay in the onset of puberty and may cause physiological and developmental problems.     

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.