Subolesin/akirin orthologs from Ornithodoros spp. soft ticks: cloning, RNAi gene silencing and protective effect of the recombinant proteins

Subolesin/akirin is a well characterized protective antigen highly conserved across vector species and thus potentially useful for the development of a broad-spectrum vaccine for the control of arthropod infestations including hard ticks, mosquitoes, sand flies and the poultry red mite Dermanyssus gallinae. Soft ticks could be also targeted by this vaccine if proved that the soft tick subolesin orthologs are conserved and induce protective immune responses too. However, to date no soft tick subolesin orthologs have been fully characterized nor tested as recombinant antigens in vaccination trials. The objectives of the present work were to clone and characterize the subolesin orthologs from two important vector species of soft ticks as Ornithodoros erraticus and O. moubata, to evaluate the effect of subolesin gene silencing by RNAi, and to test the protective value of the recombinant antigens in vaccination trials. The obtained results demonstrate that both soft tick subolesins are highly conserved showing more than 69% and 74% identity with those of hard ticks in their nucleotide and amino acid sequences, respectively. Additionally, we demonstrate that both soft ticks possess fully operative RNAi machinery, and that subolesin gene silencing by dsRNA injection inhibits oviposition indicating the involvement of subolesin in tick reproduction. Finally, vaccination with the recombinant soft tick subolesins induced a partial protective effect resulting in the reduction of the oviposition rate. These preliminary results encourage further studies on the use of recombinant subolesins as vaccines for the control of soft tick infestations, either alone or in combination with other specific molecules.     

Unrecognized diversity in New Guinean crayfish species (Decapoda,Parastacidae): The evidence from molecular data

The phylogenetic relationships among imported ornamental crayfish belonging to the genus Cherax were inferred from a combined dataset of 3 mitochondrial genes (COI, 16S and 12S) and by comparison with available GenBank sequences of 14 Cherax species. Furthermore, the concordance of previously described species obtained from a wholesaler (Cherax boesemani, C. holthuisi and C. peknyi) with available GenBank sequences was verified based on COI with special respect to comparison with sequences assigned as Cherax species. Recently described species C. gherardiae, C. pulcher and C. subterigneus belong to the northern group of Cherax species. Comparison and analysis with other GenBank COI sequences show previously unreported diversity of New Guinean species, suggesting 5 putative new species. Surprisingly, species assigned to the subgenus Astaconephrops do not form a monophyletic clade; this subgenus should be reappraised relative to the purported typical morphological characteristic of the uncalcified patch on male chelae. Increasing importation of crayfish underscores the importance of accurate species identification. Use of basic molecular methods is a necessary requisite for documenting occurrence, abundance and population trends of target species. Consequently, it helps to support eventual conservation decision‐making by stakeholders.     

Influence of Temperament Score and Handling Facility on Stress,Reproductive Hormone Concentrations,and Fixed Time AI Pregnancy Rates in Beef Heifers

The objectives were (i) to evaluate the effect of temperament, determined by modified 2‐point chute exit and gait score, on artificial insemination (AI) pregnancy rates in beef heifers following fixed time AI and (ii) to determine the effect of temperament on cortisol, substance‐P, prolactin and progesterone at initiation of synchronization and at the time of AI. Angus beef heifers (n = 967) at eight locations were included in this study. At the initiation of synchronization (Day 0 = initiation of synchronization), all heifers received a body condition score (BCS), and temperament score (0 = calm; slow exit and walk or 1 = excitable; fast exit or jump or trot or run). Blood samples were collected from a sub‐population of heifers (n = 86) at both synchronization initiation and the time of AI to determine the differences in serum progesterone, cortisol, prolactin and substance‐P concentrations between temperament groups. Heifers were synchronized with 5‐day CO‐Synch+ controlled internal drug release (CIDR) protocol and were inseminated at 56 h after CIDR removal. Heifers were examined for pregnancy by ultrasound 70 days after AI to determine AI pregnancy. Controlling for synchronization treatment (p = 0.03), facility design (p = 0.05), and cattle handling facility design by temperament score interaction (p = 0.02), the AI pregnancy differed between heifers with excitable and calm temperament (51.9% vs 60.3%; p = 0.01). The alley‐way with acute bends and turns, and long straight alley‐way had lower AI pregnancy rate than did the semicircular alley‐way (53.5%, 56.3% and 67.0% respectively; p = 0.05). The serum hormone concentrations differed significantly between different types of cattle handling facility (p < 0.05). The cattle handling facility design by temperament group interactions significantly influenced progesterone (p = 0.01), cortisol (p = 0.01), prolactin (p = 0.02) and substance‐P (p = 0.04) both at the initiation of synchronization and at the time of AI. Inter‐ and intra‐rater agreement for temperament scoring were moderate and good (Kappa = 0.596 ± 0.07 and 0.797 ± 0.11) respectively. The predictive value for calm and pregnant to AI was 0.87, and excited and non‐pregnant to AI was 0.76. In conclusion, the modified 2‐point temperament scoring method can be used to identify heifers with excitable temperament. Heifers with excitable temperament had lower AI pregnancy. Further, cattle handling facility design influenced the temperament and AI pregnancy.     

Response of gladiolus (Gladiolus spp) plants after exposure corms to chitosan and hot water treatments

The state of Morelos, Mexico has gradually become an important producer of gladiolus. Some preconditioning treatments of corms are empirically done causing uneven emergence and low quality of flowers. In this investigation, before planting, gladiolus corms var. ‘Blanca Borrego’ were dipped in chitosan (chitosan reagent and commercial chitosan Biorend^®), in hot water at various temperatures and in treatments combined with Biorend^® at 1.5% and hot water. Results indicated that the most influenced variables were corm germination, number of flowers per spike, number of cormlets and vase life. Overall, the commercial product Biorend^® at 1.5% accelerated corm emergence in approximately 4 days, the number of flowers increased by 2–7 and the vase life extended for 3 days. The number of cormlets was also duplicated. Corms dipped in the commercial chitosan Biorend^® at 1.5% at different intervals of time were not greatly affected except for the emergence and number of cormlets. However, for this experiment there were significant effects on the number of leaves and flowers because of the interactions between chitosan and the immersion time. The temperature of 55 °C affected plant development because emergence was delayed by 6 days; and there were less number of leaves, flowers and cormlets. On the other hand, the incidence of Fusarium oxysporum in naturally infected corms was 0% at temperatures of 55 °C and 50 °C. Immersion times (0, 10, 15 and 20 min) in hot water at 50 °C did not show significant effects on plant development and vase life. Corms dipped in Biorend^® at 1.5% and hot water at 50 °C accelerated their emergence for about 1–7 days, the number of flowers increased by two, extended the storage life for 1–3 days and increased the number of cormlets. The integration of these two treatments -Biorend^® and hot water- might be a good option for increasing the gladiolus plant quality and vase life.     

Persistent Hematuria as a Result of Chronic Renal Hypertension Secondary to Nephritis in a Stallion

A 13-year-old Lusitano stallion was referred to our institution with a history of severe hematuria for 8.5 months. The origin of the hematuria was determined to be the left kidney. The diagnostic workup failed to identify obstructive, infectious, or neoplastic conditions. The history and ultrasonographic examination suggested a chronic condition. The stallion was subjected to left nephrectomy because of the persistent hematuria and anemia. A histopathological examination detected lesions, which were compatible with chronic nephritis and vascular renal hypertension. It is hypothesized that changes in the normal parenchymal architecture produced a vascular aberration that led to renal hypertension, with subsequent blood extravasation and hematuria. The horse recovered completely after nephrectomy.     

Nitric oxide impacts bovine sperm capacitation in a cGMP‐dependent and cGMP‐independent manner

We aimed to elucidate whether NO acts in in vitro sperm capacitation in bovine via cGMP/PKG1 pathway. For this, cryopreserved bovine sperm were capacitated in vitro with 20 µg/ml heparin (Control) plus treatments: 1 mM L‐arginine (L‐arg, NO precursor), 50 µM Rp‐8‐Bromo‐β‐phenyl‐1,N²‐ethenoguanosine‐3′,5′‐cyclic monophosphorothioate (Rp‐8‐Br‐cGMPS, selective inhibitor of the binding site for cGMP in PKG1), 1 mM 2‐Phenyl‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl 3‐oxide (PTIO, NO scavenger), and the combinations of L‐arg + RP‐8‐Br‐cGMPS and L‐arg + PTIO. Sperm motility and vigour were determined by phase‐contrast microscopy, capacitation status by chlortetracycline staining, and the intracellular concentration of cGMP was measured by ELISA. Data were subjected to analysis of variance and means compared with SNK test at 5% probability. Motility and vigour were lower in sperm treated with PTIO when compared to Control and other treatments (p < .05). The L‐arg treatment showed the highest percentage of capacitated sperm when compared to the Control and other treatments (Rp‐8‐Br‐cGMPS, L‐arg + Rp‐8‐Br‐cGMPS and PTIO) (69.8 ± 3.4%, 51.2 ± 3.0, 51.1 ± 2.1, 51.2 ± 3.0 and 45.5 ± 2.7, respectively) (p < .05). The capacitation ratio (%) was lower in treatments with Rp‐8‐Br‐cGMPS, L‐arg + Rp‐8‐Br‐cGMPS and PTIO, respectively (p < .05). Lastly, cGMP concentration (pmol/ml) was lower in PTIO and L‐arg + PTIO (1.3 ± 0.3 and 1.6 ± 0.4) and was higher in Rp‐8‐Br‐cGMPS and L‐arg + Rp‐8‐Br‐cGMPS (3.7 ± 0.4 and 4.0 ± 0.5) treatments. We showed that during in vitro capacitation of cattle: (a) NO influences sperm motility and vigour; (b) NO is associated with cGMP synthesis through two independent pathways and (c) the cGMP/PKG1 pathway has a partial role in sperm capacitation and does not involve the L‐arg/NO.