Production and Easy One-Step Purification of Bluetongue Recombinant VP7 from Infected Sf9 Supernatant for an Immunoenzymatic Assay (ELISA).

Bluetongue (BT) is non-contagious, vector-borne viral disease of domestic and wild ruminants, transmitted by midges (Culicoides spp.) and is caused by Bluetongue virus (BTV). BTV is the type species of the Orbivirus genus within the Reoviridae family and possesses a genome consisting of 10 double-stranded RNA segments encoding 7 structural and 4 nonstructural proteins. Viral Protein 7 (VP7) is the major sera group-specific protein and is a good antigen candidate for immunoenzymatic assays for the BT diagnosis. In our work, BTV-2 recombinant VP7 (BTV-2 recVP7), expressed in Spodoptera frugiperda (Sf9) cells using a baculovirus system, was produced and purified by affinity chromatography from the supernatant of infected cell culture. The use of the supernatant allowed us to obtain a high quantity of recombinant protein with high purity level by an easy one-step procedure, rather than the multistep purification from the pellet. RecVP7-BTV2 was detected using a MAb anti-BTV in Western blot and it was used to develop an immunoenzymatic assay.

Descripción de la población canina deambulante en Villa Regina, Río Negro / Description of stray dog population in Villa Regina, Río Negro

INTRODUCCIÓN La superpoblación canina es una problemática que genera consecuencias sanitarias, ambientales, socioeconómicas y de bienestar animal. La localidad de Villa Regina no escapa a esta situación, y muestra evidencias significativas que lo demuestran. OBJETIVOS Conocer las características de la población canina deambulante en la localidad estudiada, determinando el número y la distribución geográfica. MÉTODOS Se utilizó un diseño cuantitativo, descriptivo y de corte transversal. Para facilitar la recolección de datos y el posterior análisis, se dividió a la ciudad en áreas geográficas más pequeñas llamadas subregiones y luego se hizo un muestreo al azar (sistemático estratificado) de las manzanas contenidas en esas subregiones. La recolección de datos consistió en un relevamiento observacional con recuento de la población canina deambulante. El tratamiento de los datos relevados se realizó mediante análisis cuantitativo. Resultados El total de perros deambulantes estimados para la localidad de Villa Regina en 2013 fue de 1600 (IC 95%: 1443-1763). Se observaron tres focos principales con mayor cantidad de perros deambulantes. La subregión con mayor frecuencia estimada fue la 18, correspondiente al Barrio El Sauce, con un total de 214 perros, seguida de la subregión 15, correspondiente al Barrio 25 de Mayo y Antártida, con 172 perros, y la subregión 3, correspondiente al Barrio Matadero, con 150 perros deambulantes

Geomorphology and habitat diversity in the Pantanal.

The present study deals with the inter-relations in the relief which forms the Bacia do Alto Rio Paraguay (BAP) in mid-west Brazil. The overall aim is to discuss the relationship between relief forms and the biodiversity of the Pantanal. The BAP is a natural environmental system with contrasts in two of the compartments on which it is formed: the plateau, the most elevated compartment, highly transformed by human activities, and the plain which forms the Pantanal, which is more preserved and less transformed in relation to productive activities. The analysis was performed based on publications with a geomorphologic focus, examining the different relief units of the BAP and the dynamics of the revealing processes of landscape change which the Pantanal has undergone since the end of the Pleistocene.

Hydrological cycle.

The Pantanal hydrological cycle holds an important meaning in the Alto Paraguay Basin, comprising two areas with considerably diverse conditions regarding natural and water resources: the Plateau and the Plains. From the perspective of the ecosystem function, the hydrological flow in the relationship between plateau and plains is important for the creation of reproductive and feeding niches for the regional biodiversity. In general, river declivity in the plateau is 0.6 m/km while declivity on the plains varies from 0.1 to 0.3 m/km. The environment in the plains is characteristically seasonal and is home to an exuberant and abundant diversity of species, including some animals threatened with extinction. When the flat surface meets the plains there is a diminished water flow on the riverbeds and, during the rainy season the rivers overflow their banks, flooding the lowlands. Average annual precipitation in the Basin is 1,396 mm, ranging from 800 mm to 1,600 mm, and the heaviest rainfall occurs in the plateau region. The low drainage capacity of the rivers and lakes that shape the Pantanal, coupled with the climate in the region, produce very high evaporation: approximately 60% of all the waters coming from the plateau are lost through evaporation. The Alto Paraguay Basin, including the Pantanal, while boasting an abundant availability of water resources, also has some spots with water scarcity in some sub-basins, at different times of the year. Climate conditions alone are not enough to explain the differences observed in the Paraguay River regime and some of its tributaries. The complexity of the hydrologic regime of the Paraguay River is due to the low declivity of the lands that comprise the Mato Grosso plains and plateau (50 to 30 cm/km from east to west and 3 to 1.5 cm/km from north to south) as well as the area's dimension, which remains periodically flooded with a large volume of water.

Laboratory tests for evaluating the level of attenuation of bluetongue virus.

One of the most important steps when preparing a live attenuated vaccine is the assessment of the level of attenuation in target animals. It is costly and time consuming as it requires, on each occasion, a large number of susceptible animals and contained accommodation. This study assessed the consistency of the bovine foetal aorta endothelial (BFA) cell line and newborn mice for evaluating the attenuation level of BTV4, BTV9 and BTV16 Italian field isolates. Following serial passages in BHK(21c13) or Vero cell cultures, BTV attenuated clones demonstrated a reduced replication capability in the BFA cells compared to the homologous virulent strains. Similarly, following intracerebral inoculation, the attenuated clones were completely innocuous to newborn mice contrary to the homologous virulent strains which killed all animals within 10 days. Vaccines produced with the BTV9 or BTV4 attenuated clones were safe, immunogenic and capable of preventing clinical symptoms and viraemia in sheep following challenge with homologous virulent virus. The two assays may be valuable indicators of the gradual changes occurring in the BTV population leading to virus attenuation, they can predict the safety of a BTV attenuated vaccine and, in turn, reduce the number of sheep and cattle required to assess the level of attenuation attained.

An inactivated vaccine for the control of bluetongue virus serotype 16 infection in sheep in Italy.

Because no suitable products are at the moment available to safely control the spread of BTV-16 in Europe, an inactivated vaccine was produced from the reference field isolate of bluetongue virus serotype 16. One group of six sheep was vaccinated subcutaneously with the inactivated vaccine twice, on days 0 and 28, whereas a second group of eight sheep was inoculated with saline solution and used as mock-vaccinated control animals. Seventy-eight days after the first vaccination, all sheep were inoculated subcutaneously with a suspension containing 10(6.3) TCID(50) of a virulent reference BTV-16 isolate. Apart from a transient inflammatory reaction at the injection site, no adverse effects were reported following vaccination. All vaccinated animals developed high titres (7.3-9.3log(2)(ED50%/50 microl)) of virus-specific neutralising antibodies and were resistant to challenge with BTV-16. Conversely, following challenge, control animals developed hyperthermia and long lasting high-titre viraemia.

Development and validation of a competitive ELISA kit for the serological diagnosis of ovine, caprine and bovine brucellosis.

A competitive ELISA (Brucella-Ab c-ELISA) was standardized and validated for the detection of Brucella antibodies in cattle, sheep and goat sera using a monoclonal antibody (MAb 4B5A) produced against Brucella melitensis biotype 2. The specificity and sensitivity of the assay were 100% to a 67.5% cut-off point (B/Bo%). When compared with an indirect ELISA, the Brucella-Ab c-ELISA did not demonstrate cross-reactions when testing positive sera for antibodies to some Enterobacteriaceae. A comparison was made between the Brucella-Ab c-ELISA and the complement fixation and Rose Bengal tests. Results demonstrated that the Brucella-Ab c-ELISA is a valuable tool for the serological diagnosis of bovine and ovine/caprine brucellosis.

Bluetongue laboratory diagnosis: a ring test to evaluate serological results using a competitive ELISA kit.

The occurrence of bluetongue (BT) in Italy prompted an increase in disease surveillance. Thus a competitive enzyme-linked immunosorbent assay (c-ELISA) to detect immunoglobulins to BT virus (BTV) was developed and distributed amongst 27 laboratories comprising the Italian veterinary diagnostic laboratories network to screen field sera. This ring test enabled comparison of the results and the evaluation of the reproducibility of the method. The c-ELISA developed by the National Reference Centre for Exotic Diseases (c-ELISA-IZSA&M) was compared also against a commercially available c-ELISA. In addition, results obtained by the Centre of Athens Veterinary Institutions are presented.

[Treatment of dry eye syndrome with lacrimal gel: a randomized multicenter study]. / Traitement de la sécheresse oculaire par gel lacrymal: étude multicentrique randomisée.

The appearance on the French market of a carbomer tear substitute, Gel-Larmes, an original galenic form, constitutes a real therapeutic progress in the treatment of dry eye syndrome. An open randomized multicentric trial was conducted comparing the tolerance and efficacy of Gel-Larmes with those of eye drops containing chondroitin sulphate. The statistical analysis evidences an inferior average number of daily instillations for Gel-Larmes, its better efficacy on the symptoms as well as on the objective criteria: biomicroscopic examination, break-up time, rose Bengal test. The tolerance of the product is excellent and 81% of the patients express their wish to pursue Gel-Larmes versus 54% for the reference product.

[Anatomic and functional results in fronto-laterally laryngectomized patients]. / Risultati anatomici e funzionali negli operati di larin-gectomia fronto-laterale.

The present work reports the experience acquired on 33 males who had undergone fronto-lateral laryngectomy at least three years before. The patients were divided into 4 groups according to cord neoplastic extension: 1) in 9 subjects the neoplasm was localized to the anterior half or two-thirds of the vocal cord; 2) in 9 others the entire vocal cord was involved; 3) in 8 the tumor extended forward to the median line; that is to the anterior commissure; 4) in 7 either the ventricle (4 cases) or the subglottis zone (3) were involved. In 30 cases (90%) complete clinical healing was achieved; for 14 survival has exceeded the conventional 5-year limit. Two of the three recurrences were remedied by total laryngectomy for which follow-up has proved favorable for over 5 years. In regard to functional results both phonatory and respiratory results have proved satisfactory. In regard to the latter, due to insufficient endolaryngeal calibration of the scar tissue, it proved impossible to remove the tracheal cannula in three cases. Based on the results obtained the validity of the fronto-lateral laryngectomy in terms of oncology and functional results is underlined. The authors fully agree with the caution generally taken in terms of indications. However, at the same time, they feel that careful clinical-radiological evaluation, supported by intraoperative findings as well as extemporaneous testing, makes it possible to rely on this form of surgery in cases of unilateral cord tumours of different extensions, although naturally within the limitations of oncological radicality.