A Leishmania infantum multi-component antigenic protein mixed with live BCG confers protection to dogs experimentally infected with L. infantum

The capacity of a quimeric protein, formed by the genetic fusion of five antigenic determinants from four Leishmania proteins, formulated with BCG, to protect dogs against Leishmania infantum infection is described. The data showed that after i.v. administration of 500,000 parasites of the L. infantum M/CAN/ES/96/BCN150 strain, zymodeme MON-1, the animals became infected as suggested by the humoral response against the parasite antigens. All control unvaccinated dogs had parasites in the lymph nodes at day 150 post-infection. One of these unvaccinated infected dog was parasite negative at day 634 behaving, thus, as resistant. In contrast, only 50% of the immunized dogs had parasites in the lymph nodes at day 150 post-infection. Four of these dogs became parasite negative by day 634 post-infection. The control animals developed at various times during the follow-up period clinical symptoms associated with Leishmaniasis. The control diseased dogs developed also in the liver and spleen some of the abnormal histological features associated with natural visceral Leishmaniasis. The immunized dogs, however, were not only normal at the clinical but also at the anatomo-pathological level. A positive delayed type hypersensitivity (DTH) response was observed in nine of the immunized protected dogs. The data indicated that Q+BCG confers 90% protection against infection and at least 90% protection at the clinical level.     

Time course of endocrine changes in the hypophysis-gonad axis induced by hypobaric hypoxia in male rats

Chronic hypobaric hypoxia (CHH) induces a decrease in sperm output and spermatogenesis in male rats. The mechanisms that underlie these changes in testicular function are unknown and could involve changes in the hypophysis-gonad axis. We have tested the hypothesis that changes take place in the endocrine status (FSH, follicle stimulating hormone; LH, luteinizing hormone; testosterone) of rats subjected to CHH. Male Wistar rats were maintained under normobaric or hypobaric conditions (428 torr, 4,600 m). On days 0, 5, 15 and 30 post-exposure, 12 rats were anesthetized, their body weights were measured and blood samples were collected. The testicles were fixed in 4% formaldehyde and processed for histological analysis. In this time course, the FSH levels rose by day 5 post-exposure. On subsequent days, the FSH levels decreased in rats subjected to CHH with a tendency to remain higher than the normoxic group. The LH plasma levels decreased in rats exposed to CHH. Consistent with the decrease in LH levels, the plasma testosterone level decreased significantly after 30 days of CHH exposure. Integrated analysis of hormonal changes in rats subjected to CHH and the body dehydration that occurs in HH allows us to conclude that the effects of CHH on spermatogenesis may be partially related to changes in the hypophysis-gonad hormonal axis.     

Measurement of progesterone in sheep using a commercial ELISA kit for human plasma

Determination of serum or plasma progesterone (P4) concentrations is important to recognize pregnant and non-pregnant ewes, and also to predict the number of carried lambs. The 2 most common methodologies for the detection of plasma P4 are radioimmunoassay (RIA) and enzyme immunoassay (EIA). RIA is very expensive, and not all laboratories are equipped to perform this test; EIA is commercially available for human use, but only a few companies produce species-specific kits, which are expensive. We verified for ovine plasma a less expensive and easily available ELISA kit (DiaMetra) designed to quantify P4 in humans. Pools of ovine and human plasma were used to compare repeatability, accuracy, sensitivity, and stability of P4 measured by the DiaMetra kit. Repeatability data were within 15%, and accuracy values were ~90% for both plasma matrices. Stability data showed a loss of <20% for freeze–thaw and <30% for 30-d storage. All parameters were acceptable under international guidelines for method validation. The human ELISA kit was used successfully to quantify plasma P4 in 26 ewes during pregnancy until delivery. P4 concentrations were also correlated with the number of carried lambs.     

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.     

Salmon cells SHK‐1 internalize infectious pancreatic necrosis virus by macropinocytosis

We have previously shown that infectious pancreatic necrosis virus (IPNV) enters the embryo cell line CHSE‐214 by macropinocytosis. In this study, we have extended our investigation into SHK‐1 cells, a macrophage‐like cell line derived from the head kidney of Atlantic salmon, the most economically important host of IPNV. We show that IPNV infection stimulated fluid uptake in SHK‐1 cells above the constitutive macropinocytosis level. In addition, upon infection of SHK‐1 cells, IPNV produced several changes in actin dynamics, such as protrusions and ruffles, which are important features of macropinocytosis. We also observed that the Na+/H+ pump inhibitor EIPA blocked IPNV infection. On the other hand, IPNV entry was independent of clathrin, a possibility that could not be ruled out in CHSE 214 cells. In order to determine the possible role of accessory factors on the macropinocytic process, we tested several inhibitors that affect components of transduction pathways. While pharmacological intervention of PKI3, PAK‐1 and Rac1 did not affect IPNV infection, inhibition of Ras and Rho GTPases as well as Cdc42 resulted in a partial decrease in IPNV infection. Further studies will be required to determine the signalling pathway involved in the macropinocytosis‐mediated entry of IPNV into its target cells.     

Effect of Pre- and Post-Partum Feed Supplementation on the Productive and Reproductive Performance of Grazing Primiparous Brahman Cows

This experiment studied the combined effect of pre- and post-calving feed supplementation on the productive and reproductive performance of primiparous Brahman cows. The animals were 4 years old (420 kg live weight) and pregnant, and grazed rotationally on Stargrass (Cynodon nlemfuensis) pastures. Supplementation was given before and after (T1, n = 15), only before (T2, n = 15) or only after (T3, n = 16) calving, and there was an unsupplemented control (T4, n = 16). The supplement (1.5 kg/animal per day, 13.4 MJ digestible energy/kg dry matter and 20% crude protein) was given for 45 days before and/or after calving. Changes in body weight and body condition score and the productive behaviour of the calves were similar (p>0.05) among treatments. The concentration of urea in the plasma was low in the control group (T4) before and after calving. The animals in all the groups had a poor reproductive performance. The percentage of cows in oestrus was higher (p<0.05) in T1 (73.3%) than in T3 (37.5%). The interval from calving to conception (148.7±53.7 days) and the pregnancy rate (48.5%) were similar (p>0.05) among the treatments. The level of supplementation offered before and/or after calving did not improve the pregnancy rate.     

Hysterothylacium winteri sp. n. (Nematoda: Anisakidae), a parasite of Chilean rock cod, Eleginops maclovinus (Perciformes: Eleginopidae), from South Chile

Hysterothylacium winteri sp. n. (Nematoda: Anisakidae) was collected from the intestine of a marine-estuarine fish, Eleginops maclovinus (Valenciennes) (Perciformes: Eleginopsidae), from Abtao in the Gulf of Ancud, Chile. Sixteen (51.6%) out of 31 fish were infected; the intensity was 1-10 (mean 4) worms/host. The new species belongs to the group of congeners possessing one double pair of postanal papillae. By possessing a lateral pair of phasmids situated near the tip of tail, H. winteri most closely resembles Hysterothylacium habena. The new species can be distinguished by the lip flanges forming broadly rounded points and the equal, short spicules (320-400 microm long) representing 0.9-1.7% of body length.     

Mortality in relation to growth rate and soil resistance varies by species for underplanted Nothofagus seedlings in scarified shelterwoods

This study evaluated early mortality patterns of planted Nothofagus dombeyi (Mirb.) Blume (evergreen, shade intolerant) and Nothofagus alpina (P. et E.) Oerst. (deciduous, mid-tolerant to shade) seedlings established in scarified gaps in a Nothofagus old-growth forest in the Chilean Andes that was subjected to a shelterwood cut. Soils were scarified with heavy machinery that removed up to 40 cm of the upper horizons (e.g. it completely removed the litter layer and organic horizon, and in some cases partially the A horizon) to eliminate bamboo (Chusquea culeou E. Desv) shoots and rhizomes from the scarified areas. Two years after planting, seedlings were growing free of understory competition and mortality patterns were assessed using logistic regression models with the following predictor variables: (1) basal diameter growth in the prior year, and (2) soil strength measured as resistance to penetration. The probability of mortality of both species was highly predictable based on recent basal diameter growth. N. dombeyi seedlings appeared to be less affected by mortality, as reflected in lower sensitivity of mortality at lower growth rates compared to N. alpina. High values of resistance to penetration (>2,000 kPa) induced a higher probability of mortality in N. alpina (up to 40 % at 3,500 kPa). In contrast, N. dombeyi mortality was not significantly influenced by soil strength, which illustrates the more stress-resistant and pioneer character of this species. This study showed that recent growth rates can be used as a predictor of future mortality, but these relationships need to be calibrated for each species. In addition, our results suggest that species characteristics that relate to resource limitations, such as shade tolerance, are not adequate to understand effects of other stressors, such as soil disturbance, on mortality patterns.     

Spectrophotometric and adsorptive stripping square wave voltammetric determination of iron in olive oils,as complex with 5,5-dimethylcyclohexane-1,2,3-trione 1,2-dioxime 3-thiosemicarbazone (DCDT)

Two methods for the determination of iron in olive oil by spectrophotometry and by adsorptive stripping square wave voltammetry (Ad-SSWV) have been developed. These two methods are based on the formation of a 5,5-dimethylcyclohexane-1,2,3-trione 1,2-dioxime 3-thiosemicarbazone (DCDT)-iron(II) complex in strongly acid media. In both, iron is extracted from the olive oil by using HCl. Spectrophotometric determination of iron with DCDT is based on the feature that the DCDT-Fe complex shows an absorbance maximum at 550 nm. A calibration graph has been constructed from 0 to 4000 ng mL(-)(1), and the detection limit was 115 ng mL(-)(1) (57 ng g(-)(1) in olive oil). On the other hand, the voltammetric determination of the metal is based on the appearance of a peak due to an adsorptive reductive process of the complex that it is observed when the Ad-SSWV technique is used. A calibration graph has been constructed from 0 to 30 ng mL(-)(1), and the detection limit was 0.55 ng mL(-)(1) (13.75 ng g(-)(1) in olive oil according to the proposed procedure).