Production and characterization of a panel of monoclonal antibodies for the identification of antigens of Babesia bovis

A panel of monoclonal antibodies was produced and characterized by an indirect fluorescent antibody test (IFAT), an enzyme-linked immunosorbent assay (ELISA) and Western blotting with the aim of identifying antigens of Babesia bovis. After fusion, the resultant hybrids were selected by the IFAT, cloned, maintained in culture in vitro, and cryopreserved in liquid nitrogen. Ten clones producing monoclonal antibodies were found to react against the entire merozoites, three reacted on the surface of the merozoites, and one clone reacted against the polar region of the merozoites. All monoclonal antibodies reacted in ELISA, with the optical density varying from 0.368 to 0.502 (cut off = 0.022). The bands recognized by the monoclonal antibodies in Western blotting had molecular weights ranging from 162 to 19 kDa. Four clones recognized a single band of 73 kDa, and another four did not react in Western blotting.     

OC4Influence of Seminal Plasma Added to Highly Diluted Semen on Sperm Motility of Young Boar Feed with Selenium and Vitamin E

High dilution rates have been documented as detrimental for boar spermatozoa, shortening their lifespan (Centurion et al. 2003, Biol Reprod 69: 640–646). Addition of seminal plasma (SP) to semen extenders, or selenium (Se) and vitamin E (VE) in diet of boars could increase motility of highly diluted spermatozoa (HDS). The aim of this work was to evaluate the effect of seminal plasma on sperm motility of HDS from boars feed with Se and VE. Sixteen 12 month-old boars were designed to one of four dietary treatments: (i) control, Se 0 ppm–VE 0 IU/kg; (ii) Se 0–VE 250; (iii) Se 0.5–VE 250 and (iv) Se 0.5–VE 0. Boars were treated for 8 weeks before semen collection. Sperm rich fractions from each boar were diluted to 5 × 10⁶ sperm/ml in PBS medium and incubated at 37°C with or without 10% SP. The measurements were done at 0, 2 or 5 h. Data were analyzed as a mixed model for a factorial design [2 (Se) × 2 (VE) × 2 (SP) × 3 (h)]. Percentage of sperm motility (PSM) increases significantly (p < 0.001) with addition of Se (81.3 ± 1.52), VE (81.0 ± 1.62) and SP (81.5 ± 1.57) vs control (73.4 ± 1.61). There was significant interaction Se × VE (p < 0.001) and Se × VE × SP (p < 0.05) in PSM. However, PSM was affected significantly by time (0 h 83.4 ± 1.92; 2 h 80.7 ± 1.92 and 5 h 67.9 ± 1.92; p < 0.001). There was significant interaction SP × Time (p < 0.05) in PSM. These results indicate that Se, VE and SP improve seminal viability. Addition of 10% of SP maintains PSM at least during 5 hours.     

Phenotypic characterisation of Phaeoacremonium and Phaeomoniella strains isolated from grapevines: enzyme production and virulence of extra-cellular filtrate on grapevine calluses

Extra-cellular enzyme production of different Phaeoacremonium spp. and Phaemoniella chlamydospora isolates were used to assay the possibility of inter-specific characterisation. Isolates of Phaeoacremonium aleophilum, Phaeoacremonium angustius, P. viticola and Ph. chlamydospora were grown on solid media and the activities of extra-cellular amylases, lipases, proteases, cellulases, xylanases, laccase, polygalacturonase, pectate lyase, lignin peroxidase, manganese peroxidase, urease and chitinase were assayed. Phaeoacremonium species showed activities of a larger number of enzymes and also enzyme activity was frequently higher suggesting that Phaeoacremonium can be more virulent. To assay if the produced extra-cellular enzymes could reflect the virulence capacity of the two genera, calluses of Vitis vinifera L. (cvs. Baga and Maria Gomes) and of a rootstock (R3309) were inoculated with filtrated culture liquid medium of three isolates of Ph. chlamydospora and one of P. angustius. Filtrates from all strains decreased callus growth and membrane integrity, while soluble protein content of calluses decreased with the strains CAP 054 and 1AS. P. angustius (CAP 054) induced the more severe symptoms in all genotypes. Water content decreased together with an increase of osmolality in both cultivars but not in rootstock suggesting that osmorregulatory capacity is more affected in cultivars. Data show that: (1) Phaeoacremonium and Phaeomoniella genera have different patterns of extra- cellular enzymatic production; (2) these fungi produce extra-cellular compound(s) that induce(s) senescence symptoms in plant cells inhibiting callus proliferation; (3) among the strains tested in plant calluses the most virulent isolate (CAP 054) also produced higher amounts of some extra-cellular enzymes; (5) rootstock calluses were less sensitive to inoculation than grapevine calluses.     

Uncoupling and inhibition properties of 3,4-seco-friedelan-3-oic acid isolated from Maytenus imbricata

3,4-Seco-friedelan-3-oic acid was isolated from Maytenus imbricata (Celastraceae). At low concentrations it inhibited non-cyclic electron transport and ATP synthesis in spinach chloroplasts, i.e., it behaved as a Hill reaction inhibitor, and at high concentrations it acts as an uncoupler by enhancing uncoupled electron transport and Mg²⁺-ATPase activity. 3,4-Seco-friedelan-3-oic acid did not inhibit PSII electron transport from DPC to DCPIP_ox and photosystem I activity, but it enhanced from TMQH₂ to MV, corroborating its action as uncoupler. It inhibits electron flow through PSII from water to sodium silicomolybdate. The whole results indicate that the 3,4-seco-friedelan-3-oic acid target is at the OEC complex enzyme, the donor side of PSII. The fluorescence decay data shows the formation of the K-band, which match this result, acting as inhibitor at the donor side of PSII and it as an uncoupler.     

Effects of deficit irrigation strategies on cluster microclimate for improving fruit composition of Moscatel field-grown grapevines

The grapevine plays a very important role in the economic, social and cultural sectors of many regions; however vineyards are often grown in regions under stressful conditions and thus they are vulnerable to climate change. The objective of this research was to investigate the effect of partial root-zone drying (PRD) irrigation on vine water relations, vegetative growth, plant microclimate, berry composition and yield components, compared to conventional deficit irrigation (DI, 50% ETc), full irrigation (FI, 100% of ETc) and non-irrigated vines (NI). The study was undertaken in mature ‘Moscatel’ grapevines (Vitis vinifera L.) grown in Pegões, South of Portugal. Compared to the other irrigated treatments, PRD vines showed a better microclimate at the cluster zone with higher incident photosynthetic photon flux density (PPFD). Within the more open canopies of NI and PRD treatments, berry temperatures were higher than those of denser ones (DI and FI). Compared to the conventional irrigation technique the better microclimate observed in PRD vines was a consequence of a reduction in vine growth, where lower values of leaf layer number, leaf area, canopy wideness, water shoots and shoot weight were observed. In PRD vines we observed a tendency to a development of a deeper root system, while DI and FI showed a more homogeneous root distribution throughout the different soil layers. PRD showed an improvement in berry quality with higher values of flavour precursors, and total phenols concentration without any significant yield reduction compared to DI and FI.     

Molecular analysis of resistance mechanisms to Orobanche cumana in sunflower

Resistance to the dicotyledenous parasite Orobanche cumana in sunflower is characterized by a low number of parasitic attachments and a confinement of the parasite in host tissues leading to its necrosis. To help understand what determines such resistance mechanisms, molecular, biochemical and histological approaches were employed before (early response) and after (late response) attachment of the broomrape parasite to susceptible (2603) and resistant (LR1) sunflower genotypes. The expression patterns of 11 defence-related genes known to be involved in different metabolic pathways (phenylpropanoids, jasmonate, ethylene) and/or in resistance mechanisms against microorganisms were investigated. RT-PCR and cDNA blot experiments revealed that the resistant genotype exhibited a stronger overall defence response against O. cumana than the susceptible one, involving marker genes of the jasmonate (JA) and salicylic acid (SA) pathways. Among them, the SA-responsive gene, def. (defensin), appeared to be characteristic of LR1 sunflower resistance. However, no JA accumulation and similar SA contents (250–300 ng g⁻¹ FW) were measured by GC/MS in both genotypes, parasitized or not. In addition, three cDNAs, isolated by a suppression-subtractive hybridization, were shown to be strongly induced only in the resistant genotype 8 days post-inoculation, when the first O. cumana attachments occurred. These genes, putatively encoding a methionine synthase, a glutathione S-transferase and a quinone oxidoreductase, might be involved in detoxification of reactive oxygen species, suggesting the occurrence of an oxidative burst during the incompatible interaction. Finally, host cell-wall modifications leading to parasite-confinement were correlated with more intense callose depositions in the resistant genotype, concomitant with over-expression of the callose synthase cDNA HaGSL1 .     

Variability in feeding ecology of a South American cichlid: a reason for successful invasion in mediterranean‐type rivers?

Abstract –  Ecological attributes behind the success of the South American cichlid Australoheros facetus in mediterranean-type rivers remain unclear. This study addresses this issue, by analysing its abundance and feeding patterns in the lower Guadiana drainage (Portugal), during the dry season of 2003. Despite slight spatial variations in abundance and individual size, A. facetus seemed well established in the Guadiana, Vascão and Ardila rivers. No seasonal or size-related changes in feeding intensity were found, but the diet changed across rivers, over time and throughout ontogeny. Feeding seemed highly generalist, with fish apparently reacting to both spatial and temporal changes in food supply and tending to use the most abundant and easily accessible food items. Overall prey breadth was broad relative to that of other native and non-native species. It is suggested that a generalist feeding strategy may play a significant role in the establishment of non-native fish in mediterranean-type rivers.     

The isolation and serology of Brucella melitensis in a flock of goats in central RSA

Brucella melitensis biotype 1 was isolated in pure culture from the lungs, liver, spleen, kidney, stomach contents, abomasum and brain of an aborted caprine (Boer goat) foetus in the district of Cullinan near Pretoria. The 18 does and 1 ram in the flock of Boer goates were examined serologically by means of the complement fixation (CF) test, using Brucella abortus antigen. Six weeks later they were examined again, using B. abortus as well as B. melitensis biotype 1 antigens. No significant differences were found between the 2 CF tests using B. abortus antigen, or between the results obtained by using the B. abortus and B. melitensis antigens. Twelve goats, showing CF antibody titres, were slaughtered and examined bacteriologically. No relationship was found between the serological and bacteriological results.