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.     

Effects of X-rays on Tuta absoluta for use in inherited sterility programmes

Tuta absoluta is a key pest of tomato crops originating from South America. The consequences of X-radiation on this species were studied under laboratory controlled conditions. The effect of radiation on adult emergence was evaluated exposing male and female pupae to increasing X-rays. Adult emergence decreased as doses of X-radiation increased, with the appearance of deformities such as malformed wings and bent legs at doses ≥350?Gy. Besides, males and females obtained from irradiated pupae were out crossed with untreated counterparts to explore the effects of X-radiation on inherited sterility. (a) Irradiated male?×?untreated female crosses. Both fecundity and fertility of the untreated females were reduced by radiation, and the effect was stronger as the doses increased. Neither the longevity of parental males and F1 adults nor the sex ratios of the F1 and F2 generations were affected by X-radiation (F1 and F2: first and second generation of descendants of irradiated adults). Inherited sterility effects were manifested by a significant reduction in the F1 fecundity, F1 fertility, and the amount of larvae and pupae produced. Doses of 200–250?Gy could be used to induce inherited sterility in T. absoluta males. (b) Untreated male?×?irradiated female crosses. The minimum dose at which irradiated females were completely sterile was 200?Gy. The present study is the first study in T. absoluta that provides the starting point for implementing the inherited sterility in this species.     

Differential gene expression and apoptosis markers in presymptomatic scrapie affected sheep

Bluetongue viruses (BTVs) could invade N-W Europe similar to BTV serotype 8 (BTV8/net06), since the source and route of introduction of this virus has not been solved. Therefore, the Dutch survey for Bluetongue by PCR testing was extended by further analysis of PCR positives to identify the involved BTV. In late August 2008, BTV was reported with 12 nucleotide differences in the S10 amplicon (S10 genotyping). This virus was identified as serotype 6, here named BTV6/net08. Promptly, serotype specific real-time PCR tests were developed for serotypes 1, 6, and 8 (S2 genotyping). Agreement was found between results by S10- and S2 genotyping. Further, BTV1 was identified by both S10- and S2 genotyping in one imported animal. After initial discovery of BTV6 in the Netherlands, animals from 18 holdings tested PCR positive for BTV6/net08 in 2008. Remarkably only one or two PCR positive animals per holding were found. Serum neutralization tests did not result in the discovery of more BTV6 infected animals. Retrospective studies indicated no evidence for infections by BTV6/net08 prior to the first discovery. Experimental infections with BTV6/net08 did not cause clinical disease in sheep, calves and cattle, except for a very short fever in some animals. This clearly showed that the vaccine-related BTV6/net08 is not virulent. BTV6/net08 was not found by passive and active surveys in the years after its discovery. Apparently, BTV6/net08 was not efficiently transmitted by endemic species of Culicoides in N-W Europe, and disappeared without the need of any control measure.     

Dual-label time-resolved fluoroimmunoassay for simultaneous quantification of haptoglobin and C-reactive protein in meat juice from pigs

A new method was developed to simultaneously measure 2 acute-phase proteins (APPs) by time-resolved immunofluorometry. The assay, based on double-label quantification of haptoglobin (Hp) and C-reactive protein (CRP) in meat juice samples from pigs, was constructed by use of a combination of europium and samarium chelate lanthanides as labels. Meat juice samples from 154 pigs were used for analytic and clinical validation of the assay through determination of precision, accuracy, limit of detection, and quantification. The analytic performance of the assay was satisfactory, with good intra-assay and interassay precision and accuracy. The levels of Hp and CRP were increased in the meat juice samples of diseased animals compared with healthy ones. According to the results, higher sensitivity could be achieved if the cut-off values of both proteins were taken into account for clinical relevance rather than used individually. Since the dual assay saved both time and sample, it could be used as a rapid and sensitive screening test in porcine production.     

Compositional and structural differences in two laurel forest stands (windward and leeward) on Tenerife, Canary Islands

The laurel forest of Anaga is the most emblematic community of the Canarian Archipelago. Restoration programs are being developed to increase laurel forest area on the island of Tenerife. Structural and spatial characteristics determine many aspects of the community, including regeneration patterns, disturbance level, stand history. In spite of the importance of this information for restoration, few quantitative studies have been conducted on laurel forest dynamics. We analyzed two stands of the Anaga laurel forest of different aspect. The main difference between the two sites was the wind exposure, one leeward and the other windward. Regeneration, spatial distribution of regeneration, tree species composition, asexual regeneration and environmental parameters were analyzed in three 50 × 50 m plots at each site. Both sites differ in important aspects such as species richness, species composition, asexual regeneration and dead tree composition, while they are not different in basal area, density, density of regeneration and density of dead trees. Both sites have had similar management in the last century. Asexual regeneration is able to maintain the present species composition, while sexual regeneration is able to offer future changes in the canopy composition. Regeneration strategies and the effect of some environmental characteristics should be considered in restoration programs.     

Comparative performance of tests using cytosolic or outer membrane antigens of Brucella for the serodiagnosis of canine brucellosis

Although some ELISA tests using cytoplasmic or outer membrane antigens of Brucella have been developed to improve the diagnosis of canine brucellosis, the performance of these assays has not been compared. In the present study three ELISA tests using lipopolysaccharide (LPS)-free cytoplasmic proteins (CPs) of Brucella abortus, the lumazine synthase (LS) of Brucella spp. or a hot-saline (HS) extract of Brucella canis containing outer membrane antigens were used to test sera from dogs with suspected or confirmed brucellosis (n=36) and from dogs with pathological conditions other than brucellosis (n=212). In the first group the proportion of positive results was 92, 92 and 81% for the ELISAs with HS, CP and LS, respectively, and 94% of the samples were positive by at least one ELISA test. Three dogs that were negative by agglutination (2ME-RSAT) had a positive result by at least one ELISA, and this discrepancy was attributed to the lower analytical sensitivity of agglutination tests. This hypothesis was confirmed by a serological follow-up of seven dogs recently infected with B. canis in three of which the illness was diagnosed earlier by one or more ELISA tests than by 2ME-RSAT. Among dogs having pathological conditions other than brucellosis, specificities were 94.3, 96.7 and 96.7% for the ELISAs with HS, CP and LS, respectively. This study shows that HS-ELISA and CP-ELISA are highly specific and sensitive for the diagnosis of canine brucellosis and can detect the infection by B. canis shortly after the exposure to the pathogen.     

A genetic map of an interspecific diploid pseudo testcross population of coffee

Coffee is globally one of the most important export crops and is a prominent part of the economy in more than 50 countries in Latin America, Africa and Asia. In Colombia, it has been the leading export commodity for more than a century. However, genetic research on coffee has been rather sparse and mainly focused on the two major cultivated species, Coffea arabica L. and C. canephora P., leaving unexplored the genetic potential in other species. In this study, an interspecific mapping population consisting of 101 F₁ hybrid plants from a cross between the diploid species C. liberica and C. eugenioides was evaluated for genetic segregation at 618 molecular marker loci. Of these, 168 SSRs and two ESTs exhibited polymorphic patterns that allowed segregation analysis and genetic linkage estimations. A genetic map consisting of 146 co-dominant loci and 11 predicted linkage groups was constructed using the mapping software JoinMap 3.0. The conjoined maternal/paternal map length is 798.68 cM, has an average saturation density of 6.01 cM/interval, and covers an estimated 66–86 % of the diploid coffee genome. Approximately 24 % of loci had null alleles, and 23.5 % exhibited segregation distortion. Knowledge derived from this study has important applications for quantitative trait locus analysis and marker-assisted selection in Colombian coffee breeding programs.