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.     

Ultrasonographic assessment of maternal cardiac function and peripheral circulation during normal gestation in dogs

The aim of this study was to describe changes in cardiac morphology, systolic function and some peripheral hemodynamic parameters during normal pregnancy in dogs. Twenty healthy bitches, 10 pregnant (PG) and 10 non-pregnant controls (CG), were evaluated every 10 days using echocardiography from day 0 of the estrus cycle to parturition or to day 65 for the PG and CG groups, respectively. Systolic blood pressure (SBP) and uterine artery resistance index (RI) were also assessed. Throughout the study, the shortening fraction and cardiac output increased up to 30% vs. 5% (P<0.01) and 45% vs. 2% (P<0.01) in the PG and CG groups, respectively. In contrast, SBP and RI diminished up to 20% vs. 1% (P<0.01) and 29% vs. 0% (P<0.01) in the PG and CG groups, respectively. In conclusion, a decrease in afterload, an increase in cardiac output and cardiac hypertrophy appear to be the result of the hemodynamic modifications occurring during pregnancy in dogs.     

Major outer membrane proteins of Brucella spp.: past,present and future

The major outer membrane proteins (OMPs) of Brucella spp. were initially identified in the early 1980s and characterised as potential immunogenic and protective antigens. They were classified according to their apparent molecular mass as 36–38 kDa OMPs or group 2 porin proteins and 31–34 and 25–27 kDa OMPs which belong to the group 3 proteins. The genes encoding the group 2 porin proteins were identified in the late 1980s and consist of two genes, omp2a and omp2b, which are closely linked in the Brucella genome, and which share a great degree of identity (>85%). In the 1990s, two genes were identified coding for the group 3 proteins and were named omp25 and omp31. The predicted amino acid sequences of omp25 and omp31 share 34% identity. The recent release of the genome sequence of B. melitensis 16 M has revealed the presence of five additional gene products homologous to Omp25 and Omp31. The use of recombinant protein technology and monoclonal antibodies (MAbs) has shown that the major OMPs appear to be of little relevance as antigens in smooth (S) B. abortus or B. melitensis infections i.e. low or no protective activity in the mouse model of infection and low or no immunogenicity during host infection. However, group 3 proteins, in particular Omp31, appear as immunodominant antigen in the course of rough (R) B. ovis infection in rams and as important protective antigen in the B. ovis mouse model of infection. The major OMP genes display diversity and specific markers have been identified for Brucella species, biovars, and strains, including the recent marine mammal Brucella isolates for which new species names have been proposed. Recently, Omp25 has been shown to be involved in virulence of B. melitensis, B. abortus and B. ovis. Mutants lacking Omp25 are indeed attenuated in animal models of infection, and moreover provide levels of protection similar or better than currently used attenuated vaccine strain B. melitensis Rev.1. Therefore, these mutant strains appear interesting vaccine candidates for the future. The other group 3 proteins identified in the genome merit also further investigation related to the development of new vaccines.     

The Bolivian “Altiplano” and “Valle” sheep are two different peripatric breeds

Forty-nine sheep belonged to the Andean Altiplano region (“Altiplano”) and 30 in the lowland regions of Bolivia (“Valle”), aged 1 to 4 years, were wool sampled to determine the extent of difference between these local breeds. Fibre length and the percentage of each type of fibre (long-thick, short-thin and kemp), yield and fibre diameter were measured. There was a highly significant difference between the two sheep populations that were not clearly separated in the first two principal component of a principal components analysis (PC); the first PC explained 67.1 % and the second PC explained 26.6 % of the total variation. The variables that contributed most to the separation of the sheep populations were the percentage of long-thick and short-thin fibres in the first PC and yield in the second PC. A discriminant analysis, which was used to classify individuals with respect to their breeding, achieved an accurate classification rate of 84.2 %. Thus, the Altiplano and Valle sheep must be viewed as two closely peripatric breeds rather than different “ecotypes”, as more than 80 % could be correctly assigned to one of the breeds; however, the differences are based on composition of long-thick and short-thin fibres and yield after alcohol scouring.     

Integrative approach of histopathology and histomorphometry of common carp (Cyprinus carpio L.) organs as a marker of general fish health state in pond culture

The semi‐intensive system of common carp monoculture in earthen ponds is currently the most practiced fish rearing system in several countries in Eastern Europe. This system is based on the addition of supplemented feed to ponds to achieve faster growth. In this study, the general state of the fish health was monitored when three different types of feeds were used: grain cereals (CF), commercial pelleted feed and extruded feed. The markers used were: (i) the histopathological index (HP) of the main organs: gills, liver and kidney; and (ii) the histomorphometric parameters of the hepatocytes and enterocytes to assess the state of the digestive system in fish fed different types of supplemented feed. At the end of the experiment, the fish were in good health, since irreversible lesions were not noticed to a greater extent in any of the fish organs. There were also no other severe lesions present. However, the results showed differences in the HP indices of the liver and gills, as well as the total HP index, indicating a better health status of the fish in the CF group. The histomorphometric results showed larger cytoplasmic areas of hepatocytes in fish fed CF, which is attributed to glycogen accumulation and higher numbers of goblet cells in the distal intestines of fish fed compound feeds. This can be attributed to slight inflammation.     

Characterization of Dissolved Organic Matter from Sewage Sludge using 3D-Fluorescence Spectroscopy and Chemometric Tools

The aim of the present article is to show the possibilities of chemometric tools and the parallel factor analysis (PARAFAC) model, as well as to understand the complexities of the fluorescence emission-excitation matrix (EEM) of water-soluble organic matter (WSOM) extracted from sewage sludge samples obtained with different origins and stabilization procedures. The variation in the composition of WSOM in the different sewage sludge samples could be correlated with the conditions of stabilization; therefore, the use of fluorescence spectroscopy to monitor these changes can be a useful tool to optimize the management of this residual flux, avoiding environmental pollution. However, the “contour density” EEM maps obtained with fluorescence present continuous spectra with different peaks, valleys, and shoulders where manual peak picking can be complicated and subjective, especially in complex matrixes such as sewage sludge. With the PARAFAC algorithm, it is possible to resolve the emission and excitation spectra of the different fluorescence components present in the samples and their contribution to the total fluorescence. Also, the contribution (scores) of the different fluorophors can be coupled with another chemometric tool to provide an effective classification method using the stabilization conditions of sewage sludge as main criterion. In this study, a wide survey of sewage sludge samples (287) was characterized by fluorescence emission-excitation matrix. The final molecular composition of the sewage sludge samples was not related to the stabilization (aerobic vs anaerobic) treatments. However, a sewage sludge classification based on a combined spectroscopic-chemometric approach was obtained, which could be used for the optimization of sewage sludge use in agriculture, minimizing the environmental risks.     

Genotyping of Bacillus anthracis Isolated from Croatia and Bosnia and Herzegovina

Anthrax is a serious disease caused by Bacillus anthracis. Humans can become infected by handling products from infected animals, by breathing spores and rarely by eating undercooked meat from infected animals. The genome of B. anthracis is highly monomorphic and thus shows very low DNA sequence variation. We analysed the molecular characteristics of 12 B. anthracis isolates from outbreaks in Croatia and Bosnia and Herzegovina, which have occurred during the past 10 years along with two vaccine strains. Genotyping system based on variable‐number tandem repeat analysis at six loci revealed that six isolates belong to genotype from the A1.a cluster whilst six isolates relate to the B2 cluster, compared to 89 previously described genotypes. The distribution of two evolutionarily distant clusters suggests an introduction of B. anthracis to this area in at least two separate events.     

Comparative analysis of using cereal grains and compound feed in semi-intensive common carp pond production

The semi-intensive system of carp farming is dominant in inland aquaculture. Although it appears to be simple, this system has a whole range of options, from those based on traditional rearing methods with use of plants from the region where the fish are reared to production based on the use of high-quality concentrated feeds. In this paper, we conduct a comparative analysis of the effect of using cereal grains as opposed to compound feed (pelleted and extruded) on the quantity of fish produced, flesh quality, histological changes in the digestive tract, water quality, and structure of the biocoenosis in the pond ecosystem. When the fish are fed cereal grains, production ranges from 500 to 1500 kg/ha, while using compound feed gives production that is twice as great. The proportions of protein and water are higher and that of lipids lower in flesh of common carp reared on compound feed in relation to fish fed cereal grains. Use of extruded feed gives better content of n-3 polyunsaturated fatty acids and a better n-3/n-6 ratio than when pelleted feed and cereal grains are used. Comparison of water quality in carp ponds under conditions of using various types of feed indicates that there are no significant differences. Although it was expected that feed with a higher percentage of proteins would cause disturbances in the aquatic ecosystem, their continual incorporation into the cycling of matter in the water contributes to maintenance of ecological equilibrium and even indirectly promotes growth of the common carp.     

Rough vaccines in animal brucellosis: structural and genetic basis and present status

Brucellosis control and eradication requires serological tests and vaccines. Effective classical vaccines (S19 in cattle and Rev 1 in small ruminants), however, induce antibodies to the O-polysaccharide of the lipopolysaccharide which may be difficult to distinguish from those resulting from infection and may thus complicate diagnosis. Rough attenuated mutants lack the O-polysaccharide and would solve this problem if eliciting protective immunity; the empirically obtained rough mutants 45/20 and RB51 have been used as vaccines. Strain 45/20 is reportedly unstable and it is not presently used. RB51 is increasingly used instead of S19 in some countries but it is rifampicin resistant and its effectiveness is controversial. Some controlled experiments have found good or absolute protection in adult cattle vaccinated orally (full dose) or subcutaneously (reduced dose) and in one field experiment, RB51 was reported to afford absolute protection to calves and to perform better than S19. Controlled experiments in calves, however, have shown reduced doses of RB51 to be ineffective, full doses only partially effective, and RB51 less effective than S19 against severe challenges. Moreover, other observations suggest that RB51 is ineffective when prevalence is high. RB51 is not useful in sheep and evidence in goats is preliminary and contradictory. Rough mutants obtained by molecular biology methods on the knowledge of the genetics and structure of Brucella lipopolysaccharide may offer alternatives. The B. abortus manBcore (rfbK) mutant seems promising in cattle, and analyses in mice suggest that mutations affecting only the O-polysaccharide result in better vaccines than those affecting both core and O-polysaccharide. Possible uses of rough vaccines also include boosting immunity by revaccination but solid evidence on its effectiveness, safety and practicality is not available.