Description of a novel bovine antimicrobial peptide and its antimicrobial spectrum. RNA isolation and RT-PCR were done from various tissues. DCD peptide was synthesized, and antimicrobial activity was analyzed. Bovine dermcidin gene contains five exons intermittent by 4 introns. Bovine DCD-mRNA was 398 bp with ORF 336 bp. Bovine DCD was expressed in skin and blood. Analysis of the amino acid compositions revealed that cysteine was repeated 6 times indicating the presence of 3 disulfide bonds that play role in the peptide stability. Staphylococcus epidermidis, Streptococcus bovis, and Enterococcus faecalis were affected by Bovine DCD peptide. Highest antimicrobial effect was at 50 and 100 µg/ml. The effect on Escherichia coli and Candida albicans was slightly low. In all bacteria, Bovine DCD peptide activity did not affect by varying pH values, but in Staphylococcus aureus, the activity was affected greatly at pH 4.5 and 5.5. The optimum salt concentrations were 100 and 50 mM NaCl with all bacterial strains and E. coli, respectively. In case of C. albicans, the antimicrobial activity of Bovine DCD peptide decreased with increasing the pH value regardless the NaCl concentration. The pH 6.5 of the sweat buffer was the optimum for the Bovine DCD peptide activity.

The co-circulation of duck hepatitis A virus subtypes 1 (DHAV-1) and 3 (DHAV-3) in ducklings has resulted in significant economic losses. Because ducklings infected with DHAV-1 or DHAV-3 show similar clinical signs and gross lesions, it is important to discriminate these subtypes as early as possible for better clinical management. On the basis of multiple alignments of the 5′-noncoding region sequences of strains DHAV-1 and DHAV-3, universal and type-specific primers were designed and synthesized. Using the primers in a one-tube reverse transcription-PCR (RT-PCR) assay, reference strains of DHAV-1 and DHAV-3 (isolated over a span of 60 years and covering many different countries) were successfully amplified, indicating that the primer sequences were completely conserved. The amplicon sequences results and the sizes of amplicons from reference DHAV-1 and DHAV-3 isolates correlated completely with their genotypes. Moreover, with this one-tube RT-PCR system, the amplicon sizes of liver samples of reference DHAV-1- or DHAV-3-infected birds matched perfectly with their respective genotypes, as determined by virus isolation and neutralization tests. No other RNA viruses of duck origin were detected with the synthesized primers. The sensitivity of viral RNA detection was 10 pg. With this system, 20% genotype 1, 45% genotype 3, and 9% co-infection of the two genotypes were detected in 55 clinical samples. This novel approach could be used for the rapid genotyping DHAV-1 and/or DHAV-3 infection in routine clinical surveillance or epidemiologic screening.

The application of quantitative PCR (qPCR) as a routine method to detect and enumerate Paenibacillus larvae in honey and hive debris could greatly speed up the estimation of prevalence and outbreak risk of the American foulbrood (AFB) disease of Apis mellifera. However, none of the qPCR tests described so far has been officially proposed as a standard procedure for P. larvae detection and enumeration for surveillance purposes. Therefore, in this study inclusivity, exclusivity and sensitivity in detection of P. larvae spores directly in samples of honey and hive debris were re-evaluated for the previously published qPCR methods. To this aim recently acquired P. larvae sequence data were considered to assess inclusivity in silico and more appropriate non-target species were used to verify exclusivity experimentally. This led to the modification of one of the previously described methods resulting in a new test capable to allow the detection of P. larvae spores in honey and hive debris down to 1 CFU/g. The application of the qPCR test optimized in this study can allow to reliably detect and quantify P. larvae in honey and hive debris, thus circumventing the disadvantages of late AFB diagnosis based on clinical symptoms and possible underestimation of spore numbers that is the main drawback of culture-dependent procedures.

In this paper, the active front-end (AFE) converter topology for the total harmonic distortion (THD) reduction in a wind energy system (WES) is used. A higher THD results in serious pulsations in the wind turbine (WT) output power and in several power losses at the WES. The AFE converter topology improves capability, efficiency and reliability in the energy conversion devices; by modifying a conventional back-to-back converter, from using a single voltage source converter (VSC) to use pVSC connected in parallel the AFE converter is generated. The THD reduction is done by applying a different phase shift angle at the carrier of digital sinusoidal pulse width modulation (DSPWM) switching signals of each VSC. To verify the functionality of the proposed methodology, the WES simulation in Matlab-Simulink® is analyzed, and the experimental laboratory tests using the concept of rapid control prototyping and the real-time simulator Opal-RT® Technologies is achieved. The obtained results show a type-4 WT with total output power of 6MVA, generating a THD reduction up to 5.5 times at the WES.

The airborne inoculum of Fusarium circinatum, the fungal pathogen causing Pine Pitch Canker (PPC), is one of the main means of spread of the disease in forest stands and forest nurseries. Since this world-wide known pathogen was introduced in Europe, its biology in this newly infected area still remains scarcely known. To shed more light on this topic, we set an experiment on a naturally PPC infected forest of Monterey pine in Galicia (NW Spain) with the following two goals: (i) to describe the seasonal spore dispersal pattern during one year of regular sampling and (ii) to assess the spatial spore dispersal pattern around the infested plot. Portable rotating arm spore traps were used and complemented with meteorological measurements. The abundance of F. circinatum spores in the samples was evaluated by quantitative PCR (qPCR) with hydrolysis probe. The results showed almost permanent occurrence of the air inoculum throughout the whole year, being detected in 27 of the 30 samplings. No clear temporal trends were observed, but higher air inoculum was favoured by previous lower air temperatures and lower leaf wetness. Conversely, neither rainfall nor air humidity seemed to have any significant importance. The spatial spread of the inoculum was noted to be successful up to a distance of 1000 m in the wind direction, even with winds of just 5 m s^-1. Our study shows that rotating arm spore traps combined with qPCR may be an efficient tool for F. circinatum detection.

Italian Directives recommends the good quality of natural mineral waters but literature data assert a potential risk from several microorganisms colonizing wellsprings and mineral water bottling plants. Aim of study is the identification of microorganisms from spring waters (SW) and bottled mineral waters (BMW) samples. Methods: Routine microbiological indicators, Legionella spp., Nontuberculous mycobacteria (NTM), protozoa (FLA) and physical-chemical parameters were assessed in 24 SW and 10 BMW samples performing culture methods and molecular tests as PCR and qPCR. Results: In 33 out of 34 samples no cultivable bacteria were isolated with the exception of 83 CFU/L of Mycobacterium gilvum, detected in one warm rich-mineralized SW. qPCR showed the presence of Legionella genomic units in 24% of samples (mean 2,9x10²±1,7x10² GU/L) and NTM genomic units in 18% of samples (mean 5,7x10^3±4,1x10³ GU/L). Vermamoeba vermiformis and Acanthamoeba polyphaga were recovered respectively in 70% of BMW samples (counts from 1,3x10³ to 1,2x10⁵) and 42% of SW samples (counts from 1,1x10³ to 1,3x10⁴). Vahlkampfia spp. was detected in 42% of SW and 70% of BMW samples (mean 1,3x10⁴ ±2,9x10³ GU/L). Conclusion: The study highlights a low rate of microbial risk and the importance of risk assessment in natural mineral waters.

Since chronic wasting disease (CWD) was first identified nearly 50 years ago in a captive mule deer herd in the Rocky Mountains of the United States, it has slowly spread across North America through the natural and anthropogenic movement of cervids and their carcasses. As the endemic areas have expanded, so has the need for rapid, sensitive, and cost effective diagnostic tests – especially those which take advantage of samples collected antemortem. Over the past two decades, strategies have evolved from the recognition of microscopic spongiform pathology and associated immunohistochemical staining of the misfolded prion protein to enzyme-linked immunoassays capable of detecting the abnormal prion conformer in postmortem samples. In a history that parallels the diagnosis of more conventional infectious agents, both qualitative and real-time amplification assays have recently been developed to detect minute quantities of misfolded prions in a range of biological and environmental samples. With these more sensitive and semi-quantitative approaches has come a greater understanding of the pathogenesis and epidemiology of this disease in the native host. Because the molecular pathogenesis of prion protein misfolding is broadly analogous to the misfolding of other pathogenic proteins, including Aβ and α-synuclein, efforts are currently underway to apply these in vitro amplification techniques towards the diagnosis of Alzheimer’s disease, Parkinson’s disease, and other proteinopathies. Chronic wasting disease – once a rare disease of Colorado mule deer – now represents one of the few naturally occurring protein misfolding disorders which might allow continued development and implementation of novel diagnostic strategies in an animal model.

Echinococcus granulosus is a harmful cestode parasite which could cause Cystic Echinococcosis in humans, various livestock species and wild animals. Calmodulin (CaM), a Ca²⁺ sensor protein, is widely expressed in eukaryotes and mediates a variety of cellular signaling activities. In our study, the CaM in Echinococcus granulosus (rEgCaM) was successfully cloned and the molecular and biochemical characterizations of rEgCaM were identified. The results showed that rEgCaM was a highly conserved calcium-binding protein, consisting of 149 amino acids. Immunoblot analysis revealed that rEgCaM could be identified using E. granulosus infected sheep serum. The usage of rEgCaM as antigen was evaluated by indirect ELISA which exhibited a high sensitivity of 90.3% but low specificity (47.1%). rEgCaM was mainly located in the tegument tissues and parenchymal region of protoscoleces, the tegument and inner body of adult worm and predominantly expressed in the germinal layer. The mRNA expression of rEgCaM in PSCs were gradually decreased with the increase death of PSCs. In electrophoretic mobility tests and ANS assays, rEgCaM showed a typical calcium-binding protein characteristics. This is the first report on CaM from E. granulosus and rEgCaM is considered to be involved in some important biological function of E. granulosus as a calcium-binding protein.

Pardosa pseudoannulata is one of the most common wandering spiders in agricultural fields and a potentially good bioindicator for heavy metal contamination. However, little is known about the mechanism by which spiders respond to heavy metals at the molecular level. In this study, high-throughput transcriptome sequencing was employed to characterize the de novo transcriptome of the spiders and to identify differentially expressed genes (DEGs) after cadmium exposure. We obtained 60,489 assembled unigenes, 18,773 of which were annotated in the public databases. Ultimately, 3450 cDNA simple sequence repeats were identified and validated as potential molecular markers in the unigenes. A total of 2939, 2491 and 3759 DEGs were detected among the three libraries of two Cd-treated groups and the control. Functional enrichment analysis revealed that metabolism processes and digestive system function were predominately enriched in response to Cd stress. At the cellular and molecular levels, significantly enriched pathways in lysosomes and phagosomes as well as replication, recombination and repair demonstrated that oxidative damage resulted from Cd exposure. Based on the selected DEGs, certain critical genes involved in defence and detoxification were analysed. These results may elucidate the molecular mechanism underlying spiders' responses to heavy metal stress.