Severe outbreak of Centrocestus formosanus (Trematoda: Heterophyidae) in farm-raised ornamental platies Xiphophorus maculatus.

This report describes a severe outbreak of the gill fluke Centrocestus formosanus in farm-raised platies Xiphophorus maculatus in Brazil, with mortality rate approaching 95%. Typical clinical signs of infection were observed, with microscopic examinations of fresh gills revealing multiple cysts containing a once-folded metacercaria with an X-shaped excretory bladder. The 18S subunit of the metacercariae (BR1) was amplified by PCR, sequenced and analyzed by BLAST. Subsequent phylogenetic analyses revealed that the BR1 isolate was closely related to C. formosanus from Thailand. This is the first report of C. formosanus in ornamental fish in Brazil. Our observations suggest that platies are highly sensitive to this digenetic parasite. Controlling population densities of the parasite's intermediate host, the snail Melanoides tuberculata, would help to reduce outbreaks.

The Druggable Pocketome of Corynebacterium diphtheriae: A New Approach for in silico Putative Druggable Targets.

Diphtheria is an acute and highly infectious disease, previously regarded as endemic in nature but vaccine-preventable, is caused by Corynebacterium diphtheriae (Cd). In this work, we used an in silico approach along the 13 complete genome sequences of C. diphtheriae followed by a computational assessment of structural information of the binding sites to characterize the "pocketome druggability." To this end, we first computed the "modelome" (3D structures of a complete genome) of a randomly selected reference strain Cd NCTC13129; that had 13,763 open reading frames (ORFs) and resulted in 1,253 (∼9%) structure models. The amino acid sequences of these modeled structures were compared with the remaining 12 genomes and consequently, 438 conserved protein sequences were obtained. The RCSB-PDB database was consulted to check the template structures for these conserved proteins and as a result, 401 adequate 3D models were obtained. We subsequently predicted the protein pockets for the obtained set of models and kept only the conserved pockets that had highly druggable (HD) values (137 across all strains). Later, an off-target host homology analyses was performed considering the human proteome using NCBI database. Furthermore, the gene essentiality analysis was carried out that gave a final set of 10-conserved targets possessing highly druggable protein pockets. To check the target identification robustness of the pipeline used in this work, we crosschecked the final target list with another in-house target identification approach for C. diphtheriae thereby obtaining three common targets, these were; hisE-phosphoribosyl-ATP pyrophosphatase, glpX-fructose 1,6-bisphosphatase II, and rpsH-30S ribosomal protein S8. Our predicted results suggest that the in silico approach used could potentially aid in experimental polypharmacological target determination in C. diphtheriae and other pathogens, thereby, might complement the existing and new drug-discovery pipelines.

Secretion of biologically active pancreatitis-associated protein I (PAP) by genetically modified dairy Lactococcus lactis NZ9000 in the prevention of intestinal mucositis.

BACKGROUND: Mucositis is one of the most relevant gastrointestinal inflammatory conditions in humans, generated by the use of chemotherapy drugs, such as 5-fluoracil (5-FU). 5-FU-induced mucositis affects 80% of patients undergoing oncological treatment causing mucosal gut dysfunctions and great discomfort. As current therapy drugs presents limitations in alleviating mucositis symptoms, alternative strategies are being pursued. Recent studies have shown that the antimicrobial pancreatitis-associated protein (PAP) has a protective role in intestinal inflammatory processes. Indeed, it was demonstrated that a recombinant strain of Lactococcus lactis expressing human PAP (LL-PAP) could prevent and improve murine DNBS-induced colitis, an inflammatory bowel disease (IBD) that causes severe inflammation of the colon. Hence, in this study we sought to evaluate the protective effects of LL-PAP on 5-FU-induced experimental mucositis in BALB/c mice as a novel approach to treat the disease. RESULTS: Our results show that non-recombinant L. lactis NZ9000 have antagonistic activity, in vitro, against the enteroinvasive gastrointestinal pathogen L. monocytogenes and confirmed PAP inhibitory effect against Opportunistic E. faecalis. Moreover, L. lactis was able to prevent histological damage, reduce neutrophil and eosinophil infiltration and secretory Immunoglobulin-A in mice injected with 5-FU. Recombinant lactococci carrying antimicrobial PAP did not improve those markers of inflammation, although its expression was associated with villous architecture preservation and increased secretory granules density inside Paneth cells in response to 5-FU inflammation. CONCLUSIONS: We have demonstrated for the first time that L. lactis NZ9000 by itself, is able to prevent 5-FU-induced intestinal inflammation in BALB/c mice. Moreover, PAP delivered by recombinant L. lactis strain showed additional protective effects in mice epithelium, revealing to be a promising strategy to treat intestinal mucositis.

Development of duplex real-time PCR for the detection of WSSV and PstDV1 in cultivated shrimp.

BACKGROUND: The White spot syndrome virus (WSSV) and Penaeus stylirostris penstyldensovirus 1 (previously named Infectious hypodermal and hematopoietic necrosis virus-IHHNV) are two of the most important viral pathogens of penaeid shrimp. Different methods have been applied for diagnosis of these viruses, including Real-time PCR (qPCR) assays. A duplex qPCR method allows the simultaneous detection of two viruses in the same sample, which is more cost-effective than assaying for each virus separately. Currently, an assay for the simultaneous detection of the WSSV and the PstDV1 in shrimp is unavailable. The aim of this study was to develop and standardize a duplex qPCR assay for the simultaneous detection of the WSSV and the PstDV1 in clinical samples of diseased L. vannamei. In addition, to evaluate the performance of two qPCR master mixes with regard to the clinical sensitivity of the qPCR assay, as well as, different methods for qPCR results evaluation. RESULTS: The duplex qPCR assay for detecting WSSV and PstDV1 in clinical samples was successfully standardized. No difference in the amplification of the standard curves was observed between the duplex and singleplex assays. Specificities and sensitivities similar to those of the singleplex assays were obtained using the optimized duplex qPCR. The analytical sensitivities of duplex qPCR were two copies of WSSV control plasmid and 20 copies of PstDV1 control plasmid. The standardized duplex qPCR confirmed the presence of viral DNA in 28 from 43 samples tested. There was no difference for WSSV detection using the two kits and the distinct methods for qPCR results evaluation. High clinical sensitivity for PstDV1 was obtained with TaqMan Universal Master Mix associated with relative threshold evaluation. Three cases of simultaneous infection by the WSSV and the PstDV1 were identified with duplex qPCR. CONCLUSION: The standardized duplex qPCR was shown to be a robust, highly sensitive, and feasible diagnostic tool for the simultaneous detection of the WSSV and the PstDV1 in whiteleg shrimp. The use of the TaqMan Universal Master Mix and the relative threshold method of data analysis in our duplex qPCR method provided optimal levels of sensitivity and specificity.

Identification of the antimicrobial substances produced by Solanum palinacanthum (Solanaceae)

To find out natural antimicrobial agents as alternative in therapeutics and to preserve food, the methanol extract of Solanum palinacanthum aerial parts was submitted to purification steps guided by antibacterial and antifungal assays. As a consequence, the flavonoid rutin and 3,5-dicaffeoylquinic acid were isolated by column chromatographyand high performance liquid chromatography, and identified by mass and nuclear magnetic resonance spectrometry. Minimal inhibitory concentrations (MIC) of the quinic acid derivative against Aeromonas hydrophila, Bacillus subtilis, Staphylococcus aureus and the fungus Aspergillus ochraceus were 250, 1000, 1000 and > 568µg/mL, respectively. Against the same microorganisms, MIC for rutin were 1000, > 1000, > 1000 and 35µg/mL, respectively. Rutinwas very promising for A. ochraceus control, since its MIC against such fungus was close to the one observed for benzalkonium chloride, which is used as a fungicide in Brazil.

Com vistas a descobrir antimicrobianos de origem natural para uso terapêutico ou para a preservação de alimentos, o extrato metanólico das partes aéreas de Solanum palinacanthum foi submetido a fracionamentos direcionados por testes para avaliar a atividade antibacteriana e antifúngica. Em decorrência, o flavonóide rutina e o ácido 3,5-dicafeoilquínico foram isolados por cromatografia em coluna e por cromatografia líquida de alta eficiência, para serem identificados por espectrometria de massas e de ressonância magnética nuclear. As concentrações inibitórias mínimas (CIM) do derivado do ácido cafeico contra Aeromonas hydrophila, Bacillus subtilis, Staphylococcus aureus e o fungo Aspergillus ochraceus foram 250, 1000, 1000 e > 568µg/mL, respectivamente. Contra os mesmos organismos, os valores de CIM para a rutina foram 1000, > 1000, > 1000 e 35µg/mL, respectivamente. A rutina mostrou-se muito promissora para o controle de A. ochraceus, pois seu valor de CIM contra tal fungo foi bem próximo ao observado para o cloreto de benzalcônio, que é empregado como fungicida no Brasil.

Purification of an antibacterial compound from Lantana lilacina / Purificação de um composto antibacteriano de Lantana lilacina

Observou-se, em estudo preliminar, que o extrato metanólico das folhas de L. lilacina, coletadas no município de Lavras (MG, Brasil), apresentava atividade antibacteriana. Em decorrência, buscou-se purificar e identificar a substância responsável por tal efeito, através de fracionamento do referido extrato direcionado por testes de difusão em agar com Aeromonas hydrophila, Bacillus subtilis, Pseudomonas aeruginosa e Staphylococcus aureus. Após partições com solventes e vários processos cromatográficos, isolou-se o [β-3,4-diidroxifenil)etil]-(3'-O-α-L-ramnopiranosil)-(4'- O-cafeoil)-β-D-glicopiranosídeo, que é conhecido como acteosídeo. A concentração inibitória mínima e a concentração bactericida mínima desta substância para A. hydrophila, B. subtilis, P. aeruginosa e S. aureus foram de 0,12, 1,00, 1,00 e 0,25 mg/mL, respectivamente.

Since the methanol extract of Lantana lilacina leaves collected in the city of Lavras (MG, Brazil) showed antibacterial properties in a preliminary study, a fractionation process guided by agar diffusion assays with Aeromonas hydrophila, Bacillus subtilis, Pseudomonas aeruginosa and Staphylococcus aureus was carried out to purify and identify the active compounds. After solvent partition and several chromatographic steps, [ß-3,4-dihydroxyphenyl)-ethyl]-(3'-O-α-L-rhamnopyranosyl)-(4'- O-cafeoyl)-β-D-glycopyranoside, known as acteoside, was isolated. The minimal inhibition concentration and the minimal bactericidal concentration of such substance against A. hydrophila, B. subtilis, P. aeruginosa and S. aureus were 0.12, 1.00, 1.00 and 0.25 mg/mL, respectively.