Detection of Human Enteric Viruses in Freshwater from European Countries

The transmission of water-borne pathogens typically occurs by a faecal–oral route, through inhalation of aerosols, or by direct or indirect contact with contaminated water. Previous molecular-based studies have identified viral particles of zoonotic and human nature in surface waters. Contaminated water can lead to human health issues, and the development of rapid methods for the detection of pathogenic microorganisms is a valuable tool for the prevention of their spread. The aims of this work were to determine the presence and identity of representative human pathogenic enteric viruses in water samples from six European countries by quantitative polymerase chain reaction (q-PCR) and to develop two quantitative PCR methods for Adenovirus 41 and Mammalian Orthoreoviruses. A 2-year survey showed that Norovirus, Mammalian Orthoreovirus and Adenoviruses were the most frequently identified enteric viruses in the sampled surface waters. Although it was not possible to establish viability and infectivity of the viruses considered, the detectable presence of pathogenic viruses may represent a potential risk for human health. The methodology developed may aid in rapid detection of these pathogens for monitoring

Detection of Human Enteric Viruses in Freshwater from European Countries

The transmission of water-borne pathogens typically occurs by a faecal–oral route, through inhalation of aerosols, or by direct or indirect contact with contaminated water. Previous molecular-based studies have identified viral particles of zoonotic and human nature in surface waters. Contaminated water can lead to human health issues, and the development of rapid methods for the detection of pathogenic microorganisms is a valuable tool for the prevention of their spread. The aims of this work were to determine the presence and identity of representative human pathogenic enteric viruses in water samples from six European countries by quantitative polymerase chain reaction (q-PCR) and to develop two quantitative PCR methods for Adenovirus 41 and Mammalian Orthoreoviruses. A 2-year survey showed that Norovirus, Mammalian Orthoreovirus and Adenoviruses were the most frequently identified enteric viruses in the sampled surface waters. Although it was not possible to establish viability and infectivity of the viruses considered, the detectable presence of pathogenic viruses may represent a potential risk for human health. The methodology developed may aid in rapid detection of these pathogens for monitoring

Evaluation of five commercial methods for the extraction and purification of DNA from human faecal samples for downstream molecular detection of the enteric protozoan parasites Cryptosporidium spp., Giardia duodenalis, and Entamoeba spp

High quality, pure DNA is required for ensuring reliable and reproducible results in molecular diagnosis applications. A number of in-house and commercial methods are available for the extraction and purification of genomic DNA from faecal material, each one offering a specific combination of performance, cost-effectiveness, and easiness of use that should be conveniently evaluated in function of the pathogen of interest. In this comparative study the marketed kits QIAamp DNA stool mini (Qiagen), SpeedTools DNA extraction (Biotools), DNAExtract-VK (Vacunek), PowerFecal DNA isolation (MoBio), and Wizard magnetic DNA purification system (Promega Corporation) were assessed for their efficacy in obtaining DNA of the most relevant enteric protozoan parasites associated to gastrointestinal disease globally. A panel of 113 stool specimens of clinically confirmed patients with cryptosporidiosis (n = 29), giardiasis (n = 47) and amoebiasis by Entamoeba histolytica (n = 3) or E. dispar (n = 10) and apparently healthy subjects (n = 24) were used for this purpose. Stool samples were aliquoted in five sub-samples and individually processed by each extraction method evaluated. Purified DNA samples were subsequently tested in PCR-based assays routinely used in our laboratory. The five compared methods yielded amplifiable amounts of DNA of the pathogens tested, although performance differences were observed among them depending on the parasite and the infection burden. Methods combining chemical, enzymatic and/or mechanical lysis procedures at temperatures of at least 56 °C were proven more efficient for the release of DNA from Cryptosporidium oocysts.

Biofilms Of Enterococcus Faecalis And Enterococcus Faecium Isolated From The Processing Of Ricotta And The Control Of These Pathogens Through Cleaning And Sanitization Procedures.

The biofilm formation of Enterococcus faecalis and Enterococcus faecium isolated from the processing of ricotta on stainless steel coupons was evaluated, and the effect of cleaning and sanitization procedures in the control of these biofilms was determined. The formation of biofilms was observed while varying the incubation temperature (7, 25 and 39°C) and time (0, 1, 2, 4, 6 and 8days). At 7°C, the counts of E. faecalis and E. faecium were below 2log10CFU/cm(2). For the temperatures of 25 and 39°C, after 1day, the counts of E. faecalis and E. faecium were 5.75 and 6.07log10CFU/cm(2), respectively, which is characteristic of biofilm formation. The tested sanitation procedures a) acid-anionic tensioactive cleaning, b) anionic tensioactive cleaning+sanitizer and c) acid-anionic tensioactive cleaning+sanitizer were effective in removing the biofilms, reducing the counts to levels below 0.4log10CFU/cm(2). The sanitizer biguanide was the least effective, and peracetic acid was the most effective. These studies revealed the ability of enterococci to form biofilms and the importance of the cleaning step and the type of sanitizer used in sanitation processes for the effective removal of biofilms.

Serosurvey of selected avian pathogens in brazilian commercial Rheas (Rhea americana) and Ostriches (Struthio camelus)

Ratite farming of has expanded worldwide. Due to the intensive farming methods used by ratite producers, preventive medicine practices should be established. In this context, the surveillance and control of some avian pathogens are essential for the success of the ratite industry; however, little is known on the health status of ratites in Brazil. Therefore, the prevalence of antibodies against Newcastle Disease virus, Chlamydophila psittaci, Mycoplasma gallisepticum, Mycoplasma synoviae, and Salmonella Pullorum were evaluated in 100 serum samples collected from commercial ostriches and in 80 serum samples from commercial rheas reared in Brazil. All sampled animals were clinically healthy. The results showed that all ostriches and rheas were serologically negative to Newcastle disease virus, Chlamydophila psittaci, Mycoplasma gallisepticum, and Mycoplasma synoviae. Positive antibody responses against Salmonella Pullorum antigen were not detected in ostrich sera, but were detected in two rhea serum samples. These results can be considered as a warning as to the presence of Salmonella spp. in ratite farms. Therefore, the implementation of good health management and surveillance programs in ratite farms may contribute to improve not only animal production, but also public health conditions.

Pimarane-type Diterpenes: Antimicrobial Activity against Oral Pathogens

Seven pimarane type-diterpenes re-isolated from Viguiera arenaria Baker and two semi-synthetic pimarane derivatives were evaluated in vitro against the following main microorganisms responsible for dental caries: Streptococcus salivarius, S. sobrinus, S. mutans, S. mitis, S. sanguinis and Lactobacillus casei. The compounds ent-pimara-8(14), 15-dien-19-oic acid (PA); ent-8(14), 15-pimaradien-3 beta-ol; ent-15-pimarene-8 beta, 19-diol; ent-8(14), 15-pimaradien-3 beta-acetoxy and the sodium salt derivative of PA were the most active compounds, displaying MIC values ranging from 2 to 8 mu g.mL(-1). Thus, this class of compounds seems promising as a class of new effective anticariogenic agents. Furthermore, our results also allow us to conclude that minor structural differences among these diterpenes significantly influence their antimicrobial activity, bringing new perspectives to the discovery of new natural compounds that could be employed in the development of oral care products.

Effects of protein deprivation and re-feeding on P2X(2) receptors in enteric neurons

AIM: To investigate the effects of malnutrition and refeeding on the P2X(2) receptor, nitric oxide synthase (NOS), calretinin, calbindin and choline acetyltransferase (ChAT) in neurons of the rat ileum. METHODS: We analyzed the co-localization, numbers and sizes of P2X(2)-expressing neurons in relation to NOS-IR (immunoreactive), calbindin-IR, ChAT-IR, and calretinin-IR neurons of the myenteric and submucosal plexus. The experimental groups consisted of: (1) rats maintained on normal feed throughout pregnancy until 42 d post-parturition (N); (2) rats deprived of protein throughout pregnancy and 42 d post-parturition (D); and (3) rats undernourished for 21 d post-parturition and then given a protein diet from days 22 to 42 (DR). The myenteric and submucosal plexuses were evaluated by double labeling by immunohistochemical methods for P2X(2) receptor, NOS, ChAT, calbindin and calretinin. RESULTS: We found similar P2X(2) receptor immunoreactivity in the cytoplasm and surface membranes of myenteric and submucosal neurons from the N, D and DR groups. Double labeling of the myenteric plexus demonstrated that approximately 100% of NOS-IR, calbindin-IR, calretinin-IR and ChAT-IR neurons in all groups also expressed the P2X(2) receptor. In the submucosal plexus, the calretinin-IR, ChAT-IR and calbindinIR neurons were nearly all immunoreactive for the P2X(2) receptor. In the myenteric plexus, there was a 19% increase in numbers per cm(2) for P2X(2) receptor-IR neurons, 64% for NOS-IR, 84% for calretinin-IR and 26% for ChAT-IR neurons in the D group. The spatial density of calbindin-IR neurons, however, did not differ among the three groups. The submucosal neuronal density increased for calbindin-IR, calretinin-IR and ChAT-IR neurons. The average size of neurons in the myenteric plexus neurons in the D group was less than that in the controls and, in the re-fed rats; there was a 34% reduction in size only for the calretinin-IR neurons. CONCLUSION: This work demonstrates that expression of the P2X(2) receptor is present in inhibitory, intrinsic primary afferent, cholinergic secretomotor and vasomotor neurons. Undernutrition affected P2X(2) receptor expression in the submucosal plexus, and neuronal and size. These changes were rescued in the re-fed rats. (C) 2010 Baishideng. All rights reserved.

Fungal pathogens as classical biological control agents against arthropods

Fungal entomopathogens have been used more frequently than other types of pathogens for classical biological control. Among 136 programs using different groups of arthropod pathogens, 49.3% have introduced fungal pathogens (including both the traditional fungi and microsporidia). The most commonly introduced species was Metarhizium anisopliae (Metschnikoff) Sorokin, with 13 introductions, followed by Entomophaga maimaiga Humber, Shimazu & Soper, which was released seven times. The majority of introduction programs have focused on controlling invasive species of insects or mites (70.7%) rather than on native hosts (29.4%). Almost half of the introductions of traditional fungi targeted species of Hemiptera and 75% of the microsporidia introduced have been introduced against lepidopteran species. The United States was the country where most introductions of fungi took place (n = 24). From 1993 to 2007, no arthropod pathogens were released in the US due to the rigorous regulatory structure, but in 2008 two species of microsporidia were introduced against the gypsy moth, Lymantria dispar (L.). Establishment of entomopathogenic fungi in programs introducing traditional fungi was 32.1% and establishment was 50.0% for programs introducing microsporidia. In some programs, releases have resulted in permanent successful establishment with no non-target effects. In summary, classical biological control using fungal entomopathogens can provide a successful and environmentally friendly avenue for controlling arthropod pests, including the increasing numbers of invasive non-native species.

Enteric cell proliferation in newborn lambs fed bovine and ovine colostrum

The objective of this study was to investigate immunoglobulin G (IgG) and total serum protein (TP) acquisition in newborn Santa Ines lambs fed Holstein bovine or Santa Ines ovine colostrum as well as the cell proliferation rate in the animals` intestine epithelium. At 0 h and 6 h of life, 12 newborn lambs received 250 mL of bovine 1st milking colostrum (BC) and another 12 animals received 250 mL of ovine 1st milking colostrum (OC). Blood samples were collected at 0, 6, 24. and 72 h of life. Six animals were randomly slaughtered just after birth, without colostrum intake. The other animals were randomly slaughtered at 24 and 72 h. The IgG serum concentration at 6, 24 and 72 h were significantly higher for BC, 16.32 +/- 6.19; 33.80 +/- 5.68 and 27.95 +/- 5.46 mg/mL respectively, compared with OC, 11.31 +/- 6.08, 21.02 +/- 6.53 and 19.88 +/- 7.31 mg/mL BC showed higher (P < 0.05) TP values (7.29 +/- 0.87 and 6.89 +/- 0.30 g/100 mL) at 24 and 72 h in relation to OC (5.73 +/- 1.35 and 5.69 +/- 0.57 g/100 mL). At birth, the animals showed 32.52%, 45.47% and 30.60% cells in division for the duodenum, jejunum and ileum, respectively. At 24 h, the OC animals showed lower (P < 0.0001) mitotic cell percentage in the duodenum (42.12%) and ileum (35.66%) in relation to the BC animals, 46.44% and 39.74%, respectively. At 72 h, a lower (P < 0.0001) rate of proliferation was observed in the duodenum crypts of the OC animals (36.28%) compared with BC (43.18%). The results indicate that this lacteal secretion can accelerate the epithelium renovation process and can be used as an alternative source of IgG for newborn lambs. (C) 2009 Elsevier B.V. All rights reserved.

Etiology of childhood diarrhea in the northeast of Brazil: significant emergent diarrheal pathogens

In a study conducted in Joao Pessoa, northeast of Brazil, 2344 Escherichia coli isolated from 290 infants with diarrhea and 290 healthy matched controls were analyzed for virulence traits. Enteroaggregative E. coli (EAEC) was the most prevalent pathogen associated to acute diarrhea. Based on the results of colony blot hybridization, serotyping, and HEp-2 cell adherence assays, strains were separated in categories as typical enteropathogenic E. coli (EPEC) (1.7%), atypical EPEC (a-EPEC) (9.3%), EAEC (25%), enterotoxigenic E. coli (10%), and enteroinvasive E. coli (EIEC) (1.4%). No enterohemorrhagic E. coli strains were isolated. Other enteropathogens were found, including Salmonella (7.9%), Shigella spp. (4.1%), thermophilic Campylobacter spp. (2.4%), Giardia lamblia (9.3%), and Entamoeba histolytica (5.8%). All enteropathogens were associated with diarrhea (P < 0.01). However, the association was lower for EPEC and EIEC (P < 0.03). Different pathogens associated with diarrhea may have been changing in Brazil where EAEC and a-EPEC seem to be the most prevalent pathogens among them. (C) 2010 Elsevier Inc. All rights reserved.

Ultra-Fast Gradient LC Method for Omeprazole Analysis Using a Monolithic Column: Assay Development, Validation, and Application to the Quality Control of Omeprazole Enteric-Coated Pellets

A method was optimized for the analysis of omeprazole (OMZ) by ultra-high speed LC with diode array detection using a monolithic Chromolith Fast Gradient RP 18 endcapped column (50 x 2.0 mm id). The analyses were performed at 30 degrees C using a mobile phase consisting of 0.15% (v/v) trifluoroacetic acid (TFA) in water (solvent A) and 0.15% (v/v) TFA in acetonitrile (solvent B) under a linear gradient of 5 to 90% B in 1 min at a flow rate of 1.0 mL/min and detection at 220 nm. Under these conditions, OMZ retention time was approximately 0.74 min. Validation parameters, such as selectivity, linearity, precision, accuracy, and robustness, showed results within the acceptable criteria. The method developed was successfully applied to OMZ enteric-coated pellets, showing that this assay can be used in the pharmaceutical industry for routine QC analysis. Moreover, the analytical conditions established allow for the simultaneous analysis of OMZ metabolites, 5-hydroxyomeprazole and omeprazole sulfone, in the same run, showing that this method can be extended to other matrixes with adequate procedures for sample preparation.

Antimicrobial activity of kaurane diterpenes against oral pathogens

Two kaurane diterpenes, ent-kaur-16(17)-en-19-oic acid (KA) and 15-beta-isovaleryloxy-ent-kaur-16(17)-en-19-oic acid (KA-Ival), isolated from Aspilia foliacea, and the methyl ester derivative of KA (KA-Me) were evaluated against oral pathogens. KA was the most active compound, with MIC values of 10 mu g mL(-1) against the following microorganisms: Streptococcus sobrinus, Streptococcus mutans, Streptococcus mitis, Streptococcus sanguinis, and Lactobacillus casei. However, KA did not show significant activity against Streptococcus salivarius and Enterococcus faecalis, with MIC values equal to 100 and 200 mu g mL(-1), respectively. Our results show that KA has potential to be used as a prototype for the discovery of new effective anti-infection agents against microorganisms responsible for caries and periodontal diseases. Moreover, these results allow to conclude that minor structural differences among these diterpenes significantly influence their antimicrobial activity, bringing new perspectives to studies on the structure-activity relationship of this type of metabolites with respect to caries and periodontal diseases.

Innate immune lectins kill bacteria expressing blood group antigen

The expression of ABO(H) blood group antigens causes deletion of cells that generate self-specific antibodies to these antigens but this deletion limits adaptive immunity toward pathogens bearing cognate blood group antigens. To explore potential defense mechanisms against such pathogens, given these limitations in adaptive immunity, we screened for innate proteins that could recognize human blood group antigens. Here we report that two innate immune lectins, galectin-4 (Gal-4) and Gal-8, which are expressed in the intestinal tract, recognize and kill human blood group antigen-expressing Escherichia coli while failing to alter the viability of other E. coli strains or other Gram-negative or Gram-positive organisms both in vitro and in vivo. The killing activity of both Gal-4 and Gal-8 is mediated by their C-terminal domains, occurs rapidly and independently of complement and is accompanied by disruption of membrane integrity. These results demonstrate that innate defense lectins can provide immunity against pathogens that express blood group-like antigens on their surface.

Antibacterial activity of 15-deoxygoyazensolide isolated from the stems of Minasia alpestris (Asteraceae) against oral pathogens

This work reports the isolation of the sesquiterpene lactone 15-deoxygoyazensolide from the stems of Minasia alpestris and the evaluation of its antimicrobial activity against the following oral pathogens: Enterococcus faecalis, Streptococcus salivarius, Streptococcus sobrinus, Streptococcus mutans, Streptococcus mitis, Streptococcus sanguinis, and Lactobacillus casei. Despite the cytotoxicity and genotoxicity of other sesquiterpene lactones of the furanoheliangolide-type, our results revealed that this compound exhibits low antibacterial activity against the evaluated oral pathogens; however, an interesting selectivity against E. faecalis (minimum inhibitory concentration [MIC] = 40 mu g mL(-1)) and S. sobrinus (MIC = 60 mu g mL(-1)) was observed.

Biotransformation using Mucor rouxii for the production of oleanolic acid derivatives and their antimicrobial activity against oral pathogens

The goal of this study is to produce oleanolic acid derivatives by biotransformation process using Mucor rouxii and evaluate their antimicrobial activity against oral pathogens. The microbial transformation was carried out in shake flasks at 30A degrees C for 216 h with shaking at 120 rpm. Three new derivatives, 7 beta-hydroxy-3-oxo-olean-12-en-28-oic acid, 7 beta,21 beta-dihydroxy-3-oxo-olean-12-en-28-oic acid, and 3 beta,7 beta,21 beta-trihydroxyolean-12-en-28-oic acid, and one know compound, 21 beta-hydroxy-3-oxo-olean-12-en-28-oic acid, were isolated, and the structures were elucidated on the basis of spectroscopic analyses. The antimicrobial activity of the substrate and its transformed products was evaluated against five oral pathogens. Among these compounds, the derivative 21 beta-hydroxy-3-oxo-olean-12-en-28-oic acid displayed the strongest activity against Porphyromonas gingivalis, which is a primary etiological agent of periodontal disease. In an attempt to improve the antimicrobial activity of the derivative 21 beta-hydroxy-3-oxo-olean-12-en-28-oic acid, its sodium salt was prepared, and the minimum inhibitory concentration against P. gingivalis was reduced by one-half. The biotransformation process using M. rouxii has potential to be applied to the production of oleanolic acid derivatives. Research and antimicrobial activity evaluation of new oleanolic acid derivatives may provide an important contribution to the discovery of new adjunct agents for treatment of dental diseases such as dental caries, gingivitis, and periodontitis.