An evaluation of microbial health risks to livestock fed with wastewater-irrigated forage crops.

This paper presents the results of five experiments in which animal health risks associated with the consumption of crops irrigated with domestic wastewater were evaluated. Forage maize and Tanner grass were irrigated with treated wastewater and used in goats and calves feeding trials. The irrigated crops presented high levels of surface contamination with E. coli (10(4) -10(7) 25 g(-1) ) and salmonellae (up to 1.6 × 10(4) 25 g(-1)), but none of the animals showed signs of infection or of disease. Further, the microbiological quality of animal products always complied with the Brazilian and European Union standards for food safety. It is suggested that the WHO guideline values for restricted irrigation (≤ 10(4) E. coli 100 ml(-1) and ≤ 1 helminth egg l(-1)), which were developed to protect the health of agricultural field workers, would be equally protective of the health of both animals fed with wastewater-irrigated crops and humans consuming products from such animals.

Quantitative microbial risk assessment applied to irrigation of salad crops with waste stabilization pond effluents.

A quantitative microbial risk assessment model for estimating infection risks arising from consuming crops eaten raw that have been irrigated with effluents from stabilization ponds was constructed. A log-normal probability distribution function was fitted to a large database from a comprehensive monitoring of an experimental pond system to account for variability in Escherichia coli concentration in irrigation water. Crop contamination levels were estimated using predictive models derived from field experiments involving the irrigation of several crops with different effluent qualities. Data on daily intake of salad crops were obtained from a national survey in Brazil. Ten thousand-trial Monte Carlo simulations were used to estimate human health risks associated with the use of wastewater for irrigating low- and high-growing crops. The use of effluents containing 10(3)-10(4) E. coli per 100 ml resulted in median rotavirus infection risk of approximately 10(-3) and 10(-4) pppy when irrigating, respectively, low- and high-growing crops; the corresponding 95th percentile risk estimates were around 10(-2) in both scenarios. Sensitivity analyses revealed that variations in effluent quality, in the assumed ratios of pathogens to E. coli, and in the reduction of pathogens between harvest and consumption had great impact upon risk estimates.

Could past Chlamydial vascular infection promote the dissemination of Chlamydia pneumoniae to the brain?

Chlamydia pneumoniae, a pathogen responsible for respiratory tract infections, has been associated with atherosclerosis which, along with hypertension, hyperlipidemia, cardiovascular and/or cerebrovascular ischemia and stroke, is a risk factor for chronic neurological disorders. Several studies have demonstrated the ability of C. pneumoniae to disseminate from lungs to arteries through peripheral blood mononuclear cells. Once inside the vascular tissue, C. pneumoniae infection may disseminate via peripheral monocytes to the brain over the intact blood-brain barrier, and contribute to the development of chronic neurological disorders. The aim of our study was to evaluate whether past C. pneumoniae vascular infection may promote the dissemination of this microorganism to the brain, therefore we investigated the presence of C. pneumoniae in post-mortem brain tissue specimens of patients with past chlamydial vascular infection. Seventy six post-mortem brain tissue specimens from 19 patients with past chlamydial vascular infection were investigated for the presence of C. pneumoniae by immunohistochemistry, polymerase chain reaction, in situ polymerase chain reaction and in situ reverse transcription polymerase chain reaction. As control, 28 brain tissue specimens were taken from 7 age and sex matched subjects without chlamydial infection. C. pneumoniae was detected in 16 (84.2%) out of 19 patients with chlamydial vascular infection whereas it was not detected in control subjects (p= 0.0002). In conclusion, the main result of our study is the evidence that a chlamydial vascular infection can disseminate to the brain. It will be important for current and future researches to perform large-scale prospective studies on cardiovascular patients with chlamydial vascular infection in order to evaluate the long-term pathological alterations of the brain.

Bi-directional electrical characterisation of microbial fuel cell.

The electrical performance of microbial fuel cells in steady-state is usually investigated by standard characterisation methods that reveal many important parameters e.g. maximum power. This paper introduces a novel "bi-directional" method to study how the acquisition parameters (i.e. sweep rate and sweep regime) can influence measurements and consequently performance estimations. The investigation exhibited considerable differences (hysteresis) between the forward and backward characterisation regimes, indicating a difficulty to reach steady-state under certain conditions. Moreover, it is found that fast sweep rates (time-step of 2 min) can lead to an overestimation of the short-circuit currents, while prolonged operation with high external loads leads to maximum power overestimation and extended conditioning at high currents can result in its underestimation.

UASB-polishing ponds design parameters: contributions from a pilot scale study in southeast Brazil.

This paper presents the results of five years monitoring of an experimental wastewater treatment plant in southeast Brazil, comprised of a UASB reactor followed by a submerged aerated biofilter (BF) (field scale) and shallow polishing ponds (pilot scale). Three ponds in series achieved high quality effluent standards in terms of ammonia and E.coli, but a fourth pond did not result in further efficiency. Well established models to predict ammonia and E.coli removal in facultative and/or maturation ponds were, in a way, validated for polishing ponds too. The paper also includes results of input design parameters, such as pH and E.coli die-off rate constants, and their variation along the pond series.

G2 as an emerging rotavirus strain in pediatric gastroenteritis in southern Italy.

BACKGROUND: Human rotaviruses (HRVs) represent a major cause of acute gastroenteritis in children worldwide. It is estimated that they are responsible for a large number of diarrhea-associated hospitalizations in childhood each year. In Italy, limited data are available on the patterns of distribution of HRV G and P types. We report here the results of 2 years of rotavirus strain surveillance among children with severe gastroenteritis diagnosed in the town of Portici, Campania, southern Italy. METHODS: A total of 421 stool specimens from children between 6 months and 5 years of age and presenting acute diarrhea were collected and tested by routine diagnostic tests for HRV, adenovirus, astrovirus, norovirus, and common bacterial pathogens. RESULTS: The laboratory results showed that 110 of the 225 (26.1%) virus-positive samples contained HRVs. The different G and P rotavirus genotypes were analyzed by polymerase chain reaction (PCR). Among the VP7 genotypes identified, G1 and G2 were predominant, with percentages of 48.2 and 30.9%, respectively. G4, G9, and G10 were detected in a minority of cases. Among the VP4 genotypes, P[8] occurred the most frequently (56.4%), followed by P[4] (31.8%), and only a few P[10] and P[11] at percentages of 1.8 and 0.9%, respectively. CONCLUSION: Our epidemiological data of HRV strains will contribute to assessing the magnitude of the problem of HRV in the south of Italy.

Post-treatment of UASB reactor effluent in waste stabilization ponds and in horizontal flow constructed wetlands: a comparative study in pilot scale in Southeast Brazil.

The results of a 20-month period study in Brazil were analyzed to compare horizontal-flow constructed wetlands (CW) and waste stabilization pond (WSP) systems in terms of land area requirements and performance to produce effluent qualities for surface water discharge, and for wastewater use in agriculture and/or aquaculture. Nitrogen, E. coli and helminth eggs were more effectively removed in WSP than in CW. It is indicated that CW and WSP require similar land areas to achieve a bacteriological effluent quality suitable for unrestricted irrigation (10(3) E. coli per 100 mL), but CW would require 2.6 times more land area than ponds to achieve quite relaxed ammonia effluent discharge standards (20 mg NH(3) L(-1)), and, by far, more land than WSP to produce an effluent complying with the WHO helminth guideline for agricultural use (< or =1 egg per litre).

Giardia spp. e Cryptosporidium spp. em água de manancial superficial de abastecimento contaminada por dejetos humano e animal / Giardia spp. and Cryptosporidium spp. in a fresh water supply source contaminated with human and animal excreta and waste water

Estudou-se ocorrência de cistos de Giardia spp. e oocistos de Cryptosporidium spp. - (oo)cistos - na bacia hidrográfica do Ribeirão São Bartolomeu, localizada no município de Viçosa, MG. O estudo incluiu as populações humana e animal existentes na área da bacia, bem como no efluente de uma estação de tratamento de esgoto (ETE) e de duas instalações para suínos. Os resultados indicam presença de (oo)cistos no manancial (médias geométricas: 3,92 e 3,62 (oo)cistos/l para Giardia spp. e Cryptosporidium spp., respectivamente). Propriedades com exploração bovina foram positivas ao longo de todo período (prevalência média de propriedades positivas 36,4 por cento para Giardia spp. e 18,0 por cento para Cryptosporidium spp.). O efluente da ETE apresentou elevada concentração de cistos de Giardia spp. (média geométrica na ordem de 10(4)/l), mas não foram encontrados oocistos de Cryptosporidium spp. A ocorrência de (oo)cistos apresentou comportamento sazonal, sendo que os valores médios de (oo)cistos e de pluviosidade do trimestre de coleta revelaram bom ajuste de correlação (R²=98,3 por cento; P=0,0087 para Giardia spp. e R²=91,8 por cento; P=0,0421 para Cryptosporidium spp.). A significativa ocorrência de (oo)cistos no manancial sugere que a forma de ocupação do solo interfere na qualidade parasitológica da água bruta. Os resultados apontam para a importância de adoção de medidas preventivas, como proteção de áreas de mananciais, objetivando reduzir riscos de transmissão de protozoários via água de consumo humano.

The occurrence of Giardia spp. cysts and Cryptosporidium spp. oocysts - (oo)cysts - was studied in a watershed (São Bartolomeu Stream-SB), at Viçosa city, MG. In the studied area, characterization of animal farms and identification of sewage and animal wastes discharges were undertaken. In addition, a sewage treatment plant (STP) and effluents of two swine farms were sampled. The results presented protozoan contamination (geometric averages: 3.92 and 3.62 (oo)cysts/l for Giardia spp. and Cryptosporidium spp., respectively). Positive cattle farms were detected during all the period, and the correspondent mean prevalences were: 36.4 percent for Giardia spp. and 18.0 percent for Cryptosporidium spp. STP effluent presented high concentration of Giardia spp. (geometric average of cysts in the order of 10(4)/l). However, Cryptosporidium spp. oocysts were not detected. The results showed that (oo)cysts concentration were influenced by climate factors. Precipitation and (oo)cysts concentration data were well correlated (R²=98.3 percent; P=0.0087 for Giardia spp. and R²=91.8 percent; P=0.0421 for Cryptosporidium spp.). Cysts and oocysts levels were remarkably high in the raw water source, pointing out the role of land use in an unprotected watershed as a source of protozoa. The results demonstrate the importance of preventive measures rather than relying on, sometimes unreliable, corrective measures such as water treatment, in order to minimize human health risks.

Swine infection by Streptococcus suis: a retrospective study / Infecção em suínos por Streptococcus suis: estudo retrospectivo

The epidemic aspects of swine infections caused by Streptococcus suis were studied, focusing mainly on the occurrence of several serotypes. A total of 323 samples of S. suis were isolated from clinically ill animals, serotyped according to the co-agglutination procedure, and analyzed. The serotyping revealed that S. suis was present in several Brazilian states. The largest number was isolated from the states of Minas Gerais (62.5 percent), São Paulo (10.8 percent), and Paraná (9.3 percent). Serotype 2 was the most frequent (61.0 percent), followed by the serotypes 1, 3, 4, 7, and 8. The largest number of isolations was obtained from the brain (60.1 percent), followed by the lungs (10.4 percent). About 9.4 percent of the cases were due to septicemia.

Estudaram-se os aspectos epidêmicos das infecções de suínos causadas por Streptococcus suis, enfocando, principalmente, a ocorrência de diferentes sorotipos. Foram analisadas 323 amostras isoladas de animais clinicamente doentes, as quais foram sorotipadas de acordo com o procedimento de co-aglutinação. Foi verificado que S. suis está presente em vários estados brasileiros e o maior número de isolados originou-se dos estados de Minas Gerais (62,5 por cento), São Paulo (10,8 por cento) e Paraná (9,3 por cento). O sorotipo 2 foi o mais freqüente (61.0 por cento), seguido pelos sorotipos 1, 3, 4, 7 e 8. Os isolamentos foram obtidos principalmente de cérebro (60,1 por cento) e pulmões (10,4 por cento). Os casos de septicemia representaram 9,4 por cento.

Wastewater irrigation of salad crops: further evidence for the evaluation of the WHO guidelines.

This paper presents the results of an experiment on salad crop irrigation with waste stabilisation pond effluents, conducted in Southeast Brazil. Over about 18 months several trials were carried out using different effluent qualities to irrigate lettuce, kale, arugula, spinach, and green pepper. Equations for predicting the bacterial quality of irrigated crops (E.coli per gram) based on the irrigation water quality (E.coli per 100 mL) were derived for low and high growing crops. The quantitative microbial risk analysis (QMRA) technique, using pathogen-ingestion scenarios based on these field data and on official statistics of vegetables consumption in Brazil, was used to estimate infection risks arising from the consumption of wastewater irrigated crops. It is inferred that irrigation with effluents complying with the WHO guidelines for unrestricted irrigation should result in salad crops acceptable for consumption.

Anaplastic large cell lymphomas: a study of 75 pediatric patients.

In this article, we describe the morphologic and immunophenotypic features of 75 cases of pediatric anaplastic large cell lymphoma (ALCL). According to the World Health Organization classification, 49 cases were common subtype ALCL, and respectively, 3, 6, and 17 cases were small cell, lymphohistiocytic, or mixed histologic variants. Anaplastic lymphoma kinase positivity was detected in 90.7% of the tumors and, using a panel of 9 T-cell surface markers, 88% could be assigned to the T-cell lineage. A molecular analysis for the T-cell receptor gamma (TCR- gamma) and the heavy chain of the immunoglobulin H rearrangements was performed on 6/9 ALCLs with a null immunophenotype, and a TCR clonal pattern was detected in 5/6 cases. In addition, 94.1% were immunoreactive for 1 or more cytotoxic proteins (Tia1, granzyme B, or perforin), and 15% expressed CD56. Clusterin, CD83, and Pax5, respectively, expressed in 91.3%, 1.7%, and 0% of the ALCLs, were useful biomarkers for the differential diagnosis with Hodgkin's lymphomas.

Aplicação da técnica de PCR na detecção de Yersinia enterocolitica em suínos abatidos sem inspeção / PCR technique application in Yersinia enterocolitica detection in non-inspected swine

Avaliou-se a contaminação de carcaças e tonsilas de suínos por Y. enterocolitica em estabelecimentos de abate não inspecionados, comparando a pesquisa microbiológica convencional com a técnica da reação em cadeia de polimerase (PCR) e o tipo de amostra analisada (de tonsila ou de carcaça), como subsídio ao monitoramento microbiano em sistemas de análise de perigos e pontos críticos de controle. Calcularam-se os custos dos dois testes. Não se detectou Y. enterocolitica pela técnica microbiológica convencional, mas, pela técnica de PCR foi possível detectar a bactéria em 40 por cento das carcaças e em 43 por cento das tonsilas, incluindo cepas patogênicas nas tonsilas. Não houve diferença entre os resultados positivos para as amostras de tonsilas e esfregaços de superfície das carcaças. A PCR apresentou-se como uma alternativa na detecção do agente e uma técnica aparentemente mais eficaz, econômica e rápida. Os resultados indicam a PCR como um importante recurso para o controle de qualidade da carne suína.

The contamination of swine carcasses and tonsils by Yersinia enterocolitica in slaughterhouses without inspection was evaluated. The conventional microbiological analysis was compared with the PCR technique of carcass or tonsil swabs, as a subsidy to the microbiological evaluation in the HACCP system. The costs of the two techniques were also calculated. Y. enterocolitica was not detected by the conventional microbiological analysis. Using the PCR, it was possible to detect this bacterium in carcass (40 percent) and tonsil (43 percent) samples. There was no difference between the positive results for the carcass and tonsil samples. The PCR showed to be a more effective, fast and economic alternative for the Y. enterocolitica detection, as compared to the conventional microbiological analysis.

Linkage between proton binding and folding in RNA: implications for RNA catalysis.

Small ribozymes use their nucleobases to catalyse phosphodiester bond cleavage. The hepatitis delta virus ribozyme employs C75 as a general acid to protonate the 5'-bridging oxygen leaving group, and to accomplish this task efficiently, it shifts its pKa towards neutrality. Simulations and thermodynamic experiments implicate linkage between folding and protonation in nucleobase pKa shifting. Even small oligonucleotides are shown to fold in a highly co-operative manner, although they do so in a context-specific fashion. Linkage between protonation and co-operativity of folding may drive pKa shifting and provide for enhanced function in RNA.

Mechanistic characterization of the HDV genomic ribozyme: assessing the catalytic and structural contributions of divalent metal ions within a multichannel reaction mechanism.

Hepatitis delta virus (HDV) uses genomic and antigenomic ribozymes in its replication cycle. We examined ribozyme self-cleavage over eight orders of magnitude of Mg(2+) concentration, from approximately 10(-9) to 10(-1) M. These experiments were carried out in 1 M NaCl to aid folding of the ribozyme and to control the ionic strength. The concentration of free Mg(2+) ions was established using an EDTA-Mg(2+) buffered system. Over the pH range of 5-9, the rate was independent of Mg(2+) concentration up to 10(-7) M, and of the addition of a large excess of EDTA. This suggests that in the presence of 1 M NaCl, the ribozyme can fold and cleave without using divalent metal ions. Brønsted analysis under these reaction conditions suggests that solvent and hydroxide ions may play important roles as general base and specific base catalysts. The observed rate constant displayed a log-linear dependence on intermediate Mg(2+) concentration from approximately 10(-7) to 10(-4) M. These data combined with the shape of the pH profile under these conditions are consistent with the binding of at least one structural divalent metal ion that does not participate in catalysis and binds tighter at lower pH. No evidence for a catalytic role for Mg(2+) was found at low or intermediate Mg(2+) concentrations. Addition of Mg(2+) to physiological and higher concentrations, from 10(-3) to 10(-1) M, revealed a second saturable divalent metal ion which binds tighter at high pH. The shape of the pH profile is inverted relative to that at low Mg(2+) concentrations, consistent with a general acid-base catalysis mechanism in which a cytosine (C75) acts as the general acid and a hydroxide ion from the divalent metal ion, or possibly from solvent, acts as the base. Overall, the data support a model in which the HDV ribozyme can self-cleave by multiple divalent ion-independent and -dependent channels, and in which the contribution of Mg(2+) to catalysis is modest at approximately 25-fold. Surface electrostatic potential maps were calculated on the self-cleaved form of the ribozyme using the nonlinear Poisson-Boltzmann equation. These calculations revealed several patches of high negative potential, one of which is present in a cleft near N4 of C75. These calculations suggest that distinct catalytic and structural metal ion sites exist on the ribozyme, and that the negative potential at the active site may help shift the pK(a) for N3 of C75 toward neutrality.

TINU syndrome associated with reduced complement levels.

The TINU syndrome (tubulointerstitial nephritis and uveitis) was first described by Dobrin et al. in 1975. Since then, more than 50 cases have been documented each with diverse immunopathogenetic and genetic characteristics. The aim of this report is to describe a case of TINU associated with reduced complement levels. We profile a 48-year-old white female with persistently reduced C4 complement levels during the acute phase of the pathology and with an unaltered immunologic profile. Renal biopsy evidenced a significant lymphocytic interstitial infiltration. Immunohistochemical studies of the interstitium infiltrates was positive for the presence of the T (CD3) markers (CD4 > CD8). Steroid therapy yielded a complete regression of the symptomatology with normalization of the complement levels. We suggest that it is possible to hypothesize that the various immunologic alterations associated with TINU, including the transient reduction complement levels, may be secondary to multiple inflammatory mechanisms which express themselves throughout the pathology.

Demonstration of Chlamydia pneumoniae in atherosclerotic arteries from various vascular regions.

Chlamydia pneumoniae (CP) has been reported to be a pathogenic agent in the mechanism leading to atherosclerosis. The majority of available data is focused mainly on coronary artery disease whereas the distribution of CP in different areas, associated with atherosclerotic disorders, has not been completely clarified. In this study we investigated the presence of CP in atheromasic plaques from five different vascular areas (basilary artery, coronary artery, thoracic aorta, abdominal aorta, renal arteries) using nested polymerase chain reaction (PCR) and immunohistochemical staining (IHC), in order to establish the putative association of CP with atherosclerotic disease. The same atheromasic plaques were also tested for the presence of Helicobacter pylori (HP) and cytomegalovirus (CMV), other putative agents of atherosclerosis, using a nested PCR technique. Our data indicate that the presence of CP can be demonstrated in 100% of patients tested, considering globally the five areas of analysis. On the other hand the presence of HP has been demonstrated in four out of 18 patients (22.2%), and CMV only in three out of 18 (16.6%). Our results strongly suggest an association between CP and atherosclerosis and highlight the need for the detection of CP in multiple vascular areas of the same patient.

Urbanizaçäo da leishmaniose visceral em Belo Horizonte / Urbanization of visceral leishmaniose in Belo Horizonte, Brazil

O Brasil vivencia na atualidade uma situaçäo na qual velhas edemias, que se pensava esquecidas, ressurgem com grande impacto e muitas vezes com perfis de morbi-mortalidades diferentes daqueles já conhecidos. Este é o caso da leishmaniose visceral, uma doença caracteristicamente rural e associada a condiçöes precárias de vida, que encontra no espaço urbano ambiente favorável para se estabelecer e desenvolver. O município de Belo Horizonte convive, desde 1993, com uma epidemia de calazar humano e canino. Foram feitas a reconstruçäo histórica da enfermidade e a caracterizaçäo de alguns aspectos demográficos dos casos humanos. Aparentemente a doença foi introduzida em Belo Horizonte a partir de focos existentes em municípios vizinhos, como Sabará. Os Distritos Sanitários Leste e Nordeste foram os mais acometidos. A evoluçäo espacial da epidemia mostrou que os casos caninos precederam os humanos, confirmando a importância do cäo como reservatório do calazar em áreas urbanas. O risco de adoecer foi maior em crianças menores de 15 anos. A situaçäo de Belo Horizonte ilustra muito bem o processo de urbanizaçäo de enfermidades tradicionalmente rurais, vivenciado por várias cidades brasileiras

Efficient construction of long DNA duplexes with internal non-Watson-Crick motifs and modifications.

We have developed a semi-synthetic approach for preparing long stretches of DNA (>100 bp) containing internal chemical modifications and/or non-Watson-Crick structural motifs which relies on splint-free, cell-free DNA ligations and recycling of side-products by non-PCR thermal cycling. A double-stranded DNA PCR fragment containing a polylinker in its middle is digested with two restriction enzymes and a small insert ( approximately 20 bp) containing the modification or non-Watson-Crick motif of interest is introduced into the middle. Incorrect products are recycled to starting materials by digestion with appropriate restriction enzymes, while the correct product is resistant to digestion since it does not contain these restriction sites. This semi-synthetic approach offers several advantages over DNA splint-mediated ligations, including fewer steps, substantially higher yields ( approximately 60% overall yield) and ease of use. This method has numerous potential applications, including the introduction of modifications such as fluorophores and cross-linking agents into DNA, controlling the shape of DNA on a large scale and the study of non-sequence-specific nucleic acid-protein interactions.

Straightening of bulged RNA by the double-stranded RNA-binding domain from the protein kinase PKR.

The human interferon-induced protein kinase, PKR, is an antiviral agent that is activated by long stretches of double-stranded (ds)RNA. PKR has an N-terminal dsRNA-binding domain that contains two tandem copies of the dsRNA-binding motif and interacts with dsRNA in a nonsequence-specific fashion. Surprisingly, PKR can be regulated by certain viral and cellular RNAs containing non-Watson-Crick features. We found that RNAs containing bulges in the middle of a helix can bind to p20, a C-terminal truncated PKR containing the dsRNA-binding domain. Bulges are known to change the global geometry of RNA by bending the helical axis; therefore, we investigated the conformational changes of bulged RNA caused by PKR binding. A 66-mer DNA-RNA(+/- A(3) bulge)-DNA chimera was constructed and annealed to a complementary RNA strand. This duplex forces the protein to bind in the middle. A 66-mer duplex with a top strand composed of DNA-DNA(+/-A(3) bulge)-RNA was used as a control. Gel mobility-shift changes among the RNA-protein complexes are consistent with straightening of bulged RNA on protein binding. In addition, a van't Hoff analysis of p20 binding to bulged RNA reveals a favorable DeltaDeltaH degrees and an unfavorable DeltaDeltaS degrees relative to binding to straight dsRNA. These thermodynamic parameters are in good agreement with predictions from a nearest-neighbor analysis for RNA straightening and support a model in which the helical junction flanking the bulge stacks on protein binding. The ability of dsRNA-binding motif proteins to recognize and straighten bent RNA has implications for modulating the topology of RNAs in vivo.