Evaluation of two DNA extraction methods for the PCR-based detection of eukaryotic enteric pathogens in fecal samples.

OBJECTIVE: Efficient and easy-to-use DNA extraction and purification methods are critical in implementing PCR-based diagnosis of pathogens. In order to optimize the routine clinical laboratory diagnosis of eukaryotic enteric pathogens, we compare, via quantitative PCR cycle threshold (Ct) values, the efficiency of two DNA extraction kits: the semi-automated EZ1® (Qiagen) and the manual QIAamp® DNA Stool Mini Kit (Qiagen), on six protozoa: Blastocystis spp., Cryptosporidium parvum/hominis, Cyclospora cayetanensis, Dientamoeba fragilis, Giardia intestinalis and Cystoisospora belli and one microsporidia: Enterocytozoon bieneusi. RESULTS: Whereas EZ1® (Qiagen) and QIAamp® DNA Stool Mini Kit (Qiagen) yielded similar performances for the detection of Cryptosporidium spp. and D. fragilis, significant lower Ct values (p < 0.002) pointed out a better performance of EZ1® on the five remaining pathogens. DNA extraction using the semi-automated EZ1® procedure was faster and as efficient as the manual procedure in the seven eukaryotic enteric pathogens tested. This procedure is suitable for DNA extraction from stools in both clinical laboratory diagnosis and epidemiological study settings.

A prepared mind--Ernest Edward Tyzzer's legacy of research into avian diseases.

Ernest Edward Tyzzer (1875-1965) was a physician, specializing at first (1902-1916) in cancer research and then from 1916 as a parasitologist. He was born of English parents in Wakefield, Massachusetts, where he lived all his life. Educated in Wakefield public schools, Brown University (Ph.B., A.M., Hon. Sc.D.), and Harvard University (M.D.), he established during his 40-yr career (1902-1942) an international reputation in oncology, pathology, virology, bacteriology, parasitology, and taxonomic zoology in relation to human and veterinary medicine. His contributions to knowledge of avian diseases were outstanding and wide-ranging. Seminal work included: new descriptions of tumors in chickens; the first record of Cryptosporidium in birds; studies on the biology, morphology, in vitro culture, and epizootiology of the blackhead (histomonosis) parasite and its reclassification under a new genus Histomonas; descriptions of eight new taxa of amebae and flagellates in chickens, turkeys, and ruffed grouse; descriptions of seven new species of Eimeria in chickens, turkeys, pheasants, and quail as well as studies on their biology, immunogenicity, virulence, and epizootiology; a description of the trematode Collyriclum in English sparrows; the first record of mycosis in ruffed grouse; the recognition of birds as a source of equine encephalomyelitis infections of humans; the first American record of infectious sinusitis in turkeys and discovery of a curative treatment; and studies of Newcastle disease and avian influenza during the war research program of the 1940s. Application of Tyzzer's histomonosis research to farm practice saved the Massachusetts turkey industry from extinction in the 1920s and significantly influenced the recovery of turkey farming nationally.

The struggle for iron - a metal at the host-pathogen interface.

Iron holds a central position at the host-pathogen interface because mammalian and microbial cells have an essential demand for the metal, which is required for many metabolic processes. In addition, cross-regulatory interactions between iron homeostasis and immune function are evident. While iron affects the secretion of cytokines and the activity of transcription factors orchestrating immune responses, immune cell-derived mediators and acute-phase proteins control both systemic and cellular iron homeostasis. Additionally, immune-mediated strategies aim at restricting the supply of the essential nutrient iron to pathogens, which represents an effective strategy of host defence. On the other hand, microbes have evoked multiple strategies to utilize iron because a sufficient supply of this metal is linked to pathogen proliferation, virulence and persistence. The control over iron homeostasis is a central battlefield in host-pathogen interplay influencing the course of an infectious disease in favour of either the mammalian host or the pathogenic invader. This review summarizes our current knowledge on the combat of host cells and pathogens for the essential nutrient iron focusing on the immune-regulatory roles of iron on cell-mediated immunity necessary to control intracellular microbes, the host's mechanisms of iron restriction and on the counter-acting iron-acquisition strategies employed by intracellular microbes.

Intrinsic antibody-dependent enhancement of microbial infection in macrophages: disease regulation by immune complexes.

A wide range of microorganisms can replicate in macrophages, and cell entry of these pathogens via non-neutralising IgG antibody complexes can result in increased intracellular infection through idiosyncratic Fcγ-receptor signalling. The activation of Fcγ receptors usually leads to phagocytosis. Paradoxically, the ligation of monocyte or macrophage Fcγ receptors by IgG immune complexes, rather than aiding host defences, can suppress innate immunity, increase production of interleukin 10, and bias T-helper-1 (Th1) responses to Th2 responses, leading to increased infectious output by infected cells. This intrinsic antibody-dependent enhancement (ADE) of infection modulates the severity of diseases as disparate as dengue haemorrhagic fever and leishmaniasis. Intrinsic ADE is distinct from extrinsic ADE, whereby complexes of infectious agents with non-neutralising antibodies lead to an increased number of infected cells. Intrinsic ADE might be involved in many protozoan, bacterial, and viral infections. We review insights into intracellular mechanisms and implications of enhanced pathogenesis after ligation of macrophage Fcγ receptors by infectious immune complexes.

Diarrea del viajero: prevención y tratamiento / Traveler’s diarrhea: prevention and treatment

La diarrea del viajero (DV) afecta a 34 millones de personas que viajan a países en desarrollo todos los años. El destino representa el factor de riesgo más importante para el desarrollo de DV. Por mucho, los agentes etiológicos más frecuentes son bacterias patógenas (Escherichia coli enterotoxigénica, Escherichia coli enteroagregativa, Campylobacter, Salmonella). La reducción en la tasa de diarrea sería posible al evitar el consumo de alimentos y bebestibles contaminados. Una estrategia preventiva más eficaz consiste en administrar antibióticos todos los días durante viajes a áreas en que el riesgo de DV es alto. Rifaximina, antibiótico no absorbible recientemente aprobado, puede ser usado para el tratamiento de la DV en regiones donde la E. coli no invasora es el patógeno predominante. En áreas donde un organismo invasivo como el Campylobacter y Shigella son comunes, las fluoroquinolonas sigue siendo el medicamento escogido. La azitromicina es recomendada en áreas con Campylobacter resistente a las quinolonas y para el tratamiento de niños y mujeres embarazadas.

Traveler’s diarrhea (TD) affects 34 millions of people who travel to developing countries each year. Destination represents the single most important risk factor for developing TD. By far, the most frequent etiologic agents are bacterial pathogens (enterotoxigenic Escherichia coli, enteroaggregative E. coli, Campylobacter, Salmonella). Reduction in the rate of diarrhea maybe possible by avoiding contaminated foods and beverages. A more effective preventive strategy is daily administration of antibiotics during trips to areas where the risk of TD is high. Rifaximine, a recently approved non-absorbable antibiotic, can be used for the treatment of TD in regions where non invasive E. coli is the predominant pathogen. In areas where invasive organism such as Campylobacter and Shigella are common, fluoroquinolones remain the drug of choice. Azythromycin is recommended in areas with quinolone-resistant Campylobacter and for treatment of children and pregnant women.

Adaptação ao Método de Ritchie para diagnóstico de Helmintos e Protozoários em amostras de lodo de esgoto com minimização de produtos químicos / La adaptación del Método de Ritchie para la diagnosis de Helmintos y protozoos en muestras de aguas residuales con minimización de productos químicos / Adaptation of Ritchie Method for diagnosing Helminthes and Protozoa in samples of sewage silt with chemical substances minimization

O Método de Ritchie (1948) é uma metodologia eficiente para diagnóstico parasitário em matrizes ambientais, porém apresenta como desvantagem o uso do éter etílico e formaldeído, reagentes toxicológicos para a saúde ambiental e ocupacional. O objetivo do estudo foi padronizar uma técnica adaptada do Método de Ritchie para identificação de parasitas em lodo de esgoto, que evite o uso de substâncias tóxicas e possa ser utilizada em laboratórios de parasitologia. Amostras de lodo foram submetidas ao Método Ritchie (MR) e ao Método de Ritchie Modificado por Régis Anécimo (MRMRA), no qual substancias químicas são substituídas por água a 45 graus Ce detergente neutro. Os resultados demonstraram que, no MR, a porcentagem de Ancylostoma sp foi de 19%, Hymenolepis sp 79%, Ascaris sp 2% e Trichuris trichiura 0%, em um total de 1970 helmintos visualizados. Porém, no MRMRA, a porcentagem de Ancylostoma sp foi de 56%, Hymenolepis sp 40%, Ascaris sp 4% e Tricuris trichiura 0%, em um total de 398 helmintos. Avaliando quantitativamente o MRMRA foi menos eficiente, pois esses resultados demonstraram uma diferença significativa de helmintos visualizados entre os métodos. Porém, enquanto análise qualitativa, a técnica pode ser considerada válida, uma vez que a qualidade da visualização dos parasitas é correspondente em ambos os métodos. Pode ser concluído que o MRMRA é um método simples de realizar e de fácil implementação na rotina de laboratórios e estações de tratamento de esgoto.

El Método de Ritchie (1948) es una metodología eficiente para diagnosticar organismos parásitos en matrices ambientales, pero el uso de del éter etílico y del formaldehído es una desventaja, vez que son reactivos tóxicos para el ambiente y la salud ocupacional. El objetivo de este estudio fue estandardizar una técnica conveniente del Método de Ritchie como para la identificación de parásitos en el légamo de las aguas residuales, técnica que evita el uso de sustancias tóxicas y se puede utilizar en laboratorios parasitológicos. Las muestras de légamo fueron sometidas al Método de Ritchie (MR) y al Método de Ritchie Modificado por Régis Anécimo (MMRRA), en el cual quien el agua en 45 grados C y detergente neutral substituyen las sustancias químicas. Los resultados demuestran que en RM el porcentaje de Ancylostoma sp fue 19%, Hymenolepis sp 79%, Ascaris sp 2% y Trichuris trichiura 0% de un total de 1970 helmintos, mientras que enel MRMRA el porcentaje del de Ancylostoma sp fue 56%, Hymenolepis sp 40%, Ascaris sp 4% y Trichuris trichiura 0% de un total de 398 helmintos. Cuantitativamente evaluado MRMRA fue menos eficiente, porque estos resultados demuestran una diferencia significativa de los helmintos identificados por los métodos. Sin embargo, un análisis cualitativo demuestra que la técnica es válida, puesto que la calidad de la identificación de los parásitos está igual en ambos métodos. Se puede concluir que MRMRA es un método simple y fácil como para ser utilizado en la rutina de laboratorios y de estaciones del tratamiento de aguas residuales.

Ritchie Method (1948) is an efficient methodology for diagnosing parasitic organisms in environmental matrixes, but the use of ethylic ether and formaldehyde is an disadvantage, for these are toxic reagents that damages the environment and affects occupational health. The objective of this study was to standardize a suitable technique of Ritchie Method for the identification of parasites in sewage silt, which avoids the use of toxic substances and can be used in parasitological laboratories. Samples of silt were submitted to Ritchie Method (RM) and Method of Modified Ritchie by Régis Anécimo (MMRRA), in which water at 45 degrees C and neutral detergent substitute for chemical substances. Results show that in RM the percentage of Ancylostoma sp was 19%, Hymenolepis sp 79%, Ascaris sp 2% and Trichuris trichiura 0% from a total of 1970 visualized helminthes, whereas in MRMRA the percentage of Ancylostoma sp was 56%, Hymenolepis sp 40%, Ascaris sp 4% and Tricuris trichiura 0% in a total of 398 helminthes. Evaluated quantitatively MRMRA was less efficient, for these results show a significant difference in the number of helminthes identified by the methods. However, a qualitative analysis shows the technique to be valid, since the quality of parasites identification is the same in both methods. We may conclude that MRMRA is a simple and easy method to be used in the routine of laboratories and sewage treatment stations.

varDB: common ground for a shifting landscape.

Antigenic variation is a phylogenetically widespread phenomenon thought to lead to survival benefits for the pathogen. Although governed by genetic mechanisms, antigenic variation is ultimately manifested in variant proteins. The varDB database is an attempt to gain an overview of common structures and functions of variant proteins related to enhanced survival. varDB provides a wealth of sequence data and several tools to facilitate their analysis, but current limitations preclude achievement of its full promise. A critique of this database and how it could serve the scientific community is provided here.

Microbial Cretaceous park: biodiversity of microbial fossils entrapped in amber.

Microorganisms are the most ancient cells on this planet and they include key phyla for understanding cell evolution and Earth history, but, unfortunately, their microbial records are scarce. Here, we present a critical review of fossilized prokaryotic and eukaryotic microorganisms entrapped in Cretaceous ambers (but not exclusively from this geological period) obtained from deposits worldwide. Microbiota in ambers are rather diverse and include bacteria, fungi, and protists. We comment on the most important microbial records from the last 25 years, although it is not an exhaustive bibliographic compilation. The most frequently reported eukaryotic microfossils are shells of amoebae and protists with a cell wall or a complex cortex. Likewise, diverse dormant stages (palmeloid forms, resting cysts, spores, etc.) are abundant in ambers. Besides, viral and protist pathogens have been identified inside insects entrapped in amber. The situation regarding filamentous bacteria and fungi is quite confusing because in some cases, the same record was identified consecutively as a member of these phylogenetically distant groups. To avoid these identification errors in the future, we propose to apply a more resolute microscopic and analytical method in amber studies. Also, we discuss the most recent findings about ancient DNA repair and bacterial survival in remote substrates, which support the real possibility of ancient DNA amplification and bacterial resuscitation from Cretaceous resins.

Deciphering the ubiquitin-mediated pathway in apicomplexan parasites: a potential strategy to interfere with parasite virulence.

BACKGROUND: Reversible modification of proteins through the attachment of ubiquitin or ubiquitin-like modifiers is an essential post-translational regulatory mechanism in eukaryotes. The conjugation of ubiquitin or ubiquitin-like proteins has been demonstrated to play roles in growth, adaptation and homeostasis in all eukaryotes, with perturbation of ubiquitin-mediated systems associated with the pathogenesis of many human diseases, including cancer and neurodegenerative disorders. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe the use of an HMM search of functional Pfam domains found in the key components of the ubiquitin-mediated pathway necessary to activate and reversibly modify target proteins in eight apicomplexan parasitic protozoa for which complete or late-stage genome projects exist. In parallel, the same search was conducted on five model organisms, single-celled and metazoans, to generate data to validate both the search parameters employed and aid paralog classification in Apicomplexa. For each of the 13 species investigated, a set of proteins predicted to be involved in the ubiquitylation pathway has been identified and demonstrates increasing component members of the ubiquitylation pathway correlating with organism and genome complexity. Sequence homology and domain architecture analyses facilitated prediction of apicomplexan-specific protein function, particularly those involved in regulating cell division during these parasite's complex life cycles. CONCLUSIONS/SIGNIFICANCE: This study provides a comprehensive analysis of proteins predicted to be involved in the apicomplexan ubiquitin-mediated pathway. Given the importance of such pathway in a wide variety of cellular processes, our data is a key step in elucidating the biological networks that, in part, direct the pathogenicity of these parasites resulting in a massive impact on global health. Moreover, apicomplexan-specific adaptations of the ubiquitylation pathway may represent new therapeutic targets for much needed drugs against apicomplexan parasites.

Pathogenicity of the protozoan parasite Marteilioides chungmuensis in the Pacific oyster Crassostrea gigas.

Marteilioides chungmuensis is an ovarian parasite that causes nodule-like structures to appear on the gonads of female Pacific oysters, Crassostrea gigas. It is known that the prevalence of infection increases in summer and decreases from autumn to spring. To investigate the decrease in prevalence of infection and pathogenicity of the parasite, a biopsy method was developed to detect infected oysters, which were then monitored to calculate the mortality rate. Mortality of infected oysters was recorded monthly and changes in reproductive development observed histologically. Compared with control groups, a significant difference in mortality was observed in infected oysters in September and October. Histological observations showed that infected oysters produced oocytes continuously, even in autumn when healthy oysters were reproductively inactive. This prolonged spawning activity of infected oysters resulted in nutritional wasting and mortality. From December onwards, however, almost all infected oysters survived, though the infection persisted. Infection intensity decreased gradually from December. Histological observations revealed that, in winter, infected oysters released infected and uninfected oocytes through the genital canal. The gonad subsequently degenerated and was replaced with connective tissue, as in normal, healthy spent oysters. The results revealed that prevalence of infection decreased from September to May. It is hypothesised that the decline in prevalence within the epizootic area in autumn occurred because infected oysters died and that the winter decrease was due to recovery from infection.

Control of IFN-gamma-mediated host resistance to intracellular pathogens by immunity-related GTPases (p47 GTPases).

IRG proteins (also known as p47 GTPases) are key mediators of interferon-gamma-induced resistance to pathogens. Absence of certain IRG proteins leads to profound susceptibility to protozoa and bacteria in mice. Underlying their roles in host resistance, IRG proteins regulate the processing of pathogen-containing vacuoles in host cells, and regulate hematopoiesis following infection.

Characterization of glycans in the developmental stages of Myxobolus cerebralis (Myxozoa), the causative agent of whirling disease.

Glycans and sugar-binding molecules (lectins) form an interactive recognition system, which may enable parasitic organisms to adhere to host cells and migrate into target tissues. The aim of the present study was to analyse surface-associated glycans in the developmental stages of Myxobolus cerebralis (Hofer), the causative agent of whirling disease. A panel of biotin-labelled plant lectins was used to detect a broad spectrum of glycan motifs with high specificity. Binding sites were detected histochemically in the tissue sections of infected rainbow trout, Oncorhynchus mykiss (Walbaum), and infected Tubifex tubifex (Müller), and were characterized by light, fluorescence and transmission electron microscopy. With mannose-specific lectins [Lens culinaris agglutinin, Pisum sativum agglutinin, Canavalia ensiformis agglutinin (LCA, PSA, CanA)] mannose-containing glycans were detected in all the developmental stages and host tissues. No binding sites for galactose-specific lectins were present in M. cerebralis spores but reactivity with host tissues occurred. Diversity in glycans was detected by N-acetyl-D-galactosamine-specific lectins in sporoplasm cells of M. cerebralis and triactinomyxon spores. In the group of lectins with monosaccharide-specificity for N-acetyl-D-glucosamine (GlcNAc), the reactivity of Datura stramonium agglutinin (DSA), Lycopersicon esculentum agglutinin (LEA) and Solanum tuberosum agglutinin (STA) was restricted to polar capsules whereas Griffonia simplicifolia agglutinin II (GSA II) also bound to sporoplasm cells of stages in the fish host but not in those present in infected T. tubifex. Moreover, Triticum vulgaris (wheat germ) agglutinin (WGA) and succinylated WGA indicated the presence of N-acetyl-D-glucosamine polymers in polar capsules. No specificity for spores was observed concerning 'bisected'N-glycans and no reactivity in parasitic stages was observed with the fucose-binding lectin Ulex europaeus agglutinin (UEA) I, Sambucus nigra agglutinin (SNA) (specific for alpha2,6-sialylated glycans) and Maackia amurensis agglutinin (MAAI) (specific for alpha2,3-sialylated glycans). Arachis hypogaea (peanut) agglutinin (PNA), Erythrina cristagalli agglutinin (ECA), GSA I, Sophora japonica agglutinin (SJA), Dolichos biflorus agglutinin (DBA) and GSA II detected reactive sites solely confined to the developmental stages of M. cerebralis and were not reactive in the fish host. These parasite-specific glycans may play a role in the adhesion process of the parasite to fish epidermis prior to infection, but may provide protection to the host by activating the complement system, or stimulating an adaptive immune response as putative antigens.

Current therapeutics, their problems, and sulfur-containing-amino-acid metabolism as a novel target against infections by "amitochondriate" protozoan parasites.

The "amitochondriate" protozoan parasites of humans Entamoeba histolytica, Giardia intestinalis, and Trichomonas vaginalis share many biochemical features, e.g., energy and amino acid metabolism, a spectrum of drugs for their treatment, and the occurrence of drug resistance. These parasites possess metabolic pathways that are divergent from those of their mammalian hosts and are often considered to be good targets for drug development. Sulfur-containing-amino-acid metabolism represents one such divergent metabolic pathway, namely, the cysteine biosynthetic pathway and methionine gamma-lyase-mediated catabolism of sulfur-containing amino acids, which are present in T. vaginalis and E. histolytica but absent in G. intestinalis. These pathways are potentially exploitable for development of drugs against amoebiasis and trichomoniasis. For instance, L-trifluoromethionine, which is catalyzed by methionine gamma-lyase and produces a toxic product, is effective against T. vaginalis and E. histolytica parasites in vitro and in vivo and may represent a good lead compound. In this review, we summarize the biology of these microaerophilic parasites, their clinical manifestation and epidemiology of disease, chemotherapeutics, the modes of action of representative drugs, and problems related to these drugs, including drug resistance. We further discuss our approach to exploit unique sulfur-containing-amino-acid metabolism, focusing on development of drugs against E. histolytica.

Longevity of Ceratomyxa shasta and Parvicapsula minibicornis actinospore infectivity in the Klamath River.

Infectious Ceratomyxa shasta and Parvicapsula minibicornis actinospores were present in Klamath River samples collected in April, May, and June 2005. Juvenile Chinook salmon Oncorhynchus tshawytscha exposed to river water maintained at the ambient Klamath River temperature for 0, 4, 24, 72, and 168 h (7 d) developed asymptomatic infections from both parasites. Elevated water temperature (18 degrees C) in June may have reduced actinospore viability, as both C. shasta and P. minibicornis infection markedly declined in fish exposed for over 72 h. As judged by the prevalence of infection for both parasites, the number of infectious actinospores tended to increase or remain steady through the spring. Ceratomyxa shasta infections were characterized by the presence of a few trophozoites within granulomatous foci in mesentery adipose tissue and were consistently observed outside of the intestine. Similarly, low numbers of P. minibicornis were observed in kidney glomeruli and tubules but were not associated with inflammation. Parvicapsula minibicornis DNA was consistently detected by quantitative real-time polymerase chain reaction in filtered water samples collected each month and from each time posttransfer. These data and the high prevalence of infection observed in the exposed fish indicate that P. minibicornis actinospores were at a relatively high concentration in the river during the spring. In contrast, C. shasta DNA was only detected in half of the water sample sets and its detection did not correspond well to C. shasta infectivity. An approximately threefold increase in river flow from the April to the May water collection was not associated with a decline in either the detection of actinospores (particularly for P. minibicornis) or the prevalence of infection for both parasites. Actinospores of these myxosporean parasites have the potential to infect salmonids for at least 7 d after release from the alternate polychaete host.

Cell-mediated cytotoxicity is the main innate immune mechanism involved in the cellular defence of gilthead seabream (Teleostei: Sparidae) against Enteromyxum leei (Myxozoa).

The cellular innate immune response of gilthead seabream (Sparus aurata L.) against the myxozoan Enteromyxum leei was studied. Enteromyxosis was transmitted by maintaining uninfected fish (recipients) together with infected animals. A group of fish not exposed to the infection served as controls. After 10, 22, 38, 52 and 108 days, control and recipient fish were sampled and leucocyte subpopulations and cellular immune responses (leucocyte peroxidases, phagocytosis, respiratory burst and cytotoxicity) of the head-kidney leucocytes were determined. The percentage of acidophilic granulocytes was significantly lower in non-parasitized and parasitized recipient fish than in control fish after 22 days but no significant differences were seen between non-parasitized and parasitized recipient animals. The leucocyte peroxidase content, phagocytosis and respiratory burst activity were seen to have decreased significantly at different sampling times in both non-parasitized and parasitized recipient fish with respect to the controls, whereas cytotoxic activity was up to 2.3 times higher than in control fish. Within the recipient group, little difference was observed in the studied parameters between non-parasitized and parasitized fish. These data demonstrate that cytotoxic activity may have an important role in the defence of gilthead seabream against the myxosporean E. leei. Immunological implications of E. leei infections are discussed.

Neutrophils, apoptosis and phagocytic clearance: an innate sequence of cellular responses regulating intramacrophagic parasite infections.

In complex organisms, apoptosis is a constitutive cell death process that is involved in physiological regulation of cell numbers and that can also be induced in the course of inflammatory and immune responses. Neutrophils are among the first cells recruited during inflammation. Neutrophils constitutively die by apoptosis at inflamed sites, and are ingested by macrophages. Recent studies investigated how phagocytic clearance of senescent neutrophils influences the survival of intracellular protozoan parasites that have been phagocytosed by, or have invaded phagocytes. The results indicate that neutrophil clearance plays an unexpected role in regulation of intramacrophagic protozoan parasite infection.

Imaging today's infectious animalcules.

The study of pathogens and their interactions with host cells has advanced hand-in-hand with developments in optical microscopy. Whereas microbiology benefits enormously from modern imaging technologies, for example, digital imaging and confocal microscopy, it also presents unique challenges. To overcome these, microbiologists are adept at customising imaging methods, and recently there have been studies using state-of-the-art quantitative imaging methods to probe host-pathogen interactions at the single-cell level. Of particular interest are the studies using combined light and electron microscopy methods, bi-arsenical tetra-cysteine tag labelling and automated image-acquisition and analysis for high-throughput/high-content experimentation. These applications demonstrate how imaging methodologies, adapted for microbiology, continue to open avenues for studies that previously have proven inaccessible.

Inactivation credit of UV radiation for viruses, bacteria and protozoan (oo)cysts in water: a review.

UV disinfection technology is of growing interest in the water industry since it was demonstrated that UV radiation is very effective against (oo)cysts of Cryptosporidium and Giardia, two pathogenic micro-organisms of major importance for the safety of drinking water. Quantitative Microbial Risk Assessment, the new concept for microbial safety of drinking water and wastewater, requires quantitative data of the inactivation or removal of pathogenic micro-organisms by water treatment processes. The objective of this study was to review the literature on UV disinfection and extract quantitative information about the relation between the inactivation of micro-organisms and the applied UV fluence. The quality of the available studies was evaluated and only high-quality studies were incorporated in the analysis of the inactivation kinetics. The results show that UV is effective against all waterborne pathogens. The inactivation of micro-organisms by UV could be described with first-order kinetics using fluence-inactivation data from laboratory studies in collimated beam tests. No inactivation at low fluences (offset) and/or no further increase of inactivation at higher fluences (tailing) was observed for some micro-organisms. Where observed, these were included in the description of the inactivation kinetics, even though the cause of tailing is still a matter of debate. The parameters that were used to describe inactivation are the inactivation rate constant k (cm(2)/mJ), the maximum inactivation demonstrated and (only for bacterial spores and Acanthamoeba) the offset value. These parameters were the basis for the calculation of the microbial inactivation credit (MIC="log-credits") that can be assigned to a certain UV fluence. The most UV-resistant organisms are viruses, specifically Adenoviruses, and bacterial spores. The protozoon Acanthamoeba is also highly UV resistant. Bacteria and (oo)cysts of Cryptosporidium and Giardia are more susceptible with a fluence requirement of <20 mJ/cm(2) for an MIC of 3 log. Several studies have reported an increased UV resistance of environmental bacteria and bacterial spores, compared to lab-grown strains. This means that higher UV fluences are required to obtain the same level of inactivation. Hence, for bacteria and spores, a correction factor of 2 and 4 was included in the MIC calculation, respectively, whereas some wastewater studies suggest that a correction of a factor of 7 is needed under these conditions. For phages and viruses this phenomenon appears to be of little significance and for protozoan (oo)cysts this aspect needs further investigation. Correction of the required fluence for DNA repair is considered unnecessary under the conditions of drinking water practice (no photo-repair, dark repair insignificant, esp. at higher (60 mJ/cm(2)) fluences) and probably also wastewater practice (photo-repair limited by light absorption). To enable accurate assessment of the effective fluence in continuous flow UV systems in water treatment practice, biodosimetry is still essential, although the use of computational fluid dynamics (CFD) improves the description of reactor hydraulics and fluence distribution. For UV systems that are primarily dedicated to inactivate the more sensitive pathogens (Cryptosporidium, Giardia, pathogenic bacteria), additional model organisms are needed to serve as biodosimeter.

An ultrastructural and histopathological study of Henneguya pellucida n. sp. (Myxosporea: Myxobolidae) infecting Piaractus mesopotamicus (Characidae) cultivated in Brazil.

During a study of myxosporean parasites of cultivated freshwater fish, a new myxosporean species, Henneguya pellucida n. sp., was discovered. Of the 120 Piaractus mesopotamicus sampled, only 10 specimens (8.3%) were infected. Yellow, round plasmodia measuring 0.5-3 mm were found in the serous membrane of the visceral cavity and in the tunica externa of the swim bladder. Sporogenesis was asynchronous, with the earliest developmental stages aligned prevailingly along the endoplasmic periphery and mature spores in the central zone. The mature spores were pear shaped (total length: 33.3 +/- 1.5 microm, mean +/- SD; width: 4.1 +/- 0.4 microm; body length: 11.4 +/- 0.3 microm; caudal process length: 24.1 +/- 1.5 microm). The polar capsules were elongated (length: 4.0 +/- 0.4 microm; width: 1.6 +/- 0.2 microm). The development of the parasite in the swim bladder produced thickening of the tunica externa and a granulomatous reaction. There was no correlation between the prevalence of the parasite and the chemical and physical characteristics of the water. Infection was recorded only in juvenile specimens ranging in size from 9.5 to 20 cm.