obtaining-highly-purified-toxoplasma-gondii-oocysts-discontinuous

Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient

Toxoplasma gondii is an obligate intracellular protozoan pathogen that commonly infects humans. It is a well characterized apicomplexan associated with ...

a-modified-epa-method-1623-that-uses-tangential-flow-hollow-fiber

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better ...

Long-term resistance to Toxoplasma gondii is dependent on the development of parasite-specific T cells that produce IFN-gamma. CD28 is a costimulatory molecule important for optimal activation of T cells, but CD28(-/-) mice are resistant to T. gondii, demonstrating that CD28-independent mechanisms regulate T cell responses during toxoplasmosis. The identification of the B7-related protein 1/inducible costimulator protein (ICOS) pathway and its ability to regulate the production of IFN-gamma suggested that this pathway may be involved in the CD28-independent activation of T cells required for resistance to T. gondii. In support of this hypothesis, infection of wild-type or CD28(-/-) mice with T. gondii resulted in the increased expression of ICOS by activated CD4(+) and CD8(+) T cells. In addition, both costimulatory pathways contributed to the in vitro production of IFN-gamma by parasite-specific T cells and when both pathways were blocked, there was an additive effect that resulted in almost complete inhibition of IFN-gamma production. Although in vivo blockade of the ICOS costimulatory pathway did not result in the early mortality of wild-type mice infected with T. gondii, it did lead to increased susceptibility of CD28(-/-) mice to T. gondi associated with reduced serum levels of IFN-gamma, increased parasite burden, and increased mortality compared with the control group. Together, these results identify a critical role for ICOS in the protective Th1-type response required for resistance to T. gondii and suggest that ICOS and CD28 are parallel costimulatory pathways, either of which is sufficient to mediate resistance to this intracellular pathogen.

A role for inducible costimulator protein in the CD28- independent mechanism of resistance to Toxoplasma gondii.

Eine Rolle f&#xFC;r die induzierbare Kostimulator Protein in der CD28-unabh&#xE4;ngigen Mechanismus der Resistenz gegen Toxoplasma gondii.

Costimulation through the B7-CD28 interaction is an important second signal for T-cell activation, and previous studies have shown that CD28(-/-) mice infected with Toxoplasma gondii generate suboptimal CD4(+) T-cell responses, associated with a defect in production of the T-cell growth factor interleukin-2 (IL-2). To address the role of IL-2 in the expansion of T cells during toxoplasmosis, IL-2(-/-) mice were infected with T. gondii and their ability to generate a protective T-cell response was assessed. Although IL-2(-/-) mice produced normal levels of IL-12p40, they had reduced levels of gamma interferon (IFN-gamma) in serum, had an increased parasite burden, and succumbed to infection with T. gondii within 20 days. Fluorescence-activated cell sorter analysis revealed that, although uninfected IL-2(-/-) mice had an increased number of activated T cells compared with uninfected IL-2(+/+) mice, following infection they were unable to further upregulate this population. Examination of the ability of splenocytes from uninfected and infected mice to produce IFN-gamma revealed that IL-2(-/-) mice were hyporesponsive to stimulation with anti-CD3 or parasite antigen compared with wild-type mice, and the addition of IL-2 alone or in combination with IL-12 or stimulation with phorbol myristate acetate and ionomycin did not restore the production of IFN-gamma. Together, these studies reveal that IL-2(-/-) mice are unable to generate a protective IFN-gamma response following infection with T. gondii and suggest that IL-2(-/-) mice have an intrinsic defect in their ability to activate and expand IFN-gamma-producing T cells required for resistance to T. gondii.

Susceptibility of interleukin-2-deficient mice to Toxoplasma gondii is associated with a defect in the production of gamma interferon.

Empfindlichkeit von Interleukin-2-defizienten M&#xE4;usen gegen Toxoplasma gondii ist mit einem Defekt in der Produktion von Gamma-Interferon assoziiert.

The ability of interleukin-10 (IL-10) to suppress accessory cell functions required for optimal T-cell activation makes it an important inhibitor of cell-mediated immunity. Thus, after infection with the protozoan parasite Toxoplasma gondii, IL-10 knockout (KO) mice develop a CD4(+)-T-cell-dependent shock-like reaction with high levels of IL-12 and gamma interferon (IFN-gamma) in serum, leading to death of mice during the acute phase of infection. Previous studies from this laboratory have shown that simultaneous blockade of CD28 and CD40 can prevent this lethal reaction by inhibiting the production of IFN-gamma. However, the blockade of costimulation did not affect systemic levels of IL-12. To better understand the relationship between IL-12 and the CD28 and CD40 pathways in mediating immune hyperactivity, antagonists of these factors were used to determine their effects on the development of a pathological T-cell response in IL-10 KO mice. Blockade of IL-12 or the CD28/B7 interaction alone did not affect survival; however, the combined blockade of both pathways resulted in decreased production of IFN-gamma and the survival of IL-10 KO mice. To assess the role of the two ligands for CD28, B7.1 and B7.2, IL-10 KO mice were treated with alphaIL-12 plus alphaB7.1 or alphaB7.2 or the combination of all three antibodies. These studies revealed that blockade of both B7 molecules is required for decreased production of IFN-gamma and survival of infected IL-10 KO mice, suggesting that B7.1 and B7.2 can contribute to the lethal shock-like reaction in IL-10 KO mice. In contrast, neutralization of IL-12 and blockade of the CD40/CD40 ligand (CD40L) interaction in vivo did not alter the production of IFN-gamma and only resulted in a small delay in time to death of mice. Together, these data suggest that the CD28/B7 interaction has a central role in the development of a pathological T-cell response in IL-10 KO mice, which is distinct from the role of the CD40/CD40L and IL-12 pathways.

Contribution of interleukin-12 (IL-12) and the CD28/B7 and CD40/CD40 ligand pathways to the development of a pathological T-cell response in IL-10-deficient mice.

Beitrag von Interleukin-12 (IL-12) und den CD28/B7 und CD40/CD40 Liganden-Weg zur Entwicklung eines pathologischen T-Zell-Antwort in IL-10-defizienten M&#xE4;usen.

Previous studies have suggested an important role for interleukin-15 (IL-15) in resistance to and memory for Toxoplasma gondii infection. The studies presented here reveal that IL-15 is not required for infection-induced expansion of NK or CD8+ T cells. Furthermore, IL-15-/- mice develop long-term protective immunity to this pathogen.

Interleukin-15-deficient mice develop protective immunity to Toxoplasma gondii.

Interleukin-15-defizienten M&#xE4;usen entwickeln sch&#xFC;tzende Immunit&#xE4;t gegen Toxoplasma gondii.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based analysis of Giardia lamblia and Giardia muris.

Matrix assisted Laser Desorption/Ionisation Time-of-Flight mass Spektrometrie-basierte Analyse von Giardia Lamblia und Giardia Muris.

To better characterize Cryptosporidium in the Potomac River watershed, a PCR-based genotyping tool was used to analyze 64 base flow and 28 storm flow samples from five sites in the watershed. These sites included two water treatment plant intakes, as well as three upstream sites, each associated with a different type of land use. The uses, including urban wastewater, agricultural (cattle) wastewater, and wildlife, posed different risks in terms of the potential contribution of Cryptosporidium oocysts to the source water. Cryptosporidium was detected in 27 base flow water samples and 23 storm flow water samples. The most frequently detected species was C. andersoni (detected in 41 samples), while 14 other species or genotypes, almost all wildlife associated, were occasionally detected. The two common human-pathogenic species, C. hominis and C. parvum, were not detected. Although C. andersoni was common at all four sites influenced by agriculture, it was largely absent at the urban wastewater site. There were very few positive samples as determined by Environmental Protection Agency method 1623 at any site; only 8 of 90 samples analyzed (9%) were positive for Cryptosporidium as determined by microscopy. The genotyping results suggest that many of the Cryptosporidium oocysts in the water treatment plant source waters were from old calves and adult cattle and might not pose a significant risk to human health.

Cryptosporidium source tracking in the Potomac River watershed.

Um Cryptosporidium im Einzugsgebiet Potomac Rivers besser charakterisieren zu k&ouml;nnen, wurde ein PCR-basierten Genotypisierung-Tool verwendet, um 64 Mindestdurchfluss und 28 Sturm Str&ouml;mung Proben aus f&uuml;nf Seiten des Einzugsgebietes zu analysieren. Dieser Sites enthalten zwei Wasser-Behandlung-Pflanze-Zufuhr, sowie drei vorgelagerten Standorte, jeweils eine andere Art der Landnutzung zugeordnet. Vom verwendet wird, einschlie&szlig;lich kommunales Abwasser, landwirtschaftliche (Rind) Abwasser und Wildtiere, stellte verschiedene Risiken in Bezug auf den potenziellen Beitrag von Cryptosporidium-Oozysten zum Wasser Quelle. Cryptosporidium wurde in 27 Mindestdurchfluss Wasserproben und 23 Sturm Fluss Wasserproben festgestellt. Die meisten erkannt h&auml;ufig Arten war C. Andersoni (in 41 Proben gefunden), w&auml;hrend 14 andere Arten oder Genotypen, fast alle Wildtiere im Zusammenhang, wurden gelegentlich festgestellt. Die beiden gemeinsamen menschlichen pathogenen Arten, C. Hominis und C. Parvum, wurden nicht erkannt. C. Andersoni gemeinsame an allen vier Seiten durch die Landwirtschaft gepr&auml;gt war, war es weitgehend abwesend am Standort kommunalem Abwasser. Es gab sehr wenige positive Beispiele festgelegt durch Environmental Protection Agency Methode 1623 an jeder Stelle; nur 8 von 90 Proben analysiert (9 %) waren positiv f&uuml;r Cryptosporidium durch Mikroskopie bestimmt. Die Genotypisierung-Ergebnisse legen nahe, dass viele der Cryptosporidium-Oozysten im Wasser Behandlung Pflanze Quelle Wasser aus alten K&auml;lbern und erwachsenen Rinder wurden und k&ouml;nnen kein erhebliches Risiko f&uuml;r die menschliche Gesundheit darstellen.

Cryptosporidium Quelle tracking im Einzugsgebiet Potomac Rivers.

Cryptosporidium is an important waterborne protozoan parasite that can cause severe diarrhea and death in the immunocompromised. The current methods used to monitor for Cryptosporidium oocysts in water are the microscopy-based USEPA methods 1622 and 1623. These methods assess total levels of oocysts in source waters, but do not determine oocyst viability or genotype. Recently, propidium monoazide (PMA) has been used in conjunction with molecular diagnostic tools to identify species and assess the viability of bacteria. The goal of this study was the development of a Cryptosporidium PMA-PCR (CryptoPMA-PCR) assay that includes PMA treatment prior to PCR analysis in order to prevent the amplification of DNA from dead oocysts. The results demonstrated that PMA penetrates only dead oocysts and blocks amplification of their DNA. The CryptoPMA-PCR assay can also specifically detect live oocysts within a mixed population of live and dead oocysts. More importantly, live oocysts, not dead oocysts, were detected in raw waste or surface water samples spiked with Cryptosporidium oocysts. This proof-of-concept study is the first to demonstrate the use of PMA for pre-PCR treatment of Cryptosporidium oocysts. The CryptoPMA-PCR assay is an attractive approach to specifically detect and genotype viable Cryptosporidium oocysts in the water, which is critical for human health risk assessment.

Cryptosporidium propidium monoazide-PCR, a molecular biology-based technique for genotyping of viable Cryptosporidium oocysts.

Cryptosporidium ist eine wichtige Wasserwege-Protozoen-Parasit, der heftigen Durchfall und Tod in der immungeschw&auml;chten verursachen kann. Die aktuellen Methoden zur &Uuml;berwachung der Cryptosporidium-Oozysten im Wasser sind die Mikroskopie-basierte USEPA-Methoden 1622 und 1623. Diese Methoden insgesamt Ebenen von Oozysten im Quellcode Gew&auml;sser zu beurteilen, aber bestimmen nicht Oozyste Lebensf&auml;higkeit oder Genotypus. Vor kurzem wurde Propidium Monoazide (PMA) verwendet in Verbindung mit Molekulare Diagnose-Tools Arten zu identifizieren und bewerten die Lebensf&auml;higkeit der Bakterien. Das Ziel dieser Studie war die Entwicklung einer Cryptosporidium PMA-PCR (CryptoPMA-PCR)-Assay, der PMA-Behandlung vor der PCR-Analyse umfasst, um die Verst&auml;rkung von DNA von Toten Oozysten verhindern. Die Ergebnisse zeigten, dass PMA nur Tote Oozysten und Bl&ouml;cke Verst&auml;rkung von DNA dringt. Der CryptoPMA-PCR-Assay kann auch speziell live Oozysten innerhalb einer gemischten Population von lebenden und Toten-Oozysten erkennen. Noch wichtiger ist, Leben Oozysten, nicht tot Oozysten, gefunden in roher Verschwendung oder Oberfl&auml;che Wasser Proben gespickt mit Cryptosporidium-Oozysten. Diese Proof-of-Concept-Studie ist die erste, die Verwendung von PMA f&uuml;r Pre-PCR Behandlung von Cryptosporidium-Oozysten demonstrieren. Der CryptoPMA-PCR-Assay ist ein attraktives Konzept, speziell zu erkennen und Genotyp lebensf&auml;higen Cryptosporidium-Oozysten im Wasser, das f&uuml;r menschliche Gesundheit-Risikobewertung von entscheidender Bedeutung ist.

Cryptosporidium Propidium Monoazide-PCR, ein Molekularbiologie-basierte Verfahren zur Genotypisierung von lebensf&auml;higen Cryptosporidium-Oozysten.

This study evaluates Cryptosporidium parvum oocyst production in dexamethasone suppressed CF-1 and C57BL/6 mice. Both models can yield 1 x 10(9) total oocysts over a 20-day production period; however, only 20 CF-1 mice are required to reliably achieve this goal compared to 40 C57BL/6 mice. Although oocyst yields per mouse are similar for both mouse strains, the survival rate for CF-1 mice is higher, resulting in reduced lost production time per study when compared to the C57BL/6 mice. This study presents a more efficient and cost effective dexamethasone suppressed murine model of propagating high concentrations of C. parvum oocysts.

Improved Cryptosporidium parvum oocyst propagation using dexamethasone suppressed CF-1 mice.

Diese Studie wertet Cryptosporidium Parvum Oozyste Produktion Dexamethason unterdr&uuml;ckt CF-1 und C57BL/6 M&auml;usen. Beide Modelle k&ouml;nnen &uuml;ber einen Zeitraum von 20 Tagen Produktion 1 X Neubewerber Gesamt Oozysten Ertrag; nur 20 CF-1-M&auml;use sind jedoch erforderlich, um dieses Ziel im Vergleich zu 40 C57BL/6 M&auml;usen zuverl&auml;ssig zu erreichen. Obwohl Oozyste Ertr&auml;ge per Mausklick f&uuml;r beide Maus-St&auml;mme &auml;hnlich sind, die &Uuml;berlebensrate f&uuml;r CF-1 M&auml;usen h&ouml;her, was zu reduzierter Produktionszeit pro Studie im Vergleich zu den M&auml;usen C57BL/6 verloren. Diese Studie stellt ein effizienter und kosteng&uuml;nstiger Dexamethason unterdr&uuml;ckt murinen Modell zur Vermehrung hoher Konzentrationen von C. Parvum Oozysten.

Verbesserte Cryptosporidium Parvum Oozyste Vermehrung mit Dexamethason unterdr&uuml;ckt CF-1 M&auml;usen.

From bottlenose dolphins, to walruses, to sea otters, the parasitic protozoan Toxoplasma gondii is infecting marine mammals around the world. Whereas the terrestrial transmission pathways of T. gondii are well-described, the transmission pathway by which marine mammals are being infected is unknown. We hypothesize that migratory filter feeders, specifically northern anchovies (Engraulis mordax) and Pacific sardines (Sardinops sagax), are serving as biotic vectors for T. gondii within the marine environment. By filtering oocysts from seawater, these fishes could be transporting the oocysts from nearshore to pelagic environments. In this study, we experimentally exposed northern anchovies and Pacific sardines to T. gondii oocysts under laboratory conditions. Following exposure, the fishes' alimentary canals were harvested and assayed for the presence of T. gondii by PCR. Fish exposed to as few as 1197 oocysts/L seawater tested positive for T. gondii by PCR. In total, the PCR assay detected T. gondii DNA in 66% (40/61) of the exposed fishes. Oocyst infectivity was confirmed by mouse bioassay: 30% (7/23) of mice developed toxoplasmosis when fed fish exposed to 100,000 oocysts/L. This study demonstrates that both northern anchovies and Pacific sardines can filter T. gondii oocysts out of seawater under experimental conditions. Our experiments with anchovies demonstrated that the oocysts persisted in the fish for at least 8h post-exposure and our experiments with sardines demonstrated that the oocysts remained infectious inside the fish's alimentary canals.

Uptake and transmission of Toxoplasma gondii oocysts by migratory, filter-feeding fish.

Von T&uuml;mmler, Walrosse, um Seeotter ist die Parasit&auml;re Protozoen Toxoplasma Gondii Meeress&auml;uger auf der ganzen Welt infizieren. Die terrestrische &Uuml;bertragung-Bahnen von T. Gondii beschriebenem sind, ist die &Uuml;bertragung-Pathway, die durch die Meeress&auml;uger infiziert wird, sind unbekannt. Wir vermuten, dass wandernde Filter Feeder, speziell n&ouml;rdlichen Sardellen (Engraulis Mordax) und Pacific Sardinen (Sardinops Sagax), als biotische Vektoren f&uuml;r T. Gondii in der Meeresumwelt dienen. Durch Oozysten aus Meerwasser filtern, k&ouml;nnte diese Fische die Oozysten von Nearshore pelagische Umgebungen transportieren. In dieser Studie wir versuchsweise gemachten n&ouml;rdliche Sardellen und Pacific Sardinen, T. Gondii Oozysten unter Laborbedingungen. Nach Exposition waren die Fische Alimentary Grachten geerntet und hemmende gegen das Vorhandensein von T. Gondii durch PCR. Fisch f&uuml;r so wenig wie 1197 Oozysten/L Meerwasser getestet-positiv f&uuml;r T. Gondii der PCR ausgesetzt. Insgesamt hat der PCR-Assay T. Gondii DNA bei 66 % (40/61) der exponierten Fische festgestellt. Oozyste Infektiosit&auml;t von Maus-Bioassay best&auml;tigt: 30 % (7/23) von M&auml;usen entwickelt Toxoplasmose bei 100.000 Oozysten/L. gef&uuml;ttert Fische ausgesetzt Diese Studie zeigt, dass die n&ouml;rdlichen Sardellen und Sardinen Pacific T. Gondii Oozysten aus Meerwasser unter Versuchsbedingungen filtern k&ouml;nnen. Unsere Experimente mit Sardellen gezeigt, dass die Oozysten in den Fischen f&uuml;r mindestens 8 h Expositions-beibehalten und unseren Experimenten mit Sardinen gezeigt, dass die Oozysten infekti&ouml;se innerhalb des Fisches Alimentary Kan&auml;le blieb.

Aufnahme und &Uuml;bertragung von Toxoplasma Gondii Oozysten von wandernden, Filter-F&uuml;tterung der Fische.

Toxoplasma gondii oocysts are highly resistant to many chemical sanitizers. Methods used to determine oocyst infectivity have relied primarily on mouse, chicken, and feline bioassays. Although considered gold standards, they only provide a qualitative assessment of oocyst viability. In this study, two alternative approaches were developed to quantitate viable T. gondii oocysts following treatment with several common sanitizers. The first is a quantitative reverse transcriptase real-time PCR (RT-qPCR) assay targeting the ACT1 and SporoSAG genes to enumerate viable T. gondii oocysts. RT-qPCR C(T) values between Wescodyne(R), acidified ethanol, or heat treated oocysts were not significantly different as compared with untreated controls. By contrast, treatment with formalin or Clorox(R) resulted in a 2-log(10) reduction in C(T) values. An in vitro T. gondii oocyst plaque assay (TOP-assay) was also developed to measure oocyst viability. This assay used a combination of bead milling and bile digestion, followed by culturing the excysted sporozoites in a confluent fibroblast cell monolayer. Results showed that no significant reduction in sporozoite viability was detected in acidified ethanol or Wescodyne(R) treated oocysts while at least a 2-log(10) reduction in plaques formed was observed with Clorox(R) treated oocysts. Moreover, formalin or heat treatment of oocysts resulted in at least a 5-log(10) reduction in plaques formed. This study demonstrates that an mRNA-based PCR viability assay targeting the ACT1 or SporoSAG genes is a relatively rapid technique compared to in vitro and in vivo assays. In addition, the TOP-assay proved very effective and sensitive at quantifying oocyst viability when compared with animal bioassays.

Using quantitative reverse transcriptase PCR and cell culture plaque assays to determine resistance of Toxoplasma gondii oocysts to chemical sanitizers.

Toxoplasma Gondii Oozysten sind sehr widerstandsf&auml;hig gegen viele chemische Sanitizers. Methoden zur Bestimmung der Oozyste Infektiosit&auml;t haben in erster Linie auf Maus, Huhn und katzenartige Bioassays verlassen. Obwohl Gold Standards, bieten sie nur eine qualitative Bewertung der Oozyste Lebensf&auml;higkeit. In dieser Studie wurden zwei alternative Ans&auml;tze entwickelt, um tragf&auml;hige T. Gondii Oozysten nach Behandlung mit mehreren gemeinsamen Sanitizers Quantifizierung. Die erste ist eine quantitative Reverse Transkriptase Echtzeit-PCR (RT-qPCR) Assay gezielt die Gene ACT1 und SporoSAG um tragf&auml;hige T. Gondii Oozysten aufzulisten. RT-qPCR C(T) Werte zwischen Wescodyne(R), ges&auml;uerte Ethanol oder w&auml;rmebehandelt Oozysten waren nicht signifikant im Vergleich zu unbehandelten Kontrollen. Behandlung mit Formalin oder Clorox(R) f&uuml;hrte hingegen eine Verringerung 2-log(10) C(T) Werte. Ein in-vitro-T. Gondii Oozyste Plakette Assay (TOP-Assay) wurde auch entwickelt, um Oozyste Lebensf&auml;higkeit zu messen. Diese Probe verwendet eine Kombination aus Perlen-Fr&auml;sen und Galle Verdauung, gefolgt von der Kultivierung der Excysted Sporozoites in einem konfluierende Fibroblast Zellen Monolage. Ergebnisse zeigten, dass keine signifikante Verringerung der Sporozoite Lebensf&auml;higkeit in anges&auml;uertem &Auml;thanol festgestellt oder Wescodyne(R) behandelt Oozysten w&auml;hrend mindestens einem 2-log(10) R&uuml;ckgang der Plaketten gebildet mit Clorox(R) beobachtet wurde behandelt Oozysten. Dar&uuml;ber hinaus f&uuml;hrte Formalin oder W&auml;rmebehandlung von Oozysten in mindestens einem 5-log(10) R&uuml;ckgang der Plaketten gebildet. Diese Studie zeigt, dass eine mRNA basierende PCR Entwicklungsf&auml;higkeit-Probe ACT1 oder SporoSAG Gene targeting eine relativ rasche Technik im Vergleich zur in-vitro- und in-vivo-Tests. Dar&uuml;ber hinaus erwies sich der TOP-Assay, sehr effektiv und sensibler auf die Quantifizierung der Oozyste Rentabilit&auml;t im Vergleich zu tierischen Bioassays.

Mit quantitative Reverse-Transkriptase-PCR und Zelle-Kultur-Plaque-Proben Widerstand von Toxoplasma Gondii Oozysten zu chemischen Sanitizers zu bestimmen.

The effect of UV exposure on Toxoplasma gondii oocysts has not been completely defined for use in water disinfection. This study evaluated UV-irradiated oocysts by three assays: a SCID mouse bioassay, an in vitro T. gondii oocyst plaque (TOP) assay, and a quantitative reverse transcriptase real-time PCR (RT-qPCR) assay. The results from the animal bioassay show that 1- and 3-log(10) inactivation is achieved with 4 mJ/cm(2) UV and 10 mJ/cm(2) low-pressure UV, respectively. TOP assay results, but not RT-qPCR results, correlate well with bioassay results. In conclusion, a 3-log(10) inactivation of T. gondii oocysts is achieved by 10-mJ/cm(2) low-pressure UV, and the in vitro TOP assay is a promising alternative to the mouse bioassay.

Determining UV inactivation of Toxoplasma gondii oocysts by using cell culture and a mouse bioassay.

Die Wirkung der UV-Exposition auf Toxoplasma Gondii Oozysten wurde f&uuml;r den Einsatz im Wasserdesinfektion nicht vollst&auml;ndig definiert. Diese Studie UV-bestrahltes Oozysten von drei Proben ausgewertet: ein SCID-Maus-Bioassay, eines in vitro T. Gondii Oozyste Plakette (oben) Tests und eine quantitative Reverse Transkriptase Echtzeit-PCR (RT-qPCR) Assay. Die Ergebnisse aus tierischen Bioassays zeigen, dass die Inaktivierung von 1- und 3-log(10) mit 4 mJ/cm(2), UV- und 10 mJ/cm(2) Niederdruck UV, bzw. erreicht wird. TOP Untersuchungsergebnisse, aber nicht RT-qPCR Ergebnisse korrelieren gut mit Bioassay Ergebnisse. Abschlie&szlig;end eine Inaktivierung der 3-log(10) von T. Gondii Oozysten erfolgt durch 10-mJ/cm(2) Niederdruck UV und der in-vitro-TOP-Assay ist eine vielversprechende Alternative zu den Maus-Bioassay.

Bestimmen mithilfe von Zellkultur und ein Maus-Bioassay UV Inaktivierung von Toxoplasma Gondii Oozysten.

In this study, we examined the potential for detecting fecal bacteria and microbial source tracking markers in samples discarded during the concentration of Cryptosporidium and Giardia using USEPA Method 1623. Recovery rates for different fecal bacteria were determined in sewage spiked samples and environmental waters using different group-specific and host-specific PCR assays. Bacteroidales DNA recovery ranged from 59 to 71% for aliquots of supernatant collected after the elution step. The recovery of human-specific Bacteroidales DNA from sewage spiked samples was 54% in the elution step. An additional 1-7% Bacteroidales DNA was recovered after the immunomagnetic separation step, while recovery from the pellet left after the immunomagnetic separation of protozoa parasites was substantially lower. Comparison of Bacteroidales 16S rRNA gene sequences from elution and immunomagnetic separation discarded samples indicated that the distribution of clones was not statistically different, suggesting that there were no recovery biases introduced by these steps. Human- and cow-specific Bacteroidales and fecal indicator bacteria (i.e., enterococci,) were also detected in the discarded fractions of environmental samples collected from different geographic locations. Overall, the results of this study demonstrated the potential application of leftover sample fractions that are currently discarded for the PCR detection of fecal bacterial indicators and molecular source tracking.

Application of leftover sample material from waterborne protozoa monitoring for the molecular detection of Bacteroidales and fecal source tracking markers.

In dieser Studie wurden das Potential zum Erkennen von f&auml;kalen Bakterien und mikrobielle Quelle Nachverfolgen von Markern in Proben verworfen, w&auml;hrend die Konzentration von Cryptosporidium und Giardia mit USEPA Methode 1623. Verwertungsquoten f&uuml;r verschiedene f&auml;kalen Bakterien wurden in Abwasser Spike-Proben und Gew&auml;ssern mit verschiedenen zielgruppenspezifische und Host-spezifische PCR-Assays bestimmt. Bacteroidales DNA-Erholung zwischen 59 und 71 % f&uuml;r Aliquote des &Uuml;berstands sammelte nach der Elution Schritt. Die Wiederherstellung der Mensch-spezifische Bacteroidales DNA von Abwasser Pfennigabsatz Proben war 54 % in der Elution Schritt. Eine zus&auml;tzliche 1-7 % Bacteroidales DNA wurde nach dem Immunomagnetic Trennung Schritt wiederhergestellt, w&auml;hrend der Erholung von der Pellet verlie&szlig;, nachdem die Immunomagnetic Trennung von Protozoen-Parasiten wesentlich niedriger war. Vergleich der Bacteroidales 16S rRNA-Gen-Sequenzen von Elution und Immunomagnetic Trennung verworfen Proben gaben an, dass die Verteilung der Klone war nicht statistisch anders, was darauf hindeutet, dass es keine Wiederherstellung-Vorurteile, die von diesen Schritten eingef&uuml;hrt wurden. In der verworfenen Br&uuml;che von Umweltproben aus verschiedenen geografischen Standorten gesammelt wurden auch Mensch und Kuh-spezifische Bacteroidales und f&auml;kalen Indikator Bakterien (z. B. Enterokokken,) erkannt. Insgesamt zeigten die Ergebnisse dieser Studie die m&ouml;gliche Anwendung der &uuml;brig gebliebene Beispiel Br&uuml;che, die derzeit f&uuml;r den PCR-Nachweis von f&auml;kalen Bakterien Indikatoren und Molekulare Source tracking verworfen werden.

Anwendung der &uuml;brig gebliebenen Probenmaterial aus Wasserwegen Protozoen, die &Uuml;berwachung f&uuml;r die molekulare Erkennung von Bacteroidales und f&auml;kalen Source Tracking-Marker.

ArticleTotal : 2	Year
Publication title Cited by 18	2009
Publication title Cited by 11	2012

Article	Year
A role for inducible costimulator protein in the CD28- independent mechanism of resistance to Toxoplasma gondii. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 12097399	2002
Susceptibility of interleukin-2-deficient mice to Toxoplasma gondii is associated with a defect in the production of gamma interferon. Infection and immunity\| PubMed ID: 12183516	2002
Contribution of interleukin-12 (IL-12) and the CD28/B7 and CD40/CD40 ligand pathways to the development of a pathological T-cell response in IL-10-deficient mice. Infection and immunity\| PubMed ID: 12438373	2002
Interleukin-15-deficient mice develop protective immunity to Toxoplasma gondii. Infection and immunity\| PubMed ID: 15501812	2004
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based analysis of Giardia lamblia and Giardia muris. The Journal of eukaryotic microbiology\| PubMed ID: 17169052	2006
Cryptosporidium source tracking in the Potomac River watershed. Applied and environmental microbiology\| PubMed ID: 18776033	2008
Cryptosporidium propidium monoazide-PCR, a molecular biology-based technique for genotyping of viable Cryptosporidium oocysts. Applied and environmental microbiology\| PubMed ID: 19749067	2009
Improved Cryptosporidium parvum oocyst propagation using dexamethasone suppressed CF-1 mice. Veterinary parasitology\| PubMed ID: 20036060	2010
Uptake and transmission of Toxoplasma gondii oocysts by migratory, filter-feeding fish. Veterinary parasitology\| PubMed ID: 20097009	2010
Using quantitative reverse transcriptase PCR and cell culture plaque assays to determine resistance of Toxoplasma gondii oocysts to chemical sanitizers. Journal of microbiological methods\| PubMed ID: 20385175	2010
Determining UV inactivation of Toxoplasma gondii oocysts by using cell culture and a mouse bioassay. Applied and environmental microbiology\| PubMed ID: 20543052	2010
Application of leftover sample material from waterborne protozoa monitoring for the molecular detection of Bacteroidales and fecal source tracking markers. Journal of microbiological methods\| PubMed ID: 21693138	2011
Journal of microbiological methods\| PubMed ID: 22465221	2012

Eric N. Villegas

Biography

JoVE Journal Publications

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