Antitumoral synergism between a copper(II) complex and cisplatin improves in vitro and in vivo anticancer activity against melanoma, lung and breast cancer cells.

BACKGROUND: Intrinsic resistance of cancer cells is a major concern for the success of chemotherapy, and this undesirable feature stimulates further research into the design of new compounds and/or alternative multiple drug chemotherapy protocols. METHODS: In this study, we investigated the antitumoral potential of the coordination compounds [Cu(HPClNOL)Cl]Cl (1), [Fe(HPClNOL)Cl2]NO3(2) and [Mn(HPClNOL)Cl2] (3). Using the human, MCF-7 and A549, and the murine melanoma, B16-F10, cell lines, we determined the cytotoxicity, DCFH oxidation, disruption of mitochondrial membrane potential (ΔΨm), Sub-G1 and TUNEL positive cells, and caspase 8 and 9 activities. Fractional inhibitory concentration (FIC) and xenograft models were also assessed to evaluate the efficacy of antitumoral potential. RESULTS: We observed that only complex 1 was cytotoxic. The treatment of cancer cells with complex 1 triggered ROS generation and promoted the disruption of ΔΨm. Complex 1 increased the number of Sub-G1 and TUNEL positive cells, and the measurement of caspase 8 and 9 activity confirmed that apoptosis was triggered by the intrinsic pathway. FIC demonstrated that the combination of complex 1 with cisplatin was additive for the A549 cells whilst it was synergic for MCF-7 and B16-F10. Treatment with complex 1, either alone or combined with cisplatin, reduced tumor growth on xenograft models. CONCLUSIONS: The present study brings new clues regarding the mechanism of action of [Cu(HPClNOL)Cl]Cl, either alone or in combination with cisplatin. GENERAL SIGNIFICANCE: These results indicate that complex 1, administered either singly or in combination with current drugs, has real potential for use in cancer therapy.

Functional characterization of the Aspergillus nidulans glucosylceramide pathway reveals that LCB Δ8-desaturation and C9-methylation are relevant to filamentous growth, lipid raft localization and Psd1 defensin activity.

C8-desaturated and C9-methylated glucosylceramide (GlcCer) is a fungal-specific sphingolipid that plays an important role in the growth and virulence of many species. In this work, we investigated the contribution of Aspergillus nidulans sphingolipid Δ8-desaturase (SdeA), sphingolipid C9-methyltransferases (SmtA/SmtB) and glucosylceramide synthase (GcsA) to fungal phenotypes, sensitivity to Psd1 defensin and Galleria mellonella virulence. We showed that ΔsdeA accumulated C8-saturated and unmethylated GlcCer, while gcsA deletion impaired GlcCer synthesis. Although increased levels of unmethylated GlcCer were observed in smtA and smtB mutants, ΔsmtA and wild-type cells showed a similar 9,Me-GlcCer content, reduced by 50% in the smtB disruptant. The compromised 9,Me-GlcCer production in the ΔsmtB strain was not accompanied by reduced filamentation or defects in cell polarity. When combined with the smtA deletion, smtB repression significantly increased unmethylated GlcCer levels and compromised filamentous growth. Furthermore, sdeA and gcsA mutants displayed growth defects and raft mislocalization, which were accompanied by reduced neutral lipids levels and attenuated G. mellonella virulence in the ΔgcsA strain. Finally, ΔsdeA and ΔgcsA showed increased resistance to Psd1, suggesting that GlcCer synthesis and fungal sphingoid base structure specificities are relevant not only to differentiation but also to proper recognition by this antifungal defensin.

Reduction of Toxoplasma gondii Development Due to Inhibition of Parasite Antioxidant Enzymes by a Dinuclear Iron(III) Compound.

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan that can infect a wide range of vertebrate cells. Here, we describe the cytotoxic effects of the dinuclear iron compound [Fe(HPCINOL)(SO4)]2-µ-oxo, in which HPCINOL is the ligand 1-(bis-pyridin-2-ylmethyl-amino)-3-chloropropan-2-ol, on T. gondii infecting LLC-MK2 host cells. This compound was not toxic to LLC-MK2 cells at concentrations of up to 200 µM but was very active against the parasite, with a 50% inhibitory concentration (IC50) of 3.6 µM after 48 h of treatment. Cyst formation was observed after treatment, as indicated by the appearance of a cyst wall, Dolichos biflorus lectin staining, and scanning and transmission electron microscopy characteristics. Ultrastructural changes were also seen in T. gondii, including membrane blebs and clefts in the cytoplasm, with inclusions similar to amylopectin granules, which are typically found in bradyzoites. An analysis of the cell death pathways in the parasite revealed that the compound caused a combination of apoptosis and autophagy. Fluorescence assays demonstrated that the redox environment in the LLC-MK2 cells becomes oxidant in the presence of the iron compound. Furthermore, a reduction in superoxide dismutase and catalase activities in the treated parasites and the presence of reactive oxygen species within the parasitophorous vacuoles were observed, indicating an impaired protozoan response against these radicals. These findings suggest that this compound disturbs the redox equilibrium of T. gondii, inducing cystogenesis and parasite death.

Multiscale analyses of the bone-implant interface.

Implants placed with high insertion torque (IT) typically exhibit primary stability, which enables early loading. Whether high IT has a negative impact on peri-implant bone health, however, remains to be determined. The purpose of this study was to ascertain how peri-implant bone responds to strains and stresses created when implants are placed with low and high IT. Titanium micro-implants were inserted into murine femurs with low and high IT using torque values that were scaled to approximate those used to place clinically sized implants. Torque created in peri-implant tissues a distribution and magnitude of strains, which were calculated through finite element modeling. Stiffness tests quantified primary and secondary implant stability. At multiple time points, molecular, cellular, and histomorphometric analyses were performed to quantitatively determine the effect of high and low strains on apoptosis, mineralization, resorption, and collagen matrix deposition in peri-implant bone. Preparation of an osteotomy results in a narrow zone of dead and dying osteocytes in peri-implant bone that is not significantly enlarged in response to implants placed with low IT. Placing implants with high IT more than doubles this zone of dead and dying osteocytes. As a result, peri-implant bone develops micro-fractures, bone resorption is increased, and bone formation is decreased. Using high IT to place an implant creates high interfacial stress and strain that are associated with damage to peri-implant bone and therefore should be avoided to best preserve the viability of this tissue.

Stability of surgically assisted rapid palatal expansion: a randomized trial.

The aim of this study was to evaluate the orthodontic retention of maxillary skeletal stability after surgically assisted rapid palatal expansion (SARPE). Ninety digitized plaster casts from 30 adult patients who underwent SARPE were assessed. Thirty patients were divided equally into two groups: the No Retention Group (n = 15) and the Retention Group (n = 15) with a Transpalatal Arch [TPA]). After the end of expansion, the expander appliance was stabilized and remained in place for 4 months. The additional retention period began in the Retention Group as soon as the expander was removed and replaced by a TPA. During the same period, the No Retention Group remained without retention. The casts were created pre-operatively, at 4 months and 10 months post-expansion. The models were digitized by means of a 3D Vivid 9i laser scanner. The palatal area and volume were assessed. Both variables increased after 4 months compared with pre-operative values (p < .05). At 10 months, patients' palatal areas and volumes were stable in both groups (p > .05). In conclusion, no retention other than the expander appliance is needed after SARPE.

Strategies for surgically assisted rapid maxillary expansion according to the region of transverse maxillary deficiency.

This study evaluated different techniques for surgically assisted rapid maxillary expansion (SARME) according to the type of transverse maxillary deficiency using computed tomography (CT). Six adult patients with bilateral transverse maxillary deficiencies underwent SARME. The patients were equally divided into three groups: Group I, maxillary atresia in both the anterior and posterior regions; Group II, greater maxillary atresia in the anterior region; and Group III, increased maxillary atresia in the posterior region. In Group I, a subtotal Le Fort I osteotomy was used. In Group II, a subtotal Le Fort I osteotomy was used without pterygomaxillary suture disjunction. In Group III, a subtotal Le Fort I osteotomy was used with pterygomaxillary suture disjunction and fixation of the anterior nasal spine with steel wire. The midpalatal suture opening was evaluated preoperatively and immediately after the activation period using CT. For Group I, the opening occurred parallel to midpalatal suture; for Group II, the opening comprised a V-shape with a vertex on the posterior nasal spine; and for Group III, the opening comprised a V-shape with a vertex at the anterior nasal spine. The conclusion was that the SARME technique should be individualized according to the type of transverse maxillary deficiency.

Reducing microbial contamination in storm runoff from high use areas on California coastal dairies.

High use areas are a fundamental part of California coastal dairies and grazing livestock ranches as feeding areas, nurseries, and sick pens. High stocking densities and daily use in these areas lead to soil surfaces devoid of vegetation and covered in manure, with high potential for manure transport during winter rains to receiving waters regulated for shellfish harvesting and recreation. We characterized the association between California's Mediterranean climate and a series of existing and proposed management practices on fecal coliform bacteria (FCB) transport from high use areas on dairies and ranches. Results from 351 storm runoff samples collected below 35 high-use areas indicate that removal of cattle during winter, locating high use areas on level ground, application of straw and seeding, and vegetative buffer strip implementation were significantly associated with FCB concentration and load reductions. These results complement our findings for reductions of specific pathogens in runoff from these areas. These findings have practical significance because they document surface water quality benefits that the studied management practices provide in application on working farms and ranches. This direction is critical and timely for on-farm management efforts seeking to reduce microbial pollution in runoff and comply with indicator bacteria water quality criteria.

BNCT for skin melanoma in extremities: updated Argentine clinical results.

As part of phase I/II melanoma BNCT clinical trial conducted in Argentina in a cooperative effort of the Argentine Atomic Energy Commission (CNEA) and the Oncology Institute Angel H. Roffo (IOAHR), 7 patients (6 female-1 male) received eight treatment sessions covering ten anatomical areas located in extremities. Mean age of the patients was 64 years (51-74). The treatments were performed between October 2003 and June 2007. All patients presented multiple subcutaneous skin metastases of melanoma and received an infusion containing approximately 14 gr/m(2) of (10)borophenyl-alanine (BPA) followed by the exposition of the area to a mixed thermal-epithermal neutron beam at the RA-6 reactor. The maximum prescribed dose to normal skin ranged from 16.5 to 24 Gy-Eq and normal tissue administered dose varied from 15.8 to 27.5 Gy-Eq. Considering evaluable nodules, 69.3% of overall response and 30.7% of no changes were seen. The toxicity was acceptable, with 3 out of 10 evaluable areas showing ulceration (30% toxicity grade 3).

Tumor control and normal tissue complications in BNCT treatment of nodular melanoma: a search for predictive quantities.

A previous work concerning tumor control and skin damage in cutaneous melanoma treatments with BNCT has been extended to include doses, volumes and responses of 104 subcutaneous lesions from all patients treated in Argentina. Acute skin reactions were also scored for these patients, and cumulative dose-area histograms and dose-based figures of merit for skin were calculated. Broadening the tumor response analysis with the latest data showed that the (minimum or mean) tumor dose is not a good predictor of the observed clinical outcome by itself. However, when the tumor volume was included in the model as second explicative variable, the dose increases its significance and becomes a critical variable jointly with the volume (p-values<0.05). A preliminary analysis to estimate control doses for two groups of tumor sizes revealed that for small tumor volumes (< 0.1cm(3)) doses greater than 20 Gy-Eq produce a high tumor control (> 80%). However, when tumor volumes are larger than 0.1cm(3), control is moderate (< 40%) even for minimum doses up to 40 Gy-Eq. Some quantities based on skin doses, areas and complication probabilities were proposed as candidates for predicting the severity of the early skin reactions. With the current data, all the evaluated figures of merit derived similar results: ulceration is present among the cases for which these quantities take the highest values.

Dynamic infrared imaging of cutaneous melanoma and normal skin in patients treated with BNCT.

We recently initiated a program aimed to investigate the suitability of dynamic infrared imaging for following-up nodular melanoma patients treated with BNCT. The reason that makes infrared imaging attractive is the fact that it constitutes a functional and non-invasive imaging method, providing information on the normal and abnormal physiologic response of the nervous and vascular systems, as well as the local metabolic rate and inflammatory processes that ultimately appear as differences in the skin temperature. An infrared camera, with a focal plane array of 320 x 240 uncooled ferroelectric detectors is employed, which provides a video stream of the infrared emission in the 7-14 microm wavelength band. A double blackbody is used as reference for absolute temperature calibration. After following a protocol for patient preparation and acclimatization, a basal study is performed. Subsequently, the anatomic region of interest is subjected to a provocation test (a cold stimulus), which induces an autonomic vasoconstriction reflex in normal structures, thus enhancing the thermal contrast due to the differences in the vasculature of the different skin regions. Radiation erythema reactions and melanoma nodules possess typically a faster temperature recovery than healthy, non-irradiated skin. However, some other non-pathological structures are also detectable by infrared imaging, (e.g. scars, vessels, arteriovenous anastomoses and injuries), thus requiring a multi-study comparison in order to discriminate the tumor signal. Besides the superficial nodules, which are readily noticeable by infrared imaging, we have detected thermal signals that are coincident with the location of non-palpable nodules, which are observable by CT and ultrasound. Diffuse regions of fast temperature recovery after a cold stimulus were observed between the third and sixth weeks post-BNCT, concurrent with the clinical manifestation of radiation erythema. The location of the erythematous visible and infrared regions is consistent with the 3D dosimetry calculations.

In vitro and in vivo determination of antioxidant activity and mode of action of isoquercitrin and Hyptis fasciculata.

Reactive oxygen species (ROS) are thought to underline the process of ageing and the pathogenicity of various diseases, such as neurodegenerative disorders and cancer. The use of traditional medicine is widespread and plants still present a large source of natural antioxidants that might serve as leads for the development of novel drugs. In this paper, the alcoholic extract from leaves of Hyptis fasciculata, a Brazilian medicinal plant, and isoquercitrin, a flavonoid identified in this species, showed to be active as 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavengers. The extract of Hyptis fasciculata and isoquercitrin were also able to increase tolerance of the eukaryotic microorganism Saccharomyces cerevisiae to both hydrogen peroxide and menadione, a source of superoxide. Cellular protection was correlated with a decrease in oxidative stress markers, such as levels of ROS, protein carbonylation and lipid peroxidation, confirming the antioxidant potential of Hyptis fasciculata and isoquercitrin.

Farm factors associated with reducing Cryptosporidium loading in storm runoff from dairies.

A systems approach was used to evaluate environmental loading of Cryptosporidium oocysts on five coastal dairies in California. One aspect of the study was to determine Cryptosporidium oocyst concentrations and loads for 350 storm runoff samples from dairy high use areas collected over two storm seasons. Selected farm factors and beneficial management practices (BMPs) associated with reducing the Cryptosporidium load in storm runoff were assessed. Using immunomagnetic separation (IMS) with direct fluorescent antibody (DFA) analysis, Cryptosporidium oocysts were detected on four of the five farms and in 21% of storm runoff samples overall. Oocysts were detected in 59% of runoff samples collected near cattle less than 2 mo old, while 10% of runoff samples collected near cattle over 6 mo old were positive. Factors associated with environmental loading of Cryptosporidium oocysts included cattle age class, 24 h precipitation, and cumulative seasonal precipitation, but not percent slope, lot acreage, cattle stocking number, or cattle density. Vegetated buffer strips and straw mulch application significantly reduced the protozoal concentrations and loads in storm runoff, while cattle exclusion and removal of manure did not. The study findings suggest that BMPs such as vegetated buffer strips and straw mulch application, especially when placed near calf areas, will reduce environmental loading of fecal protozoa and improve stormwater quality. These findings are assisting working dairies in their efforts to improve farm and ecosystem health along the California coast.

Effect of orthognathic surgery for class III correction on quality of life as measured by SF-36.

The purpose of this work was to evaluate the quality of life of patients with Angle's class III malocclusion submitted to orthognathic surgery. Twenty-nine patients of both sexes, ranging in age from 17 to 46 years, with Angle's class III malocclusion and indication for surgical treatment, were evaluated about 30 days before surgery and 6 months postoperatively. Surgery consisted of maxillary advancement or mandibular retrusion, or both. The generic SF-36 questionnaire was used to evaluate the following eight domains: functional capacity, physical aspects, pain, general health status, mental health, emotional aspects, social aspects and vitality. Descriptive and inferential statistical analyses were used to determine possible interactions between timing of evaluation, gender and type of surgery. With respect to physical and social aspects, a significant difference in outcomes was observed, with mean scores being higher after surgery regardless of gender or type of surgery. Regarding emotional aspects, an interaction effect was observed for timing and gender, with higher mean scores only being obtained for females after surgery. Orthognathic surgery had a positive impact on the quality of life of both male and female patients, improving physical and social aspects, and on that of female patients, improving emotional aspects.

Menadione stress in Saccharomyces cerevisiae strains deficient in the glutathione transferases.

Using S. cerevisiae as a eukaryotic cell model we have analyzed the involvement of both glutathione transferase isoforms, Gtt1 and Gtt2, in constitutive resistance and adaptive response to menadione, a quinone which can exert its toxicity as redox cycling and/or electrophiles. The detoxification properties, of these enzymes, have also been analyzed by the appearance of S-conjugates in the media. Direct exposure to menadione (20 mM/60 min) showed to be lethal for cells deficient on both Gtt1 and Gtt2 isoforms. However, after pre-treatment with a low menadione concentration, cells deficient in Gtt2 displayed reduced ability to acquire tolerance when compared with the control and the Gtt1 deficient strains. Analyzing the toxic effects of menadione we observed that the gtt2 mutant showed no reduction in lipid peroxidation levels. Moreover, measuring the levels of intracellular oxidation during menadione stress we have shown that the increase of this oxidative stress parameter was due to the capacity menadione possesses in generating reactive oxygen species (ROS) and that both GSH and Gtt2 isoform were required to enhance ROS production. Furthermore, the efflux of the menadione-GSH conjugate, which is related with detoxification of xenobiotic pathways, was not detected in the gtt2 mutant. Taken together, these results suggest that acquisition of tolerance against stress generated by menadione and the process of detoxification through S-conjugates are dependent upon Gtt2 activity. This assessment was corroborated by the increase of GTT2 expression, and not of GTT1, after menadione treatment.

Trehalose protects Saccharomyces cerevisiae from lipid peroxidation during oxidative stress.

Aiming to focus the protective role of the sugar trehalose under oxidative conditions, two sets of Saccharomyces cerevisiae strains, having different profiles of trehalose synthesis, were used. Cells were treated either with a 10% trehalose solution or with a heat treatment (which leads to trehalose accumulation) and then exposed either to menadione (a source of superoxide) or to tert-butylhydroperoxide (TBOOH). According to our results, trehalose markedly increased viability upon exposure to menadione stress, which seems to be correlated with decrease in lipid peroxidation levels. The protective effect of trehalose against oxidative damage produced by menadione was especially efficient under SOD1 deficiency. On the other hand, this sugar does not seem to participate of the mechanism of acquisition of tolerance against TBOOH, since trehalose pretreatment (addition of external trehalose) was not capable of increase cell survival. Therefore, trehalose plays a role in protecting cells, especially membranes, from oxidative injuries. However, this mechanism of defense is dependent on the type of oxidative stress to which cells are submitted.

Targets of oxidative stress in yeast sod mutants.

Eukaryotic cells have developed mechanisms to rapidly respond towards the environment by changing the expression of a series of genes. There is increasing evidence that reactive oxygen species (ROS), besides causing damage, may also fulfill an important role as second messengers involved in signal transduction. Recently, we have demonstrated that deletion of SOD1 is beneficial for the acquisition of tolerance towards heat and ethanol stresses. The present report demonstrates that a sod1 mutant was the only one capable of acquiring tolerance against a subsequent stress produced by menadione, although this mutant strain had exhibited high sensitivity to oxidative stress. By measuring the level of intracellular oxidation, lipid peroxidation as well as glutathione metabolism, we have shown that in the SOD1-deleted strain, an unbalance occurs in the cell redox status. These results indicated that the capacity of acquiring tolerance to oxidative stress is related to a signal given by one or all of the above factors.

Acquisition of tolerance against oxidative damage in Saccharomyces cerevisiae.

BACKGROUND: Living cells constantly sense and adapt to redox shifts by the induction of genes whose products act to maintain the cellular redox environment. In the eukaryote Saccharomyces cerevisiae, while stationary cells possess a degree of constitutive resistance towards oxidants, treatment of exponential phase cultures with sub-lethal stresses can lead to the transient induction of protection against subsequent lethal oxidant conditions. The sensors of oxidative stress and the corresponding transcription factors that activate gene expression under these conditions have not yet been completely identified. RESULTS: We report the role of SOD1, SOD2 and TPS1 genes (which encode the cytoplasmic Cu/Zn-superoxide dismutase, the mitochondrial Mn-isoform and trehalose-6-phosphate synthase, respectively) in the development of resistance to oxidative stress. In all experimental conditions, the cultures were divided into two parts, one was immediately submitted to severe stress (namely: exposure to H2O2, heat shock or ethanol stress) while the other was initially adapted to 40 degrees C for 60 min. The deficiency in trehalose synthesis did not impair the acquisition of tolerance to H2O2, but this disaccharide played an essential role in tolerance against heat and ethanol stresses. We also verified that the presence of only one Sodp isoform was sufficient to improve cellular resistance to 5 mM H2O2. On the other hand, while the lack of Sod2p caused high cell sensitivity to ethanol and heat shock, the absence of Sod1p seemed to be beneficial to the process of acquisition of tolerance to these adverse conditions. The increase in oxidation-dependent fluorescence of crude extracts of sod1 mutant cells upon incubation at 40 degrees C was approximately 2-fold higher than in sod2 and control strain extracts. Furthermore, in Western blots, we observed that sod mutants showed a different pattern of Hsp104p and Hsp26p expression also different from that in their control strain. CONCLUSIONS: Trehalose seemed not to be essential in the acquisition of tolerance to H2O2 stress, but its absence was strongly felt under water stress conditions such as heat and alcoholic stresses. On the other hand, Sod1p could be involved in the control of ROS production; these reactive molecules could signal the induction of genes implicated within cell tolerance to heat and ethanol. The effects of this deletion needs further investigation.

A simple HPLC-fluorescence method for the measurement of R,S-sotalol in the plasma of patients with life-threatening cardiac arrhythmias.

R,S-sotalol, a ss-blocker drug with class III antiarrhythmic properties, is prescribed to patients with ventricular, atrial and supraventricular arrhythmias. A simple and sensitive method based on HPLC-fluorescence is described for the quantification of R,S-sotalol racemate in 500 microl of plasma. R,S-sotalol and its internal standard (atenolol) were eluted after 5.9 and 8.5 min, respectively, from a 4-micron C18 reverse-phase column using a mobile phase consisting of 80 mM KH2PO4, pH 4.6, and acetonitrile (95:5, v/v) at a flow rate of 0.5 ml/min with detection at lambdaex = 235 nm and lambdaem = 310 nm, respectively. This method, validated on the basis of R,S-sotalol measurements in spiked blank plasma, presented 20 ng/ml sensitivity, 20-10,000 ng/ml linearity, and 2.9 and 4.8% intra- and interassay precision, respectively. Plasma sotalol concentrations were determined by applying this method to investigate five high-risk patients with atrial fibrillation admitted to the Emergency Service of the Medical School Hospital, who received sotalol, 160 mg po, as loading dose. Blood samples were collected from a peripheral vein at zero, 0.5, 1.0, 1.5, 2.0, 3.0, 4. 0, 6.0, 8.0, 12.0 and 24.0 h after drug administration. A two-compartment open model was applied. Data obtained, expressed as mean, were: C MAX = 1230 ng/ml, T MAX = 1.8 h, AUC T = 10645 ng h-1 ml-1, Kab = 1.23 h-1, alpha = 0.95 h-1, ss = 0.09 h-1, t((1/2))ss = 7.8 h, ClT/F = 3.94 ml min-1 kg-1, and Vd/F = 2.53 l/kg. A good systemic availability and a fast absorption were obtained. Drug distribution was reduced to the same extent in terms of total body clearance when patients and healthy volunteers were compared, and consequently elimination half-life remained unchanged. Thus, the method described in the present study is useful for therapeutic drug monitoring purposes, pharmacokinetic investigation and pharmacokinetic-pharmacodynamic sotalol studies in patients with tachyarrhythmias.

Cat flea (Ctenocephalides felis) infestation in quarantined marsh deer (Blastocerus dichotomus) populations.

Marsh deer (Blastocerus dichotomus) were captured for a research program in Brazil and maintained in quarantine stations. After 60 days, fleas were detected on animals and identified as Ctenocephalides felis felis. Elimination of the infestation was difficult. Animal treatment with a fipronil-based compound was effective, and subsequently captured animals were treated prophylactically. Some animals remained infested, and some died from the infestation.

Comparison of sensitivity of immunofluorescent microscopy to that of a combination of immunofluorescent microscopy and immunomagnetic separation for detection of Cryptosporidium parvum oocysts in adult bovine feces.

A direct immunofluorescence assay (DFA) (Merifluor; Meridian Diagnostics, Inc., Cincinnati, Ohio) was compared to an immunomagnetic separation (IMS) assay (Dynabeads; Dynal, Inc., Lake Success, N.Y.) coupled with immunofluorescent microscopy (Waterborne, Inc., New Orleans, La.) for their ability to detect low concentrations of Cryptosporidium parvum oocysts in adult bovine fecal material. IMS-DFA resulted in a 2-log-unit increase in sensitivity (10 oocysts/g) compared to DFA alone (1,000 oocysts/g). The higher sensitivity obtained with IMS-DFA resulted from testing 2 g of fecal material instead of the 13 to 19 mg of fecal material tested in the DFA; the increased sensitivity was not attributable to a higher percent recovery.