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M J Arrowood

Publications and source records attributed to M J Arrowood.

At least 37 records · Page 2Linked to original sources
PubMed1997

Isolation of the gene coding for elongation factor-1alpha in Cryptosporidium parvum.

A gene encoding for Cryptosporidium parvum (C. parvum) elongation factor 1alpha (EF-1alpha) was isolated and sequenced from a cDNA expression library. The recombinant protein cross-reacted with a monoclonal antibody that was raised to a sporozoite cell surface antigen. The gene encoded a 435 amino acid protein with a predicted molecular weight of 48.1 kDa. The predicted C. parvum EF-1alpha protein sequence showed extensive homology with the EF-1alpha proteins of other eukaryotic organisms and included three conserved sequence motifs implicated in GTP binding.

Amino Acid Sequence↗
PubMed1997

Protective monoclonal antibody defines a circumsporozoite-like glycoprotein exoantigen of Cryptosporidium parvum sporozoites and merozoites.

The apicomplexan protozoan parasite Cryptosporidium parvum causes a diarrheal disease in humans and other mammals for which specific therapy and immunoprophylaxis are unavailable. Passive immunization with Abs against whole C. parvum organisms has variable efficacy in immunocompromised or neonatal hosts. Because apical and surface-exposed zoite Ags of the Apicomplexa are critical to infectivity and targets of protective immunity, we examined the ability of mAbs generated against such Ags in C. parvum sporozoites to passively protect against infection and identify biologically relevant parasite molecules. A panel of mAbs was produced against affinity-purified native Ags using sporozoite apical- and surface-reactive mAb C4A1 as binding ligand. One resulting mAb, designated 3E2, elicited prominent morphologic changes in sporozoites and merozoites characterized by rapid and progressive formation, posterior movement, and release of membranous Ag-mAb precipitates. These changes had a striking resemblance to the malarial circumsporozoite precipitate (CSP) reaction. Sporozoite infectivity was completely neutralized after in vitro exposure to 3E2 and the CSP-like reaction. Furthermore, orally administered 3E2 completely prevented or markedly reduced infection in neonatal BALB/c mice. 3E2 bound to apical complex and surface molecules of zoites and was demonstrated in membranous precipitates by immunoelectron microscopy. In Western blots, 3E2 recognized multiple 46 to approximately 770 kDa sporozoite Ags and an approximately 1300-kDa Ag designated CSL, also expressed by merozoites. CSL was characterized as a soluble glycoprotein exoantigen released by infectious sporozoites. Further, CSL was determined to be the molecular species mechanistically involved in the CSP-like reaction by its identification in SDS-PAGE gels and Western blots of purified membranous precipitates. These findings indicate that CSL has a functional role in sporozoite infectivity and is a candidate molecular target for passive or active immunization against cryptosporidiosis.

Animals↗
PubMed1997

Laboratory diagnosis of Cyclospora infections.

The laboratory diagnosis of newly recognized infectious agents, such as Cyclospora cayetanensis, is frequently problematic because appropriate diagnostic techniques and algorithms are not available. The methods currently available for diagnosis of Cyclospora are described and compared, including concentration procedures, examination of wet preparations, various staining techniques, and the use of molecular-based assays. Because of the autofluorescent properties of the oocysts, particular attention is drawn to the role of fluorescent microscopy in providing a rapid, inexpensive, and sensitive technique for diagnosis of Cyclospora infections in stool samples. In addition to text descriptions, photomicrographs are provided to illustrate Cyclospora oocysts in wet and stained preparations and compare them with Cryptosporidium and Isospora oocysts, the other two most common coccidian infections in man.

Animals↗
PubMed1996

A cloned gene of Cryptosporidium parvum encodes neutralization-sensitive epitopes.

Two mAb, C6B6 and 7D10, each significantly reduced infection of mice by Cryptosporidium parvum and reacted with a 23-kDa glycoprotein (p23) of geographically disperse C. parvum isolates. The antibodies were used to identify plaques in a cDNA library prepared from C. parvum sporozoite mRNA. cDNA insert sequences from positive plaques were determined and used to isolate additional clones encoding p23 coding sequences. A consensus open reading frame of 333 base pairs, encoding 111 amino acids, was identified in this collection of cDNAs. The predicted amino acid sequence contained one N-glycosylation site, but lacked hydrophobic membrane spanning regions. Epitope mapping revealed that mAb 7D10 defines the linear epitope QDKPAD which occurs twice in the C terminal region of the peptide encoded by the ORF. This same C terminal peptide region contains a non-linear epitope bound by mAb C6B6. Serum from mice immunized with synthetic C terminal peptide reacted with sporozoite p23. The occurrence of neutralization-sensitive epitopes encoded by defined regions of the C. parvum genome suggests that recombinant proteins or synthetic peptides containing these epitopes may prove useful for inducing immune responses that diminish infection.

Amino Acid Sequence↗
PubMed1996

In vitro anticryptosporidial activity of dinitroaniline herbicides.

Despite the evaluation of over 100 antimicrobial drugs, the diarrheal disease cryptosporidiosis has remained refractory to treatment. We report the evaluation of five dinitroaniline herbicides including trifluralin, profluralin, nitralin, pendimethalin, and fluchloralin for anticryptosporidial activity in an in vitro cultivation model of Cryptosporidium parvum. All five compounds exhibited significant anticryptosporidial activities with no corresponding evidence of toxicity. The most active compound was pendimethalin with an IC50 of 0.19 microM while nitralin was the least active with an IC50 of 4.5 microM. These compounds should be evaluated further in an animal model of cryptosporidiosis.

Aniline Compounds↗
PubMed1996

A chemiluminescence immunoassay for evaluation of Cryptosporidium parvum growth in vitro.

A chemiluminescence immunoassay (CLIA) was developed to detect Cryptosporidium parvum growth in Madin-Darby canine kidney (MDCK) cell cultures. Optimal results were obtained when MDCK cells were plated at a density of 1 x 10(4) cells/well (96-well plate) and maintained as a monolayer for 4 days prior to infection with 2 x 10(4) parasites/well. Two compounds (paromomycin and maduramicin) were evaluated and shown to have selective activity against C. parvum in a dose-dependent manner. There was excellent correlation between CLIA and immunofluorescence assay when assessing anti-C. parvum agents in MDCK cells. CLIA offers advantages over conventional enzyme-linked immunosorbent assay and immunofluorescence assay methods in that it is more sensitive and efficient. The combination of CLIA and MDCK culture provides an efficient tool for evaluating potential anti-cryptosporidial compounds prior to testing in animal models.

Animals↗
PubMed1995

Evaluation of maduramicin and alborixin in a SCID mouse model of chronic cryptosporidiosis.

Two polyether ionophores, maduramicin and alborixin, were evaluated for anticryptosporidial activity in a severe combined immune deficient (SCID) mouse model of cryptosporidiosis. Groups of SCID mice were inoculated with 10(6) oocysts of bovine origin by oral gavage. Maduramicin or alborixin was administered beginning 4 weeks postinfection at 3 mg/kg of body weight per day. Maduramicin treatment resulted in a 96% reduction in fecal parasite load over the 3-week treatment period (P < 0.003). This reduction correlated with decreases in tissue parasite loads observed in histological sections of the small intestine (P < 0.000002) and the colon (P < 0.000006). A significant decrease in oocyst shedding was also observed after a 3-week treatment with alborixin (71% reduction, P < 0.01). Maduramicin was also evaluated in a relapsing model of cryptosporidiosis in which the infection was observed to recur after treatments were discontinued. Some toxicity, as demonstrated by weight loss, was observed with both maduramicin and alborixin. Both drugs exhibited significant anticryptosporidial activities with concomitant moderate toxicity. These polyether ionophores should be valuable as positive controls in compound evaluation studies and as lead compounds for chemical optimization (modification).

Animals↗
PubMed1995

A new method for evaluating experimental cryptosporidial parasite loads using immunofluorescent flow cytometry.

A flow cytometric method for the quantification of Cryptosporidium parvum oocysts in stool specimens was developed to replace conventional microscopic immunofluorescent assays. Fecal pellets were collected from control (uninfected) severe combined immune-deficient mice, suspended in 2.5% potassium dichromate at a ratio of 400 microliter per pellet, and homogenized by vortexing. Purified oocytes were added to the samples (10(5), 10(4), 10(3), and 10(2)/ml). Aliquots (200 microliters) of the vortexed samples were centrifuged over microscale discontinuous sucrose gradients. The oocyst-containing fractions were collected, washed, and incubated with an oocyst-specific monoclonal antibody (labeled with fluorescein isothiocyanate) for 30 min at 37 degrees C. Sample volumes were adjusted to 600 microliters with phosphate-buffered saline and assayed by using logical gating of forward/side scatter and fluorescence signal on a flow cytometer. Seeded samples showed a linear correlation with the number of oocysts recovered from the gradients. Analyses of stool samples from chronically infected mice demonstrated that the flow cytometry method was approximately 10 times more sensitive than conventional immunofluorescent assays.

Animals↗
PubMed1994

Infection dynamics and clinical features of cryptosporidiosis in SCID mice.

Cryptosporidial infections in severe combined immune deficient (SCID) mice produce a chronic disease state which in the later stages leads to extraintestinal involvement and hepatic dysfunction. To further characterize the infection dynamics in this model and monitor the changes in the hepatic system, a dose titration of the oocyst inoculum was performed and alkaline phosphatase levels in the sera were assayed. Ten SCID mice per dose were inoculated with 10(3), 10(4), 10(5), 10(6), or 10(7) oocysts. Oocyst shedding in the feces was quantified by microscopic enumeration. Mice inoculated with 10(6) oocysts and those inoculated with 10(7) oocysts demonstrated similar oocyst shedding patterns, but the 10(7)-oocyst group exhibited signs of distress (e.g., weight loss and icterus) earlier. The intensity of the infection increased markedly approximately 14 days postinoculation (p.i.) and continued to increase steadily over the next 6 weeks. Inoculation with lower oocyst doses produced a delay in patency (e.g., it occurred 7 days later with the 10(5)-oocyst inoculum and 14 days later with the 10(4)-oocyst inoculum). Mean serum alkaline phosphatase levels in the 10(7)-oocyst group were more than twice control values at 5 weeks p.i. and continued to increase over the next 8 weeks. Oocyst doses and alkaline phosphatase levels were positively correlated with hepatobiliary colonization (r = 0.71) and liver necrosis (r = 0.65) at 13 weeks p.i. A strong positive correlation between hepatobiliary colonization and liver necrosis at 13 weeks p.i. (r = 0.87) was observed.

Alkaline Phosphatase↗
PubMed1992

Effectiveness of dehydroepiandrosterone in reduction of cryptosporidial activity in immunosuppressed rats.

The aim of this study was to evaluate the efficacy of the immunomodulator dehydroepiandrosterone in the treatment of cryptosporidiosis in glucocorticoid-induced immunosuppressed rats. A significant reduction in cryptosporidial activity occurred in treated versus untreated rats as assessed by oocyst shedding intensities and examination of parasite colonization in the small intestine.

Animals↗
PubMed1991

Chronic Cryptosporidium parvum infections in congenitally immunodeficient SCID and nude mice.

Severe combined immune deficient (SCID) and athymic National Institutes of Health (NIH)-III (bg/nu/xid) mice were evaluated for susceptibility to Cryptosporidium parvum infections as neonates and as adults after challenge with inocula of 5 x 10(4) and 1 x 10(6) oocysts/mouse, respectively. SCID and NIH-III nude mice developed chronic infections that persisted over 12 weeks. Cryptosporidial organisms were observed in the small and large intestines of all mice. Colonization of the gallbladder and hepatobiliary duct epithelium occurred in a majority of the NIH-III nude mice and a smaller number of the SCID mice. Severe infections in both mouse strains were associated with clinical deterioration followed by death. Signs in these mice included dehydration, icterus, and emaciation. Deaths apparently resulted from hepatic dysfunction. Control mice experienced transient infections with no clinical signs. Immunodeficient mice should prove useful in studies of disease pathogenesis and in evaluating potential anticryptosporidial agents.

Animals↗
PubMed1991

In vitro activities of lytic peptides against the sporozoites of Cryptosporidium parvum.

Cryptosporidium parvum is a protozoan parasite that causes mild to severe diarrheal disease in animals and humans. There are currently no effective chemotherapeutic agents available for the treatment of cryptosporidiosis. Recent studies have described small, naturally occurring antimicrobial lytic peptides with antiprotozoal activities. In the present study, the anticryptosporidial activities of three synthetic lytic peptides were determined in an in vitro sporozoite susceptibility assay. Sporozoite viability was assessed microscopically by the uptake of the vital dyes fluorescein diacetate and propidium iodide. Sporozoite viability was reduced by 93.5% following a 60-min exposure to 10 microM Hecate-1 at 37 degrees C. Shiva-10 reduced sporozoite viability by approximately 74.0% after a 60-min exposure at 100 microM and 37 degrees C. The cecropin-b analog SB-37 reduced sporozoite viability by 6.0% following a 60-min exposure at 100 microM and 37 degrees C. A control peptide showed no anticryptosporidial activity.

Amino Acid Sequence↗
PubMed1989

Effects of immune colostrum and orally administered antisporozoite monoclonal antibodies on the outcome of Cryptosporidium parvum infections in neonatal mice.

A neonatal BALB/c mouse model of cryptosporidiosis was used to examine the potential passive transfer of immunity via immune colostrum and oral treatment with anticryptosporidial monoclonal antibodies. Neonates suckled by dams that recovered from Cryptosporidium parvum infections were equally susceptible to infection as their control counterparts suckled by naive dams. Parasite loads among the control and immune colostrum-fed mice were indistinguishable. Neonates receiving orally administered antisporozoite monoclonal antibodies were equally susceptible to infections compared with the control and immune colostrum-fed mice. Parasite loads among the mice receiving daily oral treatment with monoclonal antibody mixtures exhibited significantly lower parasite loads compared with the control mice (P less than 0.05).

Administration, Oral↗
PubMed1989

Comparison of conventional staining methods and monoclonal antibody-based methods for Cryptosporidium oocyst detection.

The sensitivity and specificity of seven microscopy-based Cryptosporidium oocyst detection methods were compared after application to unconcentrated fecal smears. The seven methods were as follows: (i) a commercial acid-fast (AF) stain (VOLU-SOL) method, (ii) Truant auramine-rhodamine (AR) stain method, (iii) fluorescein-conjugated C1B3 monoclonal antibody (MAb) direct fluorescence method, (iv) OW3 MAb indirect fluorescence method, (v) biotinylated OW3 indirect fluorescence method, (vi) biotinylated OW3-indirect diaminobenzidine (DAB) method, and (vii) biotinylated OW3-aminoethylcarbazole (AEC) method. A total of 281 randomly collected Formalin-fixed fecal samples (submitted to the Maricopa County Health Department, Phoenix, Ariz.) and 30 known positives (Formalin-fixed and K2Cr2O7-preserved stools from our laboratory) were examined in a blind test; 32 of 311 samples (10.3%) were confirmed positive. Of the confirmed positives, 40.6% were identified by the AF method, 93.8% were identified by the AR method, 93.8% were identified by the C1B3 method, 81.3% were identified by the OW3-DAB method, 71.9% were identified by the OW3-AEC method, 100% were identified by the OW3 indirect fluorescence method, and 100% were identified by the biotinylated OW3 indirect fluorescence method. False-positives were encountered by the AF and AR methods (52.0 and 85.7% specificity, respectively), while no false-positives encountered by the MAb-based methods. Oocysts in infected tissue sections were easily detected by the MAb-based methods.

Animals↗
PubMed1988

Antigens of Cryptosporidium sporozoites recognized by immune sera of infected animals and humans.

The humoral response of humans, calves, and horses to Cryptosporidium sporozoite antigens was evaluated using a western blot technique. Sera from calves, humans, and horses were obtained at various times following the detection of infection. Sera were reacted with detergent-solubilized, sodium dodecyl sulfate polyacrylamide gel-electrophoresed (SDS-PAGE) sporozoite antigens. The number of antigens recognized by immune sera from humans and animals increased with time postinfection. A 20-kDa antigen appears to be a major sporozoite surface determinant labeled via membrane protein biotinylation and recognized by mouse monoclonal antibodies using indirect immunofluorescence and western blotting. Detectable recognition of the 20-kDa band occurred in 3-wk postinfection (PI) sera from all species tested. Reactivity to the 20-kDa band diminished significantly in sera 5 mo PI or longer from infected humans with no known recurrence of cryptosporidial diarrhea. In contrast, 12-mo PI sera from an individual constantly exposed to oocysts under working conditions was as strongly reactive as the 3-wk convalescent sera. Serum reactivity to the 20-kDa antigen appears to be a good indicator of exposure to Cryptosporidium.

Animals↗
PubMed1988

Field inversion gel electrophoretic separation of Cryptosporidium spp. chromosome-sized DNA.

Chromosomal DNA from 5 isolates of Cryptosporidium parvum and 1 of C. baileyi were compared by field-inversion gel electrophoresis (FIGE). FIGE analyses of parasite DNA prepared from purified sporozoites versus intact oocysts showed no observable differences. Chromosomal DNA migration patterns of the 5 C. parvum isolates were indistinguishable, whereas similar but distinct differences were evident between C. baileyi and the isolates of C. parvum. Oocyst-reactive monoclonal antibodies differentiated oocysts of C. parvum from those of C. baileyi but were unable to distinguish oocysts of 1 isolate of C. parvum from another.

Animals↗
PubMed1987

Detection of Cryptosporidium in water by using polypropylene cartridge filters.

Members of the genus Cryptosporidium are protozoan parasites that cause gastroenteritis in humans and animals and appear to be spread largely by the fecal-oral route. A method was developed for the concentration and detection of Cryptosporidium oocysts in water to assess their occurrence in the environment and potential for waterborne disease transmission. This method was developed by using spun polypropylene cartridge filters. Optimal conditions for concentration, filter elution, filter porosity, and detection were determined. Fluoresceinated monoclonal antibodies were used for oocyst detection. Experiments also were conducted to study the effect of flow rate, low oocyst numbers, and the addition of detergents on recovery and retention of oocysts. The method that was developed was sensitive enough to detect oocysts at levels of less than 1 per liter. Using this method, we isolated Cryptosporidium oocysts from secondarily treated sewage.

Coccidia↗