金胺O荧光染色在石蜡组织切片麻风病诊断中的应用

目的为了验证金胺O荧光染色法应用于石蜡组织切片麻风杆菌检测的可行性。方法用金胺O荧光法对6例确诊为麻风病的病理组织切片进行染色,并与抗酸染色结果进行对比。结果荧光染色法6例结果均为阳性,在暗背景下麻风杆菌显示明亮淡绿色荧光;在菌量较少荧光染色片中寻找单根麻风杆菌,较抗酸染色片更为容易。结论金胺O荧光染色法可用于石蜡组织切片麻风病的诊断,麻风杆菌单根散在时比抗酸染色法有一定优势。     

The binding of Ca2+ to actin monomer is monitored by the fluorescence of actin-bound auramine O.

The fluorescence of the cation auramine O was substantially enhanced by the presence of actin monomer. Titrations of this fluorescence enhancement indicated that actin monomer had two auramine O binding sites, each with a dissociation constant of approx. 20 microM. Calcium ions had no effect on the number of actin monomer-bound auramine O molecules or on the dissociation constant for that interaction. However, calcium ions increased the maximum change of fluorescence that occurs when actin monomer was fully saturated with auramine O. This effect of calcium was saturable and yielded a Ca2+ dissociation constant of 1.6 mM. It was concluded that auramine O bound to sites on actin monomer and independently monitored the binding of Ca2+ ion(s) to other site(s) on actin monomer. Further, the magnitude of the Ca2+ dissociation constant suggested that this Ca2+-binding site may be representative of the multiple bivalent cation-binding sites on actin monomer which are thought to be directly involved in actin polymerization. However, the exact relationship between these sites remains unclear.     

Preparing Epoxy Sections of Insect Tissues for Light Microscopy

Sections of aldehyde-fixed and osmium-stained insect tissues embedded in various epoxy resins were affixed to glass slides by use of a slide cover and hotplate combination. A high concentration of solvent vapor over the sections was thus maintained while they dried down on the slides, resulting in excellent flatness and adhesion. Sections were then stained at an elevated temperature with a mixture of equal parts of 3 dye solutions: 1% toluidine blue O, 1% safranin O, and saturated auramine O, all made up in 1% solution of borax in water. The method resulted in excellent differentiation of all insect tissue components including lightly chitinized structures.     

Fixing coccidian oocysts is not an adequate solution to the problem of preserving protozoan type material

Fresh (36 days old) sporulated oocysts of Eimeria nieschulzi were divided into 7 groups. Control oocysts were maintained at 23 C in 2% aqueous (w/v) K2Cr2O7. The 6 experimental groups were mixed with either Bouin's solution, 10% aqueous (v/v) buffered formalin, Karnovsky's solution, glutaraldehyde, paraformaldehyde, or 70% aqueous (v/v) ethanol (EtOH). After 115 days, oocysts from all 7 groups were examined under oil immersion to determine the effect of fixation on their structural integrity. The parameters examined were lengths and widths of oocysts and sporocysts, percent sporulation (%S), and percent crenation (%C) of oocysts and sporocysts. The highest destruction (%S and %C) occurred in oocysts exposed to glutaraldehyde and Karnovsky's fixatives where 100% of both oocysts and sporocysts crenated and only 8% and 48%, respectively, remained sporulated. Of the oocysts in paraformaldehyde, 93% remained sporulated, but 95%of these oocysts and 100% of the sporocyst crenated. In Bouin's solution, 75% of the oocysts were intact structurally, but of these, only 60% were still sporulated with 70% of their sporocysts crenated. Oocysts preserved in 70% EtOH were 80% intact and 70% remained sporulated, but nearly 60% of their sporocysts collapsed even though the oocyst walls were intact. Oocysts preserved in 10% buffered formalin maintained structural integrity but had lower numbers of sporulated oocysts (84%) and greater numbers of crenated oocysts (18%) than control oocysts maintained in the dichromate solution (95% and 0%, respectively).     

Comparison between a bovine and a human enterohaemorrhagic Escherichia coli strain of serogroup O26 by suppressive subtractive hybridization reveals the presence of atypical factors in EHEC and EPEC strains

Enterohaemorrhagic Escherichia coli (EHEC) strains are responsible for food poisoning in humans in developed countries via consumption of vegetal and animal foodstuffs contaminated by ruminant feces. The clinical conditions caused by EHEC strains vary from undifferentiated diarrhea to hemorrhagic colitis with, in a few cases, the appearance of the hemolytic uremic syndrome, which can lead to death. Most EHEC strains can be found in the gut of healthy ruminants, but some of the strains, belonging to O26, O111, O118 serogroups, for example, are also responsible for digestive disorders in calves. The aim of this research was to study the genomic differences between two EHEC strains of serogroup O26 isolated from a young calf and a human with diarrhea, to identify specific sequences of the bovine strain that could be implicated in initial adherence or host specificity. No sequence implicated in host specificity was found during our study. Finally, several factors, not usually present in EHEC strains of serogroup O26, were identified in the bovine strain. One of them, the PAI I(CL3) locus initially presented as a marker for LEE-negative VTEC strains, was found in 11.3% of EPEC and EHEC strains.     

Prevalence of Cryptosporidium sp. infection in diarrheic and non-diarrheic humans in Iran

For evaluation of the prevalence of Cryptosporidium sp. infection in diarrheic and non-diarrheic humans in Iran, fecal specimens from diarrheic (n = 129) and non-diarrheic humans (n = 271) were collected and examined for the presence of Cryptosporidium sp. oocysts. The presence of Cryptosporidium sp. oocysts was determined by Ziehl- Neelsen acid-fast staining. Humans were grouped according to their age as follows: younger than 15, 16-25, 26-35, 36-50, and over 51 years. The results showed that the overall prevalence of infection in all 400 samples was 10.8%, but the prevalence (25.6%) in diarrheic humans was higher than that (3.7%) in non-diarrheic humans. Oocysts of Cryptosporidium sp. were detected in the feces of 21.4%, 9.3%, 8.8%, 6.7% and 5.7% of different age groups, respectively. The intensity of oocysts was significantly higher in diarrheic humans than in non-diarrheic ones. There was a significant association between Cryptosporidium sp. infection and occurrence of diarrhea (P < 0.05). The results indicate that Cryptosporidium sp. infection is prevalent in diarrheic humans in Iran.     

Conformational changes in liver alcohol dehydrogenase upon nucleotide binding: detection by circular dichroism of dye complexes

Michler's ketone, the ketone analog of auramine O, binds to liver alcohol dehydrogenase with an affinity comparable to that of auramine O, yields similar induced circular dichroism bands, and its binding is enhanced in the same way by binding of coenzyme fragments. These observations suggest that (a) Michler's ketone binds to liver alcohol dehydrogenase in a similar manner and at the same site as does auramine O and (b) the enhancement of auramine O and Michler's ketone binding by coenzyme fragments is not due to electrostatic interactions since both the positively charged auramine O and the neutral Michler's ketone show similar effects. Observation of similar enhancement of auramine O binding by GMP and ?-AMP as well as the activesite topology suggested by fluorescence and X-ray studies argue against any direct interaction of the dye with the purine ring. We, therefore, ascribe the enhanced binding of dye in ternary complexes to a conformational change in liver alcohol dehydogenase induced by the binding of coenzyme fragments. Studies of the circular dichroism of liver alcohol dehydrogenase complexes with 2,2′-bipyridyl are also reported.     

Prevalence of Escherichia coli O157:H7 on hides and faeces of ruminants at slaughter in two major abattoirs in Nigeria

Aim: To determine the occurrence of Escherichia coli O157: H7 in hides and faeces of slaughtered ruminants in Nigeria. Methods and Results: A total number of 320 animals were sampled from January to December covering the wet and harmattan seasons. Samples were obtained from the hides and faeces of animals at slaughter. The ISO (ISO 16654:2001, Microbiology of food and animal feedingstuffs – horizontal method for the detection of Escherichia coli O157) method for enrichment and isolation of E. coli O157 incorporating selective enrichment using modified tryptone soya broth with novobiocin (mTSBn),immunomagnetic separation and plating on sorbitol‐MacConkey agar with cefixime tellurite (CT‐SMAC) was used. Overall cattle had a prevalence rate of 49·4% followed by sheep and goats with rates of 6·3% and 2·5%, respectively. There was a significant difference in carriage of E. coli O157 among two different cattle breeds. Conclusions: The prevalence of E. coli O157: H7 is substantial from two abattoirs in the country. The carriage and shedding of E. coli O157: H7 did not differ with season but differed among groups of ruminants and among breeds of cattle in a tropical country. Significance and Impact of the Study: This is the first study on E. coli O157: H7 from abattoir operations in Nigeria. The study emphasizes the risk of E. coli O157: H7 along the meat chain and the need for concerted effort to limit it through best hygiene practices.     

Rotifers ingest oocysts of Cryptosporidium parvum

Six genera of rotifers including Philodina, Monostyla, Epiphanes, Euchlanis, Brachionus, and Asplanchna were exposed to oocysts of Cryptosporidium parvum cleaned of fecal debris. Unstained oocysts and those stained with fluorescein-conjugated monoclonal antibody were added to suspensions of viable rotifers and were examined by phase-contrast, differential interference contrast, and fluorescence microscopy. Rotifers of all six genera were observed ingesting oocysts. A maximum of 25 oocysts was observed in the stomachs of Eauchlanis and Brachionus. Euchlanis and Epiphanes were observed excreting boluses containing up to eight oocysts. It was not determined whether rotifers digested or otherwise rendered oocysts nonviable.     

A case of human cryptosporidiosis in Czechoslovakia

A case of human cryptosporidiosis, the first one reported in Czechoslovakia, is described. The disease was diagnosed by the presence of Cryptosporidium oocysts in the feces. The methods used independently to identify oocysts were the fecal flotation technique employing a saturated solution of sucrose and the microscopic examination of stained fecal smears. The patient was a 4-year-old boy with watery diarrhea of 5 days' duration who was kept on a diet and treated with a suspension of Endiaron N Spofa. Excretion positivity for Cryptosporidium oocysts in the feces was detectable at 3 and 5 days after appearance of first clinical manifestations. Bacteriological examination was repeatedly negative. This finding leaves little doubt as to existence of human cryptosporidiosis in Czechoslovakia, but what remains obscure is its overall contribution to the etiology of diarrheal diseases encountered in the population.     

Cryptosporidium parvum sporozoite staining by propidium iodide.

Modified Ziehl-Neelsen (ZN) acid-fast stain is the usual method for detection of Cryptosporidium oocysts in feces. Propidium iodide permitted us to stain free or intra-oocyst sporozoites. With the ZN method only 3-5% of the oocysts purified from three human and one experimentally infected lamb dichromate-preserved feces were stained by carbol fuchsin. These fuchsin-stained oocysts were free of intact sporozoites as identified by propidium iodide staining. Treatment with 10% formalin or 0.5% sodium hypochlorite increased the percentage of acid-fast stained oocysts and thus the sensitivity of acid-fast staining. Treatment with sodium hypochlorite induced intra-oocyst sporozoite alterations as demonstrated by flow cytometric analysis of the oocysts' DNA content. Propidium iodide staining of fixed oocysts is a simple and rapid method to visualize sporozoites and to assess oocyst preservation after different treatments.     

Quantification of Cryptosporidium parvum oocysts in mouse fecal specimens using immunomagnetic particles and two-color flow cytometry

Although single-color flow cytometry has been shown to be more sensitive than fluorescence microscopy for the quantification of Cryptosporidium parvum oocysts, this method has not been optimized. Monoclonal antibody OW50, specific to the cell wall of oocysts, was conjugated to superparamagnetic particles, to fluorescein isothiocyanate, and to r-phycoerythrin. The oocysts were then double stained with the fluorochrome-labeled OW50 and were placed in tubes with known numbers of highly fluorescent polystyrene beads, allowing quantification of the oocysts without dependence on acquired sample volume by flow cytometry. Data from 2-color flow cytometry using logical gating of the oocysts and beads showed a linear relationship between dilutions of a purified oocyst suspension and the mean numbers of oocysts detected (r2 = 1.00). An average of 15 purified oocysts/ml were counted in a dilution with a theoretical concentration of 12 oocysts/ml. Known numbers of purified oocysts were seeded into normal mouse fecal specimens, captured by OW50-labeled immunomagnetic particles, eluted with 5% potassium dichromate at low pH, and double stained with fluorochrome-labeled OW50. By flow cytometry, the mean recovery was 43.1% (+/-8.3%), and as few as 133 oocysts were detected. The captured and eluted oocysts were infective in neonatal BALB/c mice. This 2-color flow cytometry method, used in conjunction with the capture and elution of oocysts by and from immunomagnetic particles, provides a powerful tool for not only the quantification and purification of C. parvum oocysts from different sources but also for the characterization of oocysts in vitro and in vivo.     

Efficacy of hyperimmune bovine colostrum for prophylaxis of cryptosporidiosis in neonatal calves

Twelve neonatal calves were experimentally infected with oocysts of Cryptosporidium parvum. Six calves in group A fed hyperimmune colostrum at birth had significantly less diarrhea and shed oocysts for less time than did 6 calves in group B fed colostrum from cows that were not hyperimmune. Calves in group A had diarrhea for 0-4 days (means = 2.3 days), whereas calves in group B had diarrhea for 4-6 days (means = 5.0 days). Calves in group A shed oocysts for 4-9 days (means = 6.2 days), whereas calves in group B shed oocysts for 7-11 days (means = 8.5 days). These findings indicate that passive lacteal immunity conferred partial protection against cryptosporidiosis. Whether such protection was provided by the immunoglobulins that were highly elevated in the colostrum (greater than 1:200,000 for IgG1, IgM, and IgA) and constituted a large part of the circulating antibody in the calves, or by other biologically active factors, such as cytokines, is undetermined.     

Fluorescent Staining Technique for Nucleoid Regions of Streptosporangium albidum and Streptosporangium brasiliense

Fluorescent staining procedures were developed for elucidating the nucleoid region in Streptosporangium albidum and Streptosporangium brasiliense. In these procedures, plugs of nutrient agar were inoculated with the microorganims and then covered with a sterile glass slide. The growing cells adhered to the surface of the slide and remained attached throughout the staining procedures. Two separate staining methods were utilized, one with bisbenzimid H33258 and the other with auramine O. Fluorescent microscopy revealed intensely stained nucleoid regions within mycelia, spores, and sporangia.     

Examination of attachment and survival of Toxoplasma gondii oocysts on raspberries and blueberries

The consumption of Toxoplasma gondii oocysts on fresh produce may be a means of its transmission to humans. Cats shed T. gondii oocysts, which contaminate produce directly or contaminate water sources for agricultural irrigation and pesticide and fertilizer applications. Cyclospora cayetanensis is a related coccidial parasite, and outbreaks of diarrhea caused by C. cayetanensis have been associated with the ingestion of contaminated raspberries. The oocysts of these coccidians are similar in size and shape, indicating that they may attach to and be retained on produce in a similar manner. In the present study the attachment and survival of T. gondii oocysts on 2 structurally different types of berries were examined. Raspberries and blueberries were inoculated individually with 1.0 x 10(1) to 2.0 x 10(4) oocysts of sporulated T. gondii. Berries inoculated with 2.0 x 10(4) oocysts were stored at 4 C for up to 8 wk. Oocyst viability and recovery were analyzed by feeding processed material to mice. Mice fed T. gondii-inoculated berries stored at 4 C for 8 wk developed acute infections. In other experiments mice fed raspberries inoculated with > or = 1.0 x 10(1) oocysts became infected, whereas only mice fed blueberries inoculated with > or = 1.0 x 10(3) oocysts became infected. This study demonstrates that T. gondii oocysts can adhere to berries and can be recovered by bioassays in mice and that raspberries retain more inoculated oocysts than do blueberries. The results suggest that T. gondii may serve as a model for C. cayetanensis in food safety studies.     

Patterns of Cryptosporidium oocyst shedding by eastern grey kangaroos inhabiting an Australian watershed

The occurrence of Cryptosporidium oocysts in feces from a population of wild eastern grey kangaroos inhabiting a protected watershed in Sydney, Australia, was investigated. Over a 2-year period, Cryptosporidium oocysts were detected in 239 of the 3,557 (6.7%) eastern grey kangaroo fecal samples tested by using a combined immunomagnetic separation and flow cytometric technique. The prevalence of Cryptosporidium in this host population was estimated to range from 0.32% to 28.5%, with peaks occurring during the autumn months. Oocyst shedding intensity ranged from below 20 oocysts/g feces to 2.0 x 10(6) oocysts/g feces, and shedding did not appear to be associated with diarrhea. Although morphologically similar to the human-infective Cryptosporidium hominis and the Cryptosporidium parvum "bovine" genotype oocysts, the oocysts isolated from kangaroo feces were identified as the Cryptosporidium "marsupial" genotype I or "marsupial" genotype II. Kangaroos are the predominant large mammal inhabiting Australian watersheds and are potentially a significant source of Cryptosporidium contamination of drinking water reservoirs. However, this host population was predominantly shedding the marsupial-derived genotypes, which to date have been identified only in marsupial host species.     

A new filter-eluting solution that facilitates improved recovery of Cryptosporidium oocysts from water

Cryptosporidium is a zoonotic coccidian parasite associated with diarrhea, and the disinfectant-resistant oocysts are threats to public health even in industrialized countries. In order to make an accurate assessment of the risk to public health, a detection method that has a high recovery rate of oocysts in water is required. In this study, we developed a new filter-eluting solution that facilitates more efficient recovery of Cryptosporidium oocysts from different kinds of water samples. The filter-eluting solution, referred to as PET, consists of sodium pyrophosphate (0.02%), Tween 80 (0.01%) and trisodium EDTA (0.03%). By using PET instead of conventional filter-eluting solutions, the average recovery rate significantly increased from 25.5+/-15.1% to 43.1+/-13.9% (p<0.05). The improved oocyst recovery was likely due to the increased separation of the oocysts from debris trapped on the filter membrane as well as increased capture of the oocysts by immunomagnetic beads. We recommend that PET be used as the filter-eluting solution for detection of Cryptosporidium oocysts in environmental water.     

New method using sedimentation and immunomagnetic separation for isolation and enumeration of Cryptosporidium parvum oocysts and Giardia lamblia cysts

A new method for the isolation of Cryptosporidium parvum oocysts and Giardia lamblia cysts from biosolid samples has been developed that utilizes sedimentation and immunomagnetic separation. The method was used to recover stained cysts and oocysts (spike organisms) from primary settled sewage sludge, anaerobically digested sewage sludge, and bovine manure. Recovery efficiencies associated with this method were approximately 40 to 60% and were significantly greater than those associated with similar methods based on sucrose flotation (P < 0.001). The recovery efficiency of the sedimentation-based method showed no significant reduction as a result of sample storage for up to 21 days (P > 0.05). Recovery efficiencies were determined by spiking samples with prestained cysts and oocysts, allowing them to be differentiated from those naturally present in the biosolid samples. The prestained cysts and oocysts had been fixed in 5% formalin, and the recovery efficiencies associated with this method may be different from recovery efficiencies for fresh cysts or oocysts.     

An investigation of staining methods to determine total cell numbers and the number of respiring micro-organisms in samples obtained from the field and the laboratory

Abstract Dyes were evaluated in combination with 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride (INT) to enable total cell numbers and the numbers of respiring cells to be determined on the same preparation. Malachite green and 4',6-diamidino-2-phenylindole (DAPI) were unsuitable counter-stains. Cells which contained INT formazan crystals could be stained with ethidium bromide or auramine. At high concentrations of INT formazan, auramine fluorescence was reduced, although this effect was partially rectified by prior fixation with glutaraldehyde. Staining with ethidium bromide produced a strong fluorescence in cells containing crystals of INT formazan. This observation was developed into a procedure which allowed total cells to be determined and provided a useful estimate of the number of respiring cells in samples obtained from the laboratory and the field.     

Colonization, persistence, and tissue tropism of Escherichia coli O26 in conventionally reared weaned lambs

Escherichia coli O26 is recognized as an emerging pathogen associated with disease in both ruminants and humans. Compared to those of E. coli O157:H7, the shedding pattern and location of E. coli O26 in the gastrointestinal tract (GIT) of ruminants are poorly understood. In the studies reported here, an stx-negative E. coli O26 strain of ovine origin was inoculated orally into 6-week-old lambs and the shedding pattern of the O26 strain was monitored by serial bacteriological examination of feces. The location of colonization in the GIT was examined at necropsy at two time points. The numbers of O26 organisms excreted in feces declined from approximately 10(7) to 10(4) CFU per gram of feces by day 7 and continued at this level for a further 3 weeks. Beyond day 30, excretion was from few animals, intermittent, and just above the detection limit. By day 38, all fecal samples were negative, but at necropsy, O26 organisms were recovered from the upper GIT, specifically the ileum. However, no attaching-effacing (AE) lesions were observed. To identify the location of E. coli O26 within the GIT early after inoculation, two lambs were examined postmortem, 4 days postinoculation. High numbers of O26 organisms were recovered from all GIT sites examined, and approximately 10(9) CFU were recovered from 1 gram of ileal tissue from one animal. Despite high numbers of O26 organisms, AE lesions were identified on the mucosa of the ascending colon of only one animal. These data indicate that E. coli O26 readily colonizes 6-week-old lambs, but the sparseness of AE lesions suggests that O26 is well adapted to this host, and mechanisms other than those dependent upon intimin may play a role in persistence.