Freeze-Thaw Treatment of Membrane Concentrates Derived from Kraft Pulp Mill Operations

Freeze thaw was studied as a waste treatment method for concentration and volume reduction of contaminated waste concentrates that are derived from the use of membrane technology in the treatment of high strength Kraft pulp mill effluents. Unidirectional freezing experiments were conducted to simulate seminatural freezing, in which the independent variables—freezing rate, time frozen, storage temperature, concentration, liquid depth, thawing rate and method of thawing—were examined for their relative importance. Method of thawing followed by freezing rate, rate of thawing, storage temperature, and time frozen were identified as the most important independent variables that contribute significantly to treatment performance. Under ideal conditions, freeze thaw was shown to effectively concentrate and separate the constituent matter of alkaline, extraction-stage membrane concentrate to achieve color removals as high as 73% in the top 70% liquid fraction. The results suggest a new field of use for freeze thaw as a waste treatment process for the management of high strength liquid wastes amenable to mechanical coagulation by freezing.     

Optimal utilization of cryopreserved human semen for assisted reproduction: recovery and maintenance of sperm motility and viability

PURPOSE: Our purpose was to evaluate sperm motility and viability and the maintenance of these parameters in already cryopreserved semen samples following repeated freezing/thawing cycles. METHODS: Human spermatozoa were subjected to five cycles of cryopreservation/thawing. Recovery of sperm motility and viability and the proportion of viable nonmotile sperm were determined up to 6 hr after thaw. RESULTS: Sperm motilities (prefreeze motility, 70.1%; n = 9 samples) after each of five freeze/thaw cycles were 24.4, 8.0, 3.5, 1.5 and 1.8%. The recovery of sperm viability was higher than that of motility after each cycle: 39.1, 25.3, 22.6, 17.8, and 16.5%. Recoveries of motility and viability were improved if the thawed samples were left in the original cryopreservation medium prior to refreezing vs. if a washing/ resuspension step was included. The recovery of sperm motility in the first thawing cycle was indicative of the expected motile sperm recovery in the second thawing cycle. CONCLUSIONS: Cryopreserved semen that is intended to be reused in future assisted reproduction treatments should be thawed only once and aliquoted in the original freezing medium before refreezing. The recovery of sperm motility and viability in the second thawing cycle, thus the applicability of the sample in conventional in vitro fertilization or intracytoplasmic sperm injection may be anticipated in > 90% of the samples. In view of intracytoplasmic sperm injection it is important that sperm viability is maintained better than motility; after the first, second, and third thawing cycles the ratios of motile:nonmotile viable sperm were 1:1, 1:4, and 1:7, respectively.     

Enzyme-linked immunosorbent assay for the detection of Cryptosporidium parvum IgG in the serum of cats

The objective was to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of Cryptosporidium parvum IgG in the serum of cats. The ELISA was an indirect ELISA using soluble C. parvum oocyst antigens and a peroxidase-labeled anti-feline IgG secondary antibody. Sera from cats with Toxocara felis, Giardia spp., Aelurostrongylus abstrusus, Isospora felis, Isospora rivolta, Toxoplasma gondii, or Taenia spp. infections were assayed in specificity studies. Following optimization, the ELISA and fecal examination for oocysts were performed on samples from 170 client-owned or humane society source cats and 1 cat inoculated orally with C. parvum oocysts. Cryptosporidium parvum oocysts were detected in feces (4/170; 2.4%), and C. parvum IgG was detected in serum (26/170; 15.3%) from naturally exposed cats. The seroprevalence data suggest that some cats in the geographical area studied were exposed to C. parvum, but persistent oocyst shedding was less common. The ELISA is not useful for predicting oocyst shedding in individual cats.     

Simple and rapid measurement of Cryptosporidium excystation using flow cytometry

In vitro excystation is commonly used to determine the viability of samples of purified Cryptosporidium parvum oocysts. Following exposure to conditions that stimulate excystation, samples are examined microscopically to determine the number of excysted oocysts. The microscopy procedure is tedious and time consuming, and difficult to apply to most oocyst samples without a purification step. A simple flow cytometric method was developed for determining the numbers of oocysts that had excysted following the in vitro excystation procedure. Differences in light-scatter properties were used to differentiate intact, partially empty and empty oocysts. By staining samples with a monoclonal antibody specific to the oocyst wall it was possible to apply the technique to unpurified oocysts from faeces. Correlation of the flow cytometric and microscopic method was statistically significant (P < 0.05), resulting in a calculated correlation coefficient of 0.994. The flow cytometry method is faster and more sensitive than the microscopy procedure, and enables analysis of large numbers of samples and of many thousands of oocysts in each sample.     

Immunomagnetic capture PCR to detect viable Cryptosporidium parvum oocysts from environmental samples

A method to detect viable Cryptosporidium parvum oocysts was developed. Polyclonal immunoglobulin G against C. parvum oocyst and sporozoite surface antigens was purified from rabbit immune serum, biotinylated, and bound to streptoavidin-coated magnetic particles. C. parvum oocysts were captured by a specific antigen-antibody reaction and magnetic separation. The oocysts were then induced to excyst, and DNA was extracted by heating at 95 degrees C for 10 min. A 452-bp fragment of C. parvum DNA was amplified by using a pair of C. parvum-specific primers in PCR. The method detected as few as 10 oocysts in purified preparations and from 30 to 100 oocysts inoculated in fecal samples. The immunomagnetic capture PCR (IC-PCR) product was identified and characterized by a nested PCR that amplified a 210-bp fragment, followed by restriction endonuclease digestion of the IC-PCR and nested-PCR products at the StyI site and a nonradioactive hybridization using an internal oligonucleotide probe labeled with biotin. PCR specificity was also tested, by using DNAs from other organisms as templates. In the control experiments, inactivated oocysts were undetectable, indicating the ability of this method to differentiate between viable and nonviable oocysts. Thus, this system can be used to specifically detect viable C. parvum oocysts in environmental samples with great sensitivity, providing an efficient way to monitor the environment for C. parvum contamination.     

Inactivation of Cryptosporidium Oocysts in a Pilot-Scale Ozone Bubble-Diffuser Contactor. II: Model Validation and Application

The axial dispersion reactor (ADR) model developed in Part I of this study was successfully validated with experimental data obtained for the inactivation of C. parvum and C. muris oocysts with a pilot-scale ozone-bubble diffuser contactor operated with treated Ohio River water. Kinetic parameters, required to model the effect of temperature on the decomposition of ozone in treated Ohio River water and oocyst inactivation, were determined from batch and semibatch ozonation experiments. The ADR model was used to simulate the effects of operating conditions (feed-gas ozone concentration, liquid flow rate, and gas flow rate), and water quality related parameters (fast ozone demand, first and second order ozone decomposition rate constants, and temperature) on the performance of the pilot-scale contactor. The model simulation provided valuable insight into understanding the performance of ozone disinfection systems and recommendations for ozone contactor design and optimization. For example, the simulation revealed that meeting inactivation requirements for C. parvum oocysts would be more challenging at relatively lower temperatures.     

Zeta Potential, Dissolved Organic Carbon, and Removal of Cryptosporidium Oocysts by Coagulation and Sedimentation

This study evaluated the removal of viable Cryptosporidium parvum oocysts and changes in zeta potential during alum coagulation and sedimentation. Experiments were designed to evaluate oocyst removal and oocyst zeta potential at three initial dissolved organic carbon (DOC) concentrations and a wide range of alum doses and coagulation pH values. The study showed that changes in the initial DOC concentration affected the zeta potential of Cryptosporidium parvum oocysts and the removal of oocysts. Oocysts did not appear to be removed by a charge neutralization mechanism under the conditions used in this research. Sweep flocculation appeared to be the primary removal mechanism at the lowest DOC concentration tested in this study. For the highest DOC concentration tested, optimal coagulation conditions for oocyst removal coincided with optimal coagulation conditions for natural organic matter (NOM) removal, suggesting that NOM played a key role in the interaction between oocysts and the aluminum hydroxide precipitate.     

Survival and growth of Campylobacter jejuni after artificial inoculation onto chicken skin as a function of temperature and packaging conditions

Campylobacter jejuni is one of the major causes of food poisoning in humans. C. jejuni is also widespread in food animals, and meat and meat products derived from food animals are the most common vector of bacterial transmission to humans. To determine the role of packing and storage conditions on the replication of C. jejuni on chicken, the virulent strain C. jejuni 81116 was artificially inoculated onto chicken skin pieces (1 cm2) and stored at different temperatures and under various packaging conditions. C. jejuni 81116 remained viable at -20 and -70 degrees C and was able to replicate at 4 degrees C and at ambient room temperature. C. jejuni 81116 was also inoculated onto chicken skin and subjected to repeated freeze thawing and the viability of the inoculum was quantified. C. jejuni 81116 could withstand repeated freeze thawing similar to that which may occur in the domestic home. Under all freezing conditions, C. jejuni 81116 retained a high level of viability and quickly replicated to levels which exceeded Australian food authorities' permitted bacteria level on raw food products after the sample was thawed.     

Viable Cryptosporidium parvum oocysts exposed to chlorine or other oxidising conditions may lack identifying epitopes

The intestinal protozoan parasite Cryptosporidium parvum is a known cause of water-borne disease in humans. The detection of Cryptosporidium oocysts in water samples relies upon the use of fluorescently labelled antibodies, preferably using flow cytometry and epifluorescence microscopy. Here we demonstrate that four commercially available antibodies recognise a similar set of immunodominant epitopes on the oocyst wall. These epitopes appear to be carbohydrate in nature and are labile to chlorine treatment and oxidising conditions. Sodium hypochlorite and sodium meta-periodate reduced the ability of the antibodies to detect Cryptosporidium oocysts. Damage to the epitopes did not necessarily reduce the viability of oocysts. This finding may be important for the water industry, where naturally occurring oxidising conditions or sanitizing treatments could produce viable oocysts that are undetectable using standard protocols.     

A comparison of freezing and thawing methods for the cryopreservation of human semen

The purpose of this study was twofold. In the first part are compared cryosurvival rates for human semen following two different freezing and three different thawing techniques. In the second part a larger number of samples processed by the best of the six methods described were examined to ensure reproducibility. Eighteen human semen samples with initial motility greater than 60% and density greater than 20 X 10(6)/ml were mixed with a cryopreservative medium at 35 degree C and vacuumed into 0.5 ml straws. Six aliquots were prepared from as many specimens as possible. Three straws were frozen by a programmed method (P) and three by a rapid freezing technique (V). All six straws were stored in liquid nitrogen vapor (-196 degree C) for 1 week. The straws were thawed by 1 of 3 methods: (1) room temperature for 10 minutes and 37 degree C hot plate for 10 minutes, (2) ice water for 10 minutes and 37 degree C hot plate for 10 minutes, (3) 35 degree C water for 12 seconds and 37 degree C hot plate for 10 minutes. Postthaw motility was assessed for each aliquot. The freeze/thaw method P/1 was judged optimal. In the second part of the study a larger number of samples were processed by P/1. The mean +/- standard deviation postthaw motility o 57 semen specimens processed by this method was 61.4 +/- 12.1.     

Chlorine Dioxide Inactivation of Cryptosporidium Parvum in Oxidant Demand-Free Phosphate Buffer

Chlorine dioxide inactivation of Cryptosporidium parvum oocysts was studied at bench-scale in oxidant demand-free 0.05 M phosphate buffer at pH 6, 8, and 11, and temperature from 1°C to 37°C. Animal infectivity using neonatal CD-1 mice was used for evaluation of oocyst infectiousness before and after treatment. Survival curves of the oocysts following treatment declined linearly with the chlorine dioxide Cavgt product. Temperature was critical for C. parvum inactivation, while pH was found not to be a significant factor at pH 6–11. Inactivation kinetics at different temperatures was expressed as a Chick-Watson model with different reaction rate constants adjusted by van't Hoff-Arrhenius relationship. Between 1°C and 37°C, for every 10°C decrease in temperature, the reaction rate constant decreased by a factor of 2.3, corresponding to an activation energy of 54.9 kJ∕mol. Design criteria targeting 0.5 to 2.0 log-units of inactivation of C. parvum were developed for different water temperatures and their 90% confidence intervals were provided.     

Detection of Cryptosporidium oocysts and Giardia cysts in the tissues of eastern oysters (Crassostrea virginica) carrying principal oyster infectious diseases

The potential cross-reactivity of the combined Cryptosporidium/Giardia direct immunofluorescence antibodies (IFA) of MERIFLUOR and HYDROFLUOR-COMBO tests was examined against tissues containing known developmental stages of 12 pathogens causing the principal infectious diseases in oysters. Spores of Haplosporidium nelsoni and Haplosporidium costale produced positive acid-fast stain (AFS) reactions similar in intensity to Cryptosporidium parvum oocysts. Hexamia nelsoni trophozoites produced positive IFA reactions in both IFA tests; however, the intensity of fluorescence was considerably lower and the fluorescein-staining pattern different than those of Giardia cysts. The applicability of AFS for screening oysters for Cryptosporidium oocysts is low, and positive identification of Cryptosporidium oocysts cannot be accomplished based on the AFS. Presumptive IFA identification of the Cryptosporidium oocysts or Giardia cysts in the oyster tissue should fulfill 3 criteria, i.e., bright-green fluorescence of the same intensity as C. parvum oocysts and Giardia cysts in the positive control, correct size and shape of the fluorescein-stained objects, and oocyst or cyst shell clearly visible.     

Compatibility with Jet A-1 of a GCL Subjected to Freeze–Thaw Cycles

Needle-punched geosynthetic clay liner (GCL) specimens subjected to 0, 5, and 12 freeze–thaw cycles in the laboratory, and GCL specimens recovered from a composite barrier wall in the Canadian Arctic after 1 and 3 years were examined to assess the hydraulic conductivity/permeability with respect to both deionized deaired water and Jet A-l. The GCL specimens recovered from the field after 3 years had a hydraulic conductivity with respect to water that was approximately 30% less than that of the GCL specimens subjected to 12 initial freeze–thaw cycles in the laboratory, suggesting that the laboratory conditions are more severe than field conditions. The combined effects of both the freeze–thaw cycles and Jet A-l permeation increased the permeability. This increase is attributed to the creation of macropores in the GCL due to freezing and to an expansion of free-pore space due to contraction of the double layer caused by permeation of Jet A-l. Although there was an increase in permeability due to the combined effect of freeze–thaw and permeation by Jet A-l, the effect was relatively small and the results suggest that the GCL continued to exhibit good performance as a hydraulic barrier when subject to extreme climatic conditions and hydrocarbons both in the laboratory and in the field.     

Method detection limits of PCR and immunofluorescence assay for Cryptosporidium parvum in soil

We determined and compared the method detection limits (MDLalpha) of a PCR and an immunofluorescence assay (IFA) for detection of Cryptosporidium parvum oocysts in soils. Based on the MDLalpha and the quantitative nature and stability of the IFA, PCR analysis is not a useful screening step for soil studies of oocyst transport.     

Human sperm cryopreservation and reactive oxygen species (ROS) production

The aim of this work was to establish whether cryopreservation procedure can trigger the production of Reactive Oxygen Species (ROS) in selected sperm populations. Semen samples were obtained from 45 subjects attending our Department of Medical Pathophysiology. Motile sperm suspensions were obtained by swim-up in Tyrode's salt solution. After dilution with TEST yolk buffer freezing medium, they were cryopreserved in liquid nitrogen. In addition to motility assessment, in basal and freeze/thaw conditions ROS detection and the Hypoosmotic Viability Test were also carried out. In 19 subjects (42.2%) there was already evidence of ROS production prior to cryopreservation, which increased after thawing. In 9 subjects (20.0%) there was no ROS production prior to cryopreservation, however, after freezing/thawing we detected evidence of the presence of ROS. It seems, therefore, that cryoprocedure can indeed provoke or increase ROS production in some semen samples. In ROS producing subjects, the post-show recovery of sperm motility and vitality was significantly lower compared to ROS-free subjects. This was probably due to damage by oxidative stress leading to lipid peroxidation of the sperm membrane. Moreover, in some ejaculates, ROS overproduction or scavenger system failure can be regarded as a cryopathogenetic factor affecting "sperm quality" recovery.     

Effect of pasteurization on infectivity of Cryptosporidium parvum oocysts in water and milk

Cryptosporidium parvum is a major cause of diarrheal disease in humans and has been identified in 78 other species of mammals. The oocyst stage, excreted in feces of infected humans and animals, has been responsible for recent waterborne outbreaks of human cryptosporidiosis. High temperature and long exposure time have been shown to render oocysts (suspended in water) noninfectious, but for practical purposes, it is important to know if high-temperature--short-time conditions (71.7 degrees C for 15 s) used in commercial pasteurization are sufficient to destroy infectivity of oocysts. In this study, oocysts were suspended in either water or whole milk and heated to 71.7 degrees C for 15, 10, or 5 s in a laboratory-scale pasteurizer. Pasteurized and nonpasteurized (control) oocysts were then tested for the ability to infect infant mice. No mice (0 of 177) given 10(5) oocysts pasteurized for 15, 10, or 5 s in either water or milk were found to be infected with C. parvum on the basis of histologic examination of the terminal ileum. In contrast, all (80 of 80) control mice given nonpasteurized oocysts were heavily infected. These data indicate that high-temperature--short-time pasteurization is sufficient to destroy the infectivity of C. parvum oocysts in water and milk.     

Unfrozen Water in Freezing and Thawing Soils: Kinetics and Correlation

The experimental data indicate that water in freezing or thawing wet soils undergoes a gradual phase change. The mathematical forms of reported correlations of the unfrozen water content of soils varies from researcher to researcher. An improved theoretical treatment of the gradual freezing∕thawing process provides insight into the mechanism of the gradual phase change of water in wet soils and a proper means of correlating experimental measurements. Direct numerical solution of the resulting ordinary differential equation may provide accurate prediction of the unfrozen water content of wet soils, when accurate soil property data are available. Two approximate analytical solutions are derived using average thermal properties of the soil constituents. The simpler of these analytical solutions is applied to various data and shown to be sufficient for all practical purposes. The analysis presented in this article demonstrates that the unfrozen water content can be adequately correlated by means of an exponential decay function shifted by the amount of the water adsorbed over the soil grains, which cannot freeze.     

Role of immunoglobulin A monoclonal antibodies against P23 in controlling murine Cryptosporidium parvum infection

Cryptosporidium parvum is an important diarrhea-causing protozoan parasite of immunocompetent and immunocompromised hosts. Immunoglobulin A (IgA) has been implicated in resistance to mucosal infections with bacteria, viruses, and parasites, but little is known about the role of IgA in the control of C. parvum infection. We assessed the role of IgA during C. parvum infection in neonatal mice. IgA-secreting hybridomas were developed by using Peyer's patch lymphocytes from BALB/c mice which had been orally inoculated with viable C. parvum oocysts. Six monoclonal antibodies (MAbs) were selected for further study based on indirect immunofluorescence assay reactivity with sporozoite and merozoite pellicles and the antigen (Ag) deposited on glass substrate by gliding sporozoites. Each MAb was secreted in dimeric form and recognized a 23-kDa sporozoite Ag in Western immunoblots. The Ag recognized comigrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with P23, a previously defined neutralization-sensitive zoite pellicle Ag. MAbs were evaluated for prophylactic or therapeutic efficacy against C. parvum, singly and in combinations, in neonatal BALB/c mice. A combination of two MAbs given prophylactically prior to and 12 h following oocyst challenge reduced the number of intestinal parasites scored histologically by 21.1% compared to the numbers in mice given an isotype-matched control MAb (P < 0.01). Individual MAbs given therapeutically in nine doses over a 96-h period following oocyst challenge increased efficacy against C. parvum infection. Four MAbs given therapeutically each reduced intestinal infection 34.4 to 42.2% compared to isotype-matched control MAb-treated mice (P < 0.05). One MAb reduced infection 63.3 and 72. 7% in replicate experiments compared to isotype-matched control MAb-treated mice (P < 0.0001). We conclude that IgA MAbs directed to neutralization-sensitive P23 epitopes may have utility in passive immunization against murine C. parvum infection.     

Genotypic and phenotypic characterization of Cryptosporidium parvum isolates from people with AIDS

Genotypic analysis of Cryptosporidium parvum has demonstrated the presence of two subgroups within the species, whereas biochemical and antigenic characterization have shown more heterogeneity. The clinical relevance of these observations is unknown. C. parvum isolates from people with AIDS were studied with respect to parasite genotypes and virulence in cell monolayers and laboratory animals. Ten of 13 oocyst samples had a characteristic human-associated (H) genotype; 3 had a genotype typical of calf-excreted oocysts (C). Virulence in cell culture was mildly or markedly lower in the 5 isolates tested (4 H and 1 C) compared with the GCH1 reference isolate. H isolates did not infect newborn ICR mice, whereas 1 of the 2 C isolates tested did. These findings reinforce the concept of C. parvum genetic subgroupings that correlate to some extent with infectivity and suggest that additional heterogeneity is present within the subgroups.