Graft-versus-leukemia effect using mixed allogeneic bone marrow transplantation.

Previous studies have demonstrated that T cell-depleted (TCD) syngeneic marrow protects against graft-versus-host disease (GVHD) when given along with an allogeneic lymphocyte plus bone marrow (BM) inoculum to lethally irradiated mice. In spite of this anti-GVHD effect, TCD syngeneic marrow is ultimately eliminated by non-TCD allogeneic marrow, permitting complete allogeneic reconstitution. These observations suggested that allogeneic BM might also eliminate host-type leukemic cells in a model in which TCD syngeneic marrow is co-administered to provide protection from GVHD. In the present studies, we describe the establishment of a new model using the EL4 leukemia/lymphoma. Lethally irradiated B10 (H-2b) mice were given a lethal dose of EL4 cells (H-2b) along with syngeneic marrow or a mixture of TCD syngeneic plus non-TCD allogeneic (B10.D2, H-2d) marrow. Non-TCD allogeneic marrow, in contrast to TCD or unmanipulated syngeneic marrow, delayed or prevented mortality from the otherwise lethal EL4 inoculum, without producing clinically apparent GVHD. The anti-leukemic effect of allogeneic marrow alone was not attenuated by the co-administration of TCD syngeneic marrow, and such animals repopulated as completely allogeneic chimeras. Similar anti-leukemic effects of mixed marrow inocula in a haploidentical strain combination, and an anti-leukemic effect against established tumor were also demonstrated. This model may have the potential to increase the safety of clinical bone marrow transplantation across greater HLA disparities, while permitting utilization of the anti-leukemic and alloengraftment-promoting effects of T cells in allogeneic marrow inocula.

Recovery of Cryptosporidium oocysts and Giardia cysts from source water concentrates using immunomagnetic separation.

Immunomagnetic separation (IMS) procedures for the simultaneous isolation of Cryptosporidium oocysts and Giardia cysts have recently become available. We validated Dynal's GC-Combo IMS kit using source water at three turbidity levels (5000, 500 and 50 nephelometric turbidity units [ntu]) obtained from different geographical locations and spiked with approximately 9--11 (oo)cysts per ml. Mean recoveries of Cryptosporidium oocysts and Giardia cysts in deionized water were 62% and 69%, respectively. In turbid water matrices, mean recoveries of Cryptosporidium oocysts were between 55.9% and 83.1% while mean recoveries of cysts were between 61.1% and 89.6%. Marginally higher recoveries of the heat inactivated (oo)cysts were observed (119.4% Cryptosporidium oocysts and 90.9% Giardia cysts) in deionized water when compared with recoveries of viable (oo)cysts (69.7% Cryptosporidium oocysts and 79% Giardia cysts). Age of (oo)cysts on recoveries using the GC-Combo IMS kit demonstrated no effects up to 20 months old. Recovery of Giardia cysts was consistent for isolates aged up to 8 months (81.4%), however, a significant reduction in recoveries was noted at 20 months age. Recoveries of low levels (5 and 10 (oo)cysts) of Cryptosporidium oocysts and Giardia cysts in deionized water using IMS ranged from 51.3% to 78% and from 47.6% to 90.0%, respectively. Results of this study indicate that Dynal's GC-Combo IMS kit is an efficient technique to separate Cryptosporidium/Giardia from turbid matrices and yields consistent, reproducible recoveries. The use of fresh (recently voided and purified) (oo)cysts, aged (oo)cysts, viable and heat-inactivated (oo)cysts indicated that these parameters do not influence IMS performance.

Ultraviolet irradiation inactivates the waterborne infective stages of Myxobolus cerebralis: a treatment for hatchery water supplies.

The effects of ultraviolet (UV) irradiation on the viability of the waterborne triactinomyxon stages of Myxobolus cerebralis were evaluated by vital staining and the infectivity for juvenile rainbow trout Oncorhynchus mykiss. A dose of 1300 mWs cm-2 was required to inactivate 100% of the triactinomyxons held under a static collimated beam of UV as determined by vital staining. Juvenile rainbow trout were protected from infections with M. cerebralis when exposed to 14,000 or 1400 triactinomyxon spores per fish that had been treated with the collimating beam apparatus (1300 mWs cm-2). Among all fish receiving UV-treated triactinomyxons, none had clinical signs of whirling disease, or evidence of microscopic lesions or spores of M. cerebralis after 5 mo at water temperatures of 15 degrees C. In contrast, 100% of the fish receiving the higher dose of untreated triactinomyxons developed clinical signs of whirling disease and both microscopic signs of infection and spores were detected in all of the high and low dose trout receiving untreated triactinomyxon exposures. Two additional trials evaluated the Cryptosporidium Inactivation Device (CID) for its ability to treat flow-through 15 degrees C well water to which triactinomyxons were added over a 2 wk period. CID treatments of a cumulative dose exceeding 64,000 triactinomyxons per fish protected juvenile rainbow from infections with M. cerebralis. Rainbow trout controls receiving the same number of untreated triactinomyxons developed both microscopic lesions and cranial spore concentrations up to 10(4.6) per 1/2 head, although no signs of clinical whirling disease were observed. UV (126 mWs cm-2, collimated beam apparatus) was also effective in killing Flavobacterium psychrophilum, the agent causing salmonid bacterial coldwater disease, as demonstrated by the inability of bacterial cells to grow on artificial media following UV treatment.

Assessing UV reactor performance for treatment of finished water.

Recently, use of low levels of medium- and low-pressure ultraviolet light for successful inactivation of Cryptosporidium parvum oocysts has generated tremendous excitement in the water industry. Accurate delivery of the target dose, lamp performance, sensor stability and impact of water characteristics are some factors that could impact disinfection efficacy, in turn influencing decisions on application of this technology. To this end, American Water Systems, the largest investor owned water utility in the US, has responded to some of these challenges by ascertaining the long-term feasibility of applying UV for treatment of finished water. A 4 x 1 UV reactor with a 12 inch (0.3 m) diameter was installed after granular activated carbon filtration and was operated with a finished water flow rate of 600 gpm (2,700 L/min). Over a 12-month period, various chemical (THM, HAA, UV254, DOC, TOC, metals, nitrate, nitrites) and physical measurements (lamp voltage, current, sensor measurements) were monitored to define their impact (if any) on the operation of the reactor. MS2 bacteriophage challenge studies were conducted with various lamp configurations and lamp age. These inactivation data demonstrated high levels of correlation with controlled bench scale inactivation data. For C. parvum oocysts, bench scale studies were performed with a modified in vitro infectivity assay using HCT-8 cells, an enhanced infectivity protocol and with either immunofluorescence or quantitative PCR based detection. While both assays indicated increasing infections levels of HCT-8 cells with increasing oocyst inocula, UV treatment of oocysts produced markedly different infectivity responses. Based on the data generated in this study, one in vitro infectivity assay was selected to demonstrate > 3 logs inactivation with low UV doses (5 mJ/cm(20-10 mJ/cm2).

Using ultraviolet light for disinfection of finished water.

Ultraviolet light is now recognised to be very effective for inactivation of Cryptosporidium parvum oocysts; however, its application for disinfection of finished water necessitates validation of UV reactors prior to their installation. Although reactor performance will likely be assessed using non-pathogenic microorganisms as biodosimetry surrogates, it would be prudent for the water industry to simultaneously measure Cryptosporidium oocysts inactivation in controlled bench-scale studies using the water matrix intended for disinfection. The likelihood of that occurring is dependent upon the availability of infectivity measurement procedures that are more user-friendly than the mouse infectivity assays currently used. This study describes a modified cell culture procedure that would enable reliable measurement of changes in oocysts' infectivity following their UV treatment. Also, a number of different biodosimetry surrogates were examined and one selected for comparing the UV doses delivered between bench-scale and full-scale biodosimetry studies. Impacts of UV disinfection on production of disinfection byproducts, effects of lamp ageing on effectiveness of disinfection and the costs associated with employing this technology were also examined.

Development of procedures for rapid detection of E. coli O157:H7 from source and finished water samples.

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7, the causative agent for haemolytic uraemic syndrome, has become a significant health concern, due to an increasing number of cases. The focus of this project was rapid (<8 h) detection of E. coli O157:H7 from source and finished waters samples, either directly or indirectly, by dovetailing the procedures with existing total coliform procedures. Evaluation of four immunological lateral diffusion assays determined a detection range between 1 x 10(4) and 1 x 10(6) CFU, with the Reveal E. coli O157:H7 Test System (Neogen) being the most sensitive for detecting E. coli O157:H7. Evaluation of the BAX System for molecular detection determined that as few as 10 CFU could be reproducibly detected. Coupling either of these detection procedures with organism propagation using Tryptic Soy Broth (TSB) enabled sufficient quantities of E. coli O157:H7, such that the Reveal and BAX detection methods could be used with the 8 h time frame. Examination of matrix effects on the overall procedure indicated little impact on method performance.

Effect of three concentration techniques on viability of Cryptosporidium parvum oocysts recovered from bovine feces.

Bovine fecal samples (1 g) negative for Cryptosporidium sp. oocysts were seeded with 7 x 10(4) Cryptosporidium parvum oocysts and purified by either water-ether concentration, sucrose density flotation, or zinc sulfate flotation to evaluate oocyst recovery. The effect of these purification techniques on the viability of recovered oocysts was also evaluated. Significantly higher numbers of seeded oocysts were recovered by water-ether concentration (recovery rate, 46 to 75%) than by sucrose density (24 to 65%) or zinc sulfate (22 to 41%) flotation methods. In addition, water-ether concentration did not exert a significant effect on the viability of the population of oocysts recovered, whereas sucrose density flotation and zinc sulfate flotation selectively concentrated viable oocysts. The water-ether concentration procedure is recommended for use in epidemiological studies in which both oocyst enumeration and viability assessment are required.

Cryptosporidium parvum: oocyst excretion and viability patterns in experimentally infected lambs.

Cryptosporidium parvum infections of domestic animals can have a considerable economic impact and as oocysts are voided in the faeces of infected hosts, environmental contamination with agricultural waste has also become a matter of concern. Since only viable oocysts are potentially infectious, the numbers of oocysts excreted during infection can have important implications for both veterinary and public health. During the course of infection in experimentally infected lambs, oocyst viability was assessed by a fluorogenic vital dyes assay and by a maximized in vitro excystation assay. The excreted oocyst populations contained a higher proportion of viable oocysts 5-11 days post infection (d.p.i.) than later in the infection. Oocyst viability declined consistently 11-15 d.p.i. and coincided with periods when peaks in serum and intestinal anti-Cryptosporidium antibodies have been reported to occur. Infected lambs excreted a mean of 4.8 (standard error [S.E.] +/- 0.4) x 10(9) oocysts per g of faeces, of which half were non-viable and therefore of no significance for disease transmission. This study demonstrates that the numbers of viable oocysts excreted by infected lambs is smaller than previously suspected.

Recovery and viability of Cryptosporidium parvum oocysts and Giardia intestinalis cysts using the membrane dissolution procedure.

Previously, the cellulose acetate membrane filter dissolution method was reported to yield Cryptosporidium parvum oocyst recoveries of 70.5%, with recovered oocysts retaining their infectivity. In contrast, high spike doses (approximately 1 x 10(5) Cryptosporidium parvum oocysts and Giardia intestinalis cysts) yielded recoveries ranging from 0.4% to 83.9%, and 3.2% to 90.3%, respectively, in this study. Recoveries with low spike doses (approximately 100 (oo)cysts) continued to demonstrate high variability also. Efforts to optimize the method included increased centrifugation speeds, suspension of the final concentrate in deionized water for organism detection on well slides, and analysis of the entire concentrate. A comparison of two monoclonal antibodies was also conducted to identify potential differences between antibodies in detection of organisms. Archived source and finished water samples were spiked, yielding variable recoveries of C. parvum oocysts (11.8% to 71.4%) and G. intestinalis cysts (7.4% to 42.3%). Effects of organic solvents used in the membrane dissolution procedure on the viability of recovered (oo)cysts was determined using a fluorogenic vital dyes assay in conjunction with (oo)cyst morphology, which indicated > 99% inactivation. These data indicate that the membrane dissolution procedure yields poor and highly variable (oo)cyst recoveries, and also renders the majority of recovered organisms non-viable.

Immunomagnetic separation of Cryptosporidium parvum from source water samples of various turbidities.

Immunomagnetic separation (IMS) procedures which specifically capture Cryptosporidium oocysts and have the potential to isolate oocysts from debris have become commercially available. We compared two IMS kits (kit DB [Dynabeads anti-Cryptosporidium; product no. 730.01; Dynal A.S., Oslo, Norway] and kit IC1 [Crypto Scan IMS; product no. R10; Clearwater Diagnostics Company, LLC, Portland, Maine]) and a modification of kit IC1 (kit IC2 [Crypto Scan IMS; product no. R10; Clearwater Diagnostics Company, LLC]) at three turbidity levels (50, 500, and 5,000 nephelometric turbidity units [ntu]) by using water matrices obtained from different geographical locations. In deionized water, kit DB yielded recoveries between 68 and 83%, whereas the recoveries obtained with kits IC1 and IC2 were more variable and ranged from 0.2 to 74.5%. In water matrices with turbidity levels up to 500 ntu, the oocyst recoveries were more variable with kit DB; however, the recoveries were similar to those obtained in deionized water. In contrast, there were notable reductions in oocyst recoveries in the turbid matrices with kits IC1 and IC2, and the highest recovery (8.3%) was obtained with a 50-ntu sample. An examination of the effects of age on oocyst recovery with kit DB revealed that oocysts up to 16 weeks old yielded recoveries similar to the recoveries observed with fresh oocysts. These data indicate that all IMS kits do not perform equally well, and it is important to conduct in-house quality assurance work before a commercially available IMS kit is selected to replace flotation procedures for recovery of Cryptosporidium oocysts.

Inter-laboratory comparison of the CD-1 neonatal mouse logistic dose-response model for Cryptosporidium parvum oocysts.

cryptosporidium parvum oocyst viability can be determined by vital dyes, in vitro excystation, and cell culture; however, neonatal mouse infectivity assays are the reference method. Unfortunately, there have been few efforts to standardize methods for infectivity assays thus casting a veil of uncertainty over the significance and comparability of results. In order to address this issue, two laboratories proficient in measuring oocyst infectivity conducted independent dose titration studies with neonatal CD-1 mice using standardized protocols and a well-characterized isolate of Cryptosporidium parvum. The resulting independent logistic dose-response models derived by regression analysis were compared with each other and with a published model. The comparisons showed these dose-response functions to be reproducible under standardized conditions. It is important to standardize mouse strain, age of mice at inoculation and necropsy, oocyst isolate, and age of oocysts. However, other factors, including methods used to detect infectivity and to count oocyst doses, appear less critical. Adopting a standardized assay for oocyst infectivity will provide both a basis for comparing data from various oocyst disinfection studies and a suitable platform for evaluating new or existing in vitro viability surrogates such as excystation, vital dyes or cell culture.

Comparison of Cryptosporidium parvum viability and infectivity assays following ozone treatment of oocysts.

Several in vitro surrogates have been developed as convenient, user-friendly alternatives to mouse infectivity assays for determining the viability of Cryptosporidium parvum oocysts. Such viability assays have been used increasingly to determine oocyst inactivation following treatment with chemical, physical, or environmental stresses. Defining the relationship between in vitro viability assays and oocyst infectivity in susceptible hosts is critical for determining the significance of existing oocyst inactivation data for these in vitro assays and their suitability in future studies. In this study, four viability assays were compared with mouse infectivity assays, using neonatal CD-1 mice. Studies were conducted in the United States and United Kingdom using fresh (<1 month) or environmentally aged (3 months at 4 degrees C) oocysts, which were partially inactivated by ozonation before viability and/or infectivity analyses. High levels of variability were noted within and between the viability and infectivity assays in the U.S. and United Kingdom studies despite rigorous control over oocyst conditions and disinfection experiments. Based on the viability analysis of oocyst subsamples from each ozonation experiment, SYTO-59 assays demonstrated minimal change in oocyst viability, whereas 4',6'-diamidino-2-phenylindole-propidium iodide assays, in vitro excystation, and SYTO-9 assays showed a marginal reduction in oocyst viability. In contrast, the neonatal mouse infectivity assay demonstrated significantly higher levels of oocyst inactivation in the U.S. and United Kingdom experiments. These comparisons illustrate that four in vitro viability assays cannot be used to reliably predict oocyst inactivation following treatment with low levels of ozone. Neonatal mouse infectivity assays should continue to be regarded as a "gold standard" until suitable alternative viability surrogates are identified for disinfection studies.