Immunomagnetic Separation Technique Research Articles

Monitoring of Waterborne Parasites in Two Drinking Water Treatment Plants: A Study in Sarawak, Malaysia.

The occurrence of waterborne parasites coupled with water parameters at various processing sites of two drinking water treatment plants (A and B) and seven distribution system (DS) sites in Sarawak, Malaysia were studied. Ten liters of water underwent immunomagnetic separation (IMS) technique to detect the presence of Giardia and Cryptosporidium (oo)cysts. The remaining supernatant was used to detect other parasites whilst 50 mL of water sample was each used in the detection of free-living amoebae and fecal coliforms. Sampled water was positive for Giardia (32.9%; 28/85), Cryptosporidium (18.8%; 16/85) followed by Spirometra ova-like (25.9%; 22/85), Blastocystis-like (25.9%; 22/85), nematode larvae-like (8.2%; 7/85) and Taenia ova-like (1.2%; 1/85). Meanwhile, 90.2% (55/61) samples were positive for Acanthamoeba and Naegleria via cultivation and of these, 11 isolates were confirmed as Acanthamoeba genotype T3 (5/7) and T4 (2/7) followed by Naegleria sp. (4/11), Naegleria italica (2/11), Naegleria australiensis (1/11), Naegleria angularis (1/11) and Vahlkampfia sp. (3/11). Cryptosporidium, Acanthamoeba and Naegleria were also detected in one of the seven tested DS sites. Only Giardia and Cryptosporidium showed significant correlations with fluoride and fecal coliforms. These results describe the occurrence of waterborne parasites that will assist key stakeholders in mitigating contamination at the specific sites.

Efficiency and Impact of Positive and Negative Magnetic Separation on Monocyte Derived Dendritic Cell Generation.

The immunomagnetic separation technique is the basis of monocyte isolation and further generation of monocyte-derived dendritic cells. To compare the efficiency of monocyte positive and negative separation, concentration of beads, and their impact on generated dendritic cells. Monocytes were obtained using monoclonal antibody-coated magnetic beads followed the Ficoll-Paque gradient separation of mononuclear cell fraction from the peripheral blood of 6 healthy volunteers. CD14 expression was analyzed by flow cytometry. Both types of magnetic separation including recommended and reduced concentrations of beads did not affect the yield and the purity of monocytes and their surface CD14 expression. However, DCs originated from the "positively" separated monocytes had noticeable higher expression of CD80.

Rapid detection of bacteria based on homogenous immunoassay using chitosan modified quantum dots

In this study, a fast and sensitive sandwich assay has been developed with the combination of immunomagnetic separation (IMS) and fluorescence techniques to enumerate Escherichia coli (E.coli). Iron oxide core gold shell (Fe3O4@Au) magnetic nanoparticles were prepared and modified with biotinylated antibodies specific to E.coli. Fluorescence labels have been constructed by preparing chitosan coated CdTe quantum dots (CdTe QDs) with a diameter of 3±1nm. The amounts of magnetic nanoparticles and CdTe QDs are optimized to get the best sensitivity. The calibration graph fluorescence intensity versus E.coli concentration was linear in the range of 102–108cfumL−1 with a coefficient of determination (R2) of 0.9905. The limit of detection (LOD) and limit of quantification (LOQ) values are calculated as 30 and 100cfumL−1, respectively. Selectivity of the method is examined by applying the same procedure to bacteria, namely Enterobacter aerogenes (E. aerogenes), Enterobacter dissolvens (E. dissolvens), Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa), and no interference has been observed. The applicability of the developed method has also been examined in spiked urine samples. As a result, it was found that, this novel method is sensitive to target E.coli and a rapid method with a total analysis time less than 120min.

Abstract 1590: CD133-expressing circulating tumor cells might predict poor prognosis in patients with head and neck cancer

Abstract PURPOSE: The prognostic value of circulating tumor cells (CTCs) number in patients with head and neck carcinoma (HNSCC) has been reported to be positive. The hypothesis of the additional markers on CTCs might predict clinical outcomes have been proposed. This study aimed to elucidate the prognostic/predictive value of CD133-expressing CTCs in HNSCC patients. PATIENTS AND METHODS: In a prospective single-center study, CTCs were enumerated in the peripheral blood of 24 subjects, including 14 patients with HNSCC and 10 healthy donors, at baseline and 2 weeks after starting first dose of chemotherapy. CTCs were measured using an immunomagnetic separation technique by combined negative and positive selection strategies. Immunofluorescence staining methods were applied for surface marker identification. Tumor response at 12 weeks after chemotherapy of the whole cohort was performed by computed tomograpgy or magnetic resonance imaging scans following RECIST criteria 1.1. RESULTS: In the first step of efficacy evaluation phase, an average recovery rate of 85.5% and a R2 value of 0.9932 were found when compared between spiked and recovered blood samples. A CTCs detection rate of 100.0% (14/14) was found by this protocol and the number could successfully differ HNSCC patients from healthy donors (P = 0.001). The mean and median (range) of baseline CTC number were 105.0 and 87.2 (34.5-358.1) cells/mL of blood. The higher baseline CTCs number was associated with higher tumor stage (AJCC 7th edition, P = 0.003). The higher absolute number (>5cells/mL of blood) of baseline CD133-expressing CTCs could predict poor survivals (Kaplan-Meier survival curves, P = 0.013). In all assessable patients, a high correlation rate of 92.9% was found between changes of CTCs (decline or elevation) and CT responses (progressive disease [PD] and partial response [PR]/Stable disease [SD]) with a P value of 0.005 (Fisher exact test). CONCLUSION: The number of CD133-expressing CTCs might be a useful prognostic for survivals and predictors for imaging response evaluation at 12 weeks in patients with HNSCC. Further investigation in large-scale prospective trials is warranted on the basis of the findings in this study. Citation Format: Jason Chia-Hsun Hsieh, Harber Hung-Bo Shiao, Bill Yuan-Jhih Chao, Ming-Jung Lin, John Wen-Cheng Chang, Hung-Ming Wang, Tyler Min-Hsien Wu. CD133-expressing circulating tumor cells might predict poor prognosis in patients with head and neck cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1590. doi:10.1158/1538-7445.AM2015-1590

Efficacy of chemical disinfectant compounds against human norovirus

The purpose of this study was to evaluate the efficacy of various disinfectant compounds against human norovirus (NoV). We investigated the disinfection effects of ethanol, sodium hypochlorite, hydrogen peroxide, quaternary ammonium compounds, and iodine using an anti-NoV GII.4 monoclonal antibody-conjugated immuno-magnetic separation (IMS) technique combined with quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Ten-minute treatments of samples containing NoV GII.4 with 10–70% ethanol resulted in mean log10 reductions in genomic copies/μL of less than 1. In contrast, 10-min treatments with sodium hypochlorite at 200, 500, and 1000 ppm resulted in mean log10 reductions of 1.55, 1.85, and 2.45, respectively; however, 50 and 100 ppm sodium hypochlorite had no disinfection effect, as shown by the log10 reductions of less than 1. Treatment with hydrogen peroxide (200–1000 ppm), quaternary ammonium compounds (100–2000 ppm), and iodine (25–500 ppm), also exhibited no disinfection effect. The results of this study show that NoV GII.4 is remarkably resistant to most disinfectants and suggests that new disinfectant compounds are needed to inactivate foodborne bacteria and viruses, especially NoV GII.4.

Prevalence and characterization of Salmonella isolated from chicken meat in Turkey.

This study was conducted in a Turkish province to investigate the presence of Salmonella spp. in 150 chicken meat samples using 2 phenotyping techniques: classic culture technique (CCT) and immunomagnetic separation (IMS). For the confirmation of the isolates at molecular levels, invA gene was detected in these isolates. The presence of invA, class 1 (Cls1) integrons, and integrase (Int1) genes was demonstrated by PCR assay; and the resistance of the isolated Salmonella spp. strains to antibiotics was determined by disk diffusion test. All the cultural and PCR results were evaluated together; Salmonella spp. were detected in a total of 64 (42.66%) chicken meat samples. Contamination rate was higher in carcasses (53.33%, n = 75) than in meat pieces (32%, n = 75). When results of standard culture were compared with IMS technique, IMS (n = 54) showed a clear superiority over the CCT (n = 38). A very high resistance rate (≥ 89.28%) to vancomycin, tetracycline, streptomycin, or nalidixic acid was found. Trimethoprim-sulfamethoxazole resistance was present in 32.14%. Relatively lower incidence of resistance (≤ 8.33%) to gentamicin, chloramphenicol, ampicillin, and ceftriaxone was observed. Concurrent resistance to at least 4 antibiotics was detected in 92.85% of the isolates. Cls1 integrons and Int1 were positive in 80.95% and 95.23% of the isolates, respectively. However, Int1 alone was detected in 15.47% (n = 13). In conclusion, the high prevalence of Salmonella spp. in chicken meat may pose a potential public health risk, and the presence of antibiotic-resistant Salmonella spp. isolate together with Cls1 integron and/or integrase might play an important role in horizontal antibiotic gene transfer.

Micrometastatic Circulating Tumor cells; A Challenge for an Early Detection and Better Survival Rates

Micrometastasis is a health burden affecting a large population worldwide, where early stage circulating tumor cells are clinically below the detection limit of the currently used techniques in diagnosis. These cells are considered one of the sources related to the disease spread, usually associated with poor prognosis and resistant to conventional therapies. With the recent advances in technology, various molecular and biological techniques including cytological examination, RT-PCR immunocytochemistry, immuno-magnetic separation and cell-enrichment techniques have emerged to improve the early detection of circulating tumor cells in different carcinomas. However, the sensitivity and specificity of these techniques along with their prognostic influence are still contested. This review aims to discuss the role of key player molecules including cell adhesion molecules, integrins and proteases in promoting micrometastasis and the current techniques used for an early detection of these malignant cells. Understanding mechanisms underlying this invasive process, will pave the way for designing new tools to unravel difficulties associated with early detection of CTCs and will improve therapies.

Evaluation of Two Immunomagnetic Separation Techniques for the Detection and Recovery of Escherichia coli O157:H7 from Finished Composts

Two rapid immunomagnetic separation (IMS) protocols were evaluated to recover inoculated Escherichia coli O157:H7 (1–2 log colony-forming unit (CFU)/g) from 30 different commercial, finished compost samples. Both protocols detected E. coli O157:H7 in compost samples; PCR techniques required the removal of inhibitors to reduce possibility of a false negative result.

Abstract 4817: Circulating tumor cells enriched by the negative selection system PowerMag is a useful predictor for treatment response of metastatic colorectal cancer patients

Abstract Enumeration of circulating tumor cells (CTCs) provides prognostic and predictive value to guide treatment options. This study aims to (1) evaluate the merits of the PowerMag system, an immunomagnetic separation technique that we recently developed for negative selection of CTCs, in the prediction of treatment response; (2) perform analysis to investigate whether there is a correlation between the imaging response and the changes of CTCs counts before and during chemotherapy for the patients with metastatic colorectal cancer (mCRC). A total of 47 mCRC patients were enrolled in this prospective single-center study. CTCs were isolated from the peripheral blood of these patients by PowerMag at baseline and 2 weeks after starting first dose of chemotherapy. CTCs enumeration was then performed by immunofluorescence staining using the anti-epithelial cell adhesion molecule (EpCAM) antibody. Tumor response at 12 weeks after chemotherapy of the whole cohort was monitored by computed tomography (CT) scans following RECIST criteria 1.0. Our data revealed that PowerMag is a cost-effective negative selection-based CTCs isolation platform with a CTCs detection rate of 89.4% (42/47). The CTCs counts could successfully differ mCRC patients from healthy donors (P = 0.0015). The higher baseline CTCs number was associated with initially metastatic status rather than recurrent settings (P = 0.042). In all assessable patients, a high correlation rate of 86.7% was found between changes of CTCs (decline or elevation) and CT responses (progressive disease, and partial response/stable disease) with a P value of 0.005 (Fisher exact test). These data indicate that the change of CTCs counts before and during treatment is in accord with the image response evaluation at 12 weeks after initiation of treatment. In conclusion, PowerMag is a cost-effective method for negative selection of CTCs from cancer patients. Moreover, comparison of CTCs counts before and during treatment is a useful predictor for disease progression of mCRC patients that warrants further investigation in large-scale validation trial. Citation Format: Jason Chia-Hsun Hsieh, Hung-Chih Lin, Hung-Chih Hsu, John Wen-Cheng Chang, Ching-Ping Tseng. Circulating tumor cells enriched by the negative selection system PowerMag is a useful predictor for treatment response of metastatic colorectal cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4817. doi:10.1158/1538-7445.AM2014-4817

Most-Probable-Number Loop-Mediated Isothermal Amplification-Based Procedure Enhanced with K Antigen-Specific Immunomagnetic Separation for Quantifying tdh+Vibrio parahaemolyticus in Molluscan Shellfish

Although thermostable direct hemolysin-producing (tdh(+)) Vibrio parahaemolyticus is the leading cause of seafood-borne gastroenteritis, the enumeration of tdh(+) V. parahaemolyticus remains challenging due to its low densities in the environment. In this study, we developed a most-probable-number (MPN)-based procedure designated A-IS(1)-LAMP, in which an immunomagnetic separation (IMS) technique targeting as many as 69 established K antigens and a loop-mediated isothermal amplification (LAMP) assay targeting the thermostable direct hemolysin (tdh) gene were applied in an MPN format. Our IMS employed PickPen, an eight-channel intrasolution magnetic particle separation device, which enabled a straightforward microtiter plate-based IMS procedure (designated as PickPen-IMS). The ability of the procedure to quantify a wide range of tdh(+) V. parahaemolyticus levels was evaluated by testing shellfish samples in Japan and southern Thailand, where shellfish products are known to contain relatively low and high levels of total V. parahaemolyticus, respectively. The Japanese and Thai shellfish samples showed, respectively, relatively low (< 3 to 11 MPN/10 g) and considerably higher (930 to 110,000 MPN/10 g) levels of tdh(+) V. parahaemolyticus, raising concern about the safety of Thai shellfish products sold to domestic consumers at local morning markets. LAMP showed similar or higher performance than conventional PCR in the detection and quantification of a wide range of tdh(+) V. parahaemolyticus levels in shellfish products. Whereas a positive effect of PickPen-IMS was not observed in MPN determination, PickPen-IMS was able to concentrate tdh(+) V. parahaemolyticus 32-fold on average from the Japanese shellfish samples at an individual tube level, suggesting a possibility of using PickPen-IMS as an optional tool for specific shellfish samples. The A-IS(1)-LAMP procedure can be used by any health authority in the world to measure the tdh(+) V. parahaemolyticus levels in shellfish products.

Newly Established Monoclonal Antibody Diagnostic Assays for Schistosoma mansoni Direct Detection in Areas of Low Endemicity

BackgroundCurrent available methods for diagnosis of schistosomiasis mansoni lack sufficient sensitivity, which results in underreporting of infectious in areas of low endemicity.Methodology/Principal FindingsWe developed three novel diagnostic methodologies for the direct detection of schistosome infection in serum samples. These three new methods were evaluated with positive patients from a low endemicity area in southeast Brazil. The basis of the assay was the production of monoclonal antibodies against the protein backbone of heavily glycosylated Circulating Cathodic Antigen (CCA). The antibodies were also selected for having no specificity to repeating poly-Lewis x units. Assays based on the detection CCA-protein should not encounter a limitation in sensitivity due to a biological background of this particular epitope. Three diagnostic methodologies were developed and validated, (i) Immunomagnetic Separation based on improved incubation steps of non-diluted serum, (ii) Direct Enzyme-linked Immunosorbent Assay and (iii) Fluorescent Microscopy Analysis as a qualitative assay. The two quantitative assays presented high sensitivity (94% and 92%, respectively) and specificity (100%), equivalent to the analysis of 3 stool samples and 16 slides by Kato-Katz, showing promising results on the determination of cure.Conclusions/SignificanceThe Immunomagnetic Separation technique showed excellent correlation with parasite burden by Cohen coefficient. The qualitative method detected 47 positive individuals out of 50 with the analysis of 3 slides. This easy-to-do method was capable of discriminating positive from negative cases, even for patients with low parasite burden.

The same clade of Clostridium botulinum strains is causing avian botulism in southern and northern Europe

Avian botulism is a paralytic disease caused by Clostridium botulinum-produced botulinum neurotoxins (BoNTs), most commonly of type C/D. It is a serious disease of waterbirds and poultry flocks in many countries in Europe. The objective of this study was to compare the genetic relatedness of avian C. botulinum strains isolated in Spain with strains isolated in Sweden using pulsed-field gel electrophoresis (PFGE). Fifteen strains were isolated from Spanish waterbirds using an immunomagnetic separation technique. Isolates were characterized by PCR, and all were identified as the genospecies Clostridium novyi sensu lato and eight harboured the gene coding for the BoNT type C/D. PFGE analysis of the strains revealed four highly similar pulsotypes, out of which two contained strains from both countries. It also showed that outbreaks in wild and domestic birds can be caused by the same strains. These results support a clonal spreading of the mosaic C. botulinum type C/D through Europe and give relevant information for future epidemiological studies.

Abstract B40: Cytokines secreted by macrophages isolated from tumor microenvironment of inflammatory breast cancer patients possess chemotactic properties

Abstract Introduction: Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer and is characterized by rapid progression and dismal outcome. Although there is a growing literature describing the role of macrophages in non-IBC, the role of macrophages in IBC is unclear. The aims of the present study were to: 1) assess macrophages infiltration within the tumor tissues of non-IBC and IBC patients; 2) isolate and profile cytokines of macrophages drained from breast tumor axillary tributaries of non-IBC and IBC patients and 3) test whether major cytokines secreted by tumor draining macrophages induce motility of the IBC cell line SUM149. Methods: Tumor-infiltrating macrophages were measured by immunohistochemical (IHC) techniques using CD14 and CD68 monoclonal antibodies. Blood drained from axillary vein tributaries was collected from non-IBC and IBC patients during breast cancer surgery and total mononuclear cells isolated by density gradient centrifugation using Histopaque. The percentage of monocytes/macrophages (CD14+) in the total isolated leukocytes was calculated by flow cytometry using fluorochrome-labeled monoclonal antibodies (APC-CD14 and PerCP-CD3). CD14+ population was separated from total mononuclear cells using an immuno-magnetic bead separation technique. CD14+ cells were seeded overnight in appropriate culture media at 37°C in a humidified CO2 incubator and conditioned media were collected and cytokines profiled. To test whether cytokines secreted by CD14+ enhances motility and invasion of SUM149 cells, we used wound healing and invasion assays. Results: Our results revealed that there was an increase in the number of infiltrated macrophages (CD14+ cells) in IBC tumor tissues and axillary tributaries drained blood as compared to non-IBC. Cytokine profiling of IBC-CD14+ cells revealed a significant increase in tumor necrosis factor-alpha (TNF-α); monocyte chemoattractant protein-1 (MCP-1) also known as CC-chemokine ligand 2 (CCL-2); interleukin-10 (IL-10) and interleukin-8 (IL-8) as compared to non-IBC CD14+ cells. TNF-α, IL-8 and IL-10 cytokines significantly increased motility and invasion of SUM149 cells. Conclusion: IBC cancer tissues are characterized by high infiltration of monocytes/macrophages that secrete cytokines that may contribute to dissemination of IBC cells. Citation Format: Mona Mostafa Mohamed, Eslam A. El-Ghonaimy, Mohamed A. Nouh, Robert J. Schneider, Bonnie F. Sloane, Mohamed El-Shinawi. Cytokines secreted by macrophages isolated from tumor microenvironment of inflammatory breast cancer patients possess chemotactic properties. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B40.

Detection of Escherichia coli O157:H7 using immunomagnetic separation and mPCR in Turkish foods of animal origin

The present study was conducted to investigate the presence of Escherichia coli O157:H7 in food samples of animal origin and to detect its virulence genes by immunomagnetic separation technique and multiplex PCR (mPCR). A total of 500 samples (consisting of diced meat, minced meat, burger, raw cow's milk and raw cow's milk cheese) were analysed. Escherichia coli O157:H7 was detected in 5 (1%) of 500 analysed samples including two diced meat, one minced meat and two raw-milk cheese. None of the burger samples tested contained E.coli O157:H7. Three isolates obtained from minced and diced meat were found to carry stx1 , stx2 , hlyA and eaeA genes whereas two isolates from raw-milk cheese were found to harbour the stx1 , eaeA and hlyA genes. The results of this study suggest that raw meat and raw-milk cheese tested could pose public health problems in consumers with regard to their virulence factors. Escherichia coli (E.coli) O157:H7 is an important human pathogen. Escherichia coli 0157:H7 infections have been associated with consumption of uncooked meat and meat products, as well as unpasteurized dairy products. This study demonstrated that without specific tests for E.coli virulence factors raw meat and raw-milk cheese could pose public health problems to Turkish consumers.

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The main objectives of the present study were to assess the incidence of E .coli O157:H7 serotype in both raw milk and locally produced soft cheese samples and to evaluate the efficacy of new immunomagnetic separation (IMS) technique for the first time in Iraq for isolation of the same serotype from such products compared to the conventional cultural method .A total of 50 raw milk and soft cheese samples (25 samples of each) were collected randomly at weekly intervals from different retail markets in Baghdad province and its surroundings during the period of six months (from October 2011 March 2012). Each sample was divided into two equal parts where the first part was analyzed for the conventional cultural method and the second part was analyzed for the immunomagnetic separation technique. Five isolates (20%) and seven (28%) were identified as E.coli O157:H7 from the same raw milk samples by both the conventional cultural method and (IMS) technique respectively. Two (8%) and four isolates (16%) were identified as E.coli O157:H7 the same soft cheese samples using the same above methods. The detection limits by the conventional cultural method were 4x103 cfu/ml and 7x102 cfu/gm of raw milk and soft cheese samples respectively, while the detection limits by the (IMS) technique were 1x102 cfu/ml and 12x10 cfu/gm of raw milk and soft cheese sample respectively .Results obtained in this study revealed that the IMS technique has been recognized to be significantly (P&lt;0.05) more efficient in its sensitivity for the detection of low numbers of E.coli O157:H7 than the direct plating conventional method for both raw milk and soft cheese samples.

Development and evaluation of an immunomagnetic separation–ELISA for the detection of Alicyclobacillus spp. in apple juice

The immunomagnetic separation (IMS) technique was used in combination with an enzyme-linked immunosorbent assay (ELISA) procedure to shorten the total analysis time and improve the sensitivity for the detection of Alicyclobacillus spp. in apple juice samples. The specificity of IMS–ELISA for twenty strains of Alicyclobacillus spp. and eighteen strains of non-Alicyclobacillus spp. was determined and there was little cross-reaction with non-Alicyclobacillus strains. Artificially contaminated apple juice with different concentrations of Alicyclobacillus acidoterrestris was detected by IMS–ELISA, and the detection limit of the assay in apple juice was 103CFU/mL. Furthermore, the sample inoculated with 1CFU/mL of A. acidoterrestris could be detected as positive after incubation for 24h. The IMS–ELISA described, allows for the identification of suspect positive samples within 3h of testing versus 3–5days required by standard culture methods while significantly reducing the materials and labor required for the detection of Alicyclobacillus spp. in apple juice samples. As compared with the standard culture method performed concurrently on the same set of samples, the sensitivity, specificity and accuracy of IMS–ELISA for 102 naturally contaminated apple juice samples were 91.3%, 96.02% and 95.09%, respectively. These results demonstrated that the newly proposed IMS–ELISA procedure can be a potentially useful analytical method for the detection of Alicyclobacillus spp. in apple juice.

Improvement of capture efficacy of immunomagnetic beads forCampylobacter jejuniusing reagents that alter its motility

Previous studies using the immunomagnetic beads separation (IMS) technique have shown high detection limits of live campylobacters but low detection limits of formalin-killed campylobacters. The present study investigated if the addition of various concentrations of reagents that alter the motility of live Campylobacter jejuni could enhance the recovery of the organisms by IMS. The addition of 5% glycerol, 0.001% formalin, 10% polyethylene glycol, or 0.001% agarose in a buffer slowed down the movement of C. jejuni and increased the recovery of live C. jejuni, using beads coated with specific monoclonal antibodies (mAbs). The highest recovery yielded was 5.2- ± 3.3-fold with 5% glycerol at 10(5) colony-forming units (CFU)·mL(-1). The addition of 5% glycerol also improved isolation at lower concentrations of C. jejuni (10(2) to 10(4) CFU·mL(-1)) in buffer. The recovery by IMS of C. jejuni killed by 1% formalin was increased up to as high as 17-fold compared with the recovery of live organisms, as detected using a real-time polymerase chain reaction assay. The reagents investigated did not enhance the immunological reactivity of the mAbs to this organism. These results indicate that the addition of several reagents enhanced the capture of C. jejuni by IMS, which could be partially due to the slowing down of the movement or the altering of the motility of C. jejuni and to the increasing of the contact time between C. jejuni and immunomagnetic beads.

Gene expression analysis of protein synthesis pathways in bovine mammary epithelial cells purified from milk during lactation and short‐term restricted feeding

The objective of the study was to investigate selected key regulatory pathways of milk protein biosynthesis in primary bovine mammary epithelial cells (MECs) of dairy cows during the first 155days of lactation. In addition, cows were exposed to feed restriction for a short period (FR) during different stages of lactation (week 4 and 21pp) to study adjustment processes of molecular protein biosynthesis to metabolic challenge. Morning milk samples from twenty-four Holstein-Friesian cows were collected throughout the experimental period (n=10 per animal). MEC from raw milk were purified using an immunomagnetic separation technique and used for real-time quantitative PCR analyses. As was seen in transcript abundances of all major milk proteins, mRNA levels of E74-like factor 5 (ELF5), an enhancer of signal transducer and activator of transcription (STAT) action, concomitantly decreased towards mid-lactation. Expression of ELF5 as well as of all milk protein genes showed a similar increase during FR in early lactation. Occasional changes in expression could be seen in other Janus kinase (JAK)/STAT factors and in mammalian target of rapamycin (mTOR) pathway elements. Amino acid transfer and glucose transporter and the β-casein expression were also partially affected. In conclusion, our findings suggest a pivotal role of the transcription factor ELF5 in milk protein mRNA expression with complementary JAK/STAT and mTOR signalling for the regulation of protein biosynthesis in the bovine mammary gland.

Efficient monitoring of in vivo pig-a gene mutation and chromosomal damage: summary of 7 published studies and results from 11 new reference compounds.

The ability to effectively monitor gene mutation and micronucleated reticulocyte (MN-RET) frequency in short-term and repeated dosing schedules was investigated using the recently developed flow cytometric Pig-a mutation assay and flow cytometric micronucleus analysis. Eight reference genotoxicants and three presumed nongenotoxic compounds were studied: chlorambucil, melphalan, thiotepa, cyclophosphamide, azathioprine, 2-acetylaminofluorene, hydroxyurea, methyl methanesulfonate, o-anthranilic acid, sulfisoxazole, and sodium chloride. These experiments extend previously published results with seven other chemicals. Male Sprague Dawley rats were treated via gavage for 3 or 28 consecutive days with several dose levels of each chemical up to the maximum tolerated dose. Blood samples were collected at several time points up to day 45 and were analyzed for Pig-a mutation with a dual-labeling method that facilitates mutant cell frequency measurements in both total erythrocytes and the reticulocyte subpopulation. An immunomagnetic separation technique was used to increase the efficiency of scoring mutant cells. Blood samples collected on day 4, and day 29 for the 28-day study, were evaluated for MN-RET frequency. The three nongenotoxicants did not induce Pig-a or MN-RET responses. All genotoxicants except hydroxyurea increased the frequency of Pig-a mutant reticulocytes and erythrocytes. Significant increases in MN-RET frequency were observed for each of the genotoxicants at both time points. Whereas the highest Pig-a responses tended to occur in the 28-day studies, when total dose was greatest, the highest induction of MN-RET was observed in the 3-day studies, when dose per day was greatest. There was no clear relationship between the maximal Pig-a response of a given chemical and its corresponding maximal MN-RET response, despite the fact that both endpoints were determined in the same cell lineage. Taken with other previously published results, these data demonstrate the value of integrating Pig-a and micronucleus endpoints into in vivo toxicology studies, thereby providing information about mutagenesis and chromosomal damage in the same animals from which toxicity, toxicokinetics, and metabolism data are obtained.

Microfluidic applications of functionalized magnetic particles for environmental analysis: focus on waterborne pathogen detection

The continuous surveillance of drinking water is extremely important to provide early warning of contamination and to ensure continuous supplies of healthy drinking water. Isolation and detection of a particular type of pathogen present at low concentration in a large volume of water, concentrating the analyte in a small detection volume, and removing detection inhibiting factors from the concentrated sample, present the three most important challenges for water quality monitoring laboratories. Combining advanced biological detection methods (e.g., nucleic acid-based or immunology-based protocols) with microfluidics and immunomagnetic separation techniques that exploit functionalized magnetic particles has tremendous potential for realization of an integrated system for pathogen detection, in particular, of waterborne pathogens. Taking advantage of the unique properties of magnetic particles, faster, more sensitive, and more economical diagnostic assays can be developed that can assist in the battle against microbial pathogenesis. In this review, we highlight current technologies and methods used for realization of magnetic particle-based microfluidic integrated waterborne pathogen isolation and detection systems, which have the potential to comply in future with regulatory water quality monitoring requirements.