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The accurate identification of mastitis‐causing bacteria assists in effective management by both dairy farmers and veterinarians and can be used to implement the selective use of antimicrobials for treatment. The purpose of this study was to evaluate the ability of our developed anti–ribosomal protein-L7/L12 antibody–coated immunochromatographic strip (ICS) test to detect coliforms in milk by comparing the results with the bacteriological culture method. We investigated the performance of the ICS test as compared with the bacteriological culture method using 308 milk samples from clinical bovine mastitis. First, to determine the optimal ICS test cutoff point for detecting coliform mastitis, we developed a receiver-operating characteristic curve. The result showed that the cutoff point was at 0.5 of our index. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value of the ICS test were 81.3%, 84.8%, 69.2%, and 91.54%, respectively. As the clinical signs increased in severity, the F-measure, a weighted harmonic mean of the sensitivity and overall PPV performance, increased. Because it is especially important to treat clinical mastitis appropriately in the early stages of detection, the ICS test, which can be used by both dairy farmers and veterinarians on dairy farms, is considered to be a useful tool for detecting coliform mastitis, which often presents with severe signs.

Our objectives were to evaluate the diagnostic accuracy of a rapid and novel immunochromatography-based mastitis kit that includes 3 independent tests to detect coliforms (Escherichia coli or Klebsiella pneumoniae), Streptococcus spp., and Staphylococcus aureus. The kit was developed to facilitate diagnostic-based mastitis treatment. Validation of the kit was based on 154 aseptically collected mastitis samples from 2 clinical herds (clinical population) and 120 milk samples from 3 nonclinical herds (nonclinical population) without clinical cases at the time of enrollment. One herd sampled at different times was common to both populations. A 3-test in 2-population Bayesian latent class model with uniform priors for all test parameters except specificity of culture, which was modeled informatively, was used to estimate sensitivity (Se) and specificity (Sp) of the test kit, culture, and PCR at the cow level. The mastitis test kit's 96.9% Sp for Streptococcus spp. had a low false positive percentage (3.1%), which, together with the kit's rapid turnaround time for results, makes it a suitable initial screening test that producers can use to identify clinical cows to treat based on Streptococcus spp. mastitis in kit-positive results. Due to the 60.4% kit Se, producers should follow up on Streptococcus spp. kit-negative cows using a confirmatory test such as PCR (Sp of 98.4%) or culture (Sp of 99.6%). In contrast, aerobic culture had Se of 76.5% and Sp of 99.6% for Streptococcus spp. Similarly, the Sp of the kit (98.2%) and culture (99.8%) for Staph. aureus were particularly high, and even though the kit's Se (61.0%) was lower than culture (88.4%; posterior probability of difference 98%), the kit could be beneficial before use of a confirmatory test for kit-negative samples due to its ease and rapid turnaround time. Mostly, quantitative real-time (q)PCR outperformed the kit's Se (37.7%) and Sp (92.9%) for coliforms, as well as the kit's Se (60.4%) for Streptococcus spp. However, qPCR may require more technical skills and turnaround time for final results. Use of the on-farm mastitis test kit evaluated in the present study could enhance sustainable antimicrobial drug use by rapidly identifying Streptococcus mastitis for targeted treatment. Furthermore, the kit may be used in a Staph. aureus outbreak where cows can be rapidly screened to identify cases for segregation or culling during an outbreak and kit-negative cows further confirmed by milk culture or qPCR. However, the cost-effectiveness of such an approach has not been investigated.

We evaluated the relationship between the severity of coliform mastitis and bacterial load in 106 quarter milk samples. We found no significant relationship between somatic cell count and coliform bacterial load in milk in bovine clinical coliform mastitis. Results of the Cochran-Armitage test for trend in milk bacterial load proportions indicated a significant decreasing low group (P

Microchip-based genotoxic bioassay using sensing Escherichia coli strains has been performed. In this method, the assay was conducted in three-dimensional microfluidic network constructed by a silicon perforated microwell array chip and two poly(dimethylsiloxane) (PDMS) multi-microchannel chips. The sensing strains having firefly luciferase reporter gene under transcriptional control of umuD as an SOS promoter were put into the channels on one of the PDMS chips and immobilized in the silicon microwells. Samples containing genotoxic substances and substrates for luciferase were into the channels on the other PDMS chip. The optimum conditions of the assay in the on-chip format have been investigated using mitomycin C (MMC) as a genotoxic substance. As a result, the dose-dependence of bioluminescence intensity was obtained at once on the chip. Additionally, the response ratios of the bioluminescence between mutagen- and non-induced strains were successfully enhanced by improving the on-chip assay methods and conditions. Several well-known genotoxic substances were subjected to the on-chip assay, and were detected with the detection limits comparable to those in the conventional method with reduced time.

A whole-cell assay based on bacterial SOS response has been used for genotoxicity evaluation of chemical substances. In this study, for improvement of induction ratio and detection limit in the genotoxic assay, three modifications to an original tester strain, Escherichia coli harboring a multi-copy plasmid-borne fusion of firefly luciferase and the umuD promoter, were examined singly or in combinations. First, the effect of replacement of the multi-copy plasmid with a low-copy one was examined. Bioluminescence (BL) emission from the E. coli strain having the low-copy plasmid induced by mitomycin C (MMC) as a genotoxic substance was decreased compared to that from the original strain, but the BL emission ratio of the induced strain to the non-induced one was increased by two-fold at maximum due to reduced blank BL emission from the non-induced strain. Next, two modifications were made in view of control of the membrane permeability for substances by using toluene or a tolC mutant. Toluene promoted the membrane permeation of substances, while tolC mutation inhibited the efflux of them via cell membranes. The addition of toluene to the strain resulted in an increase in the BL ratio by increasing BL emission from the induced strain. On the other hand, by the use of tolC mutation, the dose-dependency curve of the BL ratio shifted to a lower concentration range of MMC, and the detection limit of MMC was therefore greatly improved. Compared to the original tester strain, in combination with the use of the low-copy plasmid, the maximum induction ratio was increased about four-fold to above 400 by the addition of toluene, whereas the detection limit was improved 1000-fold to below 10 pg ml−1 by the use of a tolC mutant. The effects of each modification and their combination on the induction ratio and detection limit were examined with various substances.

非イオン界面活性剤であるTriton X-100を用いたミセル水性二相分配系にNiIIを配位したイミノジ酢酸ポリエチレングリコール (Ni-IDA-PEG) を導入し, NiIIとのアフィニティー相互作用による膜タンパク質チトクロムb5及びオリゴペプチドの分配の制御を試みた. チトクロムb5は, Ni-IDA-PEG濃度に依存せずミセル相に分配したのに対し, ヒスチジンタグを融合したチトクロムb5及びオリゴペプチドはNi-IDA-PEG濃度の増加に伴いポリマー相に分配された. NiIIを配位していないIDA-PEG並びにイミダゾールを用いた実験から, ヒスチジンタグ融合チトクロムb5のポリマー相への分配は, ヒスチジンとNiIIとのアフィニティー相互作用によることが分かった. 更に, ヒスチジンタグ融合チトクロムb5のアフィニティー分配はpH緩衝剤の種類に大きく依存することが明らかとなった.

An on-chip whole-cell bioassay has been carried out using Escherichia coli tester strains for genotoxicity. In this assay format, the mutagen-responsive bioluminescence (BL) strains are immobilized in a chip assembly in which a silicon chip is placed between two poly(dimethylsiloxane) (PDMS) chips. In the chip assembly, microchannels fabricated on the two separate PDMS layers are connected via perforated microwells on the Si chip, and thus a three-dimensional microfluidic network is constructed. The strains mixed with agarose are loaded from the channels on one of the two PDMS layers into the wells on Si chip, followed by gelation. Induction of the expression of firefly luciferase in the tester strains and BL reaction are successively carried out by filling the channels on another PDMS layer with samples containing inducer (genotoxic substance) and then adenosine triphosphate/luciferin mixture, respectively. BL emission from each of the wells can be monitored by using a charge-coupled device camera to obtain an overall picture of the chip. The on-chip format based on a three-dimensional microfluidic network provides a combinatorial bioassay for multiple samples with multiple tester strains in a simple chip assembly. Thus, the presented method could be applied not only to various microbial sensing applications but also to other (bio)chemical analyses.

An on-chip whole-cell bioassay has been carried out using Escherichia coli tester strains for genotoxicity. In this assay format, the mutagen-responsive bioluminescence (BL) strains are immobilized in a chip assembly in which a silicon chip is placed between two poly(dimethylsiloxane) (PDMS) chips. In the chip assembly, microchannels fabricated on the two separate PDMS layers are connected via perforated microwells on the Si chip, and thus a three-dimensional microfluidic network is constructed. The strains mixed with agarose are loaded from the channels on one of the two PDMS layers into the wells on Si chip, followed by gelation. Induction of the expression of firefly luciferase in the tester strains and BL reaction are successively carried out by filling the channels on another PDMS layer with samples containing inducer (genotoxic substance) and then adenosine triphosphate/luciferin mixture, respectively. BL emission from each of the wells can be monitored by using a charge-coupled device camera to obtain an overall picture of the chip. The on-chip format based on a three-dimensional microfluidic network provides a combinatorial bioassay for multiple samples with multiple tester strains in a simple chip assembly. Thus, the presented method could be applied not only to various microbial sensing applications but also to other (bio)chemical analyses.

A whole-cell bioassay has been performed using Escherichia coli sensor strains immobilized in a chip assembly, in which a silicon substrate is placed between two poly(dimethylsiloxane) (PDMS) substrates. Microchannels fabricated on the two separate PDMS layers are connected via perforated microwells on the silicon chip, and thus, a three-dimensional microfluidic network is constructed in the assembly. Bioluminescent sensor strains mixed with agarose are injected into the channels on one of the two PDMS layers and are immobilized in the microwells by gelation. Induction of the firefly luciferase gene expression in the sensor strains can be easily carried out by filling the channels on the other layer with sample solutions containing mutagen. Bioluminescence emissions from each well are detected after injection of luciferin/ATP mixtures into the channels. In this assay format using two multichannel layers and one microwell array chip, the interactions between various types of samples and strains can be monitored at each well on one assembly in a combinatorial fashion. Using several genotypes of the sensor strains or concentrations of mitomycin C in this format, the dependence of bioluminescence on these factors was obtained simultaneously in the single screening procedure. The present method could be a promising on-chip format for high-throughput whole-cell bioassays.