Vitrification of medaka whole testis with a trehalose-containing solution and production of medaka individuals derived from the vitrified cells.

The cryopreservation of teleost eggs and embryos remains challenging, and there are no previous reports that demonstrate successful cryopreservation in medaka (Oryzias latipes). We have reported egg and sperm production, followed by the generation of donor-derived offspring by transplanting vitrified whole testes-derived testicular cells into surrogate fish. The vitrification solutions contained ethylene glycol, sucrose, and ficoll. In this study, we replaced sucrose with trehalose in the vitrification solution and medaka whole testis was vitrified with the solution. The post-vitrification survival (72.8±3.5%) was markedly improved compared with that achieved using the sucrose-containing solution (44.7±4.2%). Moreover, we demonstrated the production of eggs, sperm, and donor-derived offspring from testicular cells transplanted into surrogate recipients. The phenotype of donor-derived offspring was identical to that of transplanted testicular cells. These findings suggest that trehalose is effective for the vitrification of medaka whole testis and can be considered an effective and reliable method for the long-term preservation of their genetic resources.

A descriptive survey of porcine epidemic diarrhea in pig populations in northern Vietnam.

Porcine epidemic diarrhea (PED) virus (PEDV) is a globally emerging and re-emerging epizootic swine virus that causes massive economic losses in the swine industry, with high mortality in piglets. In Vietnam, PED first emerged in 2009 and has now developed to an endemic stage. This is the first cross-sectional survey performed to evaluate the proportion of PEDV-positive swine farms in Vietnam from January 2018 to February 2019. Fecal samples from 327 pig farms in northern Vietnam were collected and tested for PEDV infection by reverse transcription-loop-mediated isothermal amplification (RT-LAMP) method. The proportion of PEDV-positive farms was 30.9% and PEDV-positive farms were distributed throughout the study area. The highest proportion of PEDV-positive farms was 70% (7/10) among nucleus production type farms (P < 0.05). Higher proportions of PEDV-positive farms were found in the Northeast and Red River Delta areas, which are the major areas of pig production (P < 0.05). The proportion of PEDV-positive farms was higher among larger farms (P < 0.05). Our findings illustrate the high proportion of PEDV-positive farms in the Vietnamese pig population and will help to better understand the epidemiological dynamics of PED infection, to estimate impact, and establish and improve prevention and control measures.

Development of pooled testing system for porcine epidemic diarrhoea using real-time fluorescent reverse-transcription loop-mediated isothermal amplification assay.

BACKGROUND: Porcine epidemic diarrhoea (PED) is an emerging disease in pigs that causes massive economic losses in the swine industry, with high mortality in suckling piglets. Early identification of PED virus (PEDV)-infected herd through surveillance or monitoring strategies is necessary for mass control of PED. However, a common working diagnosis system involves identifying PEDV-infected animals individually, which is a costly and time-consuming approach. Given the above information, the thrusts of this study were to develop a real-time fluorescent reverse transcription loop-mediated isothermal amplification (RtF-RT-LAMP) assay and establish a pooled testing system using faecal sample to identify PEDV-infected herd. RESULTS: In this study, we developed an accurate, rapid, cost-effective, and simple RtF- RT-LAMP assay for detecting the PEDV genome targeting M gene. The pooled testing system using the RtF-RT-LAMP assay was optimized such that a pool of at least 15 individual faecal samples could be analysed. CONCLUSIONS: The developed RtF-RT-LAMP assay in our study could support the design and implementation of large-scaled epidemiological surveys as well as active surveillance and monitoring programs for effective control of PED.

The Influence of Polyploidy and Genome Composition on Genomic Imprinting in Mice.

Genomic imprinting is an epigenetic mechanism that switches the expression of imprinted genes involved in normal embryonic growth and development in a parent-of-origin-specific manner. Changes in DNA methylation statuses from polyploidization are a well characterized epigenetic modification in plants. However, how changes in ploidy affect both imprinted gene expression and methylation status in mammals remains unclear. To address this, we used quantitative real time PCR to analyze expression levels of imprinted genes in mouse tetraploid fetuses. We used bisulfite sequencing to assess the methylation statuses of differentially methylated regions (DMRs) that regulate imprinted gene expression in triploid and tetraploid fetuses. The nine imprinted genes H19, Gtl2, Dlk1, Igf2r, Grb10, Zim1, Peg3, Ndn, and Ipw were all unregulated; in particular, the expression of Zim1 was more than 10-fold higher, and the expression of Ipw was repressed in tetraploid fetuses. The methylation statuses of four DMRs H19, intergenic (IG), Igf2r, and Snrpn in tetraploid and triploid fetuses were similar to those in diploid fetuses. We also performed allele-specific RT-PCR sequencing to determine the alleles expressing the three imprinted genes Igf2, Gtl2, and Dlk1 in tetraploid fetuses. These three imprinted genes showed monoallelic expression in a parent-of-origin-specific manner. Expression of non-imprinted genes regulating neural cell development significantly decreased in tetraploid fetuses, which might have been associated with unregulated imprinted gene expression. This study provides the first detailed analysis of genomic imprinting in tetraploid fetuses, suggesting that imprinted gene expression is disrupted, but DNA methylation statuses of DMRs are stable following changes in ploidy in mammals.

Gender differences in joint torque focused on hip internal and external rotation during a change in direction while walking.

[Purpose] To investigate lower-extremity joint torque during walking direction changes, focused on gender differences in pelvic width. [Subjects and Methods] Healthy males and females (n=10) changed direction while walking by side-stepping (SS) and crossover stepping (CS), stepping the left leg to the left or right, respectively, over the right leg. Movements were recorded using four infrared cameras, and ground reaction force of each lower extremity measured using two force platforms. Joint torque of each lower extremity was calculated, and each joint peak torque among walking, SS and CS was compared between genders. Moreover, correlation between joint torque showed a gender difference and width of pelvis normalized by width of shoulder (P/S ratio) was examined. [Results] Right external and internal rotation torques during CS were higher in males. Left- and right-ankle plantar flexion torques were also higher in males during CS, while the P/S ratio was larger in females, with a positive correlation between P/S ratio and hip external and internal rotation torques. [Conclusion] No gender differences were found in joint torque during walking and SS, but only during CS. The hip external and internal rotators are necessary for smooth CS in females in terms of P/S ratio.

Restricted development of mouse triploid fetuses with disorganized expression of imprinted genes.

Eukaryotic species commonly contain a diploid complement of chromosomes. The diploid state appears to be advantageous for mammals because it enables sexual reproduction and facilitates genetic recombination. Nonetheless, the effects of DNA ploidy on mammalian ontogeny have yet to be understood. The present study shows phenotypic features and expression patterns of imprinted genes in tripronucleate diandric and digynic triploid (DAT and DGT) mouse fetuses on embryonic day 10.5 (E10.5). Measurement of crown-rump length revealed that the length of DGT fetuses (1.87 ± 0.13 mm; mean ± standard error of the mean) was much smaller than that of diploid fetuses (4.81 ± 0.05 mm). However, no significant difference was observed in the crown-rump length between diploid and DAT fetuses (3.86 ± 0.43 mm). In DGT fetuses, the expression level of paternally expressed genes, Igf2, Dlk1, Ndn, and Peg3, remained significantly reduced and that of maternally expressed genes, Igf2r and Grb10, increased. Additionally, in DAT fetuses, the Igf2 mRNA expression level was approximately twice that in diploid fetuses, as expected. These results provide the first demonstration that imprinted genes in mouse triploid fetuses show distinctive expression patterns independent of the number of parental-origin haploid sets. These data suggest that both DNA ploidy and asymmetrical functions of parental genomes separately influence mammalian ontogeny.

Change in Achilles Tendon Length after Walking on Treadmill with Gradient.

Objectives: Improving ankle joint contracture is important because stiffness in ankle dorsiflexion can lead to pain, especially when weight-bearing during walking, which tends to concentrate on the forefoot. We hypothesized that the contraction of the gastrocnemius muscle in ankle dorsiflexion would increase the Achilles tendon length and improve the dorsiflexion range of motion. We evaluated the effects of walking with and without a gradient on Achilles tendon length. Methods: This study included 23 men who underwent ultrasound imaging to measure the Achilles tendon length while they stood on an inclined table adjusted according to the dorsiflexion angle. Treadmill walking was performed for 10 min with a 10° incline (gradient condition) or without gradient (level condition). The measurements were compared using a paired t-test. Results: In the gradient condition, the range of motion for ankle dorsiflexion was significantly increased after the intervention. In the gradient condition, the Achilles tendon length while standing on an inclined surface was significantly increased after the intervention. Conclusions: Walking under gradient conditions led to the extension of the Achilles tendon in the ankle dorsiflexion position. This was accompanied by contraction of the gastrocnemius muscle, resulting in lengthening of the Achilles tendon. This finding suggests that such interventions may have clinical applications.

Analytical and clinical performances of seven direct detection assays for SARS-CoV-2.

Background: Direct detection tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that bypass complicated nucleic acid/antigen purification steps are promising tools for the rapid diagnosis of coronavirus disease 2019 (COVID-19). Methods: To determine the analytical and clinical diagnostic performances of the direct detection assays, we compared 6 direct molecular detection assays, including two loop-mediated isothermal amplification (LAMP) assays and one lateral flow antigen assay, against the reference extraction-based RT-PCR assay using 183 respiratory samples (87 nasopharyngeal swabs, 51 saliva samples, and 45 sputum samples). Results: Analytical sensitivity analysis showed that the direct RT-PCR assay of Toyobo exhibited the lowest LOD of 1,000 copies/mL. Compared with the 80 positive and 103 negative samples based on the reference assay, the Toyobo assay had the highest positive percent agreement (PPA) of 96.3%, followed by the two direct RT-PCR assays of Takara and Shimadzu and one LAMP assay of Eiken (86.3-87.5%). The Fujirebio antigen assay had the lowest PPA of 44.7% among the assays tested. The negative percent agreement of these direct detection assays was 100%, except for the Eiken assay (96.3%). Conclusions: Large differences in PPA existed among the direct detection tests. Laboratories need to take these characteristics into consideration before implementing these assays.

Cryopreservation of rat embryos at all developmental stages by small-volume vitrification procedure and rapid warming in cryotubes.

Intracellular ice formation during cryopreservation is lethal to the cell, including during warming. Here, we examined the effect of sample volume and warming rate on the cryopreservation success of 1-cell rat embryos based on successful development into blastocysts in vitro and to term in vivo following embryo transfer. Embryos were equilibrated in 5% propylene glycol solution for 10 min, held for 40 s at 0 °C in cryopreservation solution (5%PG + PEPeS), and cooled by immersion in liquid nitrogen. When 1-cell embryos were cryopreserved in a volume of 30-100 µL at a cooling rate of 5830-7160 °C/min and warmed at 35,480-49,400 °C/min by adding 1 mL of 0.3 M sucrose solution at 50 °C, 17.3-28.8% developed into blastocysts, compared with 57.0% of untreated embryos. However, when 1-cell embryos were cryopreserved in a smaller volume of 15 µl at 7950 °C/min and warmed at 68,850 °C/min, 58.8 ± 10.6% developed into blastocysts and 50.0 ± 7.4% developed to term, comparable to that of non-treated embryos (57.0 ± 5.4% and 51.4 ± 3.1%, respectively). Cryopreserved embryos at other developmental stages also showed high in vitro culture potential similar to that of the control. Using a conventional cryotube and a small-volume vitrification procedure with rapid warming, we achieved high levels of subsequent rat embryonic development at all developmental stages.

Development of a highly sensitive point-of-care test for African swine fever that combines EZ-Fast DNA extraction with LAMP detection: Evaluation using naturally infected swine whole blood samples from Vietnam.

BACKGROUND: While early detection and early containment are key to controlling the African swine fever (ASF) pandemic, the lack of practical testing methods for use in the field are a major barrier to achieving this feat. OBJECTIVES: To describe the development of a rapid and sensitive point-of-care test (POCT) for ASF, and its evaluation using swine whole blood samples for field settings. METHODS: In total, 89 swine whole blood samples were collected from Vietnamese swine farms and were performed the POCT using a combination of crude DNA extraction and LAMP (loop-mediated isothermal amplification) amplification. RESULTS: The POCT enabled crude DNA to be extracted from swine whole blood samples within 10 min at extremely low cost and with relative ease. The entire POCT required a maximum of 50 min from the beginning of DNA extraction to final judgment. Compared to a conventional real-time PCR detection, the POCT showed a 1 log reduction in detection sensitivity, but comparable diagnostic sensitivity of 100% (56/56) and diagnostic specificity of 100% (33/33). The POCT was quicker and easier to perform and did not require special equipment. CONCLUSIONS: This POCT is expected to facilitate early diagnosis and containment of ASF invasion into both regions in which it is endemic and eradicated.

Role of the X and Y Chromosomes in the Female Germ Cell Line Development in the Mouse (Mus musculus).

BACKGROUND: In eutherian mammals, the sex chromosome complement, XX and XY, determines sexual differentiation of gonadal primordia into testes and ovaries, which in turn direct differentiation of germ cells into haploid sperm and oocytes, respectively. When gonadal sex is reversed, however, the germ cell sex becomes discordant with the chromosomal sex. XY females in humans are infertile, while XY females in the mouse (Mus musculus) are subfertile or infertile dependent on the cause of sex reversal and the genetic background. This article reviews publications to understand how the sex chromosome complement affects the fertility of XY oocytes by comparing with XX and monosomy X (XO) oocytes. SUMMARY: The results highlight 2 folds disadvantage of XY oocytes over XX oocytes: (1) the X and Y chromosomes fail to pair during the meiotic prophase I, resulting in sex chromosome aneuploidy at the first meiotic division and (2) expression of the Y-linked genes during oocyte growth affects the transcriptome landscape and renders the ooplasmic component incompetent for embryonic development. KEY MESSAGE: The XX chromosome complement gives the oocyte the highest competence for embryonic development.

Very low likelihood that cultivated oysters are a vehicle for SARS-CoV-2: 2021-2022 seasonal survey at supermarkets in Kyoto, Japan.

The pandemic caused by novel coronavirus disease of 2019 (COVID-19) is a global threat. Wastewater surveillance in Japan and abroad has led to the detection of SARS-CoV-2, causing concern that SARS-CoV-2 in the feces of infected persons may contaminate the aquatic environment. Bivalves such as oysters cultivated in coastal areas are known to filter and concentrate viruses such as norovirus present in seawater in their bodies; however, whether they do so with SARS-CoV-2 is unknown. Therefore, we examined cultivated oysters sold in Japan for the presence of SARS-CoV-2 between October 2021 and April 2022 to clarify the extent of viral contamination and evaluate the risk of food-borne transmission of SARS-CoV-2. Porcine epidemic diarrhea virus (PEDV), known as pig coronavirus, was used to spike midgut-gland samples as a whole process control. The presence of SARS-CoV-2 and PEDV was investigated using a modified polyethylene glycol precipitation method and RT-qPCR. While all samples spiked with the whole process control were positive, no SARS-CoV-2 was detected in any of the 145 raw oyster samples surveyed, despite a marked increase in infections caused by the Omicron variant from January to April 2022 in Japan. Therefore, our results suggest that with well-developed sewage treatment facilities, consumption of oysters cultivated in coastal areas may not be a risk factor for SARS-CoV-2 outbreaks.

Evaluation of a loop-mediated isothermal amplification assay for rapid and simple detection of Vibrio parahaemolyticus in naturally contaminated seafood samples.

We investigated the efficacy of a loop-mediated isothermal amplification (LAMP) assay for rapid screening of seafood samples naturally contaminated with Vibrio parahaemolyticus. A total of 171 seafood samples enriched in alkaline peptone water (APW) were assessed by LAMP assay and conventional culture methods, which consist of a combination of APW enrichment culture and plating onto CHROMagar Vibrio and TCBS agars. Compared with V. parahaemolyticus isolation using the conventional culture test, LAMP results showed 100% (30/30) and 90.8% (128/141) sensitivity and specificity, respectively. The conventional culture test required more than 3 days to isolate and identify V. parahaemolyticus in the APW enrichment culture. In contrast, the LAMP assay was markedly faster, requiring less than 60 min from the beginning of DNA extraction to final detection of V. parahaemolyticus. In total, the LAMP assay required 17-19 h from the beginning of enrichment culture to final determination. This is the first report of the LAMP assay for rapid screening of seafood samples naturally contaminated by V. parahaemolyticus.

Effects of the Sex Chromosome Complement, XX, XO, or XY, on the Transcriptome and Development of Mouse Oocytes During Follicular Growth.

The sex chromosome complement, XX or XY, determines sexual differentiation of the gonadal primordium into a testis or an ovary, which in turn directs differentiation of the germ cells into sperm and oocytes, respectively, in eutherian mammals. When the X monosomy or XY sex reversal occurs, XO and XY females exhibit subfertility and infertility in the mouse on the C57BL/6J genetic background, suggesting that functional germ cell differentiation requires the proper sex chromosome complement. Using these mouse models, we asked how the sex chromosome complement affects gene transcription in the oocytes during follicular growth. An oocyte accumulates cytoplasmic components such as mRNAs and proteins during follicular growth to support subsequent meiotic progression, fertilization, and early embryonic development without de novo transcription. However, how gene transcription is regulated during oocyte growth is not well understood. Our results revealed that XY oocytes became abnormal in chromatin configuration, mitochondria distribution, and de novo transcription compared to XX or XO oocytes near the end of growth phase. Therefore, we compared transcriptomes by RNA-sequencing among the XX, XO, and XY oocytes of 50-60 µm in diameter, which were still morphologically comparable. The results showed that the X chromosome dosage limited the X-linked and autosomal gene transcript levels in XO oocytes whereas many genes were transcribed from the Y chromosome and made the transcriptome in XY oocytes closer to that in XX oocytes. We then compared the transcript levels of 3 X-linked, 3 Y-linked and 2 autosomal genes in the XX, XO, and XY oocytes during the entire growth phase as well as at the end of growth phase using quantitative RT-PCR. The results indicated that the transcript levels of most genes increased with oocyte growth while largely maintaining the X chromosome dosage dependence. Near the end of growth phase, however, transcript levels of some X-linked genes did not increase in XY oocytes as much as XX or XO oocytes, rendering their levels much lower than those in XX oocytes. Thus, XY oocytes established a distinct transcriptome at the end of growth phase, which may be associated with abnormal chromatin configuration and mitochondria distribution.

Examination of gait characteristics and related factors in elderly subjects with and without hallux valgus.

OBJECTIVE: Elderly people with hallux valgus have decreased gait speed, which can result in reduced capacity to perform the activities of daily living. Therefore, this study examined the gait ability and related factors of patients with hallux valgus. METHODS: The study participants were 10 patients with hallux valgus and 10 without. Ground reaction forces were measured as front-rear (X), lateral (Y), and vertical (Z) components from the early to late stance phases. Three-dimensional motion analysis was used to measure gait speed; touchdown distance; release distance; the angles of the limb joints and trunk at heel contact, toe-off, and peak ground reaction force; and the center of mass (COM) displacement in the sagittal plane. The height of the COM was calculated as a percentage of the body height. The hallux valgus and control groups were compared using the Mann-Whitney U-test. RESULTS: In the hallux valgus group, the ground reaction force showed a significant increase in the Y component in each stance phase and in the Z component in the late stance phase. The lowest COM position in the hallux valgus group was significantly higher than that in the control group, resulting in a smaller difference in COM height over a gait cycle. CONCLUSIONS: The hallux valgus group was found to have reduced gait speed because of a shortened touchdown distance. Moreover, the continued high COM position in the hallux valgus group meant that potential energy could not efficiently be converted to kinetic energy.

Development of a point-of-care test to detect SARS-CoV-2 from saliva which combines a simple RNA extraction method with colorimetric reverse transcription loop-mediated isothermal amplification detection.

The new coronavirus infection (COVID-19) is a major public health concern, with a high burden and risk for infection among patients and healthcare workers. Saliva droplets containing SARS-COV-2 are a major vector for COVID-19 infection, making saliva a promising alternative for COVID-19 testing using nasopharyngeal swab samples. To diagnose COVID-19 patients in the field, a point-of-care test (POCT) using saliva was conceptualized. We have developed a simple method for extracting RNA from saliva samples using semi-alkaline proteinase, a sputum homogenizer typically used for preparing samples for tuberculosis testing, and a subsequent simple heating step with no need for centrifugation or RNA extraction. Further, we newly developed a triplex reverse transcription loop-mediated isothermal amplification approach (RT-LAMP) which utilizes colorimetric readout using a heat block, with results evaluated with the unaided eye. In 44 clinical patients suspected of having COVID-19 infection, the test took 45 min, and resulted in a diagnostic sensitivity of 82.6% (19/23) and diagnostic specificity of 100% (21/21), compared to the reference standard. The limit of detection was 250 copies/reaction (25,000 copies/mL). Our newly developed POCT approach achieved simple RNA extraction and constant RT-LAMP detection. This POCT has the potential to be used for simple inspection stations in a field setting, helping reduce the risk of infection by simplifying and accelerating testing for COVID-19.

Interconnection of organic-inorganic hybrid nano-building blocks towards thermally robust mesoporous structures.

The use of organic-inorganic hybrid nanoparticles will enable a control of the characteristics of both the nanoparticles and constructed fine structures. In this study, we report the synthesis of acrylate-intercalated layered manganese, cobalt, and nickel hydroxide nanoparticles and their assembly into ordered mesoporous structures. Polymerization of the intercalated acrylates takes place by means of a radical initiator. The formed organic network improved the thermal stability of the layered hydroxides, which results in thermally robust mesoporous structures. Additionally, it is found that the polymerization can be initiated and progressed at 200 °C without any initiators for the layered nickel hydroxide system. This allows for the scalable solid-state thermal polymerization of intercalated acrylates and the formation of thermally robust hierarchically ordered meso/macroporous powders as well as mesoporous films. The electrochemical characterization reveals that the thermally robust mesoporous films having regulated mesopores allow for the effective diffusion of molecules/solvent compared with the films having collapsed mesoporous structures.

Identification of Escherichia coli and Related Enterobacteriaceae and Examination of Their Phenotypic Antimicrobial Resistance Patterns: A Pilot Study at A Wildlife-Livestock Interface in Lusaka, Zambia.

A cross-sectional study was used to identify and assess prevalence and phenotypic antimicrobial resistance (AMR) profiles of Escherichia coli and other enterobacteria isolated from healthy wildlife and livestock cohabiting at a 10,000 acres game ranch near Lusaka, Zambia. Purposive sampling was used to select wildlife and livestock based on similarities in behavior, grazing habits and close interactions with humans. Isolates (n = 66) from fecal samples collected between April and August 2018 (n = 84) were examined following modified protocols for bacteria isolation, biochemical identification, molecular detection, phylogenetic analysis, and antimicrobial susceptibility testing by disc diffusion method. Data were analyzed using R software, Genetyx ver.12 and Mega 6. Using Applied Profile Index 20E kit for biochemical identification, polymerase chain reaction assay and sequencing, sixty-six isolates were identified to species level, of which Escherichia coli (72.7%, 48/66), E. fergusonii (1.5%, 1/66), Shigella sonnei (22.7%, 14/66), Sh. flexinerri (1.5%, 1/66) and Enterobacteriaceae bacterium (1.5%, 1/66), and their relationships were illustrated in a phylogenetic tree. Phenotypic antimicrobial resistance or intermediate sensitivity expression to at least one antimicrobial agent was detected in 89.6% of the E. coli, and 73.3% of the Shigella isolates. The E. coli isolates exhibited the highest resistance rates to ampicillin (27%), ceftazidime (14.3%), cefotaxime (9.5%), and kanamycin (9.5%). Multidrug resistance (MDR) was detected in 18.8% of E. coli isolates while only 13.3% Shigella isolates showed MDR. The MDR was detected among isolates from impala and ostrich (wild animals in which no antimicrobial treatment was used), and in isolates from cattle, pigs, and goats (domesticated animals). This study indicates the possible transmission of drug-resistant microorganisms between animals cohabiting at the wildlife-livestock interface. It emphasizes the need for further investigation of the role of wildlife in the development and transmission of AMR, which is an issue of global concern.

Development of a loop-mediated isothermal amplification assay for sensitive and rapid detection of the tdh and trh genes of Vibrio parahaemolyticus and related Vibrio species.

Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are the major virulence determinants of Vibrio parahaemolyticus. TRH is further differentiated into TRH1 and TRH2 on the basis of genetic and phenotypic differences. We developed a novel and highly specific loop-mediated isothermal amplification (LAMP) assay for sensitive and rapid detection of the tdh, trh1, and trh2 genes of V. parahaemolyticus. The LAMP assay was designed for both combined and individual detection of the tdh, trh1, and trh2 genes and combined detection of the trh1 and trh2 genes. Our results showed that it gave the same results as DNA probes and conventional PCR assays for 125 strains of V. parahaemolyticus, 3 strains of Grimontia hollisae, and 2 strains of Vibrio mimicus carrying the tdh, trh1, and trh2 genes in various combinations. No LAMP products were detected for any of the 20 bacterial strains lacking the tdh, trh1, and trh2 genes. The sensitivities of the LAMP assay for detection of tdh-, trh1-, and trh2-carrying V. parahaemolyticus strains in spiked shrimp samples were 0.8, 21.3, and 5.0 CFU per LAMP reaction tube, respectively. Starting with DNA extraction from a single colony and from spiked shrimp samples, the LAMP assay required only 27 to 60 min and less than 80 min, respectively. This is the first report of a rapid and specific LAMP assay for detection and differentiation of the tdh, trh1, and trh2 genes of V. parahaemolyticus and related Vibrio species.

The mode of biofilm formation on smooth surfaces by Campylobacter jejuni.

Many microorganisms produce extracellular polymers referred to collectively as "slime" or glycocalyx, and form biofilms on solid surfaces in natural ecosystems. Campylobacter jejuni, one of the most important foodborne pathogens, also has the ability to form biofilm on stainless steel, glass, or polyvinyl chloride in vitro. However, the issue of biofilm formation by Campylobacter species has not been extensively examined. The present study was performed to examine the mode of adhesion of C. jejuni to a smooth surface. When bacterial suspensions in Brucella broth were incubated in microplate wells with a glass coverslip, microcolonies 0.5~2 mm in diameter were formed on the coverslip within 2 hr from the start of incubation. These microcolonies gradually grew and formed a biofilm of net-like connections within 6 hr. Transmission electron microscopy indicated that massive amounts of extracellular material masked the cell surface, and this material bound ruthenium red, suggesting the presence of a polysaccharide moiety. Scanning electron microscopy indicated that the flagella acted as bridges, forming net-like connections between the organisms. To determine the genes associated with biofilm formation, aflagellate (flaA(-)) and flagellate but non-motile (motA(-)) mutants were constructed from strain 81-176 by natural transformation-mediated allelic exchange. The flaA(-) and motA(-) mutants did not form the biofilm exhibited by the wild-type strain. These findings suggest that flagella-mediated motility as well as flagella is required for biofilm formation in vitro.