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.

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.

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.

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.

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.

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.

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 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.

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.

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 a Loop-Mediated Isothermal Amplification (LAMP) Assay Targeting the Citrate Synthase Gene for Detection of Ehrlichia canis in Dogs.

Canine monocytic ehrlichiosis caused by Ehrlichia canis is one of the leading tick-borne diseases of dogs, particularly in tropical countries. A highly sensitive and specific diagnostic method is essential for early detection to facilitate treatment. This study was conducted to develop E. canis loop-mediated isothermal amplification (LAMP) assay, a highly sensitive yet simple molecular technique, targeting the citrate synthase (gltA) gene of E. canis. Canine blood samples were subjected to conventional PCR targeting E. canis gltA. After analysis of the sequences of PCR amplicons, LAMP primers were generated. The optimum temperature and time for the LAMP assay were determined using eight samples-after which, the effectiveness and reproducibility of LAMP were verified by testing 40 samples, which included PCR-positive and negative samples. The detection limit was also established. The optimal condition for the assay was 61 °C for 60 min. Compared to PCR, the LAMP assay had a relative sensitivity and specificity of 92.5 and 100%, respectively. Statistical analysis using McNemar's test showed that the E. canis LAMP assay has no significant difference with PCR. Therefore, the LAMP assay developed in this study may be used as an alternative to PCR in the detection of E. canis.

Application of an Improved Micro-amount of Virion Enrichment Technique (MiVET) for the Detection of Avian Influenza A Virus in Spiked Chicken Meat Samples.

Highly sensitive detection of pathogens is effective for screening meat during quarantine inspection and export. The "micro-amount of virion enrichment technique" (MiVET) was recently developed, which is a new method combining virus concentration with immunomagnetic beads and simple RNA extraction with sodium dodecyl benzenesulfonate (SDBS) for the specific and sensitive detection of avian influenza viruses (AIVs). AIV subtypes H3N2 and H4N2 were used to spike the surface of chicken breast meat samples. The modified MiVET protocol was tested by comparing it against three different homogenate preparation conditions, as well as in samples with added α-amylase and collagenase to digest inhibitors. The performance of the modified MiVET was evaluated by real-time RT-PCR assay targeting the matrix gene. Compared with conventional RNA extraction, the modified MiVET reproducibly concentrated AIVs in chicken meat samples with 100-1000-fold improvement by 60 s-hand homogenization. The 30 s- and 60 s-stomacher homogenizations resulted 100-fold and 10-100-fold improvement, respectively. The modified MiVET required < 60 min from homogenate preparation to final RNA elution. Further, use of the modified MiVET also decreased the rate of false-negative results. The modified MiVET is effective for the rapid and highly sensitive detection of AIVs in chicken meat samples, and can be applied to quarantine and export inspection at airports and seaports.

Function of the Distal Part of the Vastus Medialis Muscle as a Generator of Knee Extension Twitch Torque.

The distal part of the vastus medialis (VM) (VM obliquus: VMO) muscle acts as the medial stabilizer of the patella. However, it has been known to facilitate VMO contraction during training of the quadriceps femoris muscle in knee joint rehabilitation. This study aimed to examine the contribution degree of VMO as a knee joint extension torque generator. Sixteen healthy male volunteers participated in this study. Electrical muscle stimulation (EMS) was performed on VMO at 60° knee angle for 20 min to induce muscle fatigue. Knee extension twitch torques (TT) at 90° and 30° knee angle evoked by femoral nerve stimulation were measured before and after EMS. Although each TT at 90° and 30° knee angle significantly decreased after EMS, the decreased TT rate in both joint angles showed no significant difference. Our results show that VMO might contribute to the generation of the knee joint torque at the same level in the range from flexion to extension. Therefore, it was suggested that the facilitating the neural drive for VMO is important during the quadriceps femoris muscle strengthening exercise.

[Development and validation of loop-mediated isothermal amplification for the detection of the Zika virus]. / Desarrollo y validación del método de amplificación isotérmica mediada en lazo para la detección del virus Zika.

A Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) method was developed to detect Zika. The primers were designed based on the NS5 region of 64 complete genomes. Lyophilized LAMP reagent was used. Initially, seven different arboviruses were tested and only Zika samples tested positive. Additionally, serial dilutions of one of Zika's RNA were compared using RT-LAMP and qRT-PCR, demonstrating that RT-LAMP is 1,000 times more sensitive. We also evaluated 300 serum samples with RT-LAMP comparing the results with standard qRT-PCR methods, and we obtained a 99.3% sensitivity, 100% specificity, 100% positive predictive value, and 99.3% negative predictive value. In conclusion, this method provides a low-cost, high-performance, viable, and reliable alternative for the rapid diagnosis of Zika in primary health-care facilities.

Se desarrolló un método de amplificación isotérmica mediada en lazo de transcriptasa inversa (RT-LAMP) para detectar Zika. Los primers se diseñaron basándose en la región NS5 de 64 genomas completos. Se usó reactivo LAMP liofilizado. Inicialmente, se probaron siete arbovirus diferentes y solo las muestras de Zika resultaron positivas. Además, las diluciones seriadas de una de los ARN de Zika se compararon mediante RT-LAMP y qRT-PCR, demostrando que RTLAMP es 1000 veces más sensible. También se evaluó 300 muestras de suero usando RT-LAMP y los resultados se compararon con los métodos de qRT-PCR estándar y obtuvimos un 99,3% (IC95%: 97,7 - 100,0) de sensibilidad, 100% (IC95%: 99,7 - 100,0) de especificidad, 100% (IC95%: 99,7 -100,0) de valor predictivo positivo y 99,3% (IC95%: 97,7 - 100,0) de valor predictivo negativo. En conclusión, este método brinda una alternativa de bajo costo, alto rendimiento, viabilidad y confiabilidad para el diagnóstico rápido de Zika en instalaciones de atención primaria de salud.

Desarrollo y validación del método de amplificación isotérmica mediada en lazo para la detección del virus Zika / Development and validation of loop-mediated isothermal amplification for the detection of the Zika virus

RESUMEN Se desarrolló un método de amplificación isotérmica mediada en lazo de transcriptasa inversa (RT-LAMP) para detectar Zika. Los primers se diseñaron basándose en la región NS5 de 64 genomas completos. Se usó reactivo LAMP liofilizado. Inicialmente, se probaron siete arbovirus diferentes y solo las muestras de Zika resultaron positivas. Además, las diluciones seriadas de una de los ARN de Zika se compararon mediante RT-LAMP y qRT-PCR, demostrando que RTLAMP es 1000 veces más sensible. También se evaluó 300 muestras de suero usando RT-LAMP y los resultados se compararon con los métodos de qRT-PCR estándar y obtuvimos un 99,3% (IC95%: 97,7 - 100,0) de sensibilidad, 100% (IC95%: 99,7 - 100,0) de especificidad, 100% (IC95%: 99,7 -100,0) de valor predictivo positivo y 99,3% (IC95%: 97,7 - 100,0) de valor predictivo negativo. En conclusión, este método brinda una alternativa de bajo costo, alto rendimiento, viabilidad y confiabilidad para el diagnóstico rápido de Zika en instalaciones de atención primaria de salud.

ABSTRACT A Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) method was developed to detect Zika. The primers were designed based on the NS5 region of 64 complete genomes. Lyophilized LAMP reagent was used. Initially, seven different arboviruses were tested and only Zika samples tested positive. Additionally, serial dilutions of one of Zika's RNA were compared using RT-LAMP and qRT-PCR, demonstrating that RT-LAMP is 1,000 times more sensitive. We also evaluated 300 serum samples with RT-LAMP comparing the results with standard qRT-PCR methods, and we obtained a 99.3% sensitivity, 100% specificity, 100% positive predictive value, and 99.3% negative predictive value. In conclusion, this method provides a low-cost, high-performance, viable, and reliable alternative for the rapid diagnosis of Zika in primary health-care facilities.

Development of a fluorescent loop-mediated isothermal amplification assay for rapid and simple diagnosis of bovine leukemia virus infection.

Bovine leukemia virus (BLV) causes enzootic bovine leukosis (EBL), a condition that threatens the sustainability of the livestock industry. A fluorescent loop-mediated isothermal amplification (fLAMP) assay targeting BLV env sequences was developed and used to evaluate 100 bovine blood samples. Compared with a conventional real-time PCR (rPCR) assay, the fLAMP assay achieved 87.3% (62/71) sensitivity and 100% (29/29) specificity. The rPCR assay took 65 min, while the fLAMP assay took 8 min to 30 min from the beginning of DNA amplification to final judgement with a comparable limit of detection. The fLAMP is a potential tool for the rapid and simple diagnosis of BLV infection to supplement ELISA testing and can be used by local laboratories and slaughterhouses without special equipment.

Improving the Detection Accuracy and Time for Campylobacter jejuni and Campylobacter coli in Naturally Infected Live and Slaughtered Chicken Broilers Using a Real-Time Fluorescent Loop-Mediated Isothermal Amplification Approach.

Rapid and accurate identification of Campylobacter-positive broiler flocks and carcasses expedites separation and control interventions before release into the food supply chain and directly facilitates a reduction in the prevalence of human campylobacteriosis. In this study, the diagnostic performance of fluorescent loop-mediated isothermal amplification (LAMP) assays for the direct detection of Campylobacter jejuni and Campylobacter coli in broiler cloacal and cecal samples were evaluated and compared with that of turbidimetric LAMP approaches investigated previously. The duplex fluorescent LAMP assay had significantly higher ( P < 0.05) diagnostic sensitivity (93.1%, 54 of 58 samples) than did the turbidimetric LAMP assay (82.8%, 48 of 58 samples) for detecting C. jejuni and C. coli in broiler cloacal samples, whereas the singleplex fluorescent LAMP assay had equivalent diagnostic sensitivity. For cecal samples, the diagnostic sensitivity of the fluorescent LAMP assay (100%, 38 of 38 samples) was the same as that of the turbidimetric LAMP. Fluorescent LAMP significantly reduced ( P < 0.05) the maximum detection time for Campylobacter-positive cloacal and cecal samples to 28 and 11 min, respectively, and reduced the influence of amplification inhibitors responsible for most false-negative results obtained for cloacal samples with the turbidimetric LAMP assay. The diagnostic accuracy of the fluorescent LAMP assay for the direct detection of C. jejuni and C. coli in cloacal and cecal samples was 97.7 and 100%, respectively. These findings indicate that fluorescent LAMP assays are robust, highly accurate, and field-applicable methods for the identification of C. jejuni and C. coli, which will allow more accurate monitoring of food safety at various stages of the food supply chain at farms and slaughter facilities.

Clinical and microbiological characteristics of patients with bacteremia caused by Campylobacter species with an emphasis on the subspecies of C. fetus.

OBJECTIVES: This study was intended to investigate the clinical and microbiological characteristics of patients with bacteremia caused by Campylobacter species. METHODS: From April 1998 to May 2014, 56 adults with bacteremia caused by Campylobacter species were evaluated. These Campylobacter species isolates were confirmed to the species level using 16S rRNA gene sequencing (all isolates) and multiplex PCR analysis (for C. fetus only). The performance of identification for Campylobacter species by the Bruker Biotyper MALDI-TOF MS was evaluated. The genetic relatedness of C. fetus isolates was analyzed by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). RESULTS: The leading underlying medical conditions of these patients were malignancy (46.4%), hypertension (35.7%), and liver cirrhosis (23.2%). The overall 30-day mortality rate was 5.4%. Using 16S rRNA sequencing analysis, 26 isolates of C. coli, 11 of C. jejuni, and 19 of C. fetus, including 15 C. fetus subsp. fetus and five C. fetus subsp. venerealis, were identified. Among the five C. fetus subsp. venerealis isolates recognized by 16S rRNA gene sequencing, only two isolates were C. fetus subsp. venerealis by multiplex PCR method. The Bruker Biotyper MALDI-TOF MS failed to correctly identify C. fetus subsp. venerealis isolates. MLST analysis of C. fetus isolates revealed three STs: ST20 (n = 12), ST11 (n = 5), and ST57 (n = 2), which were compatible with three major PFGE clusters. CONCLUSION: Database expansion of MALDI-TOF MS for the correct identification of C. fetus to subspecies levels is needed. A novel clone of ST57-PFGE Cluster C of C. fetus subsp. venerealis was noted.