BACKGROUND:Thermal imagers have been used in a number of disciplines to record animal surface temperatures and as a result detect temperature distributions and abnormalities requiring a particular course of action. Some work, with animals infected with foot-and-mouth disease virus, has suggested that the technique might be used to identify animals in the early stages of disease. In this study, images of 19 healthy cattle have been taken over an extended period to determine hoof and especially coronary band temperatures (a common site for the development of FMD lesions) and eye temperatures (as a surrogate for core body temperature) and to examine how these vary with time and ambient conditions. RESULTS:The results showed that under UK conditions an animal's hoof temperature varied from 10°C to 36°C and was primarily influenced by the ambient temperature and the animal's activity immediately prior to measurement. Eye temperatures were not affected by ambient temperature and are a useful indicator of core body temperature. CONCLUSIONS:Given the variation in temperature of the hooves of normal animals under various environmental conditions the use of a single threshold hoof temperature will be at best a modest predictive indicator of early FMD, even if ambient temperature is factored into the evaluation.

Development of small footprint, disposable, fast, and inexpensive devices for pathogen detection in the field and clinic would benefit human and veterinary medicine by allowing evidence-based responses to future out breaks. We designed and tested an integrated nucleic acid extraction and amplification device employing a loop-mediated isothermal amplification (LAMP) or reverse transcriptase-LAMP assay. Our system provides a screening tool with polymerase-chain-reaction-level sensitivity and specificity for outbreak detection, response, and recovery. Time to result is ∼90 min. The device utilizes a swab that collects sample and then transfers it to a disc of cellulose-based nucleic acid binding paper. The disc is positioned within a disposable containment tube with a manual loading port. In order to test for the presence of target pathogens, LAMP reagents are loaded through the tube's port into contact with the sample containing cellulose disc. The reagents then are isothermally heated to 63°C for ∼1 h to achieve sequence-specific target nucleic acid amplification. Due to the presence of a colorimetric dye, amplification induces visible color change in the reagents from purple to blue. As initial demonstrations, we detected methicillin resistant Staphylococcus aureus genomic DNA, as well as recombinant and live foot-and-mouth disease virus.

A lateral flow device (LFD) for the detection of foot-and-mouth disease virus (FMDV) of the SAT 2 serotype was developed using a monoclonal antibody (Mab 2H6). The performance of the LFD was evaluated in the laboratory on suspensions of vesicular epithelia: 305 positive for FMDV type SAT 2 from suspected cases of vesicular disease collected from 30 countries and 1002 samples shown to be negative for FMDV type SAT 2 collected from 67 countries between 1968 and 2008. The diagnostic sensitivity of the LFD for FMDV type SAT 2 was higher at 88% compared to 79% obtained by the reference method of antigen ELISA, and the diagnostic specificity of the LFD was approximately 99% compared to 100% for the ELISA. The device recognized FMDV strains of wide diversity within the FMDV SAT 2 serotype and gave a superior performance for their detection compared to the 1F10 LFD which had been developed previously and shown to perform less well for the detection of FMDVs of this particular serotype. Reactions in the SAT 2 2H6 LFD with the viruses of other FMDV serotypes and swine vesicular disease (which produces a clinically indistinguishable syndrome in pigs), did not occur. These data illustrate the potential for the LFD to be employed to complement the 1F10 device next to the animal in the pen-side diagnosis of FMD, for providing rapid and objective support to veterinarians in their clinical judgment of the disease and for specific confirmation of a FMDV type SAT 2 infection.

Rapid and accurate diagnosis is essential for effective control of foot-and-mouth disease (FMD). The present report describes the practical steps undertaken to deploy a real-time reverse transcription polymerase chain reaction (real-time RT-PCR) to process the samples received during the outbreaks of FMD in the United Kingdom in 2007. Two independent real-time RT-PCR assays targeting different regions (5′UTR and 3D) of the FMD virus (FMDV) genome were used to confirm the presence of FMDV in clinical samples collected from the first infected premises. Once the FMDV strain responsible had been sequenced, a single real-time RT-PCR assay (3D) was selected to test a total of 3,216 samples, including material from all 8 infected premises. Using a 96-well automated system to prepare nucleic acid template, up to 84 samples could be processed within 5 hr of submission, and up to 269 samples were tested per working day. A conservative cut-off was used to designate positive samples, giving rise to an assay specificity of 99.9% or 100% for negative control material or samples collected from negative premises, respectively. For the first time, real-time RT-PCR results were used to recognize preclinical FMD in a cattle herd. Furthermore, during the later stages of the outbreaks, the real-time RT-PCR assay supported an active surveillance program within high-risk cattle herds. To the authors' knowledge, this is the first documented use of real-time RT-PCR as a principal laboratory diagnostic tool following introduction of FMD into a country that was FMD-free (without vaccination) and highlights the advantages of this assay to support control decisions during disease outbreaks.

To test the hypothesis that relaxin is an important factor supporting implantation, two approaches have been carried out using a human‐relevant animal model, the marmoset monkey. First, uterine mRNA transcription and protein expression during the implantation phase in the conceptive and nonconceptive cycles were examined. Second, functional parameters were analyzed to assess the in vivo effects of exogenous applied relaxin throughout implantation. Relaxin and its receptor, RXFP1, were highly upregulated shortly before and during the physical process of implantation, indicating that relaxin is an important factor for remodeling and immunotolerance. The action of relaxin on the uterus was accompanied by an increase of estrogen‐associated factors and macrophage infiltration, suggesting redundant systems necessary for successful implantation. The data from relaxin‐treated animals supported those obtained from naive tissues in terms of increases in angiogenesis as well as earlier and faster growth of the uterus and placenta in the relaxin‐treated marmoset monkey group, resulting in parturition 7–10 days earlier than the control group, but not pathological. In general, relaxin is very effective in preparing the endometrium for implantation. These findings should encourage further clinical research regarding introducing relaxin for pathological pregnancies, such as early pregnancy failure or insufficient placenta.

A high-throughput multiplexed assay was developed for the differential laboratory detection of foot-and-mouth disease virus (FMDV) from viruses that cause clinically similar diseases of livestock. This assay simultaneously screens for five RNA and two DNA viruses by using multiplexed reverse transcription-PCR (mRT-PCR) amplification coupled with a microsphere hybridization array and flow-cytometric detection. Two of the 17 primer-probe sets included in this multiplex assay were adopted from previously characterized real-time RT-PCR (rRT-PCR) assays for FMDV. The diagnostic accuracy of the mRT-PCR assay was evaluated using 287 field samples, including 247 samples (213 true-positive samples and 35 true-negative samples) from suspected cases of foot-and-mouth disease collected from 65 countries between 1965 and 2006 and 39 true-negative samples collected from healthy animals. The mRT-PCR assay results were compared to those of two singleplex rRT-PCR assays, using virus isolation with antigen enzyme-linked immunosorbent assays as the reference method. The diagnostic sensitivity of the mRT-PCR assay for FMDV was 93.9% (95% confidence interval [CI], 89.8 to 96.4%), and the sensitivity was 98.1% (95% CI, 95.3 to 99.3%) for the two singleplex rRT-PCR assays used in combination. In addition, the assay could reliably differentiate between FMDV and other vesicular viruses, such as swine vesicular disease virus and vesicular exanthema of swine virus. Interestingly, the mRT-PCR detected parapoxvirus (n = 2) and bovine viral diarrhea virus (n = 2) in clinical samples, demonstrating the screening potential of this mRT-PCR assay to identify viruses in FMDV-negative material not previously recognized by using focused single-target rRT-PCR assays.

Foot-and-mouth disease (FMD) virus causes an acute vesicular disease of domesticated and wild ruminants and pigs. Identifying sources of FMD outbreaks is often confounded by incomplete epidemiological evidence and the numerous routes by which virus can spread (movements of infected animals or their products, contaminated persons, objects, and aerosols). Here, we show that the outbreaks of FMD in the United Kingdom in August 2007 were caused by a derivative of FMDV O(1) BFS 1860, a virus strain handled at two FMD laboratories located on a single site at Pirbright in Surrey. Genetic analysis of complete viral genomes generated in real-time reveals a probable chain of transmission events, predicting undisclosed infected premises, and connecting the second cluster of outbreaks in September to those in August. Complete genome sequence analysis of FMD viruses conducted in real-time have identified the initial and intermediate sources of these outbreaks and demonstrate the value of such techniques in providing information useful to contemporary disease control programmes.

This report describes the generation of novel encapsidated RNA particles and their evaluation as in-tube internal controls in diagnostic real-time reverse-transcription PCR (rRT-PCR) assays for the detection of RNA viruses. A cassette containing sequences of 2 diagnostic primer sets for foot-and-mouth disease virus (FMDV) and a set for swine vesicular disease virus (SVDV) was engineered into a full-length cDNA clone containing the RNA-2 segment of Cowpea Mosaic Virus (CPMV). After co-inoculation with a plasmid that expressed CPMV RNA-1, recombinant virus particles were rescued from cowpea plants (Vigna unguiculata). RNA contained in these particles was amplified in diagnostic rRT-PCR assays used for detection of FMDV and SVDV. Amplification of these internal controls was used to confirm that rRT-PCR inhibitors were absent from clinical samples, thereby verifying negative assay results. The recombinant CPMVs did not reduce the analytical sensitivity of the rRT-PCRs when amplification of the insert was performed in the same tube as the diagnostic target. This system provides an attractive solution to the production of internal controls for rRT-PCR assays since CPMV grows to high yields in plants, the particles are thermostable, RNase resistant and simple purification of RNA-2 containing capsids yields a preparation which is non-infectious.

The younger generation's hesitancy towards the COVID-19 vaccine in Japan received significant attention during the early stages of vaccination. However, there is a lack of a comprehensive study in Japan that analyzes the apprehension towards the third dose of vaccine, commonly known as the booster dose, and its underlying causes. Using data from an online panel survey conducted by the Hiroshima Institute of Health Economics Research at Hiroshima University, we examined the severity of booster dose aversion among youths of different ages. Our findings indicate that a sizeable proportion of the Japanese population, particularly younger men, are hesitant to receive the booster dose. Furthermore, an inter-age group difference in booster dose aversion exists only among men. According to the probit regression results, subjective health status and future anxiety are associated with the booster vaccine hesitancy of men and women of various age groups. Moreover, few socioeconomic and behavioral factors like marital status, having children, household income and assets, and having a myopic view of the future, are also associated with the booster dose aversion among youths of certain ages. Given the diverse attitude of the younger generation, our findings suggest that public health authorities should develop effective communication strategies to reduce vaccine apprehension in the society.

We describe the characterization of a foot-and-mouth disease (FMD) serotype A virus responsible for recent outbreaks of disease in Egypt. Phylogenetic analysis of VP1 nucleotide sequences demonstrated a close relationship to recent FMD virus isolates from East Africa, rather than to viruses currently circulating in the Middle East.

A novel proximity ligation assay (PLA) using a pan-serotype reactive monoclonal antibody was developed and evaluated for the detection of foot-and-mouth disease virus (FMDV) in clinical samples collected from field cases of disease. The FMDV-specific PLA was found to be 100 times more sensitive for virus detection than the commonly used antigen capture-ELISA (AgELISA). As few as five TCID50 were detected in individual assays, which was comparable with the analytical sensitivity of real-time RT-PCR. Although this assay was capable of detecting diverse isolates from all seven FMDV serotypes, the diagnostic sensitivity of the PLA assay was lower than real-time RT-PCR mainly due to a failure to detect some SAT 1, SAT 2 and SAT 3 FMDV strains. In conclusion, this new PLA format has high analytical sensitivity for the detection of FMDV in clinical samples and may prove valuable as a rapid and simple tool for use in FMD diagnosis.

Rapid and accurate diagnosis is needed for effective control of foot-and-mouth disease (FMD). Laboratory-based methods such as virus isolation, antigen-ELISA and real-time RT-PCR (rRT-PCR) can provide an objective result within a few hours of sample receipt. However, the time taken to transport suspect material to a centralized laboratory can be unacceptably long, often precluding laboratory confirmation in the event of an outbreak. There are opportunities to deploy mobile rRT-PCR assays inside a vehicle, or in local laboratories, for rapid diagnosis of suspect cases. These assays typically require pre-processing steps of samples (such as RNA extraction) which demands trained personnel. Portable PCR technology has also recently been developed with the potential for pen-side or “point of care” diagnosis. These field-based assays could be operated by untrained personnel without prior knowledge of molecular biology. In contrast to laboratory-based assays, where high sample throughput is a major prerogative, portable assays focus on speed in order to generate a diagnostic result within one hour of sample collection. To determine whether mobile and portable platforms might be used to carry out molecular diagnosis closer to suspect cases of FMD, two instruments were evaluated: (i) a “mobile” nucleic acid extraction robot (BioRobot EZ1, Qiagen) was compared with centralized robotic extraction equipment currently used to prepare RNA for FMD virus (FMDV) detection and (ii) the portable Bioseeq platform (Smiths Detection) was assessed as to whether this instrument could be used for pen-side diagnosis of FMD by rRT-PCR. Both instruments provide realistic options for performing molecular assays for FMDV away from centralised laboratories. Introduction: Rapid and accurate diagnosis is needed for effective control and eradication of FMD. Laboratory-based methods such as virus isolation (VI), antigen detection ELISA (Ferris and Dawson, 1988) and real-time RT-PCR (rRT-PCR) (Reid et al., 2003; Shaw et al., 2004) can provide an objective result within a few hours of sample receipt. However, the time taken to transport suspect material to a centralized laboratory can be unacceptably long, often precluding laboratory confirmation in the event of an outbreak. Using existing available equipment, there are opportunities to deploy mobile rRT-PCR assays inside a vehicle, or in local laboratories, for rapid diagnosis of suspect cases (Callahan et al., 2002; Hearps et al., 2002). These assays typically require pre-processing of samples (such as RNA extraction) which demands trained personnel. Portable PCR technology has also recently been developed with the potential for pen-side or “point of care” diagnosis. Field-based assays like these could be operated by untrained personnel without prior knowledge of molecular biology. In contrast to laboratory-based assays, where high sample throughput is a major prerogative, portable assays focus on speed in order to generate a diagnostic result within one hour of sample collection. To determine whether mobile and portable platforms might be used to carry out molecular diagnosis closer to suspect cases of FMD, two instruments were evaluated. Firstly, the performance of a “mobile” nucleic acid extraction robot (BioRobot EZ1, Qiagen) was compared with centralized robotic extraction equipment currently used to prepare RNA for FMDV detection. Secondly, the portable Bioseeq platform (Smiths Detection, Watford, UK) was assessed as to whether this instrument could be used for pen-side diagnosis of FMD by rRTPCR. Materials and methods: Comparison of the BioRobot EZ1 RNA extraction instrument with other extraction methods for diagnosis of FMD An initial evaluation of the Qiagen BioRobot EZ1 was performed by comparing RNA extracted by this potential mobile device with other robotic instruments, namely the Qiagen BioRobot 9604 and the MagNA Pure LC (Roche) (Fig. 1). The QIAamp Viral RNA Mini Kit (Qiagen) was also included to compare the performance of a manual extraction method. Decimal dilutions of an ~10% epithelial suspension of the reference FMDV strain O1 Manisa (TUR 8/69) were prepared in suspensions of uninfected negative bovine epithelium and 200μl of each dilution was lysed in 300μl of the accompanying lysis buffer as appropriate (Table 1). For the manual RNA extractions, 140μl of the dilutions were lysed in 560μl of AVL

This Brief Report describes a patient with the rare syndrome of tumor-induced osteomalacia, which is accompanied by phosphaturia and hypophosphatemia. Renal phosphate wasting, presumably caused by phosphatonins, was abolished by octreotide, a synthetic somatostatin analogue, before successful surgical removal of a benign hemangiopericytoma.

Glycoprotein B (gB) is involved in cell to cell transmission of human cytomegalovirus (HCMV) and may be a critical factor in tissue tropism and viral pathogenesis. We analyzed the distribution of the four known gB genotypes of HCMV in 99 HIV-positive patients. 29 patients had HCMV retinitis, and 70 patients had asymptomatic HCMV infection. DNA was isolated from blood, urine, and aqueous humor, and gB genotypes were determined by PCR and restriction analysis. Infections with gB type 1 were less frequent in patients with retinitis than in patients with asymptomatic HCMV infection (17% versus 37%; p = 0.05). Furthermore, the gB type was correlated with dissemination of infection. In patients with HCMV detected in only one compartment (blood or urine) the gB type 1 was found more frequently than in patients with HCMV detected in at least two compartments (p = 0.01). The data show that gB genotypes differ in their association with clinical disease, and indicate that the gB genotype may contribute to the course of HCMV infection.

Alateralflowdevice (LFD) forthepensidediagno sisofFMDVhasbeendevelopedusinga monoclonalantibody(Mab1F10)andevaluatedinthe laboratoryforitssensitivityandspecificity forthedetectionofFMDV.Suspensionsof1288vesi cularepitheliaandtwovesicularfluidsfrom worldwidesuspectcasesofFMDwereevaluatedbyth eLFD. Thecollectionrepresented 304 samplesofdifferentgeographicaloriginandantige nicandmolecularvariationwithineachofthe FMDVserotypesand986samplesinwhichFMDVhadno tbeendetected.Additionally,fivesamples containingvirusesoftheothervesiculardiseases ofSVDandfiveofVSplussevenfromtrue negativesampleswerealsotested.Simplesampleho mogenisers(adisposablepelletpestlewith microtubeandaplasticrodwithdisposablebijou) wereevaluatedfortheirpotentialabilitiesto prepareepithelialsuspensionsunderfieldconditio nsforLFDuse. TheLFDdetectedantigensofFMDVofwidediversity