A habitat-based framework to predict the effects of agricultural drain maintenance on imperiled fishes.

One third of the total global land viable for agricultural production has artificial drainage systems. These drainage systems can provide important habitat for fishes and, in some cases, imperiled fish species vulnerable to impact by drainage maintenance activities. A framework to provide quantitative assessments of the effects of maintenance activities on imperiled fish species is needed. In this study, a six-step habitat-based framework was developed to predict suitable habitat for two at-risk species in an agricultural drain: the Endangered Pugnose Shiner (Notropis anogenus) and the Special Concern Blackstripe Topminnow (Fundulus notatus). Using the framework, spatial models were developed to assess the effects of proposed drain maintenance on the overall amount of suitable habitat, habitat patch size, and connectivity of habitat patches. Maintenance had a significant impact on habitat connectivity, but did not significantly reduce the habitat size of isolated patches. The amount of suitable habitat available after maintenance fell below the minimum area for population viability (MAPV) for the Pugnose Shiner, but not the Blackstripe Topminnow. Future impact assessments of drain maintenance should incorporate population viability analysis, coupled with habitat patch analysis (patch size and connectivity), to quantitatively test consequences of proposed alteration to the viability of spatially structured populations.

Exploring quality of life of children with cerebral palsy and intellectual disability: What are the important domains of life?

BACKGROUND: Although it is estimated that half of all children with cerebral palsy also have comorbid intellectual disability, the domains of quality of life (QOL) important for these children are not well understood. The aim of this study was to identify important domains of QOL for these children and adolescents. METHODS: Due to the children's communication impairments, qualitative semi-structured interviews were conducted with 18 parents. The children (9 males) had a median age of 12 (range 7 to 17) years at interview and nearly two thirds were classified as Gross Motor Function Classification System IV or V. A grounded theory approach was used to identify domains of QOL. RESULTS: The 11 domains identified as important to QOL were physical health, body comfort, behaviour and emotion, communication, predictability and routine, movement and physical activity, nature and outdoors, variety of activity, independence and autonomy, social connectedness, and access to services. CONCLUSIONS: The domains of QOL that emerged from this study will be useful for professionals who support children with cerebral palsy and their families. They will also be important for developing a QOL instrument essential for informing the development of interventions and their monitoring and evaluation.

Tertiary paediatric hospital admissions in children and young people with cerebral palsy.

BACKGROUND: Many previous studies that have investigated hospital admissions in children and young people with cerebral palsy lack information on cerebral palsy severity and complexity. Consequently, little is known about factors associated with the frequency and type of hospital admissions in this population. This study used hospital admission data available for all children and young people known to a population-based cerebral palsy register to describe the patterns of use of tertiary paediatric hospital services over a 5-year period. METHODS: This was a retrospective cohort analysis of routinely collected admission data from the two tertiary paediatric hospitals in the Australian state of Victoria. Data on admissions of individuals born between 1993 and 2008 registered on the Victorian Cerebral Palsy Register were analysed (n = 2183). RESULTS: Between 2008 and 2012, 53% of the cohort (n = 1160) had at least one same-day admission, and 46% (n = 996) had one or more multi-day admissions. Those with a moderate to severe motor impairment and those with a co-diagnosis of epilepsy had more admissions, and for multi-day admissions, longer lengths of stay, P < 0.05. Across all severity levels, respiratory and musculoskeletal diseases were the most frequently reported reasons for medical and surgical admissions, respectively. All-cause readmission rates for urgent multi-day stays within 7, 30 and 365 days of an index admission were 10%, 23% and 63%, respectively. CONCLUSIONS: The reasons for hospital admissions reported here reflect the range of comorbidities experienced by children and young people with cerebral palsy. This study highlights priority areas for prevention, early diagnosis and medical management in this group. Improved primary and secondary prevention measures may decrease non-elective hospital admissions and readmissions in this group and reduce paediatric inpatient resource use and healthcare expenditure attributable to cerebral palsy.

Design and optimization of a novel reverse transcription linear-after-the-exponential PCR for the detection of foot-and-mouth disease virus.

AIMS: A novel molecular assay for the detection of foot-and-mouth disease virus (FMDV) was developed using linear-after-the-exponential polymerase chain reaction (LATE-PCR). METHODS AND RESULTS: Pilot experiments using synthetic DNA targets demonstrated the ability of LATE-PCR to quantify initial target concentration through endpoint detection. A two-step protocol involving reverse transcription (RT) followed by LATE-PCR was then used to confirm the ability of the assay to detect FMDV RNA. Finally, RT and LATE-PCR were combined in a one-step duplex assay for co-amplification of an FMDV RNA segment and an internal control comprised of an Armored RNA. In that form, each of the excess primers in the reaction mixture hybridize to their respective RNA targets during a short pre-incubation, then generate cDNA strands during a 3-min RT step at 60°C, and the resulting cDNA is amplified by LATE-PCR without intervening sample processing. CONCLUSIONS: The RT-LATE-PCR assay generates fluorescent signals at endpoint that are proportional to the starting number of RNA targets and can detect a range of sequence variants using a single mismatch-tolerant probe. SIGNIFICANCE AND IMPACT OF THE STUDY: In addition to offering improvements over current laboratory-based molecular diagnostic assays for FMDV, this new assay is compatible with a novel portable ('point-of-care') device, the BioSeeq II, designed for the rapid diagnosis of FMD in the field.

Clinical and laboratory investigations of the outbreaks of foot-and-mouth disease in southern England in 2007.

A case of foot-and-mouth disease (fmd) on a cattle farm in Normandy, Surrey, was confirmed on Friday August 3, 2007, the first case in the uk since 2001. The infection was detected nearby on a second farm on August 6. On September 12, fmd was confirmed on a farm approximately 20 km from Normandy in Egham, and this was followed by cases on five more farms in that area in the next three weeks. The majority of the infected farms consisted of multiple beef cattle holdings in semi-urban areas. In total, 1578 animals were culled on the infected farms, and fmd virus infection was confirmed in 278 of them by the detection of viral antigen, genome or antibodies to the virus, or by clinical signs. This paper describes the findings from animal inspections on the infected farms, including the estimated ages of the fmd lesions and the numbers of animals infected. It also summarises the test results from samples taken for investigation, including the detection of preclinically viraemic animals by using real-time reverse transcriptase-pcr.

Evaluation of laboratory tests for SAT serotypes of foot-and-mouth disease virus with specimens collected from convalescent cattle in Zimbabwe.

During a field study in Zimbabwe, clinical specimens were collected from 403 cattle in six herds, in which the history of foot-and-mouth disease (FMD) vaccination and infection appeared to be known with some certainty. Five herds had reported outbreaks of disease one to five months previously but clinical FMD had not been observed in the sixth herd. A trivalent vaccine (South African Territories [SAT] types 1, 2 and 3) had been used in some of the herds at various times either before and/or after the recent outbreaks of FMD. The primary aim of this study was to evaluate the performance of serological tests for the detection of SAT-type FMD virus infection, particularly elisas for antibodies to non-structural proteins (NSPs) of FMD virus and solid phase competition ELISAS (SPCEs) for serotypes SAT1 and SAT2. Secondary aims were to examine NSP seroconversion rates in cattle that had been exposed to infection and to compare virus detection rates by virus isolation and real-time reverse transcriptase-PCR (rtRT-PCR) tests on both oesophagopharyngeal fluids and nasopharyngeal brush swabbings. In addition, the hooves of sampled animals were examined for growth arrest lines as clinical evidence of FMD convalescence. Laboratory tests provided evidence of FMD virus infection in all six herds; SAT2 viruses were isolated from oesophagopharyngeal fluids collected from two herds in northern Zimbabwe, and SAT1 viruses were isolated from three herds in southern Zimbabwe. Optimised rtRT-PCR was more sensitive than virus isolation at detecting FMD virus persistence and when the results of the two methods were combined for oesophagopharyngeal fluids, between 12 and 35 per cent of the cattle sampled in the convalescent herds were deemed to be carriers. In contrast, nasopharyngeal swabs yielded only two virus-positive specimens. The overall seroprevalence in the five affected herds varied with the different NSPS from 56 per cent to 75 per cent, compared with 81 per cent and 91 per cent by homologous SPCE and virus neutralisation tests respectively. However, if serological test results were considered only for the cattle in which persistent infection with FMD virus had been demonstrated, 70 to 90 per cent scored seropositive in the different NSPs.

Foot-and-mouth disease virus: a first inter-laboratory comparison trial to evaluate virus isolation and RT-PCR detection methods.

Five European reference laboratories participated in an exercise to evaluate the sensitivity and specificity of their routinely employed RT-PCR tests and cell cultures for the detection and isolation of foot-and-mouth disease (FMD) virus. Five identical sets of 20 coded samples were prepared from 10 vesicular epithelia, which were derived from submissions from suspect cases of FMD or swine vesicular disease (SVD). Sixteen samples were derived from six FMD virus positive epithelia representing four different serotypes (two each of types O and A and one each of types Asia 1 and SAT 2), two from samples which had been found to be negative by antigen ELISA and virus isolation (VI) in cell culture and two from SVD virus positive epithelia. Some of the FMD virus positive samples were prepared from 10-fold serial dilutions of three of the initial suspensions. Each laboratory tested the samples by one or more of its available RT-PCR procedures and inoculated cell cultures that it routinely uses for FMD diagnosis in attempts to isolate virus, the specificity of which was confirmed by antigen ELISA. The best of the RT-PCR assays used in each laboratory gave comparable results while the sensitivity of cell cultures was variable from high in one laboratory, moderate in two and low in two others. This prototype panel of samples would appear suitable for external quality assurance of these tests but would benefit from the inclusion of more negative samples and an extension in the serial dilution range of one or more of the FMD positive sample titration series.

Comparisons of original laboratory results and retrospective analysis by real-time reverse transcriptase-PCR of virological samples collected from confirmed cases of foot-and-mouth disease in the UK in 2001.

There were 2030 designated cases of foot-and-mouth disease (FMD) during the course of the epidemic in the UK in 2001 (including four from Northern Ireland). Samples from 1720 of the infected premises (IPs) were received in the laboratory and examined for either the presence of FMD virus (virological samples from 1421 IPs) or both FMD virus and antibody (virological and serological samples from 255 IPs) or antibody alone (from 44 IPs). The time taken to issue final diagnostic results ranged from a few hours in cases in which positive results were obtained by ELISA on epithelia containing sufficient virus to be detected, to several days for samples containing small amounts of virus requiring amplification through cell culture, negative samples or samples tested for antibody. Two subsets of samples were analysed retrospectively by real-time reverse transcriptase-PCR (RT-PCR); first, epithelia that were negative by both ELISA and virus isolation (VI) in cell culture, and secondly, samples that were negative by ELISA on epithelial suspension but positive by VI. There was broad agreement between the RT-PCR and VI/ELISA combined, except that the RT-PCR procedure did not detect a group of related virus isolates from Wales. These viruses had evidently evolved during the epidemic and had a nucleotide substitution in the RT-PCR probe site, which prevented them from being detected by the routine diagnostic probe. No evidence of FMD virus, antibody or nucleic acid was found in approximately 23 per cent (390 of 1730) of IPs from which samples were received, suggesting that the incidence of FMD during the outbreak may have been over-reported.

Highly Pathogenic Avian Influenza H5N8 Clade 2.3.4.4 Virus: Equivocal Pathogenicity and Implications for Surveillance Following Natural Infection in Breeder Ducks in the United Kingdom.

Since early 2014, several outbreaks involving novel reassortant highly pathogenic avian influenza (HPAI) A(H5N8) viruses have been detected in poultry and wild bird species in Asia, Europe and North America. These viruses have been detected in apparently healthy and dead wild migratory birds, as well as in domestic chickens, turkeys, geese and ducks. In this study, we describe the pathology of an outbreak of H5N8 HPAIV in breeder ducks in the UK. A holding with approximately 6000 breeder ducks, aged approximately 60 weeks, showed a gradual reduction in egg production and increased mortality over a 7-day period. Post-mortem examination revealed frequent fibrinous peritonitis, with severely haemorrhagic ovarian follicles and occasional splenic and pancreatic necrosis and high incidence of mycotic granulomas in the air sacs and lung. Low-to-moderate levels of HPAI H5N8 virus were detected mainly in respiratory and digestive tract, with minor involvement of other organs. Although histopathological examination confirmed the gross pathology findings, intralesional viral antigen detection by immunohistochemistry was not observed. Immunolabelled cells were rarely only present in inflamed air sacs and serosa, usually superficial to granulomatous inflammation. Abundant bacterial microcolonies were observed in haemorrhagic ovaries and oviduct. The limited viral tissue distribution and presence of inter-current fungal and bacterial infections suggest a minor role for HPAIV H5N8 in clinical disease in layer ducks.

Primary diagnosis of foot-and-mouth disease by reverse transcription polymerase chain reaction.

Universal and serotype-specific primer sets were used in simple reverse transcription polymerase chain reaction (RT-PCR) assays on field samples of epithelium and vesicular fluid to determine their suitability for primary diagnosis of all seven serotypes of foot-and-mouth disease (FMD). The specificity of reactions was confirmed by using other vesicular disease viruses, namely: swine vesicular disease virus, vesicular stomatitis virus and three vesiviruses. This resulted in the identification of a universal O/A/C/Asia 1 primer set (1F/1R) located in the 5' untranslated region (UTR) of the FMD virus genome for the successful detection of virus of these serotypes in clinical samples although this primer set detected FMD virus of the SAT1/2/3 serotypes less efficiently. The 5' UTR universal primer set could be used for the primary diagnosis of FMD in conjunction with the routine diagnostic methods of virus isolation in cell culture and ELISA, although a more favourable reaction would be expected with FMD viruses of the O/A/C/Asia 1 group than with those of the SAT serotypes. The other examined universal and serotype-specific primer sets, located principally in the P1 capsid-coding region, were generally inferior to the 5' UTR universal primer set. It is envisaged that this evaluation of primers will lead to the development of alternative PCR strategies, for example nested PCR formats, with concomitant improvement in the speed of primary diagnosis of FMD which under present procedures can be lengthy.

Comparison of reverse transcription polymerase chain reaction, enzyme linked immunosorbent assay and virus isolation for the routine diagnosis of foot-and-mouth disease.

A reverse transcription polymerase chain reaction (RT-PCR) method was compared with virus isolation in cell culture and the antigen detection ELISA for the primary diagnosis of foot-and-mouth disease (FMD) on 166 clinical samples from the field. Eighty samples were positive by virus isolation/ELISA and 78 by RT-PCR. The RT-PCR detected FMD viral RNA in 11 of the 86 samples assessed as negative by virus isolation/ELISA but conversely failed to diagnose 13 samples identified as positive by the latter procedures. This RT-PCR is not serotype-specific so a cDNA product is indicative of the presence of FMD viral RNA only. Confirmation of the specificity of the cDNA product and the identification of the serotype requires nucleotide sequence analysis. The value of the RT-PCR is that it can rapidly facilitate the molecular analysis of field isolates and thus provide important epidemiological information regarding the source of outbreaks. However, it is a sophisticated technique requiring specialised equipment, expertise and refined reagents and has to be used in conjunction with current procedures for FMD diagnosis.

Diagnosis of foot-and-mouth disease by RT-PCR: evaluation of primers for serotypic characterisation of viral RNA in clinical samples.

Multiple primers designed from the 1D and 2AB regions of the foot-and-mouth disease (FMD) viral genome were evaluated extensively for the detection of all seven serotypes of the virus by reverse transcription polymerase chain reaction (RT-PCR) at the OIE/FAO World Reference Laboratory for FMD (WRL), Pirbright. The primers had been characterised previously elsewhere on a relatively small number of cell culture grown isolates and epithelial suspensions and had been shown to identify and differentiate all seven serotypes of FMD virus. The extended study evaluated several RT-PCR protocols on epithelial suspensions and supernatant fluids, resulting from their passage in cell culture, derived from clinical samples of diverse molecular characteristics. Each of the serotype-specific primers in selected RT-PCR protocols demonstrated suitable specificity and detected cell culture passaged isolates with some success but were not adequate for the serotyping of suspensions prepared from clinical samples of epithelium. The results showed that the primers can be used in RT-PCR procedures in conjunction with the routine detection methods of virus isolation and ELISA for the diagnosis and serotyping of FMD virus.

Development of a rapid chromatographic strip test for the pen-side detection of foot-and-mouth disease virus antigen.

Foot-and-mouth disease (FMD) is the most contagious animal virus disease of cloven-hoofed livestock and requires reliable and accurate diagnosis for the implementation of measures to control effectively its spread. Routine diagnosis of FMD is carried out at the OIE/FAO World Reference Laboratory for Foot-and-Mouth Disease (WRL for FMD), Pirbright by the combined use of ELISA and virus isolation in cell culture supplemented by reverse transcription polymerase chain reaction (RT-PCR) methods. These techniques require skilled personnel and dedicated laboratory facilities which are expensive. The development of a rapid and simple test for the detection of FMD virus antigen using Clearview chromatographic strip test technology for field application is described. This device detected FMD viral antigen in nasal swabs, epithelial suspensions and probangs from clinical samples submitted from the field, from animals infected experimentally and in supernatant fluids resulting from their passage in cell culture. The test system was more sensitive than ELISA for the diagnosis of all seven serotypes of FMD virus in the epithelial suspensions and nasal swabs and had equivalent sensitivity to the ELISA for the detection of contemporary virus strains in cell culture supernatant fluids. The study demonstrated the potential for this device to confirm a clinical diagnosis at the site of a suspected FMD outbreak, thereby offering the possibility of implementing control procedures more rapidly. Such pen-side diagnosis would have particular benefits in FMD emergencies, relevance to FMD control programmes which operate in endemic regions of the world such as South East Asia and for increasing disease awareness in other areas where efforts to control disease may be difficult. In each circumstance the availability of a pen-side device for diagnosis would reduce the necessity for sending routine diagnostic samples to an FMD laboratory and thereby reduce the delay in diagnosis, which can in some areas be considerable.

Development of a reverse transcription polymerase chain reaction procedure for the detection of marine caliciviruses with potential application for nucleotide sequencing.

A reverse transcription polymerase chain reaction (RT-PCR) procedure is described for the detection of marine caliciviruses including vesicular exanthema of swine virus (VESV), San Miguel sea lion virus (SMSV), bovine Tillamook virus (BCV Bos-1) and caliciviruses (CV) isolated from dolphin (Cetacean CV), gorilla (Primate CV) and rattlesnake (Reptile CV) using primers (1F and 1R) designed from the capsid-coding region of the viral genome. These primers were compared with those described by Neill, J.D. and Seal, B.S., 1995: Development of PCR primers for specific amplification of two distinct regions of the genomes of San Miguel sea lion and vesicular exanthema of swine viruses, Mol. Cell. Probes 9, 33-38 (Hel1/Hel2), which had been designed from the 2C-like region of the calicivirus genome. Both sets proved to be extremely useful diagnostic tools for all of the known marine calicivirus serotypes with the exception of three: SMSV-8 and -12 and mink CV suggesting that these three caliciviruses may belong to a different group. Neither of the two primer sets reacted with strains of the vesicular disease viruses of foot-and-mouth disease (FMD), swine vesicular disease (SVD) or vesicular stomatitis (VS) nor with two feline caliciviruses (FCV). The 1F/1R primer set has the advantage over the Hel1/Hel2 set in that it generates a larger PCR product for nucleotide sequence investigations and so provides greater opportunity for identifying molecular differences between the viruses.

Use of automated real-time reverse transcription-polymerase chain reaction (RT-PCR) to monitor experimental swine vesicular disease virus infection in pigs.

Automated real-time RT-PCR was evaluated as a diagnostic tool for swine vesicular disease virus (SVDV) infection on a range of samples (vesicular epithelium, serum, nasal swabs, faeces) from four inoculated and three in-contact pigs over a period of 28 days. Traditional diagnostic procedures (virus isolation, and ELISAs for antigen and antibody) were used in parallel. Each inoculated pig developed a significant viraemia and clinical disease, and excreted virus, which was transmitted to the in-contact animals. The latter, however, developed only a short-lived, low-level viraemia and no clinical disease. The RT-PCR and virus isolation were generally comparable in detecting SVDV in the serum and nasal swabs from inoculated and in-contact pigs up to day 6 after infection; it was possible, however, to isolate virus for a longer period from the faeces of a few pigs. This suggested that further optimization of the template extraction method was required to counteract the effects of RT-PCR inhibitors in faeces. It was concluded that the automated real-time RT-PCR is a useful diagnostic method for SVD in clinically or subclinically affected pigs and contributed to the study of the pathogenesis of SVD in the pigs.

Enhanced laboratory diagnosis of foot and mouth disease by real-time polymerase chain reaction.

The performance of an automated real-time reverse transcription polymerase chain reaction (RT-PCR) was compared to virus isolation (VI) in cell culture and antigen detection enzyme-linked immunosorbent assay (ELISA) for the laboratory diagnosis of foot and mouth disease (FMD). The World Reference Laboratory for FMD in Woking, the United Kingdom, examined a collection of 334 epithelia received from eighteen countries between August 2002 and January 2004. The results showed that all VI positive (n = 195) and VI and ELISA positive samples combined (n = 204) were also positive by RT-PCR. Depending on the cut-off used, FMD virus genome was detected in a minimum of an additional 60 samples (18% of all samples tested). Furthermore, the RT-PCR generated results in less than one day from test commencement in contrast to up to 4 days to define some positive and all negative samples by VI. The study demonstrates that real-time RT-PCR provides an extremely sensitive and rapid procedure for improved laboratory diagnosis of FMD.

A neurological disorders workshop for school nurses.

The Neurological Disorders Workshop was a continuing education experience for school nurses in central Vermont. It examined neurologically-related clinical problems commonly encountered by school nurses such as neurological screening, mental retardation, seizure disorders, motor system disorders, attention and behavior disorders, diagnostic screening tools, and referral sources. The six-day workshop (five consecutive days plus a follow up four months later) used didactic presentations, demonstrations, and practicum experiences. After the workshop, written evaluations were conducted.

First reported detection of a low pathogenicity avian influenza virus subtype H9 infection in domestic fowl in England.

In December 2010, infection with a H9N1 low pathogenicity avian influenza (LPAI) virus was detected in a broiler breeder flock in East Anglia. Disease suspicion was based on acute drops in egg production in two of four sheds on the premises, poor egg shell quality and evidence of diarrhoea. H9N1 LPAI virus infection was confirmed by real-time reverse transcription PCR. Sequencing revealed high nucleotide identity of 93.6 per cent and 97.9 per cent with contemporary North American H9 and Eurasian N1 genes, respectively. Attempted virus isolation in embryonated specific pathogen free (SPF) fowls' eggs was unsuccessful. Epidemiological investigations were conducted to identify the source of infection and any onward spread. These concluded that infection was restricted to the affected premises, and no contacts or movements of poultry, people or fomites could be attributed as the source of infection. However, the infection followed a period of extremely cold weather and snow which impacted on the biosecurity protocols on site, and also led to increased wild bird activity locally, including waterfowl and game birds around the farm buildings. Analysis of the N1 gene sequence suggested direct introduction from wild birds. Although H9 infection in poultry is not notifiable, H9N2 LPAI viruses have been associated with production and mortality episodes in poultry in many parts of Asia and the Middle East. In the present H9N1 outbreak, clinical signs were relatively mild in the poultry with no mortality, transient impact on egg production and no indication of zoonotic spread. However, this first reported detection of H9 LPAI virus in chickens in England was also the first H9 UK poultry case for 40 years, and vindicates the need for continued vigilance and surveillance of avian influenza viruses in poultry populations.