Longitudinal study of Staphylococcus aureus genotypes isolated from bovine clinical mastitis.

Bovine clinical mastitis is an important problem for the dairy industry, and Staphylococcus aureus is a common mastitis-causing pathogen in many countries. Detailed knowledge on genetic variation of Staph. aureus strains within the bovine population, including changes over time, can be useful for mastitis control programs, because severity of disease and effects on milk production are at least partly strain-associated. Therefore, the major aim of this study was to compare sequence types of Staph. aureus isolated from cases of bovine clinical mastitis from 2002 to 2003 with sequence types of a more recent set of isolates collected from 2013 to 2018, using core genome multi-locus sequence typing (cgMLST). We also wanted to compare antibiotic resistance genes of isolates from the 2 sets, to identify changes that may have occurred over time in the Staph. aureus population. A total of 157 isolates of Staph. aureus, almost equally distributed between the 2 time periods, were subjected to high-throughput sequencing and cgMLST. The results showed that the most prevalent sequence types found among the 2002 to 2003 isolates belonged to the clonal complexes CC97, CC133, and CC151, and that those complexes still dominated among the isolates from 2013 to 2018. However, a population shift from CC133 to CC97 and CC151 over time was observed. Likewise, no important differences in prevalence of antibiotic resistance genes were found between the 2 sets of isolates. As expected, genes belonging to the major facilitator superfamily of transporter proteins, and multidrug and toxic compound extrusion transporters, were very common. Moreover, several genes and mutations conferring resistance to fosfomycin were present, but not in CC97 isolates. The ß-lactamase gene blaZ was found in only 3 out of 81 isolates from 2002 to 2003 and 1 out of 76 isolates in 2013 to 2018. In conclusion, the results indicate that mastitis-associated Staph. aureus strains circulating among dairy cows in Sweden exhibit a remarkable genotypic persistence over a time frame of close to 15 yr.

Emergence of a new rhabdovirus associated with mass mortalities in eelpout (Zoarces viviparous) in the Baltic Sea.

We report the first description of a new Rhabdoviridae tentatively named eelpout rhabdovirus (EpRV genus Perhabdovirus). This virus was associated with mass mortalities in eelpout (Zoarces viviparous, Linnaeus) along the Swedish Baltic Sea coast line in 2014. Diseased fish showed signs of central nervous system infection, and brain lesions were confirmed by histology. A cytopathogenic effect was observed in cell culture, but ELISAs for the epizootic piscine viral haemorrhagic septicaemia virus (VHSV), infectious pancreas necrosis virus (IPNV), infectious haematopoietic necrosis virus (IHNV) and spring viraemia of carp virus (SVCV) were negative. Further investigations by chloroform inactivation, indirect fluorescence antibody test and electron microscopy indicated the presence of a rhabdovirus. By deep sequencing of original tissue suspension and infected cell culture supernatant, the full viral genome was assembled and we confirmed the presence of a rhabdovirus with 59.5% nucleotide similarity to the closest relative Siniperca chuatsi rhabdovirus. The full-genome sequence of this new virus, eelpout rhabdovirus (EpRV), has been deposited in GenBank under accession number KR612230. An RT-PCR based on the L-gene sequence confirmed the presence of EpRV in sick/dead eelpout, but the virus was not found in control fish. Additional investigations to characterize the pathogenicity of EpRV are planned.

Assessment of Preparation of Samples Under the Field Conditions and a Portable Real-Time RT-PCR Assay for the Rapid On-Site Detection of Newcastle Disease Virus.

Newcastle disease virus (NDV), also known as virulent forms of avian paramyxovirus serotype 1 (AMPV-1), is the causative agent of Newcastle disease affecting many species of birds and causing heavy losses to the poultry industry worldwide. Early, rapid and sensitive detection of the viruses or the viral nucleic acids is very important for disease diagnosis and control. This study aimed to evaluate sample preparation under field conditions and the application of a real-time RT-PCR method in the portable T-COR4 platform for the rapid, on-site detection of NDV on a farm. In the laboratory setting, the portable real-time RT-PCR assay had a similar performance compared with that obtained with a larger, stationary Rotor Gene real-time thermocycler. In the field conditions, viral nucleic acids were manually extracted just outside of animal units with minimal equipment and real-time RT-PCR detection was performed with the portable thermocycler T-COR4 placed in a nearby room. The portable assay at the farm detected viral RNA in 15 samples and reached an agreement of 83% (39/47) when the same RNA preparations were tested in the Rotor Gene thermocycler under the laboratory setting. The results demonstrated the feasibility of performing field detection but also the need to improve and further simplify sample preparation procedures.

Tick-borne encephalitis.

Tick-borne encephalitis (TBE), a zoonotic arbovirosis caused by tick-borne encephalitis virus (TBEV), is an increasing public health concern. Infections result in neurological symptoms in humans and the virus has rapidly expanded to new geographical areas. Three subtypes are currently present in different parts of Europe and Asia. The virus is transmitted by ticks, mainly Ixodes spp., between small mammals such as rodents, which serve as virus amplifying hosts. Humans are infected sporadically, either by a tick bite or by ingestion of infected milk or milk products. Other mammals (e.g. ruminants) can also be infected, but most of the time do not show clinical signs. In contrast to rodents, other wild and domestic mammals probably play only a very small direct role in maintaining TBEV in an area, but they might play an important role as hosts in sustaining a large tick population. Therefore, the virus prevalence and the occurrence of TBE can be influenced by several environmental, genetic and behavioural factors associated with the virus, the vectors or the hosts, and understanding these factors is essential for implementation of effective control measures. This article reviews virus characteristics and the epidemiological and clinical aspects of TBEV infections and examines pathogenesis, diagnostic approaches and control measures.

Development of improved analytical methods for use in animal health and in foodborne disease surveillance for source attribution.

Considering the 'One Health' principles, the links between animal and human health are very strong. Both domestic and wild animals are sources of infectious agents that cause diseases in humans. Poor animal health may also indirectly affect human health, through reduced access to food. A large number of infectious diseases of animals, the transboundary animal diseases, spread rapidly across borders. Robust and accurate diagnostic assays are needed to detect the infectious agents rapidly and to limit their spread. A large arsenal of novel assays has been developed during the last three decades, with a tremendous impact on the detection of infectious agents. The new diagnostic methods are mostly laboratory-based and expensive, requiring sophisticated equipment and special skills. However, rapid and cheap field-based assays have also been developed. Herein, the authors give several examples of the development of novel assays, with special focus on the 'One Health' principles.

High-throughput sequencing in veterinary infection biology and diagnostics.

Sequencing methods have improved rapidly since the first versions of the Sanger techniques, facilitating the development of very powerful tools for detecting and identifying various pathogens, such as viruses, bacteria and other microbes. The ongoing development of high-throughput sequencing (HTS; also known as next-generation sequencing) technologies has resulted in a dramatic reduction in DNA sequencing costs, making the technology more accessible to the average laboratory. In this White Paper of the World Organisation for Animal Health (OIE) Collaborating Centre for the Biotechnology-based Diagnosis of Infectious Diseases in Veterinary Medicine (Uppsala, Sweden), several approaches and examples of HTS are summarised, and their diagnostic applicability is briefly discussed. Selected future aspects of HTS are outlined, including the need for bioinformatic resources, with a focus on improving the diagnosis and control of infectious diseases in veterinary medicine.

Development and comparison of a Primer-Probe Energy Transfer based assay and a 5' conjugated Minor Groove Binder assay for sensitive real-time PCR detection of infectious laryngotracheitis virus.

In this study the design and development of two real-time PCR assays for the rapid, sensitive and specific detection of infectious laryngotracheitis virus (ILTV) DNA is described. A Primer-Probe Energy Transfer (PriProET) assay and 5' conjugated Minor Groove Binder (MGB) method are compared and contrasted. Both have been designed to target the thymidine kinase gene of the ILTV genome. Both PriProET and MGB assays are capable of detecting 20 copies of a DNA standard per reaction and are linear from 2×10(8) to 2×10(2)copies/µl. Neither PriProET, nor MGB reacted with heterologous herpesviruses, indicating a high specificity of the two methods as novel tools for virus detection and identification. This study demonstrates the suitability of PriProET and 5' conjugated MGB probes as real-time PCR chemistries for the diagnosis of respiratory diseases caused by ILTV.

Does a physical activity referral scheme improve the physical activity among routine primary health care patients?

Physical activity referral (PAR) schemes or concepts occur in varying forms. Because few physical activity intervention studies have been carried out in routine health care settings, it is difficult to translate research findings into daily practice. The aim of this study was to analyze the effectiveness of a PAR scheme implemented in routine primary health care. The study did not include a control group and was based on the ordinary staff's work efforts and follow-up measures. During a 2-year period, 6300 PARs were issued. Effectiveness was measured by an increase in self-reported physical activity. Half of the patients reached reported increased physical activity both at 3 months (49%) and at 12 months (52%). The proportion of inactive patients decreased from 33% at baseline to 17% at 3 months and 20% at 12 months. The proportion of patients who were physically active on a regular basis increased from 22% at baseline to 33% at 3 months and 32% at 12 months. Neither the patient's age nor the profession of the prescriber was associated with differences in effectiveness. The patient's activity level at baseline, the type of physical activity as well as the reason for the prescription were associated with increased physical activity.

Computerized lifestyle intervention in routine primary health care: evaluation of usage on provider and responder levels.

OBJECTIVE: The aim of this study was to evaluate the use of a computerized concept for lifestyle intervention in routine primary health care (PHC). METHODS: Nine PHC units were equipped with computers providing a lifestyle test and tailored printed advice regarding alcohol consumption and physical activity. Patients were referred by staff, and performed the test anonymously. Data were collected over a period of 1 year. RESULTS: During the study period 3,065 tests were completed, representing 5.7% of the individuals visiting the PHC units during the period. There were great differences between the units in the number of tests performed and in the proportion of patients referred. One-fifth of the respondents scored for hazardous alcohol consumption, and one-fourth reported low levels of physical activity. The majority of respondents found the test easy to perform, and a majority of those referred to the test found referral positive. CONCLUSION: The computerized test can be used for screening and intervention regarding lifestyle behaviours in PHC. Responders are positive to the test and to referral. PRACTICE IMPLICATIONS: A more widespread implementation of computerized lifestyle tests could be a beneficial complement to face-to-face interventions in PHC.

Physical activity referrals in Swedish primary health care - prescriber and patient characteristics, reasons for prescriptions, and prescribed activities.

BACKGROUND: Over the past decade, practitioners in primary health care (PHC) settings in many countries have issued written prescriptions to patients to promote increased physical activity or exercise. The aim of this study is to describe and analyse a comprehensive physical activity referral (PAR) scheme implemented in a routine PHC setting in Ostergötland County. The study examines characteristics of the PARs recipients and referral practitioners, identifies reasons why practitioners opted to use PARs with their clients, and discusses prescribed activities and prescriptions in relation to PHC registries. METHODS: Prospective prescription data were obtained for 90% of the primary health care centres in Ostergötland County, Sweden, in 2004 and 2005. The study population consisted of patients who were issued PARs after they were deemed likely to benefit from increased physical activity, as assessed by PHC staff. RESULTS: During the two-year period, a total of 6,300 patients received PARs. Two-thirds of the patients were female and half of the patients were 45-64 years. Half of the patients (50.8%) who received PARs were recommended a home-based activity, such as walking. One third (33%) of the patients issued PARs were totally inactive, reporting no days of physical activity that lasted for 30 minutes, and 29% stated that they reached this level 1-2 days per week. The number of PARs prescribed per year in relation to the number of unique individuals that visited primary health care during one year was 1.4% in 2004 and 1.2% in 2005. Two-thirds of the combined prescriptions were issued by physicians (38%) and nurses (31%). Physiotherapists and behavioural scientists issued the highest relative number of prescriptions. The most common reasons for issuing PARs were musculoskeletal disorders (39.1%) and overweight (35.4%), followed by high blood pressure (23.3%) and diabetes (23.2%). CONCLUSION: Ostergötland County's PAR scheme reached a relatively high proportion of physically inactive people visiting local PHC centres for other health reasons. PAR-related statistics, including PAR-rates by individual PHC centres and PAR- rates per health professional category, show differences in prescribing activities, both by patient categories, and by prescribing professionals.

Physical activity on prescription in primary health care: a follow-up of physical activity level and quality of life.

To examine whether physical activity on prescription in routine primary care patients would influence physical activity level and quality of life 6 months later. In 2001-2003, 13 Swedish primary health care units took part in an uncontrolled clinical study. If a patient in primary health care needed physical activity preventively or for treatment of a disease and patient-centered motivational counseling found physical activity to be suitable, individualized physical activity could be prescribed. Patients (n=481) of both sexes and all ages [75% women, mean age 50 (12-81)] participated in the study. Self-reported physical activity, readiness to change to a more physically active lifestyle and quality-of-life data were collected through questionnaires. The follow-up rate was 62% at 6 months. Intention-to-treat analysis showed a significant increase (P<0.01) in self-reported physical activity level, the stages of action and maintenance of physical activity as well as quality of life. Physical activity level, stages of change and quality of life increased analogically, indicating that physical activity on prescription may be suitable as a conventional treatment in an ordinary primary health care setting to promote a more physically active lifestyle.

Adding functional entities to plasmids.

Non-viral gene therapy constitutes an alternative to the more common use of viral-mediated gene transfer. Most gene transfer methods using naked DNA are based upon non-sequence-specific interactions between the nucleic acid and cationic lipids (lipoplex) or polymers (polyplex). We have developed a technology in which functional entities hybridize in a sequence-specific manner to the nucleic acid (bioplex). This technology is still in its infancy, but has the potential to become a useful tool, since it allows the construction of highly defined complexes containing a variety of functional entities. In its present form the bioplex technology is based upon the use of peptide/nucleic acids (PNA) as anchors. Single, or multiple, functional entities are directly coupled to the anchors. By designing plasmids, or oligonucleotides, with the corresponding anchor target sequence, complexes with desired composition can easily be generated. The long-term aim is to combine functional entities in order to achieve optimal, synergistic interactions allowing enhanced gene transfer in vivo.

Imino proton exchange in DNA catalyzed by ammonia and trimethylamine: evidence for a secondary long-lived open state of the base pair.

The base-pair opening kinetics of the self-complementary oligomer d(CGCGAATTCGCG)(2) has been derived from NMR measurements of the imino proton exchange. In general, it has previously been found that imino proton exchange in duplex DNA is limited by the proton-transfer step from the open state and that the dependence of the exchange times on the inverse concentration of an added exchange catalyst is linear. In the present study, a curvature is observed for, in particular, the innermost AT base pair with both ammonia and trimethylamine (TMA) as exchange catalysts. The two catalysts act on the same open states, but the accessibility of TMA is reduced by a factor of 2-3 compared to ammonia. Assuming that ammonia accesses the imino proton equally in the open state of the base pair and in the mononucleoside, the curvature is consistent with 7-9% of the openings ending in open states with lifetimes of about 1 micros while the bulk of open-state lifetimes fall in the nanosecond range. A curvature is also found for the exchange times of the imino protons in the A-tract sequence CGCA(8)CGC/GCGT(8)GCG. This curvature becomes increasingly pronounced from the 5'-end toward the center of the tract and hereby seems to be correlated with the contraction of the minor groove. Thus, while the base-pair lifetimes deduced from the present study are in accordance with previous measurements, a substantial fraction of the open states formed by the central AT-base pairs in the two oligomers exhibits microsecond lifetimes in contrast to previous estimates in the nanosecond range. These findings may be of relevance for the way sequence specific recognition is accomplished by proteins and ligands.

High base pair opening rates in tracts of GC base pairs.

Sequence-dependent structural features of the DNA double helix have a strong influence on the base pair opening dynamics. Here we report a detailed study of the kinetics of base pair breathing in tracts of GC base pairs in DNA duplexes derived from 1H NMR measurements of the imino proton exchange rates upon titration with the exchange catalyst ammonia. In the limit of infinite exchange catalyst concentration, the exchange times of the guanine imino protons of the GC tracts extrapolate to much shorter base pair lifetimes than commonly observed for isolated GC base pairs. The base pair lifetimes in the GC tracts are below 5 ms for almost all of the base pairs. The unusually rapid base pair opening dynamics of GC tracts are in striking contrast to the behavior of AT tracts, where very long base pair lifetimes are observed. The implication of these findings for the structural principles governing spontaneous helix opening as well as the DNA-binding specificity of the cytosine-5-methyltransferases, where flipping of the cytosine base has been observed, are discussed.

Gender trends in sick-listing with musculoskeletal symptoms in a Swedish county during a period of rapid increase in sickness absence.

UNLABELLED: Sickness absence and disability pension due to musculoskeletal diagnoses has increased in Sweden. STUDY OBJECTIVE: To study gender trends in sickness absence with musculoskeletal diagnoses and its changes in 1985-87. DESIGN: A prospective population-based study of all new sick-leave spells exceeding seven days in 1985-87. Sickness absence with "all diagnoses" was compared to "all musculoskeletal diagnoses", the latter group was also divided into three sub-groups. SETTING: The county of Ostergötland, Sweden. PARTICIPANTS: All sick-leave insured aged 16-65; 107,000 women and 100,000 men. RESULTS: More women than men were sick-listed in "all diagnoses" in 1985. There were corresponding gender differences in sickness absence with musculoskeletal diagnoses except with the diagnosis "low back pain". Sick-listing with musculoskeletal diagnoses increased for both women and men from 1985 to 1987, but the increase was consistently much higher for women, especially for younger women.

Unique base-pair breathing dynamics in PNA-DNA hybrids.

Kinetic and thermodynamic parameters, derived from 1H-NMR measurements of the imino proton exchange rates upon titration with the exchange catalyst ammonia, are reported for two mixed-sequence peptide nucleic acid (PNA)-DNA hybrids and their counterpart DNA duplex. The exchange times of the imino protons in the PNA strands extrapolate to very short base-pair lifetimes in the limit of infinite exchange catalyst concentration. This is not due to generally less stable base-pairs in PNA-DNA hybrids, since the lifetimes, apparent dissociation constants and thermodynamic stability (DeltaG degrees ) of the innermost DNA guanine imino protons are similar in the hybrid duplexes and in the DNA duplex. In addition, the apparent dissociation constants determined for PNA bases of the hybrids are of the same order as those of the corresponding bases in the DNA duplex. An exchange process from the closed state was found to be inconsistent with the experimental data. From these results, we conclude that opening and closing rates of the PNA guanine and thymine bases are at least two orders of magnitude higher than those of the corresponding bases in the DNA duplex. Unusual kinetics in the hybrids is also evident from the destabilization of the complementary DNA strand thymine bases, which exhibit base-pair dissociation constants increased by approximately two orders of magnitude compared to what is observed in the DNA duplex, while the DNA strand guanine bases are largely unaffected. The general pattern of the base-pair dynamics in the hybrids obtained when using trimethylamine as an exchange catalyst is the same as when using ammonia. However, the long base-pair lifetimes i. e. those of the DNA duplex and the guanine bases of the DNA strands in the hybrids, are approximately three to five times longer than when using ammonia. Thus, all opening events sensed by ammonia are not accessible to trimethylamine. These observations are discussed in regard to the mechanism of base-pair opening and the nature of the open state.

Long-term economic effects of team-based clinical case management of patients with chronic minor disease and long-term absence from working life.

OBJECTIVES: To examine the socio-economic effects of team-based clinical case management of patients with chronic minor disease bound for early retirement. DESIGN: Marginal analysis of programme costs and benefits to society compared with no-programme baseline of costs occurring in society due to productivity loss. Prospective patient data collection on admission, discharge, and at one year and five years after discharge to determine programme effectiveness. SETTING: Out-patient clinic at the department of social medicine in tertiary care hospital. SUBJECTS: 239 patients with minor disease and long-term vocational absence consecutively admitted to the study. At the one-year evaluation, 17 patients had been readmitted to the team, 7 could not be found, 6 declined the interview and 2 were deceased. At the five-year evaluation of 49 patients who were active after one year, one was deceased and 10 were unable to be found. MAIN OUTCOME MEASURES: Vocational activity. Programme costs. Benefits to society measured by decrease in indirect costs. RESULTS: The one-year vocational rehabilitation rate from the program was 20.5% and the five-year rehabilitation rate was 11.3%. The total discounted cost for case management of the 239 patients was 7.3 MSEK (600,000 Pounds). The decrease in the indirect costs to society from the 28 patients found active after five years was 35.1 MSEK (2,500,000 Pounds). The net present value of the programme at the 1991 price level was 27.5 MSEK (2,365,000 Pounds). CONCLUSIONS: Tertiary care level team-based clinical case management for vocational rehabilitation of patients with chronic minor disease has a positive cost-benefit ratio. A cross-boundary awareness at a health policy level is needed of the societal costs involved for this group of patients who fall between the traditional services in health care and social work.

Internal motions of nucleic acid structures and the determination of base-pair lifetimes.

Over the years, imino proton exchange measurements have provided a description of base-pair opening and of properties of internal motions of nucleic acids such as the individual opening of base pairs and the insensitivity of base-pair lifetimes to the nature and stability of neighboring pairs. A recent determination of base-pair lifetimes in d(CGCGATCGCG) conflicts with the original measurements and their interpretation. This question is analyzed in the present work. We emphasize the importance of high concentrations of exchange catalyst (eg 1 M NH3) for the accurate determination of base-pair lifetimes. These concentrations entail a high ionic strength, which can lead to aggregation, enhanced magnetic relaxation and underestimation of base-pair lifetimes if exchange is measured by its effect on the proton relaxation rate. Magnetization transfer which provides a more direct method for the measurement of proton exchange rates is therefore preferred. We show that the lack of measurements at high catalyst concentration is responsible for the discrepancy mentioned above. Measurements by the magnetization transfer method validate the original interpretation and inversion recovery experiments illustrate the effect of the ionic strength on the relaxation rate of the imino protons.

Proton exchange rates measured by saturation transfer using delayed randomization of the solvent magnetization.

The spin-lock saturation transfer experiment introduced by B. Adams and L. Lerner (J. Magn. Reson. 96, 604-607, 1992) is analyzed in terms of the Bloch equations. It is shown that the T1rho relaxation of the solvent is introduced in the decay of the exchangeable protons under conditions of relatively rapid exchange. An alternative experiment is suggested that randomizes the solvent magnetization with a pulsed field gradient before the observe pulse. This gives a single exponential intensity decay for the exchanging protons at all exchange rates. In addition, efficient water suppression and an even excitation profile are obtained.

Sex differences in sickness absence in relation to parental status.

The aim of this study was to analyse sex differences in medically certified sickness absence with special regard to parental status and age. All new sick-leave spells exceeding seven days and certified by a physician in a county of Sweden were registered in 1985-1987. The data-base was related to the population at risk by using data from the Swedish Official Population and Housing Census 1985. The analysis concerned the employed population in the age group from 16 to 54 with and without custody of children younger than ten years. In all ages, women with children had more sickness absence than men with children. Corresponding sex differences did not appear for persons without children, except for the oldest age-groups. Men with children had the lowest sickness absence of all groups. Young women with children had the highest. These findings remained the same when pregnancy-related sickness absence was excluded. The common finding of sex differences in sickness absence is here highly interrelated to custody of small children. The results suggest that the often reported positive health effects of multiple roles in women may be counteracted by inequality between the sexes in the responsibility as parents.