Region-wise analysis of dairy cow movements in Japan.

BACKGROUND: Animal movement is considered the most significant factor in the transmission of infectious diseases in livestock. A better understanding of its effects would help provide a more reliable estimation of the disease spread and help develop effective control measures. If the movement pattern is heterogeneous, its characteristics should be considered in epidemiological analyses, such as when using simulation models to obtain reliable outputs. In Japan, following the bovine spongiform encephalopathy epidemic, a traceability system for cattle was established in 2003, and the registration of all cattle movements in the national database began. This study is the first to analyze cattle movements in Japan. We examined regional and seasonal heterogeneity in dairy cow movements, which accounted for most Japanese breeding cattle. RESULTS: In the 14 years from April 2005 to March 2018, 4,577,709 between-farm movements of dairy cows were recorded, and the number of movements was counted by month and age for both inter- and intra-regional movements. As a result, two characteristic round-trip movements were observed: one was non-seasonal and inter-regional movements related to cattle-breeding ranches in Hokkaido (the northern region of Japan), which consists of the movement of cows around ages 6 to 8 and 21 to 23 months old. In addition, the seasonal movement of heifers for summer grazing within Hokkaido occurred in May and October at the peak ages of 13 to 14 and 19 to 20 months old, respectively. The observed heterogeneity seemed to reflect the suitability of raising the Holstein breed in Hokkaido and the shortage of supply of replacement heifers and available farming areas outside Hokkaido. CONCLUSIONS: Understanding the patterns of dairy cow movements will help develop reliable infectious disease models and be beneficial for developing effective control measures against these diseases.

Participatory epidemiology: Reviewing experiences with contexts and actions.

This paper reviews experiences with participatory epidemiology (PE) and focuses in pastoralist areas of Africa where much of the early development and institutionalization of PE occurred. The paper discusses the importance of context when designing PE activities and framing study questions, and uses the example of changing livelihoods in pastoralist areas, and marked wealth and gender differences within communities. By reference to a recently proposed definition of PE that includes community empowerment, the paper describes the marked socio-economic and gender differentiation in pastoralist communities. In these contexts, questions of "disease control for who?" and "empowerment for who?" arise, as does the need to move beyond the concept of communities as homogenous social and economic units. By reference to the persistent complex emergencies and other humanitarian crises, the paper discusses community participation and how functional participation in programmes and PE can contribute to successful livestock disease control. In contrast, empowering forms of participation are difficult to achieve in humanitarian contexts and might not be needed to achieve effective livestock disease control. In non-humanitarian contexts, and secure countries with stable governments, national control programmes for important transboundary or zoonotic diseases usually require control strategies that can be applied consistently across areas, and with disease policy, funding, coordination and implementation controlled centrally. In contrast, empowerment in PE implies local, community-level decision-making and control of resources. The paper also discusses importance of designing PE studies that focus on action, and participatory evaluation of new or adapted disease control methods with communities.

"Can we agree on that"? Plurality, power and language in participatory research.

Participatory epidemiology (PE) is a method that gathers data from groups through focus group interviews and participatory visual and scoring exercises. The method is often used in poor communities in low-income countries where it is hard to obtain conventional epidemiological data. This paper draws on research on the public sphere and democratic deliberation, along with research on language and interpretation, to suggest how PE research could be better equipped to account for diversity in local knowledge, include minority views and acknowledge power dynamics. These aspects are discussed under the three themes of 'plurality', 'power' and 'language'. A review of highly-cited PE literature suggests that PE research engages with plurality and power to a very limited extent, and only marginally more so with language and translation. Examples are taken from the authors' own PE research on African swine fever in -Uganda, classical swine fever in Germany, peste des petits ruminants (PPR) in Eastern Europe, and Ugandan pastoralists' understanding of cattle disease to provide more detail as to why conventional PE studies might fail to record issues of plurality, power and language, and also to suggest how this can be addressed. With reference to the literature on the public sphere and democratic deliberation, and on language and interpretation, this paper concludes with some suggestions as to how to take plurality, power and language into greater consideration in PE studies in future, thus improving the validity and reliability of PE data.

Power, participation and their problems: A consideration of power dynamics in the use of participatory epidemiology for one health and zoonoses research.

The use of Participatory Epidemiology in veterinary research intends to include livestock keepers and other local stakeholders in research processes and the development of solutions to animal health problems, including potentially zoonotic diseases. It can also be an attempt to bring some of the methods and insights of social science into a discipline largely shaped by natural science methods and ways of seeing the world. The introduction of participatory methodologies to veterinary epidemiology and disease surveillance follows a wider movement in development thinking, questioning the top-down nature of much post-second world war development efforts directed from the Global North towards the Global South. In the best cases, participatory methods can help to empower the poor and marginalised to participate in and have some control over research and interventions which affect them. Compiled from experience in multi-disciplinary One Health projects, this paper briefly traces the rise of participatory epidemiology before examining some of the limitations observed in its implementation and steps that might be taken to alleviate the problems observed. The three areas in which the operationalisation of Participatory Epidemiology in veterinary and One Health research could be improved are identified as: broadening the focus of engagement with communities beyond quantitative data extraction; taking note of the wider power structures in which research takes place, and questioning who speaks for a community when participatory methods are used. In particular, the focus falls on how researchers from different disciplines, including veterinary medicine and the social sciences, can work together to ensure that participatory epidemiology is employed in such a way that it improves the quality of life of both people and animals around the world.

Practising co-production and interdisciplinarity: Challenges and implications for one health research.

We review the nature of interdisciplinary research in relation to One Health, a perspective on human-animal health which would appear to merit close interdisciplinary cooperation to inform public health policy. We discuss the relationship between biological sciences, epidemiology and the social sciences and note that interdisciplinary work demands attention be given to a range of often neglected epistemological and methodological issues. Epidemiologists may sometimes adopt social science techniques as "bolt-ons"1 to their research without having a complete understanding of how the social sciences work. The paper introduces a range of social science concepts and applies them to the challenges of understanding and practicing participatory and local epidemiology. We consider the problem of co-production of knowledge about One Health and zoonotic diseases in relation to funding structures, working in large international teams and explore some of the often-neglected realities of working across disciplines and cultures. We do this in part by applying the concept of value-chain to the research process.

Building the foundation for veterinary register-based epidemiology: A systematic approach to data quality assessment and validation.

Epidemiological studies often use data from registers. Data quality is of vital importance for the quality of the research. The aim of this study was to suggest a structured workflow to assess the quality of veterinary national registers. As an example of how to use the workflow, the quality of the following three registers was assessed: the Central Husbandry Register (CHR), the database for movement of pigs (DMP) and the national Danish register of drugs for veterinary use (VetStat). A systematic quantitative assessment was performed, with calculation the proportion of farms and observations with "poor quality" of data. "Poor" quality was defined for each measure (variable) either as a mismatch between and/or within registers, registrations of numbers outside the expected range, or unbalanced in- and outgoing movements. Interviews were conducted to make a complementary qualitative assessment. The proportion of farms and observations within each quality measure varied. This study highlights the importance of systematic quality assessment of register data and suggests a systematic approach for such assessments and validations without the use of primary data.

Managing complexity: Simplifying assumptions of foot-and-mouth disease models for swine.

Compartmental models have often been used to test the effectiveness and efficiency of alternative control strategies to mitigate the spread of infectious animal diseases. A fundamental principle of epidemiological modelling is that models should start as simple as possible and become as complex as needed. The simplest version of a compartmental model assumes that the population is closed, void of births and deaths and that this closed population mixes homogeneously, meaning that each infected individual has an equal probability of coming into contact with each susceptible individual in the population. However, this assumption may oversimplify field conditions, leading to conclusions about disease mitigation strategies that are suboptimal. Here, we assessed the impact of the homogeneous mixing/closed population assumption, which is commonly assumed for within-farm models of highly contagious diseases of swine, such as foot-and-mouth disease (FMD), on predictions about disease spread. Incorporation of farm structure (different barns or rooms for breeding and gestation, farrowing, nursery and finishing) and demography (piglet births and deaths, and animal movement within and off of the farm) resulted in transmission dynamics that differed in the latter portion of an outbreak. Specifically, farm structure and demography, which were included in the farrow to finish and farrow to wean farms, resulted in FMD virus persistence within the population under certain conditions. Results here demonstrate the impact of incorporating farm structure and demography into models of FMD spread in swine populations and will ultimately contribute to the design and evaluation of effective disease control strategies to mitigate the impact of potential incursions.

What's in a Name? The Incorrect Use of Case Series as a Study Design Label in Studies Involving Dogs and Cats.

BACKGROUND: Study design labels are used to identify relevant literature to address specific clinical and research questions and to aid in evaluating the evidentiary value of research. Evidence from the human healthcare literature indicates that the label "case series" may be used inconsistently and inappropriately. OBJECTIVE: Our primary objective was to determine the proportion of studies in the canine and feline veterinary literature labeled as case series that actually corresponded to descriptive cohort studies, population-based cohort studies, or other study designs. Our secondary objective was to identify the proportion of case series in which potentially inappropriate inferential statements were made. DESIGN: Descriptive evaluation of published literature. PARTICIPANTS: One-hundred published studies (from 19 journals) labeled as case series. METHODS: Studies were identified by a structured literature search, with random selection of 100 studies from the relevant citations. Two reviewers independently characterized each study, with disagreements resolved by consensus. RESULTS: Of the 100 studies, 16 were case series. The remaining studies were descriptive cohort studies (35), population-based cohort studies (36), or other observational or experimental study designs (13). Almost half (48.8%) of the case series or descriptive cohort studies, with no control group and no formal statistical analysis, included inferential statements about the efficacy of treatment or statistical significance of potential risk factors. CONCLUSIONS: Authors, peer-reviewers, and editors should carefully consider the design elements of a study to accurately identify and label the study design. Doing so will facilitate an understanding of the evidentiary value of the results.

Molecular epidemiology of Anaplasma platys, Ehrlichia canis and Babesia vogeli in stray dogs in Paraná, Brazil / Epidemiologia molecular de Anaplasma platys, Ehrlichia canis e Babesia vogeli em cães não domiciliados do Paraná

Hemoparasitic infections are tick-borne diseases, which affect animals and humans. Considering the importance of canine hemoparasitic infections in veterinary clinics, this study aimed to determine the occurrence of Anaplasma platys, Ehrlichia canis and Babesia vogeli in blood samples from 182 dogs not domiciled in the city of Pato Branco, southwestern region of Paraná State, Brazil, using polymerase chain reaction (PCR). The prevalence of A. platys and B. vogeli was 32.9% and 10.9% respectively, and A. platys infection prevailed (p<0.001). The number of dogs positive for A. platys was larger in Winter (p<0.05). All blood samples were negative for E. canis. In the dogs, infestation by Amblyomma cajennense predominated over that by Rhipicephalus sanguineus (p<0.001); but there was no significant association between PCR and the variables presence of ticks, sex and age. Dogs infected by A. platys and B. vogeli showed thrombocytopenia, lymphopenia and leukocytosis; but there was no correlation between such hematological changes and infection by hemoparasites. This appears to be the first molecular study that demonstrates the existence of A. platys and B. vogeli in dogs from the southwestern region of Paraná.(AU)

As hemoparasitoses são enfermidades transmitidas por carrapatos que afetam os animais e os humanos. Considerando a importância das hemoparasitoses caninas na clínica médica veterinária, este estudo objetivou determinar a ocorrência de Anaplasma platys, Ehrlichia canis e Babesia vogeli em amostras de sangue de 182 cães não domiciliados do município de Pato Branco, região sudoeste paranaense, Brasil, utilizando a reação em cadeia da polimerase (PCR). A prevalência de A. platys e B. vogeli foi de 32,9% e 10.9%, respectivamente, predominando a infecção por A. platys (p<0,001). Constatou-se um maior número de cães positivos para A. platys no período do inverno (p<0.05). Todas as amostras de sangue foram negativas para E. canis. Nos cães, a infestação por Amblyomma cajennense prevaleceu sobre a infestação por Rhipicephalus sanguineus (p<0,001), mas não foi observada associação significativa entre a PCR e as variáveis presença de carrapatos, sexo e idade. Cães infectados por A. platys e B. vogeli apresentaram trombocitopenia, linfopenia e leucocitose, porém não houve correlação destas alterações hematológicas com a infecção pelos hemoparasitas. Este é o primeiro estudo molecular que demonstra a existência de A. platys e de B. vogeli em cães da região sudoeste paranaense.(AU)

Inferring infection hazard in wildlife populations by linking data across individual and population scales.

Our ability to infer unobservable disease-dynamic processes such as force of infection (infection hazard for susceptible hosts) has transformed our understanding of disease transmission mechanisms and capacity to predict disease dynamics. Conventional methods for inferring FOI estimate a time-averaged value and are based on population-level processes. Because many pathogens exhibit epidemic cycling and FOI is the result of processes acting across the scales of individuals and populations, a flexible framework that extends to epidemic dynamics and links within-host processes to FOI is needed. Specifically, within-host antibody kinetics in wildlife hosts can be short-lived and produce patterns that are repeatable across individuals, suggesting individual-level antibody concentrations could be used to infer time since infection and hence FOI. Using simulations and case studies (influenza A in lesser snow geese and Yersinia pestis in coyotes), we argue that with careful experimental and surveillance design, the population-level FOI signal can be recovered from individual-level antibody kinetics, despite substantial individual-level variation. In addition to improving inference, the cross-scale quantitative antibody approach we describe can reveal insights into drivers of individual-based variation in disease response, and the role of poorly understood processes such as secondary infections, in population-level dynamics of disease.

Veterinary epidemiology: Forging a path toward one health.

The One Health concept has been extensively used to describe those practices that support transdisciplinary collaborations involving animal and human health and the environment. During the past International Society of Veterinary Epidemiology and Economics (ISVEE) conference in Merida (Mexico) in November 2015, a panel discussion was held to identify gaps and needs required for success with such approaches. Key messages included further development of dynamic, transdisciplinary collaborations, new mechanisms for obtaining, integrating and interpreting data from diverse sources, the identification of One Health joint priorities and resources for the veterinary and public health professions, and operationalization and institutionalization of One Health. Additionally, all abstracts that were presented at ISVEE containing the term "One Health" were identified. There has been an increase in the use of the term over time. One Health research has been presented at ISVEE since at least as early as 2009 and has been highlighted at the conference for work carried out in at least 41 countries from 69 research centers or institutions, with highly diversified articles, which reveals the richness of this field of research.

The application of epidemiology in national veterinary services: Challenges and threats in Brazil.

The application of epidemiology in national veterinary services must take place at the interface between science and politics. Animal health policy development and implementation require attention to macro-epidemiology, the study of economic, social and policy inputs that affect the distribution and impact of animal or human disease at the national level. The world has changed fast over the last three decades including the delivery of veterinary services, their remit and the challenges addressed by public and animal health policies. Rethinking the role of public services and how to make public programs more efficient has been at the heart of the political discussion. The WTO through its SPS Agreement has changed the way in which national veterinary services operate and how trade decisions are made. Most low and middle income countries are still struggling to keep up with the new international scene. Some of these countries, such as Brazil, have very important livestock industries and are key to the global food systems. Over the last two decades, Brazil became a leading player in exports of livestock products, including poultry, and this created a strong pressure on the national veterinary services to respond to trade demands, leading to focus animal health policies on the export-driven sector. During the same period, Brazil has gone a long way in the direction of integrating epidemiology with veterinary services. Epidemiology groups grew at main universities and have been working with government to provide support to animal health policy. The scope and quality of the applied epidemiological work improved and focused on complex data analysis and development of technologies and tools to solve specific disease problems. Many public veterinary officers were trained in modern epidemiological methods. However, there are important institutional bottlenecks that limit the impact of epidemiology in evidence-based decision making. More complex challenges require high levels of expertise in veterinary epidemiology, as well as institutional models that provide an appropriate environment for building and sustaining capacity in national veterinary services. Integrating epidemiology with animal health policy is a great opportunity if epidemiologists can understand the real issues, including the socio-economic dimensions of disease management, and focus on innovation and production of knowledge. It may be a trap if epidemiologists are restricted to answering specific decision-making questions and policy makers perceive their role exclusively as data analysts or providers of technological solutions. Fostering solutions for complex issues is key to successful integration with policy making.

Approaches to Teaching Biometry and Epidemiology at Two Veterinary Schools in Germany.

In a thematically broad and highly condensed curriculum like veterinary medicine, it is essential to pay close attention to the didactic and methodical approaches used to deliver that content. The course topics ideally should be selected for their relevance but also for the target audience and their previous knowledge. The overall objective is to improve the long-term availability of what has been learned. For this reason, an evaluation among lecturers of German-speaking veterinary schools was carried out in 2012 to consider which topics in biometry and epidemiology they found relevant to other subject areas. Based on this survey, two veterinary schools (Berlin and Hannover) developed a structured approach for the introductory course in biometry and epidemiology. By means of an appropriate choice of topics and the use of adequate teaching methods, the quality of the lecture course could be significantly increased. Appropriately communicated learning objectives as well as a high rate of student activity resulted in increased student satisfaction. A certain degree of standardization of teaching approaches and material resulted in a comparison between the study sites and reduced variability in the content delivered at different schools. Part of this was confirmed by the high consistency in the multiple-choice examination results between the study sites. The results highlight the extent to which didactic and methodical restructuring of teaching affects the learning success and satisfaction of students. It can be of interest for other courses in veterinary medicine, human medicine, and biology.

Multiple imputation in veterinary epidemiological studies: a case study and simulation.

The problem of missing data occurs frequently in veterinary epidemiological studies. Most studies use a complete case (CC) analysis which excludes all observations for which any relevant variable have missing values. Alternative approaches (most notably multiple imputation (MI)) which avoid the exclusion of observations with missing values are now widely available but have been used very little in veterinary epidemiology. This paper uses a case study based on research into dairy producers' attitudes toward mastitis control procedures, combined with two simulation studies to evaluate the use of MI and compare results with a CC analysis. MI analysis of the original data produced results which had relatively minor differences from the CC analysis. However, most of the missing data in the original data set were in the dependent variable and a subsequent simulation study based on the observed missing data pattern and 1000 simulations showed that an MI analysis would not be expected to offer any advantages over a CC analysis in this situation. This was true regardless of the missing data mechanism (MCAR - missing completely at random, MAR - missing at random, or NMAR - not missing at random) underlying the missing values. Surprisingly, recent textbooks dealing with MI make little reference to this limitation of MI for dealing with missing values in the dependent variable. An additional simulation study (1000 runs for each of the three missing data mechanisms) compared MI and CC analyses for data in which varying levels (n=7) of missing data were created in predictor variables. This study showed that MI analyses generally produced results that were less biased on average, were more precise (smaller SEs), were more consistent (less variability between simulation runs) and consequently were more likely to produce estimates that were close to the "truth" (results obtained from a data set with no missing values). While the benefit of MI varied with the mechanism used to generate the missing data, MI always performed as well as, or better than, CC analysis.

Ecological approaches in veterinary epidemiology: mapping the risk of bat-borne rabies using vegetation indices and night-time light satellite imagery.

Rabies remains a disease of significant public health concern. In the Americas, bats are an important source of rabies for pets, livestock, and humans. For effective rabies control and prevention, identifying potential areas for disease occurrence is critical to guide future research, inform public health policies, and design interventions. To anticipate zoonotic infectious diseases distribution at coarse scale, veterinary epidemiology needs to advance via exploring current geographic ecology tools and data using a biological approach. We analyzed bat-borne rabies reports in Chile from 2002 to 2012 to establish associations between rabies occurrence and environmental factors to generate an ecological niche model (ENM). The main rabies reservoir in Chile is the bat species Tadarida brasiliensis; we mapped 726 occurrences of rabies virus variant AgV4 in this bat species and integrated them with contemporary Normalized Difference Vegetation Index (NDVI) data from the Moderate Resolution Imaging Spectroradiometer (MODIS). The correct prediction of areas with rabies in bats and the reliable anticipation of human rabies in our study illustrate the usefulness of ENM for mapping rabies and other zoonotic pathogens. Additionally, we highlight critical issues with selection of environmental variables, methods for model validation, and consideration of sampling bias. Indeed, models with weak or incorrect validation approaches should be interpreted with caution. In conclusion, ecological niche modeling applications for mapping disease risk at coarse geographic scales have a promising future, especially with refinement and enrichment of models with additional information, such as night-time light data, which increased substantially the model's ability to anticipate human rabies.

An approach for de-identification of point locations of livestock premises for further use in disease spread modeling.

We describe a method for de-identifying point location data used for disease spread modeling to allow data custodians to share data with modeling experts without disclosing individual farm identities. The approach is implemented in an open-source software program that is described and evaluated here. The program allows a data custodian to select a level of de-identification based on the K-anonymity statistic. The program converts a file of true farm locations and attributes into a file appropriate for use in disease spread modeling with the locations randomly modified to prevent re-identification based on location. Important epidemiological relationships such as clustering are preserved to as much as possible to allow modeling similar to those using true identifiable data. The software implementation was verified by visual inspection and basic descriptive spatial analysis of the output. Performance is sufficient to allow de-identification of even large data sets on desktop computers available to any data custodian.

Complex system modelling for veterinary epidemiology.

The use of mathematical models has a long tradition in infectious disease epidemiology. The nonlinear dynamics and complexity of pathogen transmission pose challenges in understanding its key determinants, in identifying critical points, and designing effective mitigation strategies. Mathematical modelling provides tools to explicitly represent the variability, interconnectedness, and complexity of systems, and has contributed to numerous insights and theoretical advances in disease transmission, as well as to changes in public policy, health practice, and management. In recent years, our modelling toolbox has considerably expanded due to the advancements in computing power and the need to model novel data generated by technologies such as proximity loggers and global positioning systems. In this review, we discuss the principles, advantages, and challenges associated with the most recent modelling approaches used in systems science, the interdisciplinary study of complex systems, including agent-based, network and compartmental modelling. Agent-based modelling is a powerful simulation technique that considers the individual behaviours of system components by defining a set of rules that govern how individuals ("agents") within given populations interact with one another and the environment. Agent-based models have become a recent popular choice in epidemiology to model hierarchical systems and address complex spatio-temporal dynamics because of their ability to integrate multiple scales and datasets.

Detection of Trypanosoma evansi infection in wild capybaras from Argentina using smear microscopy and real-time PCR assays.

Trypanosoma evansi is a flagellated protozoan that parasitizes a wide variety of mammals, occasionally including humans. In South America, it infects horses, cattle, buffaloes, dogs and wild mammals, causing a disease known as "Mal de Caderas", which results in important economic losses due to a wide range of pathological expressions. Argentina represents the southern limit of its distribution. The capybara (Hydrochoerus hydrochaeris) is a large rodent found in tropical to temperate freshwater wetlands of South America. As capybaras infected with T. evansi present no clinical signs of disease, withstanding high parasitaemia, this species was proposed as a reservoir host. In this study we investigated the prevalence and parasitaemic intensity of T. evansi in samples obtained from 60 free-ranging capybaras of Esteros del Iberá (Corrientes province, northeastern Argentina) using smear microscopy and real-time PCR assays. All the cases of capybaras infected with T. evansi were found during one of the years studied, with no evidence of seasonality. The overall infection prevalence was 10%, but between years it ranged from 0% to 17% (in 2011). This is the first confirmation of T. evansi infection in Argentina by molecular biology techniques. Our results showed no differences between the methods used to detect the presence of T. evansi in capybaras, which indicates that simple methods like microscopy can generate important data on the ecoepidemiology of this parasite. Both techniques used in this study represent a viable tool for ecoepidemiological studies, and can be used to produce good estimates of prevalence and parasitaemic level of the infection, which inform for the implementation of strategies for the control of the disease.

A high resolution melting real time PCR for mapping of filaria infection in domestic cats living in brugian filariosis-endemic areas.

We present here a real time PCR with high resolution melting (HRM) analysis for determining the prevalence and distribution of filarial species in domestic cats residing in brugian filariosis endemic areas of Narathiwat province, Thailand. Filarial species can be clearly distinguished in a single well using a single pair of primers. Blood samples were taken from a total of 2039 domestic cats living in endemic areas. Microfilariae were detected in 5.7% of the sample, while the overall prevalence of filaria infection by HRM analysis was 6.6%. The filariae species found in the infected cats were Brugia malayi, Dirofilaria immitis, D. repens as well as Acanthocheilonema (Dipetalonema) reconditum. This is the first report of A. reconditum infection from Thailand. The study also observed an overlapping of the distribution areas of animal and human filariae. From a public health perspective, the distribution and prevalence of these nematodes warrant an appropriate drug-based prophylaxis to be administered to cats in the endemic areas to reduce the number of diseased carriers. Furthermore, this molecular approach is more sensitive than microfilariae detection, enables species identification and greatly facilitates the collection of epidemiological data. Thus, the present study may help to bridge human-animal interface by coordinating research outcomes with the control of zoonoses that is vitally important for human and veterinary public health.