Clinical assessment of a point-of-care assay to determine protective vaccinal antibody titers to canine viral diseases.

Guidelines recommend that dogs are vaccinated for canine distemper virus (CDV), canine parvovirus (CPV), and canine adenovirus (CAV) every 3 years. Alternatively, their antibody titers are measured and vaccines given when titers fall below a protective threshold. In this study, a point-of-care (POC) assay was compared to hemagglutination inhibition (for CPV) and virus neutralization (for CAV and CDV) assays to predict the need for revaccination Ninety-two dogs presented for vaccination were enrolled. The POC assay indicated protective titers against CDV in 79/80, CPV in 89/90, and CAV in 91/91 dogs with reference standard antibody measurements that were over a protective threshold. The sensitivity of the POC assay for to detect protective concentrations of CDV antibodies was 99% (95% confidence interval [CI 95%], 93.3-99.9%). Ten dogs were falsely considered protected against CDV by the POC assay with a specificity of 17% (CI 95%, 3.0-44.8%). The sensitivity of the POC assay for protective concentrations of CPV titers was 99% (CI 95%, 93.9-99.9%). The sensitivity of the POC assay to detect protective concentrations of CAV antibodies was 100% (CI 95%, 95.9-100%). Only classifying high-positive CDV and CPV titers on the POC assay as protective improved assay specificity to 100%, but sensitivity decreased to 51% and 76% respectively. This POC assay had a high sensitivity for the detection of protective antibody titers; however, some dogs were falsely categorized as protected, especially for CDV.

Assessment of the Hemotek® system for the in vitro feeding of field-collected Culicoides imicola (Diptera: Ceratopogonidae) in South Africa.

The optimising and standardisation of in vitro blood feeding protocols for field-collected Culicoides species (Diptera: Ceratopogonidae) will be of essence for the comparison of the vector competencies of various populations of viruses of veterinary importance and the establishment of laboratory colonies of putative vector species. A custom-made feeding chamber to accommodate the small size of Culicoides imicola Kieffer was designed for the commercially available Hemotek® system and compared to existing membrane and cotton pledge feeding methods. High feeding rates coupled to higher mean blood meal volume than that of the existing OVI device indicated that the Hemotek system will be suitable for the feeding of field-collected Culicoides. The Hemotek system was subsequently used to identify factors that may affect feeding success in the laboratory. Evaluated factors were the source (host) and temperature of the blood meal, time of the day of feeding, the position of the blood reservoir in relation to the midges and exposure time to the blood. While only feeding orientation and the temperature of the blood source seems to significantly affect the feeding rate, all the factors did influence the volume of blood consumed.

Longitudinal monitoring of honey bee colonies reveals dynamic nature of virus abundance and indicates a negative impact of Lake Sinai virus 2 on colony health.

Honey bees (Apis mellifera) are important pollinators of plants, including those that produce nut, fruit, and vegetable crops. Therefore, high annual losses of managed honey bee colonies in the United States and many other countries threaten global agriculture. Honey bee colony deaths have been associated with multiple abiotic and biotic factors, including pathogens, but the impact of virus infections on honey bee colony population size and survival are not well understood. To further investigate seasonal patterns of pathogen presence and abundance and the impact of viruses on honey bee colony health, commercially managed colonies involved in the 2016 California almond pollination event were monitored for one year. At each sample date, colony health and pathogen burden were assessed. Data from this 50-colony cohort study illustrate the dynamic nature of honey bee colony health and the temporal patterns of virus infection. Black queen cell virus, deformed wing virus, sacbrood virus, and the Lake Sinai viruses were the most readily detected viruses in honey bee samples obtained throughout the year. Analyses of virus prevalence and abundance revealed pathogen-specific trends including the overall increase in deformed wing virus abundance from summer to fall, while the levels of Lake Sinai virus 2 (LSV2) decreased over the same time period. Though virus prevalence and abundance varied in individual colonies, analyses of the overall trends reveal correlation with sample date. Total virus abundance increased from November 2015 (post-honey harvest) to the end of the almond pollination event in March 2016, which coincides with spring increase in colony population size. Peak total virus abundance occurred in late fall (August and October 2016), which correlated with the time period when the majority of colonies died. Honey bee colonies with larger populations harbored less LSV2 than weaker colonies with smaller populations, suggesting an inverse relationship between colony health and LSV2 abundance. Together, data from this and other longitudinal studies at the colony level are forming a better understanding of the impact of viruses on honey bee colony losses.

The NC229 multi-station research consortium on emerging viral diseases of swine: Solving stakeholder problems through innovative science and research.

The NC229 research consortium was created in 1999 in response to the emergence of porcine reproductive and respiratory syndrome virus (PRRSV), a viral agent responsible for devastating economic losses to the swine industry. The project follows the traditional "consortium" approach for Multistate Agricultural Research driven through the US State Agricultural Experiment Stations (SAES), wherein stakeholder-driven needs to combat swine infectious diseases are identified and scientific solutions pursued by combining funds from federal, state, commodity groups, and the animal health industry. The NC229 consortium was the main driving force in successfully competing for a USDA multi-station Coordinated Agricultural Project (PRRS CAP-I) in 2004-2008, immediately followed by a renewal for 2010-2014 (PRRS CAP-II)-, resulting in an overall record achievement of almost $10 million dollars. The CAP funding was not only useful for quality research, extension, and education in PRRS and related diseases, but also instrumental in enabling the group to leverage swine industry funding of more than $34 million dollars, distributed between creative research and extension on PRRS during the last 20 years. The North American/International PRRS Symposium, now recognized by the community as a highly effective platform for the exchange of basic research findings and fundamental translational technology, is directly derived from the NC229 consortium. Other significant offshoots from NC229 include the PHGC (PRRS Host Genomic Consortium), a platform for discoveries on the role of host genetics during PRRSV infection, since 2007. Since 2009, the NC229 consortium has expanded its collective research interests beyond PRRSV to include nine other emerging viral diseases of swine. In the current project (2019-2024), African Swine Fever Virus (ASFV) retains a central focus, with the goal of harnessing the group's expertise in promoting preparedness for the global control of ASFV.

Characterization of viral, bacterial, and parasitic causes of disease in small-scale chicken flocks in the Mekong Delta of Vietnam.

In the Mekong Delta region of Vietnam, small-scale chicken farming is common. However, high levels of disease or mortality in such flocks impair economic development and challenge the livelihoods of many rural households. We investigated 61 diseased small-scale flocks (122 chickens) for evidence of infection with 5 bacteria, 4 viruses, and helminths. Serological profiles (ELISA) were also determined against 6 of these pathogens. The aims of this study were the following: (1) to investigate the prevalence of different pathogens and to compare the probability of detection of bacterial pathogens using PCR and culture; (2) to investigate the relationship between detection of organisms in birds' tissues and the observed morbidity and mortality, as well as their antibody profile; and (3) to characterize risk factors for infection with specific viral or bacterial pathogens. We used PCR to test for viral (viruses causing infectious bronchitis [IB], highly pathogenic avian influenza [HPAI], Newcastle disease, and infectious bursal disease [IBD]) and bacterial pathogens (Mycoplasma gallisepticum, Pasteurella multocida, Avibacterium paragallinarum, and Ornithobacterium rhinotracheale [ORT]). The latter two were also investigated in respiratory tissues by conventional culture. Colisepticemic Escherichia coli was investigated by liver or spleen culture. In 49 of 61 (80.3%) flocks, at least one bacterial or viral pathogen was detected, and in 29 (47.5%) flocks, more than one pathogen was detected. A. paragallinarum was detected in 62.3% flocks, followed by M. gallisepticum (26.2%), viruses causing IBD (24.6%) and IB (21.3%), septicemic E. coli (14.8%), ORT (13.1%), and HPAI viruses (4.9%). Of all flocks, 67.2% flocks were colonized by helminths. Mortality was highest among flocks infected with HPAI (100%, interquartile range [IQR]: 81.6-100%) and lowest with flocks infected with ORT (5.3%, IQR: 1.1-9.0%). The results indicated slight agreement (kappa ≤ 0.167) between detection by PCR and culture for both A. paragallinarum and ORT, as well as between the presence of cestodes and ORT infection (kappa = 0.317). Control of A. paragallinarum, viruses causing HPAI, IBD, and IB, M. gallisepticum, and gastrointestinal helminths should be a priority in small-scale flocks.

Prioritization of livestock transboundary diseases in Belgium using a multicriteria decision analysis tool based on drivers of emergence.

During the past decade, livestock diseases have (re-)emerged in areas where they had been previously eradicated or never been recorded before. Drivers (i.e. factors of (re-)emergence) have been identified. Livestock diseases spread irrespective of borders, and therefore, reliable methods are required to help decision-makers to identify potential threats and try stopping their (re-)emergence. Ranking methods and multicriteria approaches are cost-effective tools for such purpose and were applied to prioritize a list of selected diseases (N = 29 including 6 zoonoses) based on the opinion of 62 experts in accordance with 50 drivers-related criteria. Diseases appearing in the upper ranking were porcine epidemic diarrhoea, foot-and-mouth disease, low pathogenic avian influenza, African horse sickness and highly pathogenic avian influenza. The tool proposed uses a multicriteria decision analysis approach to prioritize pathogens according to drivers and can be applied to other countries or diseases.

HEALTH ASSESSMENT OF FREE-RANGING CHELONIANS IN AN URBAN SECTION OF THE BRONX RIVER, NEW YORK, USA.

The Bronx River in Bronx, New York, US spans an area of significant human development and has been subject to historic and ongoing industrial contamination. We evaluated the health of freeranging native common snapping turtles ( Chelydra serpentina) and nonnative invasive red-eared sliders ( Trachemys scripta) in a segment of the Bronx River between May and July 2012. In 18 snapping turtles and nine sliders, complete physical examinations were performed, ectoparasites collected, and blood was analyzed for contaminants (mercury, thallium, cadmium, arsenic, lead, selenium, oxychlordane, alpha-chlordane, dieldrin, DDD, DDE, polychlorinated biphenyls). Complete blood counts and the presence of hemoparasites were determined in 16 snapping turtles and nine sliders. Swabs of the choana and cloaca were screened for ranavirus, adenovirus, herpesvirus, and Mycoplasma spp. by PCR in 39 snapping turtles and 28 sliders. Both turtle species exhibited bioaccumulation of various environmental contaminants, particularly organochlorines and polychlorinated biphenyls. Molecular screening revealed a unique herpesvirus in each species. A Mycoplasma sp. previously isolated from emydid turtles was detected in red-eared sliders while a unique Mycoplasma sp. was identified in common snapping turtles. Ranaviruses and adenoviruses were not detected. Our study established a baseline health assessment to which future data can be compared. Moreover, it served to expand the knowledge and patterns of health markers, environmental contaminants, and microorganisms of freeranging chelonians.

Global trends in infectious diseases of swine.

Pork accounts for more than one-third of meat produced worldwide and is an important component of global food security, agricultural economies, and trade. Infectious diseases are among the primary constraints to swine production, and the globalization of the swine industry has contributed to the emergence and spread of pathogens. Despite the importance of infectious diseases to animal health and the stability and productivity of the global swine industry, pathogens of swine have never been reviewed at a global scale. Here, we build a holistic global picture of research on swine pathogens to enhance preparedness and understand patterns of emergence and spread. By conducting a scoping review of more than 57,000 publications across 50 years, we identify priority pathogens globally and regionally, and characterize geographic and temporal trends in research priorities. Of the 40 identified pathogens, publication rates for eight pathogens increased faster than overall trends, suggesting that these pathogens may be emerging or constitute an increasing threat. We also compared regional patterns of pathogen prioritization in the context of policy differences, history of outbreaks, and differing swine health challenges faced in regions where swine production has become more industrialized. We documented a general increasing trend in importance of zoonotic pathogens and show that structural changes in the industry related to intensive swine production shift pathogen prioritization. Multinational collaboration networks were strongly shaped by region, colonial ties, and pig trade networks. This review represents the most comprehensive overview of research on swine infectious diseases to date.

Seroprevalence of economically important viral pathogens in swine populations of Trinidad and Tobago, West Indies.

The objective of this study was to evaluate the seroprevalence and identify the strains of swine influenza virus (SwIV), as well as the seroprevalence of porcine parvovirus (PPV), transmissible gastroenteritis virus (TGEV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine respiratory coronavirus (PRCV), porcine circovirus type 2 (PCV-2), and classical swine fever virus (CSFV) in pigs in Trinidad and Tobago (T&T). Blood samples (309) were randomly collected from pigs at farms throughout T&T. Serum samples were tested for the presence of antibodies to the aforementioned viruses using commercial ELISA kits, and the circulating strains of SwIV were identified by the hemagglutination inhibition test (HIT). Antibodies against SwIV were detected in 114 out of the 309 samples (37%). Out of a total of 26 farms, 14 tested positive for SwIV antibodies. HI testing revealed high titers against the A/sw/Minnesota/593/99 H3N2 strain and the pH1N1 2009 pandemic strain. Antibodies against PPV were detected in 87 out of the 309 samples (28%), with 11 out of 26 farms testing positive for PPV antibodies. Antibodies against PCV-2 were detected in 205 out of the 309 samples tested (66%), with 25 out of the 26 farms testing positive for PCV-2 antibodies. No antibodies were detected in any of the tested pigs to PRRSV, TGEV, PRCV, or CSFV.

Effect of Cage-Wash Temperature on the Removal of Infectious Agents from Caging and the Detection of Infectious Agents on the Filters of Animal Bedding-Disposal Cabinets by PCR Analysis.

Efficient, effective cage decontamination and the detection of infection are important to sustainable biosecurity within animal facilities. This study compared the efficacy of cage washing at 110 and 180 °F on preventing pathogen transmission. Soiled cages from mice infected with mouse parvovirus (MPV) and mouse hepatitis virus (MHV) were washed at 110 or 180 °F or were not washed. Sentinels from washed cages did not seroconvert to either virus, whereas sentinels in unwashed cages seroconverted to both agents. Soiled cages from mice harboring MPV, Helicobacter spp., Mycoplasma pulmonis, Syphacia obvelata, and Myocoptes musculinus were washed at 110 or 180 °F or were not washed. Sentinels from washed cages remained pathogen-free, whereas most sentinels in unwashed cages became infected with MPV and S. obvelata. Therefore washing at 110 or 180 °F is sufficient to decontaminate caging and prevent pathogen transmission. We then assessed whether PCR analysis of debris from the bedding disposal cabinet detected pathogens at the facility level. Samples were collected from the prefilter before and after the disposal of bedding from cages housing mice infected with both MPV and MHV. All samples collected before bedding disposal were negative for parvovirus and MHV, and all samples collected afterward were positive for these agents. Furthermore, all samples obtained from the prefilter before the disposal of bedding from multiply infected mice were pathogen-negative, and all those collected afterward were positive for parvovirus, M. pulmonis, S. obvelata, and Myocoptes musculinus. Therefore the debris on the prefilter of bedding-disposal cabinets is useful for pathogen screening.

The spread of non-OIE-listed avian diseases through international trade of chicken meat: an assessment of the risks to New Zealand.

Twelve avian diseases are listed by the World Organisation for Animal Health (OIE), although more than 100 infectious diseases have been described in commercial poultry. This article summarises a recent assessment of the biosecurity risks posed by non-listed avian diseases associated with imports of chilled or frozen chicken meat and meat products into New Zealand. Following the guidelines described in Chapter 2.1 of the OIE Terrestrial Animal Health Code, avian adenovirus splenomegaly virus, avian paramyxovirus-2 (APMV-2), Bordetella avium, Mycoplasma spp., Ureaplasma spp., Ornithobacterium rhinotracheale, Riemerella anatipestifer, and Salmonella arizonae have been identified as hazards. However, of all the non-listed avian diseases discussed here, only APMV-2 and S. arizonae are assessed as being risks associated with the commercial import of chicken meat into New Zealand. Specific control measures may have to be implemented to mitigate such risks. This conclusion is likely to reflect both the high-health status of New Zealand poultry and the threat posed by these infectious agents to New Zealand's unique population of native psittacine species.

Infectious diseases affect marine fisheries and aquaculture economics.

Seafood is a growing part of the economy, but its economic value is diminished by marine diseases. Infectious diseases are common in the ocean, and here we tabulate 67 examples that can reduce commercial species' growth and survivorship or decrease seafood quality. These impacts seem most problematic in the stressful and crowded conditions of aquaculture, which increasingly dominates seafood production as wild fishery production plateaus. For instance, marine diseases of farmed oysters, shrimp, abalone, and various fishes, particularly Atlantic salmon, cost billions of dollars each year. In comparison, it is often difficult to accurately estimate disease impacts on wild populations, especially those of pelagic and subtidal species. Farmed species often receive infectious diseases from wild species and can, in turn, export infectious agents to wild species. However, the impact of disease export on wild fisheries is controversial because there are few quantitative data demonstrating that wild species near farms suffer more from infectious diseases than those in other areas. The movement of exotic infectious agents to new areas continues to be the greatest concern.

Health assessment of wild lowland tapir (Tapirus terrestris) populations in the Atlantic Forest and Pantanal biomes, Brazil (1996-2012).

Abstract The lowland tapir (Tapirus terrestris) is found in South America and is listed as Vulnerable to Extinction by the International Union for Conservation of Nature, Red List of Threatened Species. Health issues, particularly infectious diseases, are potential threats for the species. Health information from 65 wild tapirs from two Brazilian biomes, Atlantic Forest (AF) and Pantanal (PA), were collected during a long-term study (1996-2012). The study included physic, hematologic and biochemical evaluations, microbiologic cultures, urinalysis, and serologic analyses for antibodies against 13 infectious agents (viral and bacterial). The AF and PA tapirs were significantly different for several hematologic and biochemical parameters. Ten bacteria taxa were identified in the AF and 26 in the PA. Antibodies against five viruses were detected: Bluetongue virus, eastern equine encephalitis virus, western equine encephalitis virus, infectious bovine rhinotracheitis virus, and porcine parvovirus. A high prevalence of exposure to Leptospira interrogans (10 serovars: Autumnalis, Bratislava, Canicola, Copenhageni, Grippotyphosa, Hardjo, Hebdomadis, Icterohaemorrhagiae, Pomona, and Pyrogenes) was detected in both the AF and PA sites. A greater diversity of serovars and higher antibody titers were found in the PA. Statistically significant differences between sites were found for L. interrogans, equine encephalitis virus, and porcine parvovirus. Based on physical evaluations, both AF and PA populations were healthy. The differences in the overall health profile of the AF and PA tapir populations appear to be associated with environmental factors and infectious diseases ecology. The extensive datasets on hematology, biochemistry, urinalysis, and microbiology results from this paper can be used as reference values for wild tapirs.

Development of an EvaGreen-based multiplex real-time PCR assay with melting curve analysis for simultaneous detection and differentiation of six viral pathogens of porcine reproductive and respiratory disorder.

Concurrent infection of pigs with two or more pathogens is common in pigs under intensive rearing conditions. Porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), Japanese encephalitis virus (JEV) and pseudorabies virus (PRV) are all associated with reproductive or respiratory disorders or both and can cause significant economic losses in pig production worldwide. An EvaGreen-based multiplex real-time PCR (EG-mPCR) with melting curve analysis was developed in this study for simultaneous detection and differentiation of these six viruses in pigs. This method is able to detect and distinguish PCV2, PPV, PRRSV, CSFV, JEV and PRV with the limits of detection ranging from 100 to 500 copies/µL, high reproducibility, and intra-assay and inter-assay variation ranging from 0.11 to 3.20%. After validation, a total of 118 field samples were tested by the newly developed EG-mPCR. PCV2 was identified in 23%, PPV in 15%, PRRSV in 17% and PRV in 5% of the samples. Concurrent PCV2 and PRRSV infection was detected in 6.7%, PCV2 and PPV in 5% and PPV2 and PRRSV infection was detected in 5% of the cases. The agreement of the EG-mPCR and conventional PCR tests was 99.2%. This EG-mPCR will be a useful, rapid, reliable and cost-effective alternative for routine surveillance testing of viral infections in pigs.

Socio-economic, industrial and cultural parameters of pig-borne infections.

The pork-processing industry has been possibly the fastest growing sector of the food industry in recent years. Specialization, genetic homogenization of the pig population, high density of the breeding population, reduced human-animal interactions, slaughter at a lower age and increased international trade of live animals and pork are parameters that affect, positively or negatively, the emergence of novel pig-borne pathogens, many of which are pig-specific, and many of which have significant zoonotic potential, as observed in recent outbreaks of Nipah virus and Streptococcus suis in Southeast Asia and China, respectively. Numerous other pathogens are transmitted to humans through direct contact with or consumption of pig products, and globalization trends in trade and human population movements have resulted in outbreaks of pig-borne diseases even in Muslim countries and in Israel, where pork consumption is religiously prohibited. The role of pigs as potential reservoirs of antibiotic-resistant pathogens or genes encoding resistance, and the role of feral pigs as a reservoir of zoonotic disease, are scientific fields in direct need of further research.

Biologics industry challenges for developing diagnostic tests for the National Veterinary Stockpile.

Veterinary diagnostic products generated ~$3 billion US dollars in global sales in 2010. This industry is poised to undergo tremendous changes in the next decade as technological advances move diagnostic products from the traditional laboratory-based and handheld immunologic assays towards highly technical, point of care devices with increased sensitivity, specificity, and complexity. Despite these opportunities for advancing diagnostic products, the industry continues to face numerous challenges in developing diagnostic products for emerging and foreign animal diseases. Because of the need to deliver a return on the investment, research and development dollars continue to be focused on infectious diseases that have a negative impact on current domestic herd health, production systems, or companion animal health. Overcoming the administrative, legal, fiscal, and technological barriers to provide veterinary diagnostic products for the National Veterinary Stockpile will reduce the threat of natural or intentional spread of foreign diseases and increase the security of the food supply in the US.

Intervention strategies for emerging respiratory virus infections: policy and public health considerations.

Respiratory viruses have emerged and re-emerged in humans for hundreds of years. In the recent past avian and animal influenza viruses have caused human disease ranging from conjunctivitis to respiratory illnesses, including the 2009-10 A(H1N1)pdm09 pandemic. Coronaviruses, human metapneumovirus (hMPV) and enteroviruses have also impacted humans globally. Since the likely public health impacts are common, plans and policies for intervention strategies can be developed, encompassing early detection through surveillance and diagnostics, as well as treatment and prevention through clinical and non-clinical interventions. The global comprehensiveness of these varies according to differing resources, competing health priorities and the causative agent, yet, irrespective of this, activities must be proportional to the threat. Pandemics and severe epidemics enable policies to be tested and gaps identified.

Production of viral vaccines for veterinary use.

This review provides inside information on the production of vaccines for veterinary use. The vaccines against rinderpest as well as foot and mouth disease are considered milestones in the history of veterinary vaccine production. Modern vaccines are based on the scientific progress in virology, cell biology and immunology. While naturally occurring attenuated viruses or viruses obtained after passage in different animal species or cell culture were used as vaccine strains in the early vaccines, nowadays targeted mutagenesis can be applied to generate vaccine virus strains. In principle, the antigen production process is the same for live and inactivated vaccines. The vaccine virus is usually grown in cell culture, either in roller bottles or bioreactors. Most live vaccines are freeze-dried in order to enable storage in the refridgerator for a longer period. To this end, a so-called stabilizer is added to the culture medium. The inactivation of the vaccine virus for the production of killed vaccines is done by physical or chemical treatments that lead to denaturation of the proteins or damage of the nucleic acids. The inactivated antigen may be further purified and mixed with an adjuvant. The quality standards for vaccines are layed down in international regulations and laws. Numerous tests are performed during the different production steps and on the final product in order to warrant the quality of each batch.

Vaccines and global health.

Vaccines have made a major contribution to global health in recent decades but they could do much more. In November 2011, a Royal Society discussion meeting, 'New vaccines for global health', was held in London to discuss the past contribution of vaccines to global health and to consider what more could be expected in the future. Papers presented at the meeting reviewed recent successes in the deployment of vaccines against major infections of childhood and the challenges faced in developing vaccines against some of the world's remaining major infectious diseases such as human immunodeficiency virus (HIV), malaria and tuberculosis. The important contribution that development of more effective veterinary vaccines could make to global health was also addressed. Some of the social and financial challenges to the development and deployment of new vaccines were reviewed. The latter issues were also discussed at a subsequent satellite meeting, 'Accelerating vaccine development', held at the Kavli Royal Society International Centre. Delegates at this meeting considered challenges to the more rapid development and deployment of both human and veterinary vaccines and how these might be addressed. Papers based on presentations at the discussion meeting and a summary of the main conclusions of the satellite meeting are included in this issue of Philosophical Transactions of the Royal Society B.