Reproductive, productive and financial consequences of chronic Trypanosoma vivax infection in a dairy cattle herd in a region without a cyclic vector.

This study evaluated the reproductive, productive and financial consequences of chronic Trypanosoma vivax infection in a dairy cattle herd located in a region without the cyclic vector during two years. Animals were categorized as either positive (chronically infected) or negative for T. vivax antibodies using a commercial rapid test. Additionally, serum samples from cows were analyzed for the presence of anti-Neospora caninum antibodies. Pregnancy diagnoses were performed through rectal palpation and ultrasonography after 30, 60 and every 21 days until the 144th day of pregnancy. If an abortion occurred in the final trimester, serology and cPCR were performed on calves for T. vivax and N. caninum. The breeding period, calving interval and pregnancy losses were recorded. The milk production of each animal during the 305 days of lactation was measured, and the annual financial impact of milk production was calculated using a revenue minus feed cost (RMFC) indicator. Out of 177 cows, 71.75 % were chronically infected, and 13.50 % were T. vivax-negative. No correlation (p = 0.8854) of co-infection between T. vivax and N. caninum was observed. Negative cows required fewer (p≤0.05) artificial inseminations than chronically infected ones. T. vivax was not significantly associated (p = 0.7893) with pregnancy loss up to 81 days of pregnancy. Cows chronically infected by T. vivax had 4-fold greater chance (p = 0.0280) of experiencing pregnancy loss between 82 and 144 days of gestation. Eighteen cows aborted, two were positive for T. vivax antibodies, and one for N. caninum antibodies. The calves were negative for T. vivax and N. caninum. Chronically infected cows and negative cows for T. vivax that experienced pregnancy loss (82-144 days of pregnancy) had a longer (p≤0.05) breeding period to become pregnant, and consequently a longer calving interval compared to cows that maintained pregnancy. The difference (p≤0.05) in milk production was evident when pregnancy loss occurred between 82 and 144 days of gestation in cows chronically infected by T. vivax. The RMFC indicated a negative impact of 38.2 % on the farm's annual milk revenue due to the presence of chronically infected cows.

Re-calculating the cost of coccidiosis in chickens.

Coccidiosis, caused by Eimeria species parasites, has long been recognised as an economically significant disease of chickens. As the global chicken population continues to grow, and its contribution to food security intensifies, it is increasingly important to assess the impact of diseases that compromise chicken productivity and welfare. In 1999, Williams published one of the most comprehensive estimates for the cost of coccidiosis in chickens, featuring a compartmentalised model for the costs of prophylaxis, treatment and losses, indicating a total cost in excess of £38 million in the United Kingdom (UK) in 1995. In the 25 years since this analysis the global chicken population has doubled and systems of chicken meat and egg production have advanced through improved nutrition, husbandry and selective breeding of chickens, and wider use of anticoccidial vaccines. Using data from industry representatives including veterinarians, farmers, production and health experts, we have updated the Williams model and estimate that coccidiosis in chickens cost the UK £99.2 million in 2016 (range £73.0-£125.5 million). Applying the model to data from Brazil, Egypt, Guatemala, India, New Zealand, Nigeria and the United States resulted in estimates that, when extrapolated by geographical region, indicate a global cost of ~ £10.4 billion at 2016 prices (£7.7-£13.0 billion), equivalent to £0.16/chicken produced. Understanding the economic costs of livestock diseases can be advantageous, providing baselines to evaluate the impact of different husbandry systems and interventions. The updated cost of coccidiosis in chickens will inform debates on the value of chemoprophylaxis and development of novel anticoccidial vaccines.

Estimating the total economic costs of Neospora caninum infections in dairy cows in Turkey.

The aim of this study was to estimate the total cost of Neospora caninum infection in cows in Turkey. According to present literature, the mean seroprevalence of infection was estimated as 14.7%. The seropositivity rate of N. caninum in aborted cows was found to be 18%. The financial costs were estimated in US$ at 2019 prices. The total cost of N. caninum infection was estimated at US$710 (438-1043) per a dairy cow and the annual economic costs were estimated at US$40.5 (24.6-60.3) million for Turkey. In this study, the distribution of economic costs caused by neosporosis per cow was determined to be 67.3% for abortion, 16.8% prolonged calving interval, 4.6% milk loss, 3.5% additional artificial insemination, and 7.7% veterinary and diagnostic costs, respectively. All studies that have been conducted so far on Neosporosis are focused on the prevalence and the diagnosis of the disease in Turkey. In this study, we firstly determined the economic costs due to N. caninum infections and try to attract attention on the economic impacts of the disease. As a result, awareness among breeders regarding the epidemiology, control, and eradication of neosporosis should be raised through training activities; coordination between the relevant institutions and organizations should be ensured; emergency action plans should be prepared; and further spread of the disease should be prevented.

A potential vaccine candidate towards chicken coccidiosis mediated by recombinant Lactobacillus plantarum with surface displayed EtMIC2 protein.

Eimeria tenella (E. tenella) has caused severe economic loss in chicken production, especially after the forbidden use of antibiotics in feed. Considering the drug resistant problem caused by misuse of chemoprophylaxis and live oocyst vaccines can affect the productivity of chickens, also it has the risk to reversion of virulence, the development of efficacious, convenient and safe vaccines is still deeply needed. In this study, the EtMic2 protein of E. tenella was anchored on the surface of Lactobacillus plantarum (L. plantarum) NC8 strain. The newly constructed strain was then used to immunize chickens, followed by E. tenella challenge. The results demonstrated that the recombinant strain could provide efficient protection against E. tenella, shown by increased relative body weight gains, percentages of CD4+ and CD8+ T cells, humoral immune response and inflammatory cytokines. In addition, decreased cecum lesion scores and fecal oocyst shedding were also observed during the experiment. In conclusion, this study proves the possibility to use L. plantarum as a vessel to deliver protective antigen to protect chickens against coccidiosis.

Combined control evaluation for Neospora caninum infection in dairy: Economic point of view coupled with population dynamics.

Neospora caninum infection is regarded as one of the most important infectious causes of abortion in dairy cattle. To intervene in its spread, four potential controls including test-and-cull, medication, vaccination, and selective breeding are considered and assessed in this study. The cost of each control, together with the inevitable annual loss due to population dynamics, is adopted as an assessment criterion from an economic point of view. By performing simulation and sensitivity analysis, our results demonstrate that compared with each single control, combined controls are worthwhile with better financial outcomes. For farm affected with significant prevalence (equal to or greater than 30%), vaccine treatment is the most effective and economical option among all control strategies. On the other hand, for farm where prevalence is relatively low (around 10%), combined control, by applying vaccination followed with test-and-cull, medication or selective breeding, could be alternative treatment to provide better financial outcome against single control in an observed period.

Identification of a Short DNA Bar Code in the 18S rDNA for Improved Differentiation of Common Eimeria Species Infecting Chickens.

The coccidian species Eimeria is a parasitic protozoan that causes the gastrointestinal disease coccidiosis in numerous vertebrate species. Incidence of the disease in commercial chickens produces drastic economic losses. Traditionally, detection of Eimeria has been performed using classical methods such as observation of oocyst morphology. However, molecular methods to detect and speciate Eimeria are becoming more prevalent. The 18S ribosomal gene, in particular, has been a widely used DNA amplification target for detection of Eimeria. Although the full-length gene is typically used for this purpose, newer research targeting shorter regions of the gene is being performed. This study investigated the suitability of a 120-base pair (bp) DNA bar code within the 18S gene for species differentiation. When comparing sequence variation from the Eimeria species infecting chickens, shortening the 18S gene to the 120-bp highly variable region provided increased species differentiation, while also reducing intraspecies variation. This DNA bar code is useful for distinction of the Eimeria species infecting chickens and should be considered for future molecular detection assays and metagenomic sequencing.

Assessing the prevalence and economic significance of coccidiosis individually and in combination with concurrent infections in Pakistani commercial poultry farms.

Coccidiosis is one of the most economically important diseases of the poultry, around the globe. In order to assess seasonal and age-wise prevalence of coccidiosis individually and with concurrent infections, this study was conducted on commercial poultry farms in and around Multan division, province of Punjab, Pakistan. A total of 28,126 boilers, 4,052 layers and 7,699 golden bird samples, provided by regional farmers and consultants, were examined by microscopy for the diagnosis of coccidiosis. Based on postmortem lesions, several infections including coccidiosis, viral, and miscellaneous diseases were catalogued, whereas samples were cultured to identify concurrent bacterial diseases. Cumulative analysis of this large set of samples revealed a prevalence of 14.16, 11.01, and 19.57% in broiler, layer and golden birds, respectively. Ascaridia galli (A. galli) showed a higher prevalence in layer (2.47%) compared to golden (0.21%) birds (P < 0.01). Amongst all concurrent infections investigated, bacterial infections were identified in highest percentage of samples (59.24%; P < 0.05). The age-wise and season-wise prevalence of coccidiosis and A. galli was found to be significant (P < 0.05). During the study period, an estimated of 10.69 (coccidiosis) and 0.29 (A. galli) million poultry birds were treated or provided prophylaxis and supportive therapy at Pakistani commercial poultry farms. Depending upon the prevailing market conditions, the total economic losses (treatment, prophylaxis, and supportive therapy) from coccidiosis and A. galli were estimated to be US $45,405.00 and 2,638.50, respectively, while production (weight and eggs) losses for broiler (US$ 2,750,779.00), layer and golden, young (US$ 13,974.98 and 50,228.76) and adult (US$ 104.74 and 203.77) were estimated. Unit loss of coccidiosis with concurrent diseases and intestinal worm was estimated to be US$ 0.005 and 0.01, respectively. These results highlight the potential impact of coccidiosis individually and with concurrent infections on the poultry productivity and will inform farmers, policy makers, and other governmental and non-governmental stakeholders on the use of control and management measures in containing these infections.

Efficacy and economic analysis of two treatment regimens using toltrazuril in lambs naturally infected with Eimeria spp. on pasture.

This study evaluated the efficacy and the economic viability of two anticoccidial treatment regimens tested in lambs naturally exposed to Eimeria spp. re-infections in a grazing system during a 140-day period. Twenty-four suckling lambs were distributed into three groups based on the individual count of oocysts per gram of feces (OPG) and body weight. Animals were treated with toltrazuril 5% (20 mg/kg) at 14- (GI) or 21-day (GII) intervals, and GIII was kept as untreated control. A cost-benefit analysis of each treatment regimen was calculated. Additionally, economic analysis was performed on four hypothetical scenarios, in which lambs could be having 10, 25, 50, or 85% decrease in their expected body weight gain due to clinical. Efficacy of toltrazuril against Eimeria spp. was 96.9-99.9% (GI) and 74.2-99.9% (GII). E. ovinoidalis was most frequently identified, but no clinical signs of coccidiosis were observed in lambs. There were no differences in weight gain among the groups. The cost of treatment per lamb was $13.09 (GI) and $7.83 (GII). The estimation model showed that the cost-benefit ratio favored treatment with toltrazuril when lambs fail to gain weight. In the studied flock, the break-even point for toltrazuril administered at 14-day intervals was reached with 85% decrease in mean weight gain. In conclusion, toltrazuril can be used at 14-day intervals to control Eimeria spp. (re)-infection in lambs raised on pasture. This treatment regimen was not economically feasible for subclinical coccidiosis; however, it may be feasible when used to prevent weight loss caused by clinical coccidiosis.

Caprine besnoitiosis: an emerging threat and its relationship to some other infections of ungulates by Besnoitia species.

Caprine besnoitiosis, caused by the cyst-forming protozoal apicomplexan Besnoitia caprae appears to be endemic in Kenya, Nigeria and Iran, but has yet to be detected in other parts of the world. The infection causes an important parasitic disease of goats in affected developing countries. Bovine besnoitiosis, is a widespread disease of cattle in Africa, Asia (but not Iran) and southern Europe. Recent epidemiological data confirm that the incidence and geographical range of bovine besnoitiosis in Europe is increasing, which is why growing attention has been given to the condition during the past decade. This paper reviews pertinent information on the biology, epidemiology, pathology, clinical signs, diagnosis and control of caprine besnoitiosis, together with its similarities to, and differences from, bovine besnoitiosis. The serious economic consequences of besnoitiosis on goat breeding and local meat and hide industries is also considered.

Epidemiology and economic benefits of treating rabbits coccidiosis in small farms from West Pomerania province, Poland.

The studies were carried out on 9 farms which deals with meat race of rabbits breeding. The basic herds had from 28 to 63 rabbits (total 275 rabbits) on Choszczno and Recz district in West Pomerania province. The study was based on two flotation methods:Willis-Schlaaf for qualitative and McMaster for quantitative analysis, which helped to establish the number of oocysts in 1g of feces (OPG). Ten species of coccidian were found in which 9 were intestinal (E. exigua, E. perforans, E. media, E. magna, E. irresidua, E. coecicola, E. flavescens, E. piriformis, E. intestinalis) and one was living in liver (E. stiedai). Baycox (Bayer) was used in drinking water in a dose of 25 ppm in 1 liter of water for 2 days. In rabbits from the experimental groups higher weight gain and lower mortality compared to the control group animals were observed. The results highlight the beneficial effects of coccidiostats used in the production effects in rabbits.

Neospora caninum causes severe economic losses in cattle in the humid pampa region of Argentina.

This work estimates the economic losses due to Neospora abortions in the humid pampa region of Argentina.The total dairy and beef cattle population at risk of abortion is 1,771,326 and 9,726,684 head, respectively. In dairy cattle, there was an 8 % risk of experiencing abortion due to a variety of causes, but 16.5 % of them were due to Neospora caninum. The economic losses were estimated at US$1,415 (1,400-1,431) per abortion, which equates to a total loss of US$33,097,221 (15,622,600-119,349,693) for the dairy industry at the humid pampa region of Argentina. In beef cattle, the overall risk of abortion was estimated to be 4.5 % for all pregnancies,whereas 6.7 % are specifically due to N. caninum, with an economic loss of US$440 (range, 150-730) per abortion.This amounts to an annual loss to the beef industry of US$12,903,440 (range, 1,130,700-42,070,630) in the same area. The results of this study show that Neospora infections and thus abortions cause severe economic impacts in the dairy and beef industries in the humid pampa region of Argentina, which is one the most important areas of cattle production in the world [corrected].

What is the global economic impact of Neospora caninum in cattle - the billion dollar question.

Neospora caninum is regarded as one of the most important infectious causes of abortions in cattle worldwide, yet the global economic impact of the infection has not been established. A systematic review of the economic impact of N. caninum infections/abortions was conducted, searching PubMed with the terms 'cattle' and 'Neospora'. This yielded 769 publications and the abstracts were screened for economically relevant information (e.g. abortion prevalence and risk, serological prevalence). Further analysis was restricted to countries with at least five relevant publications. In total, 99 studies (12.9%) from 10 countries contained data from the beef industry (25 papers (25.3%)) and 72 papers (72.8%) from the dairy industry (with the remaining two papers (2.0%) describing general abortion statistics). The total annual cost of N. caninum infections/abortions was estimated to range from a median US $1.1 million in the New Zealand beef industry to an estimated median total of US $546.3 million impact per annum in the US dairy population. The estimate for the total median N. caninum-related losses exceeded US $1.298 billion per annum, ranging as high as US $2.380 billion. Nearly two-thirds of the losses were incurred by the dairy industry (US $842.9 million). Annual losses on individual dairy farms were estimated to reach a median of US $1,600.00, while on beef farms these costs amounted to just US $150.00. Pregnant cows and heifers were estimated to incur, on average, a loss due to N. caninum of less than US $20.00 for dairy and less than US $5.00 for beef. These loss estimates, however, rose to ∼US $110.00 and US $40.00, respectively, for N. caninum-infected pregnant dairy and beef cows. This estimate of global losses due to N. caninum, with the identification of clear target markets (countries, as well as cattle industries), should provide an incentive to develop treatment options and/or vaccines.

Estimation of the economical effects of Eimeria infections in Estonian dairy herds using a stochastic model.

In this study, a stochastic predictive model stimulating a constant infection pressure of Eimeria was used to estimate production outcome, economic, and effects of treatment decisions in a dairy herd of 100 cows. The intestinal parasite cause problems mainly in calves, and is known to have long term effects on the growth rate, and in severe cases can result in mortalities. Due to the inconspicuous nature of the parasite, the clinical signs and sub-clinical manifestations it may produce can be overlooked. Acquired data from literature and Estonian dairy farms were implemented in the SimHerd IV model to simulate three scenarios of symptomatic treatment: no calves treated (NT), default estimate of the current treatment strategy (DT), and all calves treated (AT). Effects of metaphylactic treatment were studied as a lowering of the infection pressure. Delay in the age for beginning of insemination of heifers was the effect with the largest economic impact on the gross margin, followed by calf mortality and reduction in growth rate. Large expenses were associated with the introduction of replacement heifers and feeding of heifers as a result of the delay in reaching a specific body weight at calving. Compared to the control scenarios, with no effects and treatments of Eimeria, dairy farmers were estimated to incur annual losses ranging 8-9% in the balanced income. Providing metaphylactic drugs resulted in an increased gross margin of 6-7%. Purchase of new heifers compensated for some production losses that would otherwise have enhanced expenses related to Eimeria. The simulation illustrates how effects of Eimeria infections can have long lasting impact on interacting management factors. It was concluded that all three simulated symptomatic treatment regimes provided only small economic benefits if they were applied alone and not in combination with lowering of infection pressure.

Neospora caninum--how close are we to development of an efficacious vaccine that prevents abortion in cattle?

Neospora caninum is a protozoan parasite that causes abortion in cattle around the world. Although the clinical signs of disease in both dogs and cattle have now been recognised for over 20years, treatment and control options are still limited, despite the availability of a commercial vaccine in some countries of the world. The case for an efficacious vaccine has not been convincingly waged by farmers, veterinarians and other members of the agricultural and rural communities. In recent times, however, economic modelling has been used to estimate the industry losses due to Neospora-associated abortion, providing, in turn, the business case for forms of control for this parasite, including the development of vaccines. In this review, we document progress in all areas of the vaccine development pipeline, including live, killed and recombinant forms and the animal models available for vaccine evaluation. In addition, we summarise the main outcomes on the economics of Neospora control and suggest that the current boom in the global dairy industry increases the specific need for a vaccine against N. caninum-associated abortion.

Genomic analysis of Eimeria spp. populations in relation to performance levels of broiler chicken farms in Arkansas and North Carolina.

The impact of coccidiosis outbreaks on the productivity of broiler chicken farms can be substantial, depending on the severity of disease caused by particular species and strains of Eimeria. We examined the genetic diversity of Eimeria species present in commercial broiler farms in relation to their performance level. Four groups of broiler chicken farms in Arkansas (AR) and North Carolina (NC), having either high or low performance levels, were sampled for Eimeria spp. oocysts. We amplified gDNA from oocysts by using genus-specific primers targeting 18S ribosomal RNA, the first and second internal transcribed spacer regions, and cytochrome c oxidase subunit I as the established species-specific primers. Eimeria spp. diversity was not homogenous among the 4 farm groups, with less-pathogenic species (E. mitis and E. mivati-like) associated with AR and NC high-performance farms, respectively, and a pathogenic species (E. brunetti) associated with AR low-performance farms. Sequence analyses identified multiple E. maxima and E. mitis genetic variants, from which 2 E. maxima variants were unique to low-performance farms. Distinct populations of sequences at the NC high-performance farms were identified as E. mivati-like, based on homology searches. Our study demonstrated the utility of analyzing multiple genomic loci to assess composition and polymorphisms of Eimeria spp. populations.

[Epidemiological and financial considerations for the control of Neospora caninum on Swiss dairy farms]. / Epidemiologische und finanzielle Entscheidungsgrundlagen zur Kontrolle von Neospora caninum auf Schweizer Milchviehbetrieben.

Neospora caninum is widely recognized as one of the most important abortifacients in cattle and causes substantial financial losses to bovine livestock production. This study aimed to calculate the losses caused by N. caninum on Swiss dairy farms and to evaluate the efficacy and profitability of the control strategies culling, not breeding replacements and chemotherapy of calves on farm level. Three different farm sizes with high, medium and low herd prevalences were defined. Epidemiological and financial models were used to simulate the effect of control strategies on the prevalence over time and to perform a cost-benefit analysis. The median annual losses on farm level ranged between CHF 3094.- (= Euro 1875; 60 dairy cattle, high prevalence) and CHF 134.- (= Euro 81; 15 dairy cattle, low prevalence). Culling of animals that had any abortion or a N. caninum abortion, or not breeding replacements from such animals, respectively, were neither effective nor profitable. Only the strategy "not breeding replacements from N. caninum seropositive cows" on farms with a high prevalence was financially attractive. The strategy "chemotherapy of calves" should be re-evaluated as soon as new data regarding the efficacy of treatment and a corresponding protocol have been scientifically validated.

A survey of the economic impact of subclinical Eimeria infections in broiler chickens in Norway.

The objective of this work was to examine the impact of subclinical coccidial infection on commercial performance, expressed as a modified European Production Index, in broilers. Performance data, and litter and faecal samples, were collected from two independent observational surveys of Norwegian broilers receiving in-feed narasin during 2000 to 2004. Numbers of oocysts per gram (OPG) of litter collected during rearing (Study 1) or faecal samples collected at slaughter (both studies), and relative frequencies of Eimeria species categories (both studies) were calculated. Polymerase chain reaction-based identification of Eimeria species was performed in Study 2. A definition of flocks at risk of impaired performance associated with coccidia ("risk flock"), using the predominant species and OPG level as criteria, was tested. Coccidia had a significant effect on performance in the first, but not the second study. In Study 1 the following coccidia variables were found to be associated with impaired performance in multivariate models: OPG at slaughter (ordinal), mean OPG during rearing (ordinal) and "risk flock" (binomial). The European Production Index was approximately 9% lower in flocks with infection levels >50 000 OPG at slaughter in Study 1. The composition of coccidial populations shifted between Study 1 and Study 2, from a dominance of medium and large oocysts to a dominance of small oocysts. There was a substantial increase in prevalence of coccidial infection from Study 1 to Study 2, but mean infection levels were similar in the two surveys. The "risk flock" definition was useful as an indicator of coccidia-associated performance loss in Study 1, where subclinical coccidiosis was an important factor. The results suggest that the economic importance of subclinical coccidiosis may vary substantially with time, and they emphasize the need for population studies on the importance and dynamics of specific coccidial infections under different field conditions.

Financial analysis of various strategies for the control of Neospora caninum in dairy cattle in Switzerland.

The present study was conducted to estimate the direct losses due to Neospora caninum in Swiss dairy cattle and to assess the costs and benefits of different potential control strategies. A Monte Carlo simulation spreadsheet module was developed to estimate the direct costs caused by N. caninum, with and without control strategies, and to estimate the costs of these control strategies in a financial analysis. The control strategies considered were "testing and culling of seropositive female cattle", "discontinued breeding with offspring from seropositive cows", "chemotherapeutical treatment of female offspring" and "vaccination of all female cattle". Each parameter in the module that was considered to be uncertain, was described using probability distributions. The simulations were run with 20,000 iterations over a time period of 25 years. The median annual losses due to N. caninum in the Swiss dairy cow population were estimated to be euro 9.7 million euros. All control strategies that required yearly serological testing of all cattle in the population produced high costs and thus were not financially profitable. Among the other control strategies, two showed benefit-cost ratios (BCR) >1 and positive net present values (NPV): "Discontinued breeding with offspring from seropositive cows" (BCR=1.29, NPV=25 million euros ) and "chemotherapeutical treatment of all female offspring" (BCR=2.95, NPV=59 million euros). In economic terms, the best control strategy currently available would therefore be "discontinued breeding with offspring from seropositive cows".

The efficacy and economic benefits of Supercox, a live anticoccidial vaccine in a commercial trial in broiler chickens in China.

The efficacy and economic benefits of Supercox, a live anticoccidial vaccine were examined and compared with an anticoccidial drug in a trial in broiler chickens under modern commercial conditions in China. In total, 40,660 chickens were used in the present study, half of which were vaccinated with the Supercox vaccine comprising a precocious line of Eimeria tenella and non-attenuated lines of Eimeria maxima and Eimeria acervulina, and the other half were medicated with Diclazuril delivered as feed additive at the dosage of 1mg/kg of feed. The vaccine was administered orally to 7-day-old chickens. No clinical diseases were diagnosed in any of the vaccinated birds. However, clinical coccidiosis occurred in a large proportion of medicated control birds and these chickens had to be treated with anticoccidial drugs (Diclazuril and Toltrazuril). Comparison of production performance between vaccinated birds and medicated control birds revealed that the vaccine Supercox performed better than anticoccidial drugs in terms of mortalities, costs and overall economic benefits (profits). These findings demonstrated that the use of the Supercox vaccine could control clinical coccidiosis in broilers and achieve production performance superior to that using anticoccidial drugs, particularly where drug resistance might result in failure to control clinical diseases.