Controversies in therapy of infections caused by Rhodococcus equi in foals

Pneumonia caused by Rhodococcus equi is one of the most important causes of disease and death in foals. R. equi can also be cultured from a large variety of extrapulmonary sites of infection. In the absence of an effective vaccine, ultrasonographic screening for early detection of pulmonary lesions has become routine practice at many farms endemic for pneumonia caused by R. equi. Consequently, the most frequently recognised form of R. equi infection at such farms is a subclinical form in which foals develop sonographic evidence of peripheral pulmonary consolidation or abscessation without necessarily manifesting clinical signs. Evidence exists that not all foals with ultrasonographic lesions will progress to develop clinical signs, and treating a large proportion of foals based on subclinical ultrasonographic findings has been linked to emergence of macrolide‐ and rifampin‐resistant R. equi at a horse farm. Selectively treating only those foals with larger lesion scores and monitoring foals with daily physical inspections and weekly thoracic ultrasonography offers an approach that could decrease antimicrobial drug use without significantly increasing mortality. Current evidence continues to support the combination of rifampin with a macrolide (azithromycin, clarithromycin or erythromycin) for treating clinical infections caused by R. equi despite recently described pharmacological interactions between these drugs. When infection with a macrolide‐resistant isolate is confirmed, limited effective alternatives exist.     

Role of antibody to extracellular proteins of Rhodococcus equi in protection against R. equi pneumonia in foals

Rhodococcus equi produces two exoenzymes (REE), a cholesterol oxidase in large amounts and a phospholipase C, which cause lysis of sheep red blood cells (SRBC) sensitized with Staphylococcus aureus beta toxin. Two immunization studies were done in foals to determine the role of antibody to REE in protection against R. equi pneumonia. In the first study, three foals (mean age 10 days) were vaccinated four times at 2-week intervals with over 1 million units of partially purified exoenzymes (PREE). In the second study, three foals (mean age 19 days) were administered plasma from an adult horse vaccinated with PREE. Relatively low titres (16-32) of neutralizing antibody were detected in the foals of the former group, and passive transfer of neutralizing antibody (titres 32-64) occurred in the latter. Following immunization, principal foals and an equal number of similarly aged nonimmunized foals were challenged by aerosol with 1 x 10(10) live R. equi per day for 5 consecutive days. No severe clinical pneumonia developed in either group and, with one exception, only minor and resolving lung abscesses developed in these foals. These studies showed that antibody response of foals to immunization with PREE was poor, antibody to PREE did not prevent foals from developing lung abscesses following experimental infection, and that foals even as young as 3 weeks of age may be largely refractory to aerosol challenge with virulent R. equi.     

Rhodococcus equi foal pneumonia: protective effects of immune plasma in experimentally infected foals

The immunoprophylactic capacity of specific immune plasma was evaluated in pony foals infected experimentally with Rhodococcus equi. Immune plasma, produced by repeated parenteral administration of viable R. equi to adult horses, was harvested and frozen. Group I (six control foals) and Group II (six principal foals) received lactated Ringers solution and immune plasma respectively at three and five days of age. R. equi were aerosolised into a caudal lung lobe of all foals at seven days of age. Clinical signs, haematological alterations, immune responses, thoracic radiographs and technetium99m pulmonary perfusion scans were monitored. All foals were destroyed and complete post mortem examinations performed. All foals developed pneumonia as evidenced by clinical, radiographic and perfusion alterations, but the survival rate of principal foals was significantly (P less than 0.01) greater than that of control foals. Five control foals developed terminal disease, whereas all principal foals recovered. There was no significant (P greater than 0.05) difference in temperature response, or peripheral blood leucocyte, neutrophil or fibrinogen concentrations between groups. ELISA values for R. equi antibody were significantly (P less than 0.001) greater in principal foals following treatment, but there was no significant (P greater than 0.05) difference in IgG or IgM concentrations between groups. Results of the haemolysis inhibition assay indicated that equi factor neutralising antibodies were transferred by immune plasma to the principal foals. Post mortem examinations of five control foals destroyed at approximately three weeks post infection because of terminal disease, revealed severe pyogranulomatous pneumonia. One control and all principal foals were either free of lesions or had resolving lesions and/or minimal scar formation at three months post infection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recognition of a B-cell epitope of the VapA protein of Rhodococcus equi in newborn and experimentally infected foals

The aim of this study was to evaluate the usefulness of the previously identified B-cell epitope TSLNLQKDEPNGRASDTAGQ of the VapA protein of Rhodococcus equi and its association with R. equi pneumonia. A modified peptide designated PN11-14 corresponding to the epitope was recognized by all sera from experimentally infected foals with virulent R. equi ATCC103+ containing the virulence plasmid but not by its plasmid-cured derivative ATCC103- strain. Marked levels of VapA-specific immunoglobulin (Ig)G were detected in all sera from the ATCC103+ infected foals at 2 weeks after the infection. One control animal had high titres as determined by the peptide enzyme-linked immunosorbent assay (ELISA), indicating the ELISA may not absolutely differentiate between foals with R. equi pneumonia and healthy exposed foals in farms where the prevalence of disease is high. However, numbers of animals used were small. Further evaluation of the peptide ELISA with field samples is necessary to determine whether the assay is diagnostically useful. This study showed that levels of passive transfer of maternal IgG antibodies to the epitope in newborn foals could be measured. Interestingly, the maternally derived antibodies were found to significantly (P<0.05 by Student's t-test) decline 2 weeks after birth. Seroconversion against naturally occurring VapA expressing R. equi could be detected in some foals at 4 weeks of age. Antibodies to the epitope peaked and were significantly (P<0.05) greater in foals aged between 6 and 8 weeks. These results indicated that the peptide ELISA could be used to monitor anti-VapA antibodies in foals, particularly those at the age of 4-6 weeks. It is possible that the ELISA may be of some use as a diagnostic test on farms where R. equi is non-endemic. Further studies using large number of field samples are needed to verify this assumption.     

Effect of hyperimmune plasma on the severity of pneumonia caused by Rhodococcus equi in experimentally infected foals

This study evaluated the prophylactic effectiveness of hyperimmune plasma (HIP) as an aid in the prevention of pneumonia caused by experimental infection with Rhodococcus equi. Thirty neonatal foals were administered R. equi HIP or saline at 2 days of age and were infected with virulent R. equi at 7 days. All foals developed signs or symptoms of respiratory disease. Radiographic scores on day 28 and neutrophil concentrations on day 49 were significantly greater in control foals, and time to respiratory effort score of 2 or higher was significantly shorter for control foals. Three foals, all in the principal group, died or were euthanized before the end of the study, but there was no significant difference in mortality between groups. VapA titers were significantly greater in principal foals. Administration of R. equi HIP decreased the severity of radiographic lesions and prolonged time to increased respiratory effort due to R. equi-induced pneumonia.     

Association of soil concentrations of Rhodococcus equi and incidence of pneumonia attributable to Rhodococcus equi in foals on farms in central Kentucky

OBJECTIVE: To determine whether soil concentrations of total or virulent Rhodococcus equi differed among breeding farms with and without foals with pneumonia caused by R equi. SAMPLE POPULATION: 37 farms in central Kentucky. Procedures-During January, March, and July 2006, the total concentration of R equi and concentration of virulent R equi were determined by use of quantitative bacteriologic culture and a colony immunoblot technique, respectively, in soil specimens obtained from farms. Differences in concentrations and proportion of virulent isolates within and among time points were compared among farms. RESULTS: Soil concentrations of total or virulent R equi did not vary among farms at any time point. Virulent R equi were identified in soil samples from all farms. Greater density of mares and foals was significantly associated with farms having foals with pneumonia attributable to R equi. Among farms with affected foals, there was a significant association of increased incidence of pneumonia attributable to R equi with an increase in the proportion of virulent bacteria between samples collected in March and July. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that virulent R equi were commonly recovered from soil of horse breeding farms in central Kentucky, regardless of the status of foals with pneumonia attributable to R equi on each farm. The incidence of foals with pneumonia attributable to R equi can be expected to be higher at farms with a greater density of mares and foals.     

Increased pulmonary activation of nuclear factor-kappaB (NF-kappaB) in foals inoculated with Rhodococcus equi is associated with increased expression of inflammatory cytokines

Previous studies revealed that foals inoculated with virulent Rhodococcus equi had significantly higher pulmonary levels of interleukin-1beta, interleukin-12 p40, interferon-gamma, and tumor necrosis factor-alpha mRNA compared to foals inoculated with an avirulent plasmid-cured derivative. The purpose of this study was to determine if the increases in cytokine expression were associated with increased pulmonary activation of nuclear factor-kappaB (NF-kappaB). Electrophoretic mobility shift assays were performed on pulmonary nuclear protein extracted from foals treated with phosphate-buffered saline, or inoculated with either virulent or avirulent R. equi. NF-kappaB activation was increased in the nuclear extracts from foals inoculated with virulent R. equi at 14 days after inoculation when increased cytokine expression was also observed. Southwestern histochemistry revealed activated NF-kappaB in multinucleated giant cells that often contained bacteria. These results indicate that the cytokine response to R. equi is at least partially mediated by NF-kappaB activation.     

Farm characteristics and management practices associated with development of Rhodococcus equi pneumonia in foals

OBJECTIVE: To identify farm characteristics and management practices associated with development of Rhodococcus equi pneumonia in foals. DESIGN: Prospective case-control study. ANIMALS: 5230 foals on 138 breeding farms with 9136 horses. PROCEDURE: During 2003, participating veterinarians provided data from 1 or 2 farms with > or =1 foal with R equi pneumonia and unaffected farms. Data from affected and unaffected farms were compared by use of logistic regression analysis. RESULTS: A number of variables relating to farm size and desirable management practices were significantly associated with increased odds of farms being affected with R equi pneumonia. By use of multivariate logistic regression, affected farms were determined significantly more likely to have raised Thoroughbreds, housed > or =15 foals, used concrete floors in foaling stalls, and tested foals for passive transfer of immunity than unaffected farms. These results remained significant even after accounting for exposure of foals to other breeding farms during the first month of life. Conclusions and Clinical Relevance-Breeding farms with large acreage and a large number of mares and foals have greater odds of being affected by R equi pneumonia. Clinical relevance of associations with Thoroughbred breed and concrete flooring in foaling stalls remains uncertain. Desirable management factors commonly used on farms were not effective for controlling or preventing development of R equi pneumonia. This finding indicates a need to focus on host factors that influence disease development.     

Effects of inoculum size on cell-mediated and humoral immune responses of foals experimentally infected with Rhodococcus equi: A pilot study

The objective of this pilot study was to compare the cytokine profile as well as cell-mediated and antibody responses of foals infected with a low inoculum of virulent Rhodococcus equi resulting in subclinical pneumonia to that of foals infected with a high inoculum resulting in severe clinical pneumonia. The mean (±SD) ratio of post-infection to pre-infection anti-R. equi IgG(T) concentration was significantly (P = 0.002) higher in foals infected with the high inoculum (195 ± 145; range 62–328) compared to foals infected with the low inoculum (3.9 ± 4.5; range 0.5–11). Similarly, mean (±SD) ratio of post-infection to pre-infection IgM concentration was significantly (P = 0.002) higher in foals infected with the high inoculum (12 ± 4.0; range 7.4–14) compared to foals infected with the low inoculum (2.5 ± 1.5; range 1.2–4.7). Proliferative responses to R. equi antigens as well as expression of mRNA for IL-2, IL-4, IL-10, and IFN-γ in BLN were not significantly different between the two groups. There was a tendency (P = 0.073) towards a higher IFN-γ/IL-4 ratio in the low inoculum group. This study demonstrates that the size of inoculum modulates the IgG subisotype response and possibly the cytokine profile of foals.     

Diagnosis and therapy of Rhodococcus equi infection in the horse

Infection with Rhodococcus equi is an important cause of pneumonia in foals, but other organ systems may also be affected. The intracellular presence of R. equi and the formation of granulomatous and suppurative inflammatory tissue mean that prolonged treatment is needed. The pharmacological properties of the combination of erythromycin and rifampicin have improved the survival of foals infected with R. equi; however, erythromycin can cause adverse reactions in foals and mares, which has prompted the search for alternative therapies. The combination of azithromycin or clarithromycin with rifampicin seems to be a promising alternative. However these combinations are expensive and adverse effects remain to be determined, especially in the dams of treated foals. Thus correct diagnosis and appropriate use of drugs are essential for the treatment of R. equi infection in foals.     

Diagnosis, treatment, control, and prevention of infections caused by Rhodococcus equi in foals

Rhodococcus equi, a gram-positive facultative intracellular pathogen, is one of the most common causes of pneumonia in foals. Although R. equi can be cultured from the environment of virtually all horse farms, the clinical disease in foals is endemic at some farms, sporadic at others, and unrecognized at many. On farms where the disease is endemic, costs associated with morbidity and mortality attributable to R. equi may be very high. The purpose of this consensus statement is to provide recommendations regarding the diagnosis, treatment, control, and prevention of infections caused by R. equi in foals.     

Air sampling in the breathing zone of neonatal foals for prediction of subclinical Rhodococcus equi infection

Reasons for performing the study: Disease caused by Rhodococcus equi is a significant burden to the horse breeding industry worldwide. Early detection of rhodococcal pneumonia, albeit important to minimise treatment costs, is difficult because of the insidious nature of the disease and the lack of definitive diagnostic tests. Objectives: To investigate air sampling from the breathing zone of neonatal foals as a predictor of subsequent rhodococcal pneumonia. Methods: Air samples were collected from the breathing zone of 53 neonatal foals (age ≤10 days) and again at the time of routine ultrasonographic screening for R. equi pneumonia (age 1–2 months). Results: Pneumonia was diagnosed ultrasonographically in 23% of foals. Virulent R. equi was detected in air from the breathing zone of 19% of neonatal foals and 45% of foals at age 1–2 months. There was no association between virulent R. equi in the breathing zone of foals and the subsequent ultrasonographic diagnosis of rhodococcal pneumonia. The median concentration of virulent R. equi in the breathing zone of both neonates (0 [range 0–4] colony‐forming units [cfu]/250 l) and older foals (0 [range 0–3] cfu/250 l) was not significantly different from that in background air samples (0 [range 0–6] cfu/250 l). There was no difference in the concentration of virulent R. equi in the breathing zone of older foals that were diagnosed with rhodococcal pneumonia or clinically normal foals. Conclusion: Detection of virulent R. equi in air from the breathing zone was not a positive predictor of rhodococcal pneumonia in foals up to age ≤2 months. Potential relevance: Selective culture of air samples from the breathing zone of young foals is not better at diagnosing rhodococcal pneumonia than early ultrasonographic screening. However, culture of air samples from the breathing zone of older foals remains a useful herd‐based epidemiological tool.     

Evaluation of equine immunoglobulin specific for Rhodococcus equi virulence-associated proteins A and C for use in protecting foals against Rhodococcus equi-induced pneumonia

OBJECTIVE: To determine whether purified equine immunoglobulin specific for Rhodococcus equi virulence-associated proteins A and C (VapA and VapC) can confer passive protection against R. equi-induced pneumonia in foals. ANIMALS: Twenty-eight 3-week-old mixed-breed pony foals. PROCEDURE: 7 foals received IV injections of equine hyperimmune plasma (HIP) against whole-cell R. equi, and 7 received purified equine immunoglobulin specific for VapA and VapC 1 day prior to intrabronchial infection with R. equi strain 103+. Eleven foals were not treated prior to infection, and 3 control foals were neither treated nor infected. Heart rate, respiratory rate, and rectal temperature were recorded twice daily, and serum fibrinogen concentration and WBC count were determined every other day following infection. Foals were euthanatized 14 days following infection, and lung lesions and concentration of R. equi in lungs were assessed. RESULTS: The onset of clinical signs of pneumonia was significantly delayed in the HIP- and immunoglobulin-treated groups, compared with the untreated infected group. Moreover, pulmonary lesions were less severe in the treated groups, and significantly fewer R. equi organisms were cultured from the lungs of treated foals. CONCLUSIONS AND CLINICAL RELEVANCE: Degree of protection against R. equi-induced pneumonia provided by purified immunoglobulin specific for VapA and VapC was similar to that provided by commercially available HIP. Results not only suggest that immunoglobulin is the primary component of HIP that confers protection against R. equi-induced pneumonia in foals but also indicate that antibodies against R. equi VapA and VapC are protective.     

Review of Corynebacterium (Rhodococcus) equi lung abscesses in foals: pathogenesis, diagnosis and treatment

Corynebacterium (Rhodococcus) equi is becoming increasingly significant as a cause of bronchopneumonia and lung abscessation in foals. The organism can survive within macrophages and may thus escape normal pulmonary defence mechanisms, particularly in immunocompromised animals. The disease has hitherto been associated with mortality rates as high as 80 per cent, partly as a result of inappropriate therapy. The selection of lipid-soluble antibiotics capable of intracellular penetration is critical for the successful treatment of C equi lung abscesses. A combination of two such antibiotics, erythromycin (25 mg/kg three times daily) and rifampicin (5 mg/kg twice daily) has been used on foals since 1981. Most of these animals had radiographic evidence of extensive lung abscessation, and in all cases the presence of C equi was confirmed on culture of tracheal aspirates. The duration of therapy ranged from four to nine weeks. Mild gastritis and diarrhoea were occasionally noted, but never such as to require termination of the therapy. No other adverse side effects were encountered. The success rate, as judged by a return to normal of chest radiographs and plasma fibrinogen concentrations, has exceeded 80 per cent.     

Pulmonary disposition of tilmicosin in foals and in vitro activity against Rhodococcus equi and other common equine bacterial pathogens

The objectives of this study were to determine the serum and pulmonary disposition of tilmicosin in foals and to investigate the in vitro activity of the drug against Rhodococcus equi and other common bacterial pathogens of horses. A single dose of a new fatty acid salt formulation of tilmicosin (10 mg/kg of body weight) was administered to seven healthy 5- to 8-week-old foals by the intramuscular route. Concentrations of tilmicosin were measured in serum, lung tissue, pulmonary epithelial lining fluid (PELF), bronchoalveolar lavage (BAL) cells, and blood neutrophils. Mean peak tilmicosin concentrations were significantly different between sampling sites with highest concentrations measured in blood neutrophils (66.01+/-15.97 microg/mL) followed by BAL cells (20.1+/-5.1 microg/mL), PELF (2.91+/-1.15 microg/mL), lung tissue (1.90+/-0.65 microg/mL), and serum (0.19+/-0.09 microg/mL). Harmonic mean terminal half-life in lung tissue (193.3 h) was significantly longer than that of PELF (73.3 h), bronchoalveolar cells (62.2 h), neutrophils (47.9 h), and serum (18.4 h). The MIC90 of 56 R. equi isolates was 32 microg/mL. Tilmicosin was active in vitro against most streptococci, Staphylococcus spp., Actinobacillus spp., and Pasteurella spp. The drug was not active against Enterococcus spp., Pseudomonas spp., and Enterobacteriaceae.     

Characterization of virulence plasmid types in Rhodococcus equi isolates from foals,pigs, humans and soil in Hungary

Rhodococcus equi isolates (204) obtained from foals (lung abscesses, lymph nodes, nasal discharge, rectal swabs) bred in 15 studs located throughout Hungary, isolates from soil samples, lymph nodes of pigs and from lesions of human patients were examined to determine genotypic diversity of virulence-associated plasmids. Isolates were tested for the presence of 15-17 kDa virulence-associated protein antigen (VapA) and 20k Da (VapB) genes by polymerase chain reaction (PCR). Plasmid DNAs were isolated and analysed by digestion with restriction endonucleases for estimation of size and comparison of polymorphisms. Of 146 clinical isolates from foals in 15 studs, 129 (88.3%) gave positive results for the VapA gene, showing a 564 bp product of the expected size in the PCR amplification. Of the 129 clinical isolates from foals, 123 contained an 85 kb type I plasmid and the remaining six contained an 87 kb type I plasmid. Of 48 soil isolates from two horse studs, 26 (54.2%) were positive for VapA gene and contained an 85 kb type I plasmid. Of three pig isolates, one was positive for VapA gene and contained an 85 kb type I plasmid, and the remaining two were positive for the VapB gene, showing a 827 bp product of the expected size in the PCR amplification and were R. equi of intermediate virulence which contained a 95 kb type S5 plasmid. Of the seven human isolates, five were positive for VapB gene by PCR, these were R. equi of intermediate virulence, which contained a 95 kb type S5 plasmid. These results revealed that virulent R. equi strains harbouring a virulence plasmid of 85 kb type I or 87 kb type I, which have been found in clinical isolates from Europe and North and South America, are widespread in Hungary. Furthermore, same intermediately virulence plasmid type was found in both human and pig isolates.     

Retrospective comparison of azithromycin, clarithromycin, and erythromycin for the treatment of foals with Rhodococcus equi pneumonia

The objective of this retrospective study was to compare the efficacy of azithromycin-rifampin, clarithromycin-rifampin, and erythromycin-rifampin for the treatment of pneumonia caused by Rhodococcus equi in foals. Eighty-one foals with naturally acquired pneumonia caused by R. equi were included in the study. Information on age, sex, breed, physical examination findings, laboratory testing, and thoracic radiography was abstracted from each medical record. Foals were divided in 3 groups based on the antimicrobial agent selected for therapy. Short-term (discharge from the hospital) and long-term (apparently healthy as a yearling) success rates, days of hospitalization, days with fever, days with tachypnea, and percentage of radiographic improvement were compared among groups. Foals treated with clarithromycin-rifampin had significantly (P = .02) higher odds of overall short-term (odds ratio [OR] = 12.2) and long-term (OR = 20.6) treatment success and significantly fewer days with fever than foals treated with erythromycin-rifampin. Foals treated with clarithromycin-rifampin had a significantly (P = .03) higher percentage of radiographic improvement and a tendency (P = .06) toward higher odds of overall short-term (OR = 8.1) and long-term (OR = 11.8) treatment success compared to foals treated with azithromycin-rifampin. Among foals with severe radiographic lesions, the success rates of foals treated with clarithromycin-rifampin both short-term (88%) and long-term (83%) were significantly (P = .02) higher than that of foals treated with azithromycin-rifampin (0%). For each treatment group, the only reported adverse effect was diarrhea that was mild and self-limiting in most cases. The combination clarithromycin-rifampin is superior to azithromycin-rifampin or erythromycin-rifampin for the treatment of pneumonia caused by R. equi in foals in a referral population.     

Evaluation of equine breeding farm management and preventative health practices as risk factors for development of Rhodococcus equi pneumonia in foals

OBJECTIVE: To determine whether foal management practices, environmental management, and preventative health practices are risk factors for development of Rhodococcus equi pneumonia in foals. DESIGN: Prospective matched case-control study. ANIMALS: 2,764 foals on 64 equine breeding farms with 9,991 horses. PROCEDURE: During 1997, participating veterinarians completed paired data collection forms for comparison; 1 for an affected farm (containing > or = 1 foal with pneumonia caused by R equi) and 1 for a control farm. Information collected pertained to stabling facilities, environmental management, foal husbandry, and preventative equine health practices. RESULTS: Matched farm data compared by use of conditional logistic regression indicated that personnel on affected farms were more likely to attend foal births, test foals for adequacy of passive immunity, administer plasma or other treatments to foals to supplement serum immunoglobulin concentrations, administer hyperimmune plasma prophylactically to foals, vaccinate mares and foals against Streptococcus equi infection, and use multiple anthelmintics in deworming programs. Affected farms were also more likely to have foals that developed other respiratory tract disorders and were approximately 4 times as likely to have dirt floors in stalls used for housing foals as were control farms. CONCLUSIONS AND CLINICAL RELEVANCE: Rhodococcus equi pneumonia does not appear to be associated with poor farm management or a lack of attention to preventative health practices. Housing foals in stalls with dirt floors may increase the risk for development of R equi pneumonia.     

Differential expression of inflammatory and immune response genes in sheep infected with Anaplasma phagocytophilum

Anaplasma phagocytophilum infects a wide variety of host species and causes the diseases tick-borne fever (TBF) in ruminants and granulocytic anaplasmosis in humans, horses and dogs. TBF in sheep has become one of the more prevalent tick-borne diseases in some regions of Europe. A. phagocytophilum infection modifies host gene expression and immune response. The objective of this research was to characterize differential gene expression in sheep experimentally and naturally infected with A. phagocytophilum by microarray hybridization and real-time RT-PCR. The results of these studies demonstrated in sheep the activation of inflammatory and innate immune pathways and the impairment of adaptive immunity during A. phagocytophilum infection. The characterization of the genes and their expression profiles in sheep in response to A. phagocytophilum infection advances our understanding of the molecular mechanisms of pathogen infection and the pathogenesis of TBF. Collectively, these results expand current information on the mammalian host response to A. phagocytophilum infection.