A survey of hemoparasite infections in free-ranging mammals and reptiles in French Guiana.

Blood smears of 1,353 free-ranging mammals (35 species) and 112 reptiles (31 species) from French Guiana were examined for hemoparasites. Parasites from 3 major groups were recorded: Apicomplexa (including hemogregarines, piroplasms, and Plasmodium spp.), Trypanosomatidae, and Filaroidea. Fifty percent of the individuals (86% of the species) were infected by parasites from at least 1 group. Hemogregarines, identified as Hepatozoon sp., infected numerous snakes with high prevalences (30-100%); infection is reported for the first time in 5 host genera of snakes: Clelia, Oxybelis, Pseustes, Rhinobotryum, and Bothriopsis. Infections were also observed in 4 marsupial species and 1 rodent. Hepatozoon spp. recorded in Didelphis albiventris (Marsupialia) and Coendou prehensilis (Rodentia) may be new species. Plasmodium sp. were observed in 2 snake species, Dipsas indica (Colubridae) and Bothrops atrox (Viperidae). Plasmodium brasilianum was recorded in all 5 primate species examined. Piroplasms were observed in all mammal orders except primates. Large terrestrial rodents were the main hosts of members of the Babesidae; 42% of Myoprocta acouchy, 36% of Dasyprocta agouti, and 44% of Agouti paca were infected. Trypanosomes were common in mammals and were recorded in 70% of the examined genera. Trypanosoma cruzi-like infections were reported in 21 mammal species, including sloths, rodents, carnivores, and primates. Microfilariae were also widespread, with higher prevalences in sloths, anteaters, and porcupines (>40% of the individuals infected) and in tamarins (95% infected). This survey highlights some potential anthropozoonotic risks due to the recent further evidence of Plasmodium brasilianum and P. malariae as a single species and to the increased diversity of hosts for Trypanosoma cruzi.

Chemotherapy of haemobartonellosis in squirrel monkeys (Saimiri sciureus).

Splenectomised Saimiri sciureus squirrel monkeys are being used increasingly as an experimental host for human malarial studies, notably for the assessment of candidate vaccines against Plasmodium falciparum blood-stage infection. Recently, we have reported that colony-reared S. sciureus monkeys are asymptomatic carriers of Haemobartonella sp. and that patent Haemobartonella infection, activated following splenectomy, may interfere with the course of P. falciparum parasitaemia in these animals. For several years, splenectomised S. sciureus monkeys were routinely submitted to oxytetracycline therapy before their use in malarial studies in order to prevent a possible spontaneous Heamobartonella infection. However, we report here that such antibiotic therapy is often ineffective and that neoarsphenamine chemotherapy may be considered as an alternative to cure both latent and patent haemobartonellosis in S. sciureus monkeys.

Haemobartonellosis in squirrel monkeys (Saimiri sciureus): antagonism between Haemobartonella sp. and experimental Plasmodium falciparum malaria.

A hemotropic parasite of the genus Haemo bartonella (rickettsial parasite of the Family Anaplasmataceae) is responsible for latent asymptomatic infection in colony-born Saimiri monkeys. Indeed, many of these animals develop a patent Haemobartonella infection following splenectomy. Such patent parasitism is characterized by an intense Haemobartonella parasitemia which peaks between days 12 and 14 after removal of the spleen and then decreases to become undetectable between days 25 and 30. During the resolving phase of parasitemia, a moderate anemia associated with monocytosis and erythrophagocytosis is observed. In certain Saimiri monkeys, Haemobartonella parasitemia remains latent following removal of the spleen. This indicates that the spleen plays a role but is not necessary to maintain latent Haemobartonella parasitism. It also suggests the existence of heterogeneity in the host immune reactivity to the parasite. Latent or patent haemobartonellosis might raise a problem when Saimiri monkeys are used as experimental hosts of Plasmodium falciparum asexual blood stages, as already noticed with "rodent malaria." Thus we investigated the relationship between Haemobartonella and P. falci parum in splenectomized monkeys. When animals harboring latent Haemobartonella sp. were infected with P. falciparum, the former remained latent and exerted no influence on the course of the P. falciparum parasitemia. In constrast, when P. falciparum was initiated in animals which were in the process of developing patent haemobarto nellosis, the course of the former was protracted and either the animal resisted longer, or it self-cleared the P. falciparum infection. Conversely, patent haemobartonellosis was delayed when splenectomy was performed at different times after initiation of P. falciparum infection in intact monkeys. Our results do not allow us to draw conclusions as to the mechanism(s) of the antagonism between the two parasites, but they emphasize the need to monitor the presence of Haemobartonella when splenectomized Saimiri monkeys are used as experimentals hosts for P. falciparum parasitism.

Plasmodium falciparum: immune pressure in Saimiri sciureus monkeys can select for a parasite population inducing a protective immunity that is not controlled by antibody.

Protective immunity against a Plasmodium falciparum blood infection can be passively transferred by antibodies in humans and in the primate experimental malaria model Saimiri sciureus. We report here the emergence of a novel virulent parasite population after such passive transfer of hyperimmune serum in splenectomized monkeys. These FUP-2 parasites have been partially genotyped and phenotyped. Although no genotypic variation was detected for four polymorphic loci compared to the original FUP-1 parasite population, FUP-2-infected erythrocytes exhibit little or no detectable surface determinants, including those reacting with antibodies raised against FUP-1 surface antigens. In addition, FUP-2-infected erythrocytes exhibit no rosetting or autoagglutination. Interestingly, although Saimiri monkeys control efficiently FUP-2 parasites after repetitive infections, this protection cannot be passively transferred to naive recipients. Our results suggest that antibody-mediated and antibody-independent T-cell-mediated protective responses may cooperate in controlling P. falciparum infection in splenectomized Saimiri monkeys.

Immunogenicity and efficacy trials with Plasmodium falciparum recombinant antigens identified as targets of opsonizing antibodies in the naive squirrel monkey Saimiri sciureus.

We have previously shown that Plasmodium falciparum recombinant antigens PfEB200, R23, and Pfi72 consistently inhibit opsonization of infected red blood cells by protective hyperimmune Saimiri sera, indicating that they present target epitopes involved in the phagocytosis of infected red blood cells. We report here an analysis of the immune response elicited in naive squirrel monkeys injected with the individual recombinant antigens or with a mixture of the three antigens combined with a synthetic peptide. In the three administration protocols investigated, there was no evidence for the production of antibody contributing to the phagocytosis of infected red blood cells, contrasting with the increase of opsonizing antibodies elicited by these antigens in monkeys with a prior (> or = 500 days) experience with malaria infection. However, the recombinant antigens were highly immunogenic, inducing specific antibody responses to P. falciparum and to the recombinant antigens. When the monkeys immunized with the antigen combination were challenged with blood-stage parasites, there was substantial protection: three of seven immunized animals self-cured and two others experienced a delayed peak of parasitemia. Taken together with our previous findings, these results suggest that PfEB200, R23, and Pfi72 constitute interesting vaccine candidates, and show that the presence of antibodies promoting phagocytosis of infected red blood cells is not a prerequisite for protection after immunization with these antigens in the Saimiri model.

Induction of opsonizing antibodies after injection of recombinant Plasmodium falciparum vaccine candidate antigens in preimmune Saimiri sciureus monkeys.

We have previously shown that Plasmodium falciparum recombinant antigens PfEB200, R23, and Pfi72 inhibit opsonization of infected erythrocytes by hyperimmune Saimiri sera, indicating that they contain target epitopes involved in the phagocytosis of infected erythrocytes. We have investigated in this study the immune response of Saimiri monkeys with previous experience of malaria infections (preimmune monkeys) after injection of these recombinant antigens, administered alone or simultaneously. The humoral response to the recombinant antigens was monitored by radioimmunoassay, and the response to P. falciparum blood stages was assayed by immunofluorescence. The relative proportion of protective versus nonprotective immunoglobulin subtypes was investigated by using 3A2/G6 and 3E4/H8 monoclonal antibodies, and the capacity of the antisera to promote in vitro phagocytosis of infected erythrocytes was evaluated. The antigens evoked in most cases a secondary-type antibody response, resulting in important increases in antigen-specific antibody titers and concomitantly in anti-P. falciparum titers. The ratio of 3A2/G6 to 3E4/H8 immunoglobulin subtypes varied with the immunogen used. Opsonizing antibodies were boosted in several animals, the most promising combination being the mixture of PfEB200 and R23 that induced long-lasting production in five of five animals. The detectable opsonizing activity appearing after immunization of the animals was antigen specific, as it was lost after adsorption of the recombinant antigens. The challenge of the animals with blood stage parasites confirmed previous findings showing a correlation between the presence of detectable opsonizing antibodies in serum and protection.

Flow cytometry detection of surface antigens on fresh, unfixed red blood cells infected by Plasmodium falciparum.

The immunofluorescence detection of parasite-specific antigens on the surface of red blood cells infected by Plasmodium falciparum parasites is usually performed by visual detection under a fluorescence microscope. We describe here a technique permitting the analysis of surface immunofluorescence labelling by flow cytometry. Infected red blood cells are selected on the basis of their parasitic DNA and RNA content by Hoechst and Thiazole Orange vital dyes. Cytometric analysis of these labels, as well as general erythrocyte characteristics assessed by analysis of forward and side scatter allows the selection of viable intact infected erythrocytes from other blood cells. The integrity of these selected erythrocytes was confirmed by the absence of labelling with antibodies directed against internal components such as spectrin. This technique permits the detection of specific surface immunofluorescence staining on red blood cells infected with mature stages of P. falciparum by antibodies in sera from hyperimmune Saimiri monkeys. Using Thiazole Orange dye for detection of parasitised cells, this analysis was performed on a FACSscan apparatus equipped with a single laser.

Manipulating blood T cells and B cells from squirrel monkeys: some technical considerations.

The squirrel monkey Saimiri sciureus is an experimental host for a range of human pathogens, and for the assessment of vaccine candidate antigens and vaccine strategies. This experimental host is thus particularly suitable for the follow-up of humoral responses. To understand some of the mechanisms that underlie the defense against experimental pathogens, there is a need of basic knowledge on cellular immune effectors also. The authors report here their experience in characterizing squirrel monkey blood T and B lymphocytes, and in studying in vitro induced activation and proliferation of T and B cells. Particular emphasis is given to the in vitro differentiation of squirrel monkey B cells into immunoglobulin secreting cells, with respect to Plasmodium falciparum antigens.

Squirrel monkey (Saimiri sciureus) B lymphocytes: secretion of IgG directed to Plasmodium falciparum antigens, by primed blood B lymphocytes restimulated in vitro with parasitized red blood cells.

Blood B lymphocytes obtained from Plasmodium falciparum-immune Saimiri monkeys were assayed for their in vitro differentiation in immunoglobulin-secreting cells upon restimulation with P. falciparum-parasitized Saimiri red blood cells. Selected culture conditions enabled appropriately stimulated blood B cells to secrete 3F11/G10+ IgG, detected in the supernatants by means of a dot immunobinding assay. Primed blood B lymphocytes from P. falciparum-immune Saimiri monkeys were thus able to secrete IgG when restimulated by parasitized red blood cells in the presence of T cell- and monocyte-derived cytokines (recombinant human cytokines). These primed blood B cells, which were able to differentiate, were shown to secrete antibodies reactive with P. falciparum-infected red blood cells, as detected by means of an indirect immunofluorescence assay, and reactive with P. falciparum-infected red blood cell extracts, as detected by means of Western blot analysis. Furthermore, due to the possibility of discriminating between IgG subtypes in the squirrel monkey (3F11/G10+::3A2/G6+ IgG [associated with protection against the blood stages of P. falciparum] vs. 3F11/G10+::3E4/H8+ IgG [usually not functionally associated with protection]), we have attempted to estimate the respective proportions of each IgG subtype. In defined culture conditions, Saimiri monkey blood B cells preferentially secrete 3F11/G10+::3E4/H8+ IgG in response to parasitized red blood cells. We therefore discuss the conditions that would render this assay suitable for the selection, among P. falciparum blood stage antigens, of those that have major B-cell epitopes.

Antibodies and reactive T cells against the malaria heat-shock protein Pf72/Hsp70-1 and derived peptides in individuals continuously exposed to Plasmodium falciparum.

Pf72/Hsp70-1, a heat-shock protein of m.w. 72 kDa from Plasmodium falciparum is one of the Ag of interest to be included in a polyvalent vaccine against malaria. It is one of the major immunogens present in a fraction of purified blood stage parasites that elicited protection against experimental infection of Saimiri monkeys with blood stages of P. falciparum. It is present at all blood stages and one of its B cell epitopes is also detected on the surface of the infected hepatocyte. Moreover, Pf72 appears to be well conserved among different isolates of P. falciparum. We have examined the immune response against Pf72/Hsp70-1 in individuals from different age groups living in a holoendemic area (West Africa). The immune response against the native Ag (purified from schizonts and called Pf/Hsp70) was analyzed both at the humoral level by ELISA and at the cellular level by assessing in vitro proliferation and IFN-gamma production of PBMC. Of the individuals studied 52% had a statistically significant level of anti-Pf/Hsp70 antibodies as compared with unexposed individuals. These positive individuals showed a heterogeneous distribution because significant levels of antibodies were found in 70% of the adults but in only 26% of the children. The presence of Pf/Hsp70-specific reactive T cells in the blood was detected in 32% of the individuals. The total anti-Pf/Hsp70 antibody level (IgG+IgM) appeared strongly age related and correlated positively with parasite exposure, whereas the T cell response failed to correlate either with the antibody level or with age. Moreover, PBMC of donors responded to the Pf/Hsp70 in a dissociated way, namely, by either T cell proliferation or IFN-gamma production. Ten synthetic peptides based on sequences found in the C-terminal part of Pf72/Hsp70-1 were further tested as potential T cell epitopes. The proliferative response of PBMC from individuals continuously exposed to the parasite showed that three peptides more frequently trigger significant T cell proliferation (in 21% to 27% of the individuals) and three others less frequently (10%). None of these peptides allowed detection of reactive T cells in PBMC of Europeans with no previous exposure to malaria. Some of the stimulating peptides are highly similar to human heat-shock Hsc and Hsp70 with large stretches of identical amino acids.(ABSTRACT TRUNCATED AT 400 WORDS)

M. leprae- and BCG-induced chemiluminescence response of monocytes from leprosy patients and healthy subjects: effects of gamma-interferon and GM-CSF.

Mycobacterium leprae, in contrast to BCG, failed to trigger any chemiluminescence (CL) response in mononuclear cells from either leprosy patients or healthy subjects, a deficit not reversed by either interferon-gamma or GM-CSF. Chemiluminescence responses induced without mycobacteria or with BCG were found to be lower in leprosy patients than in controls. M. leprae were also less well phagocytosed than BCG. However, there was a significant difference in phagocytosis between healthy and tuberculoid leprosy subjects. Phagocytosis was not altered by the addition of either lymphokine, and no major differences between healthy subjects and patients were observed. Preincubating mononuclear cells with anti-mycobacteria antibodies (lepromatous patients' sera) did not increase the CL response nor the phagocytosis of M. leprae or BCG.

Opsonic activity of ascitic fluids from Plasmodium falciparum-infected Saimiri monkey: positive correlation with protection in passive transfer assay.

An investigation into the protective activity of ascitic fluids from Saimiri monkeys infected with Plasmodium falciparum and the role played by opsonins in that activity was undertaken. P. falciparum-parasitized blood was collected from splenectomized Saimiri (when parasitaemia reached at least 20% or more) and used in an in vitro phagocytic assay including ascitic fluid and cultures of peripheral blood monocytes from normal Saimiri. Under the conditions of this in vitro assay, we found that ascitic fluid phagocytosis-promoting factors were opsonic rather than cytophilic. The opsonic activity was highly specific for parasitized red blood cells and was effective against all stages of development of the parasite. A highly positive relationship between in vitro opsonizing activity and in vivo protective capacity of immune ascitic fluid was found.

[Inflammation and resistance of mice against "Plasmodium berghei" (author's transl)]. / Incidence d'une réaction inflammatoire sur la résistance des souris envers Plasmodium berghei.

An inflammatory reaction induced in mice by a subcutaneous injection of magnesium silicate embedded in a calcium phosphate gel is followed by an increased resistance against Plasmodium berghei. The occurrence of this increased resistance against Plasmodium berghei. The occurrence of this increased resistance is related to the time elapsed between the induction of the inflammatory process and the infection. The delayed mortality is correlated with a slowed development of parasitemia. It is hypothesized that the protective effect may be related to the capacity of the inflammatory reaction to promote in mice both specific and non-specific antiparisitic immune responses.

Absence of correlation between delayed-type hypersensitivity and protection in experimental systemic candidiasis in immunized mice.

We found that in mice which had been immunized intraperitoneally with 2 x 10(8) heat-killed Candida albicans cells there was a striking temporal relationship between resistance to systemic challenge with 10(6) living C. albicans cells and a number of measurable cellular parameters of the host response. These included the emergence of delayed-type hypersensitivity and the development of granulocytosis. Since it had been shown in previous work that granulocytosis was associated with an increase in resistance when nonspecific immunostimulation was used, we performed experiments to induce delayed-type hypersensitivity without any measurable modification of the granulocyte population. Adoptive transfer of delayed-type hypersensitivity with spleen cells from immune and resistant donor mice did not produce any increase in resistance in normal recipients. When separate groups of mice were immunized intraperitoneally or subcutaneously with varying doses of heat-killed C. albicans, we found that doses of less than 10(8) cells did induce significant delayed-type hypersensitivity without any increase in granulocytosis. In such mice, as well as in animals pretreated with immunomodulators before immunization with heat-killed C. albicans, the presence of cell-mediated immunity, as measured by the delayed-type hypersensitivity test, was not associated with an increase in resistance against systemic candidiasis. On the contrary, the results suggested that cell-mediated immunity was associated with an increase in the susceptibility of these mice. The same effect on candidiasis susceptibility was observed when animals were immunized with heat-killed filamentous C. albicans.

Residual immunogenicity of irradiated spleen cell suspensions after a single intravenous injection of sheep red blood cells in mice.

Studies were carried out to characterize the residual immunogenicity located in spleen cells of mice after one single intravenous injection of SRBC. An in vivo system was applied for initiation and expression of the immune response consisting of one intraperitoneally injection of spleen cell suspension from SRBC-injected mice in separate groups of recipients in which DTH and secondary humoral response were measured, respectively: six days or four days after the transfer of irradiated spleen cell suspension, the separate groups of recipients being either pretreated with cyclophosphamide (200 mg/kg) or primed with 5 X 10(5) SRBC. The residual immunogenicity was found to be located in a population of radio-resistant (10,000 rads) adherent spleen cells. The same population derived from naive mice injected together with the native antigen did not modify the immune response as compared with those induced by the antigen alone. Furthermore, irradiated spleen cell suspensions from SRBC-injected mice were not able to transfer adoptively DTH sensitivity in naive recipients. Accordingly to the test used to evaluate the residual immunogenicity, it was found that the kinetics of residual immunogenicity able to induce DTH was different from those which stimulate the secondary humoral response. Furthermore, it was found that distinct variations of residual immunogenicity for DTH and antibody formation were induced by varying host manipulations, such as non-specific stimulation of the reticulo-endothelial system, specific activation of T cells involved in DTH or helper function or induction of a state of anergy. It appears, therefore, that the same population of radio-resistant adherent spleen cells are able to induce an immune response and to dispatch information to cells involved in DTH or those involved in antibody synthesis. Moreover, depending upon conditions of immunization, varying subsets of T cells are able to modify the dispatching function of these cells in the processing or presentation of antigen to committed or non-committed lymphocytes.

Delayed-type hypersensitivity to sheep red blood cells in selected lines of mice with high or low antibody responses.

Interline differences in DTH to SRBC which developed in high antibody producer (H/Ab) and low antibody producer (L/Ab) genetically selected lines of mice were observed after intravenous (IV) or subcutaneous (SC) sensitization. This interline difference was more marked in female that in male mice, and with the optimal dose for DTH sensitization. The kinetics of the local inflammatory reaction after local challenge, the development and decay of the 24-h DTH reaction tested at varying intervals after sensitization, were always statistically significantly higher in H/Ab than in L/Ab mice. This higher DTH was associated with a higher capacity of these mice to produce a secondary humoral response, and the more marked difference was observed for the production of 2-mercaptoethanol-resistant specific antibodies. Higher DTH reaction in H/Ab mice could not be explained by the participation of either B lymphocytes or humoral antibodies, since high dose (200 mg/kg) of CY injected before IV-SRBC, sensitization, in order to inhibit B-cell activation, did not alter the DTH-interline difference. Moreover, IV injection of specific antibodies from H/Ab immune mice did not enhance DTH in adoptively immunization L/Ab mice. Immune serum alone was not able to adoptively transfer DTH either in H/Ab or in L/Ab mice. Also, a low dose (40 mg/kg) known to inhibit T-cell suppression, injected before SC-SRBC sensitization, was not able to modify the DTH reaction in L/Ab mice. The persisting higher 48-h local inflammatory reaction in H/Ab mice after CY pretreatment was also related to a high blood monocyte rebound. When the afferent arc of the immune response to SRBC was studied in these mice, as tested by the lymphoproliferative response in draining popliteal node after footpad sensitization and by the evaluation of the ability of the T-activated cells located in these draining nodes to transfer locally an adoptively DTH reaction into naive recipients, it was shown that in immunized mice more specifically activated T cells were produced in H/Ab than in L/Ab mice. Moreover, the H/Ab recipients had a higher capacity to develop a local inflammatory reaction than the L/Ab recipients. The mechanisms of these observed interline differences are discussed.

Systemic candidiasis in mice. II.--Main role of polymorphonuclear leukocytes in resistance to infection.

Cyclophosphamide (CY) increased whereas the talc embedded in a calcium phosphate gel (TCP) decreased the susceptibility of mice to systemic candidiasis estimated by measuring mean survival time and "renal infectivity" 12 h after challenge. Transfers of plasma from CY- and TCP-treated mice did not modify cnadidiasis susceptibility of recipient mice. Granulopenia and granulocytosis induced respectively by CY and TCP were significantly correlated with susceptibility or resistance to candidiasis. Nevertheless, TCP produced significant reticuloendothelial stimulation which could be also correlated with TCP protection. Reticuloendothelial stimulation with associated granulopenia in TCP-CY-treated mice gave protection against Listeria monocytogenes challenge but not against Candida albicans. Thus, blood polymorphonuclear leukocytes seem to play the main role in natural resistance of mice to candidiasis. This was corroborated after injection of immunostimulants; a good correlation was found between C. albicans resistance and the induced granulocytosis.

Systemic candidiasis in mice. I.--Correlation between kidney infection and mortality rate.

After intravenous inoculation of mice with large doses of living Candida albicans, the kidneys are the only organs where multiplication of fungi occurs. After systemic challenge performed with varying doses of yeasts, kidney infection shows an identical pattern during the first 12 h, independently of the magnitude of the inoculum. Destruction of parasites occurs during the first 3 h and then C. albicans begins to multiply. Death occurs when the number of microorganisms in the kidneys exceed 10(5) viable units and the time of death is correlated with renal infection measured 12 h after challenge. An identical correlation is found when mice are pretreated with an immunosuppressor or immunostimulants. No statistical significance is observed in counts of C. albicans in right or left kidney, in males or females, but a slightly higher total count and a lower mean survival time are noted in female mice. These results show that counting C. albicans in kidneys 12 h after systemic infection provides a rapid screening test. In addition, this fact taken with the mortality may explain the nature of the pathological processes which occur during systemic candidiasis in mice.

Modulation by malaria infection of the induction of T lymphocyte-dependent delayed-type hypersensitivity and antibody formation to sheep erythrocytes in mice.

A profound alteration of the inductive phase of delayed-type hypersensitivity and antibody formation to SRBC was found in malaria infected mice when sensitization with this antigen was performed intravenously at a critical time of the disease, but not after subcutaneous immunization, suggesting a major role for the spleen in the mechanism of immunodepression.

[Effect of protein additives in mouse food on immune response to sheep red blood cells in mice (author's transl)]. / Influence de l'alimentation sur l'induction d'une hypersensibilité de type retardé chez la souris.

Protein additives used in mouse food (fish meal, bovine blood meal, milk powder) interfere with the specific immune response induced with sheep red blood cells. Bovine blood meal reduces delayed type hypersensitivity (DTH) reactions and delays IgG production. Blood derivatives suppress mostly the central induction and not the peripheral expression of DTH since depression can be shown after systemic and not after subcutaneous immunization. The suppression of DTH is specific, it did not appear when DTH was produced with antigens presenting no antigenic cross-reactivity with protein additives (chicken red blood cells, tuberculin). This depressive effect was related neither to the presence of inhibitory serum factor nor to the production after immunization of specific antigen-antibody-blocking factor. Blood derivatives seem to act on the afferent arc of immune response especially on the splenic immunogenicity of the injected antigen to produce DTH.