Influence of rodent malaria on the course of Leishmania enriettii infection of hamsters.

Plasmodium yoelii infection was established in hamsters, and the effect of this type of malaria on concurrent Leishmania enriettii infection was examined. It was found that the course of the L. enriettii infection was affected by P. yoelii and that this effect depended on the relative timing of the two infections. A chronic malarial infection with Plasmodium berghei was also established in hamsters, and this was found to affect the course of a concurrent L. enriettii infection in a similar manner to P. yoelii. These results are discussed in relation to current knowledge of the immunosuppressive effects of plasmodia.     

The quantification of viable Leishmania enriettii from infected guinea-pig tissues.

Results are presented demonstrating that in vitro cultivation of Leishmania enrietti can be used to determine the level of viability in suspensions of L. enrietti used for infection and also to quantify the number of viable organisms in infected tissues.     

Histological studies of the elimination of Leishmania enriettii from skin lesions in the guinea-pig.

Nineteen guinea-pigs were each inoculated intradermally with 10(6) amastigotes of Leishmania enriettii, and the development of the lesions was followed from Weeks 4 to 10 with a view to elucidating the histological mechanisms involved with the elimination of parasites. Electron microscopic observations were made in 1 animal. Extensive necrosis of the parasite-laden macrophages was observed in 7 out of 7 animals at 4 and 5 weeks. In the ulcerated core of the lesion at 4 weeks no intact macrophages could be identified. Very many amastigotes were extracellular. Others were present in the cytoplasm of residual macrophages the cell walls of which had disintegrated. Necrosis was less marked at 8 weeks and absent in the resolving lesions at 10 weeks. Signs of stimulation or maturation of macrophages were only apparent when parasites were few. At 4 weeks macrophages were almost all of the non-stimulated form, but cytological evidence of activation became progressively more definite and widespread from 5 to 8 weeks, starting at the periphery of the lesion. Ultrastructural observations of amastigotes suggested that there might be more than one mechanism of degradation. It appeared that the majority of parasites were released through necrosis and discharged through the ulcer, and that intracellular degradation of the remaining parasites was important mainly in the later phase before resolution. The first phase was associated mainly with plasma-cell production, the second mainly with lymphocytes.     

Isolation and characterization of an alpha-tubulin gene from Leishmania enriettii

An alpha-tubulin gene of Leishmania enriettii has been identified in genomic Southern blots by hybridization with a heterologous alpha-tubulin gene from Drosophila melanogaster. A clone containing this gene has been isolated from a plasmid library of size-selected L. enriettii DNA. It was identified by hybridization with the D. melanogaster tubulin gene. The cloned DNA fragment was characterized by restriction analysis and partial DNA sequence analysis. The cloned DNA fragment is 2 kb in length, bounded by Pst I sites, and appears to contain the entire coding region of the alpha-tubulin gene.     

Experimental cutaneous leishmaniasis. II. Effects of immunosuppression and antigenic competition on the course of infection with leishmania enriettii in the guinea-pig

Intradermal inoculation of the guinea-pig with Leishmania enriettii results in a self-healing cutaneous lesion which provides a laboratory model of human cutaneous leishmaniasis and which is dominated by cell-mediated immunological responses (Bryceson et al., 1970). In this study we sought to design experimental situations resembling non-healing forms of cutaneous leishmaniasis in man and to determine whether these experimental situations were accompanied by abnormalities in the immunological response to infection. This paper describes three procedures which impair the resistance of guinea-pigs to leishmanial infection: (i) induction of partial immunological tolerance to leishmanial antigen; (ii) systemic injection of anti-lymphocyte serum (ALS); and (iii) regional antigenic competition produced by multiple injections of bacterial adjuvants.

Injection of soluble leishmanial antigen (PSA) during the third week of foetal life suppressed resistance to neonatal infection with L. enriettii; local infections were severe and were accompanied by metastatic spread and by impaired development of delayed hypersensitivity (DH). Injection of PSA into the 6-week foetus and into the adult guinea-pig led to impaired DH after leishmanial infection, but resistance to infection was only slightly suppressed. Transient impairment of DH was induced by a short injection course of adult animals with ALS during the first 3 weeks of infection, which resulted in large primary lesions with temporary metastatic spread.

Multiple regional injections of tubercle- and corynebacterial-adjuvant emulsions markedly suppressed resistance to subsequent leishmanial infection; large ulcerative lesions were accompanied by widespread nodular metastases, the unusual appearance of haemagglutinating antibody, and death of some animals.

There was no impairment of DH to PSA; in fact, this was temporarily enhanced during the first 6 weeks of leishmanial infection of the tubercle-adjuvant group. This suppression of resistance could not be attributed to systemic desensitization of DH to bacterial antigen or to the local sclerosing effects of adjuvant emulsion; the term `regional antigenic competition' was therefore employed. Both the induction of partial tolerance and the injection of ALS selectively impaired paracortical responses in lymph nodes draining leishmanial infections; however, in regional antigenic competition the lymph nodes were infiltrated with macrophages but both follicular and paracortical responses were prominent.

Suppression of resistance to L. enriettii was not related simply to impairment of DH; for there was overall dissociation of protective, allergic and antibody responses in these suppressed animals. The relationship of cellular immune mechanisms to cutaneous leishmaniasis was viewed in two ways. First, it was inferred that resistance of guinea-pigs to L. enriettii requires both successful induction of cell-mediated immunity and functional integrity of the regional lymphoid system at the time of infection. Second, it was suggested that nonhealing forms of human cutaneous leishmaniasis may arise from defective coordination of surveillance, inflammatory and adjuvant functions of the cellular immune response to leishmanial antigens. It was concluded that interference with cellular immune responses in the guinea-pig led to experimental infections which resembled some features of more generalized cutaneous leishmaniasis in man.

     

Experimental cutaneous leishmaniasis. IV. Selective suppression of cell-mediated immunity during the response of guinea-pigs to infection with leishmania enriettii

The purpose of this study was to determine whether suppression of acquired, cell-mediated immunity occurred during the course of cutaneous leishmaniasis of the guinea-pig. In order to do this, groups of animals were infected with amastigotes of Leishmania enriettii in doses ranging from 10¹ to 2 × 10⁸ organisms. The clinical course of primary and metastatic infection was followed for up to 30 weeks and the immunological response was examined by skin tests of hypersensitivity to soluble leishmanial antigens, by detection of circulating antibody, and by determining the resistance of animals to reinfection with fresh organisms.     

Selective lectin reactions of two stocks of Leishmania enriettii with differing pathogenicity

Five days old promastigote culture forms of two stocks of Leishmania enriettii pathogenic and non-infective for Cavia procellus, were tested with the lectins of Canavalia ensiformis, Ricinus communis-120, Soja hispida (Glycine maxima), Arachis hypogaea, Ulex europaeus, Ulex europaeus I, Ulex europaeus II, Laburnum alpinum, Lotus tetragonolobus, Euonymus europaeus and with a monoclonal antibody against blood group H. The pathogenic stock reacted with the anti-H lectin of Lotus tetragonolobus and the monoclonal anti-H and the monoclonal anti-H but not with anti-H of U. europaeus I and E. europaeus. The non-infective stock reacted with none of these anti-H specific agglutinins. They had strong agglutination reactions to C. ensiformis (500 g/ml, 1,000 g/ml, 10 mg/ml) R. communis-120 (110), S. hispida (1,000 g/ml) and A. hypogaea (500 g/ml, 1,000 g/ml, 2,000 g/ml). They showed moderate agglutinations to U. europaeus, L. alpinum and U. europaeus II. The non-infective stock showed only moderate reactions to C. ensiformis (10 mg/ml), R. communis-120 (110), A. hypogaea (2,000 g/ml) and S. hispida (1,000 g/ml). Neither N-acetylneuraminic acid nor neuraminidase was detected on the cell surface of both stocks. This result demonstrates clearly that both stocks of L. enriettii differ in their cell surface carbohydrates. The agglutination reactions with lectins of the non-infective stock of L. enriettii are very similar to L.t. major.Supported in the context of a Research Programme of the Commission of the European Communities and the Bernhard-Nocht-Institute for Nautical and Tropical Medicine, Department of Protozoology, Hamburg, TSD-005-D(B)The work was supported by the Ministry for Youth, Family Affairs and Health     

Testicular amyloidosis in hamsters experimentally infected with Leishmania donovani.

Thirty hamsters were inoculated intraperitoneally with Leishmania donovani. Testes were examined grossly and histologically by light and electron microscopy. Progressive testicular atrophy developed. Spermatogenic cells of the seminiferous tubules showed vacuolar degeneration and decreased in number leading to a total azoospermia in the final weeks of the pathological process. Lymphoplasmocytic infiltrates with macrophages containing leishmanias appeared in the intertubular space. Amyloid deposits in the intertubular space and tubular basement membrane were identified by optical and ultrastructural methods. It has been suggested that testicular amyloidosis may have a pathogenic mechanism related to a dysfunction of plasma cells and stimulation of the reticuloendothial system, due to the antigenic character of the parasite.     

Migration-inhibitory factor gene-deficient mice are susceptible to cutaneous Leishmania major infection

To determine the role of endogenous migration-inhibitory factor (MIF) in the development of protective immunity against cutaneous leishmaniasis, we analyzed the course of cutaneous Leishmania major infection in MIF gene-deficient mice (MIF(-/-)) and wild-type (MIF(+/+)) mice. Following cutaneous L. major infection, MIF(-/-) mice were susceptible to disease and developed significantly larger lesions and greater parasite burdens than MIF(+/+) mice. Interestingly, antigen-stimulated lymph node cells from MIF(-/-) mice produced more interleukin-4 (IL-4) and gamma interferon (IFN-gamma) than those from MIF(+/+) mice, although the differences were statistically not significant. IFN-gamma-activated resting peritoneal macrophages from MIF(-/-) mice showed impaired macrophage leishmanicidal activity and produced significantly lower levels of nitric oxide and superoxide in vitro. The macrophages from MIF(-/-) mice, however, produced much more IL-6 than macrophages from wild-type mice. These findings demonstrate that endogenous MIF plays an important role in the development of protective immunity against L. major in vivo. Furthermore, they indicate that the susceptibility of MIF(-/-) mice to L. major infection is due to impaired macrophage leishmanicidal activity rather than dysregulation of Th1 and Th2 responses.     

Visceral spreading depletion of thymus-dependent regions and amyloidosis in mice and hamsters infected intradermally with Leishmania isolated from Sudanese cutaneous leishmaniasis.

Eighteen outbred mice and 21 golden hamsters were each inoculated intradermally with 2 X 10(6) Leishmania amastigotes obtained from 1 case of Sudanese cutaneous leishmaniasis. The skin lesions, spleen, lymph nodes, liver and kidney were examined by light-, polarizing-, and electron microscope at 5, 9 and 18 weeks after inoculation. The aim of the investigations was to follow the development of the inflammatory reaction and the change of the morphology of the lymphoid organs during the infection. In all the mice and in the majority of the hamsters visceral leishmaniasis developed which was characterized by a "noncure" type of cellular reaction, a selective T-cell depletion in the lymph nodes and the spleen, and the development of a reactive, systemic amyloidosis. These findings point to the failure of the acquired resistance against Leishmania to develop. In some of the hamsters the response was of the "cure" type without the development of amyloidosis. At the site of the inoculation the lesions healed suggesting the positive role of necrosis and the elimination of the parasites through the ulcer in the healing process. Electron microscopy showed erythrophagocytosis in the spleen of the 2 mice examined presenting an experimental evidence of the destruction of the red blood cells, which is a common feature of human kala-azar.     

Different genetic control of cutaneous and visceral disease after Leishmania major infection in mice

The mouse strains BALB/cHeA (BALB/c) and STS/A (STS) are susceptible and resistant to Leishmania major-induced disease, respectively. We analyzed this difference using recombinant congenic (RC) BALB/c-c-STS/Dem (CcS/Dem) strains that carry different random subsets of 12.5% genes of the strain STS in a BALB/c background. Previously, testing the resistant strain CcS-5, we found five novel Lmr (Leishmania major response) loci, each associated with a different combination of pathological and immunological reactions. Here we analyze the response of RC strain CcS-16, which is even more susceptible to L. major than BALB/c. In the (CcS-16 x BALB/c)F(2) hybrids we mapped three novel loci that influence cutaneous or visceral pathology. Lmr14 (chromosome 2) controls splenomegaly and hepatomegaly. On the other hand Lmr15 (chromosome 11) determines hepatomegaly only, and Lmr13 (chromosome 18) determines skin lesions only. These data confirm the complex control of L. major-induced pathology, where cutaneous and visceral pathology are controlled by different combinations of genes. It indicates organ-specific control of antiparasite responses. The definition of genes controlling these responses will permit a better understanding of pathways and genetic diversity underlying the different disease phenotypes.     

Parameters of Mycoplasma pneumoniae infection in Syrian hamsters.

An animal model for evaluating the potency of Mycoplasma pneumoniae vaccines was developed with hamsters. Factors that influence hamster infection by M. pneumoniae were defined, and parameters for assessment of intensity of pulmonary disease were established. Colonization of hamster lungs was determined by culture, and intensity of lung disease was assessed histopathologically and expressed numerically as a lung pathological score. Intratracheal inoculation of the challenge was superior to the intranasal or aerosol route for inducing a consistent degree of lung disease. A challenge dose of 10(6) CFU inoculated intratracheally produced lung colonization and significant reproducible lung pathological scores in essentially all unvaccinated animals. The peak of infection, as determined by these criteria, was at about 2 weeks after challenge. Animals over 6 weeks of age were preferable for the test, since younger animals exhibited a lower lung pathological score even though they showed the same degree of lung colonization. The hamster assay developed provides a dependable experimental system for testing the protective potency of M. pneumoniae vaccines.     

Testicular amyloidosis in hamsters experimentally infected with Leishmania donovani

Thirty hamsters were inoculated intraperitoneally with Leishmania donovani. Testes were examined grossly and histologically by light and electron microscopy. Progressive testicular atrophy developed. Spermatogenic cells of the seminiferous tubules showed vacuolar degeneration and decreased in number leading to a total azoospermia in the final weeks of the pathological process. Lymphoplasmocytic infiltrates with macrophages containing leishmanias appeared in the intertubular space. Amyloid deposits in the intertubular space and tubular basement membrane were identified by optical and ultrastructural methods. It has been suggested that testicular amyloidosis may have a pathogenic mechanism related to a dysfunction of plasma cells and stimulation of the reticuloendothial system, due to the antigenic character of the parasite.     

Effect of lipopolysaccharide on intracellular killing of Leishmania enriettii and correlation with macrophage oxidative metabolism

The effect of lipopolysaccharide (LPS) on the lymphokine (LK)-dependent activation of murine peritoneal macrophages for intracellular killing of Leishmania enriettii parasites was investigated. Exposure to LPS alone did not induce macrophages to kill the parasite. In the presence of LK or recombinant interferon-gamma, however, which by themselves rendered the macrophages only weakly cytotoxic, considerable stimulation of intracellular parasite killing was achieved already at a LPS concentration of 1 ng/ml. The response to LPS was of the same magnitude in macrophages tested for intracellular killing as in parallel assays of extracellular cytolysis of target cells. Acquisition of leishmanicidal activity by macrophages exposed to LK and LPS correlated with stimulation of the respiratory burst, as shown by increased hexose monophosphate shunt levels, and priming for elevated chemiluminescence and O2- and H2O2 production. Polymyxin B blocked both this LPS-dependent metabolic activity and intracellular parasite destruction. Intracellular killing was, however, not solely dependent on oxidative metabolism of macrophages since in the absence of LK, LPS stimulated respiratory burst activity, yet no intracellular killing was observed, and triggering of the respiratory burst by phorbol myristate acetate or zymosan did not affect intracellular parasite survival. These results suggest that, in this experimental model, efficient intracellular parasite killing depends both on increased production of oxygen metabolites and on the availability of so far unidentified factor(s), the synthesis of which requires exposure of macrophages to both LK and LPS.     

Polyclonal B cell activation in hamsters infected with parasites of the genus Leishmania.

Mesocricetus auratus (golden hamsters) infected with leishmania developed characteristic B cell immune responses that depended on the infecting species of leishmania. Thus, hamsters infected with viscerotropic leishmania (Leishmania donovani) developed antileishmania antibodies and hypergammaglobulinemia due to polyclonal activation of B cells as measured by reverse hemolytic plaque assay. In contrast, dermotropic leishmanias (L. braziliensis braziliensis and L. mexicana amazonensis) stimulated antileishmania antibodies with no increase in either serum immunoglobulin concentration or in the number of antibody-forming cells per spleen. The dermotropic leishmanias were unable to stimulate polyclonal activation even in hamsters in which visceralization had occurred with high splenic parasitization. These findings suggest that species-specific leishmania antigens (or factors) might be the modulators of the altered immune response present in these diseases.     

Dynamics of immune granuloma formation in a Leishmania braziliensis-induced self-limiting cutaneous infection in the primate Macaca mulatta

In order to unravel the physiopathology of leishmaniasis in humans, it is necessary to better understand how Leishmania are able to survive for years within immunologically active granulomas. In the present study, we used a macaque (Macaca mulatta) model of infection with Leishmania braziliensis as a means of assessing the usefulness of this primate system. This model more closely mirrors human protective immunity to Leishmania than the murine model; therefore, we used it to study the host inflammatory granulomatous response involved in the control of cutaneous leishmaniasis. Infected primates developed localized long-term skin ulcerations, but complete spontaneous clinical healing occurred in all infected animals. The infection induced the recruitment and activation of inflammatory mast cells, granulocytes, mononuclear phagocytes, and lymphocytes at the site of infection. During the acute reaction, polymorphonuclear leukocytes were more prominent than other cell types and apparently destroyed many parasites; macrophages then rapidly engulfed dying neutrophils together with their parasitic cargo. In the chronic phase, persisting parasites induced a typical T helper (Th) cytokine, type 1-mediated, immunity-induced granulomatous reaction. By this time, more or less differentiated macrophage accumulations were found, and these evolved to become mature tissue granulomas consisting of all the specific cell types found within human granulomas. In the healing stage, fibroblasts proliferated at the periphery and finally invaded the granulomas with fibrotic substitution. These findings point to the feasibility of using this model to elucidate the potentially disabling Th1-cell mechanisms that may eventually render the host granulomatous response inadequate for fighting L. braziliensis infections.     

Latent infection and measles meningoencephalitis in Syrian hamsters.

A strain designated 42-Edmonston was isolated from the brain of a Syrian hamster 30 days after inoculation during the neonatal period with the reference "wild" measles virus strain Edmonston which produced a latent infection accompanied by production of measles antibody and insignificant pathomorphological changes. Intracerebral inoculation with the 42-Edmonston strain of newborn, one day-and 5-7-day-old Syrian hamsters resulted in the development of acute measles encephalitis with typical clinical symptoms and pathomorphological changes. It is suggested that selection and adaptation of a neurotropic measles virus variant probably had occurred in the course of virus persistence in the Syrian hamster brain.     

Leishmania pifanoi amastigote antigens protect mice against cutaneous leishmaniasis.

In the search for a leishmaniasis vaccine, extensive studies have been carried out with promastigote (insect stage) molecules. Information in this regard on amastigote (mammalian host stage) molecules is limited. To investigate host immune responses to Leishmania amastigote antigens, we purified three stage-specific antigens (A2, P4, and P8) from in vitro-cultivated amastigotes of Leishmania pifanoi by using immunoaffinity chromatography. We found that with Corynebacterium parvum as an adjuvant, three intraperitoneal injections of 5 micrograms of P4 or P8 antigen provided partial to complete protection of BALB/c mice challenged with 10(5) to 10(7) L. pifanoi promastigotes. These immunized mice developed significantly smaller or no lesions and exhibited a 39- to 1.6 x 10(5)-fold reduction of lesion parasite burden after 15 to 20 weeks of infection. In addition, P8 immunization resulted in complete protection against L. amazonensis infection of CBA/J mice and partial protection of BALB/c mice, suggesting that this antigen provided cross-species protection of mice with different H-2 haplotypes. At different stages during infection, vaccinated mice exhibited profound proliferative responses to parasite antigens and increased levels of gamma interferon production, suggesting that a Th1 cell-mediated immune response is associated with the resistance in these mice. Taken together, the data in this report indicate the vaccine potential of amastigote-derived antigens.