Experimental infection of canine peritoneal macrophages with visceral and dermotropic Leishmania strains

A study was carried out using macrophages cultured from the peritoneal exudate of dogs infected in vitro with three species of Leishmania: L. (L.) chagasi, L. (Viannia) braziliensis and L. (L.) amazonensis with the aim of investigating the growth kinetics and infectivity of these species in the host cell. Results were expressed as the percentage of macrophages infected measured at 24 hr intervals over six days in RPMI - 1640 culture medium at a temperature of 34-35oC. The findings open the possibility of using canine peritoneal cells as a model for the screenning of leishmanicide drugs and to study the pathogenesis of these species.

Experimental toxoplasmosis in Balb/c mice. Prevention of vertical disease transmission by treatment and reproductive failure in chronic infection

In a study of congenital transmission during acute infection of Toxoplasma gondii, 23 pregnant Balb/c mice were inoculated orally with two cysts each of the P strain. Eight mice were inoculated 6-11 days after becoming pregnant (Group 1). Eight mice inoculated on the 10th-15th day of pregnancy (Group 2) were treated with 100 mg/kg/day of minocycline 48 h after inoculation. Seven mice inoculated on the 10th-15th day of pregnancy were not treated and served as a control (Group 3). Congenital transmission was evaluated through direct examination of the brains of the pups or by bioassay and serologic tests. Congenital transmission was observed in 20 (60.6%) of the 33 pups of Group 1, in one (3.6%) of the 28 pups of Group 2, and in 13 (54.2%) of the 24 pups of Group 3. Forty-nine Balb/c mice were examined in the study of congenital transmission of T. gondii during chronic infection. The females showed reproductive problems during this phase of infection. It was observed accentuated hypertrophy of the endometrium and myometrium. Only two of the females gave birth. Our results demonstrate that Balb/c mice with acute toxoplasmosis can be used as a model for studies of congenital T. gondii infection. Our observations indicate the potential of this model for testing new chemotherapeutic agents against congenital toxoplasmosis.

Comparison of dengue infection in human mononuclear leukocytes with mosquito C6/36 and mammalian Vero cells using flow cytometry to detect virus antigen

Fluorescent activated cell sorter (FACS) analysis is useful for the detection of cellular surface antigens and intracellular proteins. We used this methodology in order to detect and quantify dengue antigens in highly susceptible cells such as clone C6/36 (Aedes albopictus) and Vero cells (green monkey kidney). Additionally, we analyzed the infection in vitro of human peripheral blood mononuclear leukocytes (PBML). FACS analysis turned out to be a reliable technique to quantify virus growth in traditional cell cultures of C6/36 as well as Vero cells. High rates of infection were achieved with a good statistical correlation between the virus amount used in infection and the percentage of dengue antigen containing cells detected in infected cultures. We also showed that human monocytes (CD14+) are preferred target cells for in vitro dengue infection among PBML. Monocytes were much less susceptible to virus infection than cell lines but they displayed dengue antigens detected by FACS five days after infection. In contrast, lymphocytes showed no differences in their profile for dengue specific immunofluorescence. Without an animal model to reproduce dengue disease, alternative assays have been sought to correlate viral virulence with clinical manifestations and disease severity. Study of in vitro interaction of virus and host cells may highlight this relationship.

Study of the safety, immunogenicity and efficacy of attenuated and killed Leishmania (Leishmania) major vaccines in a rhesus monkey (Macaca mulatta) model of the human disease

We have compared the efficacy of two Leishmania (Leishmania) major vaccines, one genetically attenuated (DHFR-TS deficient organisms), the other inactivated [autoclaved promastigotes (ALM) with bacillus Calmete-Guérin (BCG)], in protecting rhesus macaques (Macaca mulatta) against infection with virulent L. (L.) major. Positive antigen-specific recall proliferative response was observed in vaccinees (79% in attenuated parasite-vaccinated monkeys, versus 75% in ALM-plus-BCG-vaccinated animals), although none of these animals exhibited either augmented in vitro gamma interferon (IFN-g) production or positive delayed-type hypersensitivity (DTH) response to the leishmanin skin test prior to the challenge. Following challenge, there were significant differences in blastogenic responses (p < 0.05) between attenuated-vaccinated monkeys and naïve controls. In both vaccinated groups very low levels of antibody were found before challenge, which increased after infective challenge. Protective immunity did not follow vaccination, in that monkeys exhibited skin lesion at the site of challenge in all the groups. The most striking result was the lack of pathogenicity of the attenuated parasite, which persisted in infected animals for up to three months, but were incapable of causing disease under the conditions employed. We concluded that both vaccine protocols used in this study are safe in primates, but require further improvement for vaccine application.

Leishmania braziliensis: partial control of experimental infection by interleukin-12 p40 deficient mice

Resistance to infection by Leishmania major has been associated with the development of a Th1 type response that is dependent on the presence of interleukin 12 (IL-12). In this work the involvement of this cytokine in the response to infection by L. braziliensis, a less virulent species in the mouse model, was evaluated. Our results show that while interferon (IFN-g) deficient (-/-) mice inoculated L. braziliensis develop severe uncontrolled lesions, chronic lesions that remained under control up to 12 weeks of infection were observed in IL-12p40 -/- mice. IL 12p40 -/- mice had fewer parasites in their lesions than IFN-g-/- mice. Lymph node cells from IL-12p40 -/- were capable of producing low but consistent levels of IFN-g suggestive of its involvement in parasite control. Furthermore, as opposed to previous reports on L. major-infected animals, no switch to a Th2 response was observed in IL-12p40 -/- infected with L. braziliensis.

Experimental lagochilascariosis: histopathological study of inflammatory response to larval migration in the Murine model

The goal of this study was to investigate the pattern of inflammatory response induced by Lagochilascaris minor in murine experimental model. For this purpose 115 mice were given 1000-3000 L. minor infective eggs "per os" and 51 uninfected mice were considered as controls. Four hours post-inoculation (PI), 3rd stage larvae were seen passing through the mucosa of terminal ends of small intestine. Six hours PI larvae were observed as an embolus inside the portal vein and also migrating through the liver parenchyma. During the first 24 h larvae-containing eggs of L. minor were observed in the lumen of intestinal tract. Two days PI larvae were seen migrating through lung parenchyma associated with an initial neutrophilic perivasculitis. From the 13th day of this experimental study, L. minor larvae were found mainly in skeletal muscles, in the center of granulomas. Concentric fibrosis with mixed inflammatory infiltrate involved the larvae after the 47th day PI, persistently. This experimental murine study with L. minor indicated that the 3rd stage larvae penetrated via ileum-cecal mucosa reaching the liver and probably other tissues through the hematogenic via. Throughout its pathway the larvae induced a granulomatous reaction, with abundant polimorphonuclear cells.

Infection of Calomys callosus (Rodentia Cricetidae) with strains of different Trypanosoma cruzi biodemes: pathogenicity, histotropism, and fibrosis induction

The influence of different Trypanosoma cruzi biodemes on the evolution of the infection and on the histopathological lesions of the heart and skeletal muscles, during the experimental infection of Calomys callosus, was investigated. Three groups of C. callosus were infected, respectively, with parasite strains representative of three different Biodemes: Type I (Y strain), Type II (21 SF strain), and Type III (Colombian strain). For each group, normal C. callosus were also used as controls. Marked differences have been detected in the responses of C. callosus to the infection with the three strains in this model. The strains Types I and II (Y and 21 SF) determined moderate lesions, mostly in the myocardium, with low parasitism, a rapid course, and total regression of the lesions by the 60th day of infection. Differently, Type III strain (Colombian), was more pathogenic for C. callosus and induced necrotic-inflammatory lesions in skeletal muscles and myocardium, in correspondence to intracellular parasitism. Proliferation of fibroblasts and amorphous matrix deposits, followed by interstitial fibrosis were present. Progressive regression of the inflammatory changes and collagen deposits occurred spontaneously. The progression and regression of both inflammation and fibrosis induced by the Colombian strain were further submitted to quantitative evaluation by morphometry. Results of the morphometric studies presented good correlation with the histopathological findings. The results confirm the importance of the different biodemes in the determination of tissue lesions and the peculiarities of response of C. callosus to infection with T. cruzi.

Experimental infection of Leishmania (L.) chagasi in a cell line derived from Lutzomyia longipalpis (Diptera:Psychodidae)

The present work describes the in vitro infection of a cell line Lulo, derived from Lutzomyia longipalpis embryonic tissue, by Leishmania chagasi promastigotes. This infection process is compared with a parallel one developed using the J774 cell line. The L. chagasi MH/CO/84/CI-044B strain was used for experimental infection in two cell lines. The cells were seeded on glass coverslips in 24-well plates to reach a final number of 2 x 10(5) cells/well. Parasites were added to the adhered Lulo and J774 cells in a 10:1 ratio and were incubated at 28 and 37ºC respectively. After 2, 4, 6, 8, and 10 days post-infection, the cells were extensively washed with PBS, fixed with methanol, and stained with Giemsa. The number of internalized parasites was determined by counting at least 400 cultured cells on each coverslip. The results showed continuous interaction between L. chagasi promastigotes with the cell lines. Some ultrastructural characteristics of the amastigote forms were observed using transmission electron microscopy. The highest percentage of infection in Lulo cells was registered on day 6 post-infection (29.6%) and on day 4 in the J774 cells (51%). This work shows similarities and differences in the L. chagasi experimental infection process in the two cell lines. However, Lulo cells emerge as a new model to study the life-cycle of this parasite.

Plasmodium berghei ANKA infection induces thymocyte apoptosis and thymocyte depletion in CBA mice

Immune responses to malaria infections are characterized by strong T and B cell activation, which, in addition of potentially causing immunopathology, are of poor efficacy against the infection. It is possible that the thymus is involved in the origin of immunopathological reactions and a target during malaria infections. This work was developed in an attempt to further clarify these points. We studied the sequential changes in the thymus of CBA mice infected with Plasmodium berghei ANKA, a model in which 60-90% of the infected animals develop cerebral malaria. During the acute phase of infection, different degrees of thymocyte apoptosis were recorded: (1) starry-sky pattern of diffuse apoptosis with maintenance of cortical-medullary structure; (2) intense apoptosis with cortical atrophy, with absence of large cells; (3) severe cortical thymocyte depletion, resulting in cortical-medullary inversion. In the latter, only residual clusters of small thymocytes were observed within the framework of epithelial cells. The intensity of thymus alterations could not be associated with the degree of parasitemia, the expression of clinical signs of cerebral malaria or intensity of brain lesions. The implications of these events for malaria immunity and pathology are discussed.

Schistosome vaccine testing: lessons from the baboon model

The high level of protection elicited in rodents and primates by the radiation-attenuated schistosome vaccine gives hope that a human vaccine relying on equivalent mechanisms is feasible. In humans, a vaccine would be undoubtedly administered to previously or currently infected individuals. We have therefore used the olive baboon to investigate whether vaccine-induced immunity is compromised by a schistosome infection. We showed that neither a preceding infection, terminated by chemotherapy, nor an ongoing chronic infection affected the level of protection. Whilst IgM responses to vaccination or infection were short-lived, IgG responses rose with each successive exposure to the vaccine. Such a rise was obscured by responses to egg deposition in already-infected animals. In human trials it would be necessary to use indirect estimates of infection intensity to determine vaccine efficacy. Using worm burden as the definitive criterion, we demonstrated that the surrogate measures, fecal eggs, and circulating antigens, consistently overestimated protection. Regression analysis of the surrogate parameters on worm burden revealed that the principal reason for overestimation was the threshold sensitivity of the assays. If we extrapolate our findings to human schistosomiasis mansoni, it is clear that more sensitive indirect measures of infection intensity are required for future vaccine trials.

Trypanosoma cruzi high infectivity in vitro is related to cardiac lesions during long-term infection in Beagledogs

Trypanosoma cruzi is a hemoflagelate parasite associated with heart dysfunctions causing serious problems in Central and South America. Beagle dogs develop the symptoms of Chagas disease in humans, and could be an important experimental model for better understanding the immunopathogenic mechanisms involved in the chagasic infection. In the present study we investigated the relation among biological factors inherent to the parasite (trypomastigote polymorphism and in vitro infectivity) and immunoglobulin production, inflammation, and fibrosis in the heart of Beagle dogs infected with either T. cruzi Y or Berenice-78 strains. In vitro infectivity of Vero cells as well as the extension of cardiac lesions in infected Beagle was higher for Y strain when compared to Berenice-78 strain. These data suggested that in vitro infectivity assays may correlate with pathogenicity in vivo. In fact, animals infected with Y strain, which shows prevalence of slender forms and high infectivity in vitro, presented cardiomegaly, inflammation, and fibrosis in heart area. Concerning the immunoglobulin production, no statistically significant difference was observed for IgA, IgM or IgG levels among T. cruzi infected animals. However, IgA together IgM levels have shown to be a good marker for the acute phase of Chagas disease.

Vaccination with epimastigotes of different strains of Trypanosoma rangeli protects mice against Trypanosoma cruzi infection

In our laboratory, we have developed a model of vaccination in mice with Trypanosoma rangeli, a non-pathogenic parasite that shares many antigens with Trypanosoma cruzi. The vaccinated mice were protected against infection with virulent T. cruzi. The goal of the present work was to study the protective activity of strains of T. rangeli of different origin, with the aim of analysing whether this protective capacity is a common feature of T. rangeli. BALB/c mice were vaccinated with live or fixed epimastigotes of two T. rangeli strains, Choachi and SC-58. Vaccinated (VM) and control mice (CM) were infected with virulent T. cruzi, Tulahuen strain. The results showed that the levels of parasitemia of VM, vaccinated with the two strains of T. rangeli were significantly lower than those developed in CM. The survival rate of VM was higher than that CM. Histological studies revealed many amastigote nests and severe inflammatory infiltrates in the heart and skeletal muscles of CM, whereas in the VM only moderate lymphomonocytic infiltrates were detected. Altogether, the results of the present work as well as previous studies show that the antigens involved in the protection induced by T. rangeli are expressed in different strains of this parasite. These findings could prove useful in vaccine preparation.

Toxoplasma gondii infection induces lipid metabolism alterations in the murine host

Host lipids have been implicated in the pathogenesis of Toxoplasma gondiiinfection. To determine if Toxoplasmainfection influences the lipid status in the normal host, we assessed serum lipids of Swiss-Webster mice during infection with the BGD-1 strain (type-2) at a series of time points. Mice were bled at days zero and 42 post-infection, and subgroups were additionally bled on alternating weeks (model 1), or sacrificed at days zero, 14 and 42 (model 2) for the measurement of total cholesterol (Chl), high density lipoproteins (HDL), low density lipoproteins (LDL) and triglycerides and adiponectin. At day 42, brains were harvested for cyst enumeration. A significant decrease (p = 0.02) in HDL and total Chl was first noted in infected vs. control mice at day 14 and persisted to day 42 (p = 0.013). Conversely, LDL was unaltered until day 42, when it increased (p = 0.043). Serum LDL levels at day 42 correlated only with cyst counts of above 300 (found in 44% mice), while the change in HDL between days zero and 42 correlated with both the overall mean cyst count (p = 0.041) and cyst counts above 300 (p = 0.044). Calculated per cyst, this decrease in HDL in individual animals ranged from 0.1-17 µmol/L, with a mean of 2.43 ± 4.14 µmol/L. Serum adiponectin levels remained similar between infected and control mice throughout the experiment.

Circulating natural killer and γδ T cells decrease soon after infection of rhesus macaques with lymphocytic choriomeningitis virus

Rhesus macaques infected with the WE strain of lymphocytic choriomeningitis virus (LCMV-WE) serve as a model for human infection with Lassa fever virus. To identify the earliest events of acute infection, rhesus macaques were monitored immediately after lethal infection for changes in peripheral blood mononuclear cells (PBMCs). Changes in CD3, CD4, CD8 and CD20 subsets did not vary outside the normal fluctuations of these blood cell populations; however, natural killer (NK) and γδ T cells increased slightly on day 1 and then decreased significantly after two days. The NK subsets responsible for the decrease were primarily CD3-CD8+ or CD3-CD16+ and not the NKT (primarily CD3+CD56+) subset. Macaques infected with a non-virulent arenavirus, LCMV-Armstrong, showed a similar drop in circulating NK and γδ T cells, indicating that this is not a pathogenic event. V³9 T cells, representing the majority of circulating γδ T cells in rhesus macaques, displayed significant apoptosis when incubated with LCMV in cell culture; however, the low amount of cell death for virus-co-cultured NK cells was insufficient to account for the observed disappearance of this subset. Our observations in primates are similar to those seen in LCMV-infected mice, where decreased circulating NK cells were attributed to margination and cell death. Thus, the disappearance of these cells during acute hemorrhagic fever in rhesus macaques may be a cytokine-induced lymphopenia common to many virus infections.

Dengue-2 infection and the induction of apoptosis in human primary monocytes

Monocytes/macrophages are important targets for dengue virus (DENV) replication; they induce inflammatory mediators and are sources of viral dissemination in the initial phase of the disease. Apoptosis is an active process of cellular destruction genetically regulated, in which a complex enzymatic pathway is activated and may be trigged by many viral infections. Since the mechanisms of apoptotic induction in DENV-infected target cells are not yet defined, we investigated the virus-cell interaction using a model of primary human monocyte infection with DENV-2 with the aim of identifying apoptotic markers. Cultures analyzed by flow cytometry and confocal microscopy yielded DENV antigen positive cells with rates that peaked at the second day post infection (p.i.), decayed afterwards and produced the apoptosis-related cytokines TNF-α and IL-10. Phosphatidylserine, an early marker for apoptosis, was increased at the cell surface and the Fas death receptor was upregulated at the second day p.i. at significantly higher rates in DENV infected cell cultures than controls. However, no detectable changes were observed in the expression of the anti-apoptotic protein Bcl-2 in infected cultures. Our data support virus modulation of extrinsic apoptotic factors in the in vitro model of human monocyte DENV-2 infection. DENV may be interfering in activation and death mechanisms by inducing apoptosis in target cells.