Trypanosoma lewisi: Enhanced resistance in naive lactating rats and their suckling pups

Lactating rats and their suckling offspring were shown to have a naturally occurring resistance to Trypansoma lewisi which was greater than that seen in normal adult rats and was not dependent on previous exposure to the parasite. In the case of the pups maximum protection was dependent on suckling being the sole source of nutriment and also on the peak of the parasitemia falling within the nursing period. Supplementary solid food reduced survival rates in concurrent Hemobartonella muris—T. lewisi infections: pups infected at 10 days of age and totally dependent on nursing showed 82.5% survival but only 6.6% survival when solid food was allowed to supplement milk. However, supplementary food did not reduce survival in pups infected with only T. lewisi. When pups were totally dependent upon nursing until the normal time of weaning (21 days of age), infection with H. muris—T. lewisi at the following days of age allowed the indicated mean survival rates: 10 days (82.5%), 20 (32.5%), 30 (14.3%), and 40 (100%). Infection with T. lewisi alone at the following days of age allowed the indicated mean survival rates: 10 days (100%), 15 (76%), 17 (52%), 20 (100%), and 30 (100%). Lactating rat serum agglutinated “adult forms” of T. lewisi, which correlated well with the observed sudden resolution of parasitemia in lactating animals at the time of the antigenic transition from “juvenile” to “adult” type. The “adult” stage parasitemia in suckling pups was selectively reduced when compared to that of nonlactating adult rats. The lactating rat serum factor could be passively transferred with lactating rat serum to animals already weaned.     

Cell cycle and cleavage events during in vitro cultivation of bloodstream forms of Trypanosoma lewisi,a zoonotic pathogen

Trypanosoma (Herpetosoma) lewisi is a globally distributed rat trypanosome, currently considered as a zoonotic pathogen; however, a detailed understanding of the morphological events occurring during the cell cycle is lacking. This study aimed to investigate the cell cycle morphology and cleavage events of Trypanosoma lewisi (T. lewisi) during in vitro cultivation. By establishing in vitro cultivation of T. lewisi at 37°C, various cell morphologies and stages could be observed. We have provided a quantitative analysis of the morphological events during T. lewisi proliferation. We confirmed a generation time of 12.14 ± 0.79 hours, which is similar to that in vivo (12.21 ± 0.14 hours). We also found that there are two distinct cell cycles, with a two-way transformation connection in the developmental status of this parasite, which was contrasted with the previous model of multiple division patterns seen in T. lewisi. We quantified the timing of cell cycle phases (G1_n, 0.56 U; S_n, 0.14 U; G2_n, 0.16 U; M, 0.06 U; C, 0.08 U; G1_k, 0.65 U; S_k, 0.10 U; G2_k, 0.17 U; D, 0.03 U; A, 0.05 U) and their morphological characteristics, particularly with respect to the position of kinetoplast(s) and nucleus/nuclei. Interestingly, we found that both nuclear synthesis initiation and segregation in T. lewisi occurred prior to kinetoplast, different to the order of replication found in Trypanosoma brucei and Trypanosoma cruzi, implicating a distinct cell cycle control mechanism in T. lewisi. We characterized the morphological events during the T. lewisi cell cycle and presented evidence to support the existence of two distinct cell cycles with two-way transformation between them. These results provide insights into the differentiation and evolution of this parasite and its related species.     

The kinetoplast DNA of the Australian trypanosome,Trypanosoma copemani,shares features with Trypanosoma cruzi and Trypanosoma lewisi

Kinetoplast DNA (kDNA) is the mitochondrial genome of trypanosomatids. It consists of a few dozen maxicircles and several thousand minicircles, all catenated topologically to form a two-dimensional DNA network. Minicircles are heterogeneous in size and sequence among species. They present one or several conserved regions that contain three highly conserved sequence blocks. CSB-1 (10?bp sequence) and CSB-2 (8?bp sequence) present lower interspecies homology, while CSB-3 (12?bp sequence) or the Universal Minicircle Sequence is conserved within most trypanosomatids. The Universal Minicircle Sequence is located at the replication origin of the minicircles, and is the binding site for the UMS binding protein, a protein involved in trypanosomatid survival and virulence. Here, we describe the structure and organisation of the kDNA of Trypanosoma copemani, a parasite that has been shown to infect mammalian cells and has been associated with the drastic decline of the endangered Australian marsupial, the woylie (Bettongia penicillata). Deep genomic sequencing showed that T. copemani presents two classes of minicircles that share sequence identity and organisation in the conserved sequence blocks with those of Trypanosoma cruzi and Trypanosoma lewisi. A 19,257?bp partial region of the maxicircle of T. copemani that contained the entire coding region was obtained. Comparative analysis of the T. copemani entire maxicircle coding region with the coding regions of T. cruzi and T. lewisi showed they share 71.05% and 71.28% identity, respectively. The shared features in the maxicircle/minicircle organisation and sequence between T. copemani and T. cruzi/T. lewisi suggest similarities in their process of kDNA replication, and are of significance in understanding the evolution of Australian trypanosomes.     

PCR identification of Trypanosoma lewisi,a common parasite of laboratory rats

Trypanosoma (Herpetosoma) lewisi is a trypanosome of the sub-genus Herpetosoma (Stercoraria section), parasite of rats (Rattus rattus and Rattus norvegicus) transmitted by fleas. T. lewisi has a stringent species specificity and cannot grow in other rodents such as mice. Rats are infected principally by oral route, through contamination by flea faeces or ingestion of fleas. Trypanosoma lewisi infections in rat colonies can interfere with research protocols and fleas of wild rats are often the source of such infections. Currently, diagnosis of T. lewisi in rats is performed by microscopic observation of stained blood smears. In the course of a research project at CIRDES, a T. lewisi infection was detected in the rat colony. In this study we evaluated PCR primer sets for their ability to diagnose multiple species of trypanosomes with a single amplification. We show that the use of ITS1 sequence of ribosomal DNA provides an efficient and sensitive assay for detection and identification of T. lewisi infection in rats and recommend the use of this assay for monitoring of T. lewisi infections in rat colonies.     

Trypanosoma lewisi: antibody classes which mediate precipitation, agglutination, and the inhibition of reproduction

Sera from Trypanosoma lewisi-infected and uninfected rats were applied to Protein A-Sepharose CL-4B columns. The absorbed fractions of antisera which contained only IgG molecules were reacted in microimmunodiffusion analyses with the exoantigens of T. lewisi in plasma collected from irradiated infected rats, and formed one precipitin line. These sera were also applied to T. lewisi extract immunoabsorbent columns and bound proteins were eluted and analyzed by immunodiffusion against antisera specific for rat immunoglobulins. IgG₁, IgG_2a, IgG_2b, IgG_2c, and IgM were absorbed by the immuno-absorbent columns. Absorption of the rat antisera with anti-rat IgG or anti-rat IgM removed one of the two precipitin lines against extracts prepared from parasites collected from irradiated infected animals. The absorbed IgG fractions and nonabsorbed fractions of antisera which were collected after Protein A-Sepharose CL-4B column chromatography agglutinated trypanosomes. After treatment of antisera with 2-mercaptoethanol, the agglutinin titers were lower than those of the control antisera suggesting both IgG and IgM are involved in the agglutination. The ablastic activity of the fractions eluted from Protein A-Sepharose CL-4B Chromatographic columns was assayed in cultures of bloodstream forms ofT. lewisi. Ablastic activity of proteins of antisera absorbed by the columns was demonstrated indicating they belonged to the IgG class of antibodies.     

Mechanism of Acquired Immunity Induced by “Leptomonas pessoai” against Trypanosoma cruzi in Mice*

Living culture forms of “Leptomonas pessoai” cross protected mice against T. cruzi challenge infection. Circulating antibodies have been detected in the immunized mice by immunodiffusion analysis, passive hemagglutination, complement fixation test and antibody binding assay; these antibodies cross reacted with T. cruzi extracts. A cellular immune response was indicated by leucocyte migration inhibition using L. pessoai and T. cruzi antigens, strongly suggesting a role for cell-mediated immunity in the mechanism of protection induced by L. pessoai.     

Further Studies on Antigenic Changes in Trypanosoma lewisi*

SYNOPSIS. An investigation of day-to-day changes in adult form antigens of T. lewisi in the rat shows that adult antigens, 2 of which are present in trace amounts in young forms, suddenly appear in large amounts in 9-day-old trypanosomes. An additional antigen is formed on the 13th day. In addition, 2-day-old trypanosomes contain some antigens not present in 6-day-old forms. The combined results of the present paper and a previous one indicate that there are antigenically 3 distinct stages of T. lewisi in the rat: 1- to 3-day-old, 4- to 8-day-old, and 9- to 14-day-old forms. From 9- to 11-day-old trypanosomes, there is a form containing both young and adult antigens.     

The Effects of Antilymphocytic Serum (ALS) on Trypanosoma lewisi Infection

SYNOPSIS. ALS was produced in rabbits immunized with cells collected from spleens and mesenteric lymph nodes of normal female Holtzman rats. The ALS was pooled and leucoagglutination tests were performed to determine the ability of the antiserum to agglutinate rat white blood cells in vitro. ALS employed in the study had a titer of 1 :512. Treatment of rats with ALS prior to and during Trypanosoma lewisi infection resulted in suppression of the immune response. Ablastin production was inhibited. The numbers of dividing forms of the parasites were higher in ALS-treated rats during the course of infection than in controls injected with saline or normal rabbit serum. ALS-treated rats continued to have a higher parasitemia than the controls and died 5–12 days post-infection. Hematocrit readings were lower in the ALS-treated rats infected with T. lewisi than in rats infected with T. lewisi or normal animals treated with ALS.     

Trypanosoma cruzi: Multiplex PCR to detect and classify strains according to groups I and II

A multiplex PCR was developed for simultaneous detection of Trypanosoma cruzi DNA and classification of the parasite strain into groups I and II. As little as 10 fg of T. cruzi DNA could be detected by multiplex PCR. The technique was shown to be specific for T. cruzi DNA, since no PCR amplification products were obtained with DNA from other tripanosomatid species. Multiplex PCR was validated by assaying genomic DNA from 34 strains of T. cruzi that had been previously characterized; 24 blood samples from experimentally-infected mice and non-infected controls; 20 buffy coat samples from patients in the acute phase of Chagas disease and non-infected individuals, and 15 samples of feces from naturally-infected Triatoma infestans. T. cruzi samples from patients and from Y strain-infected mice were classified by multiplex PCR as T. cruzi II and samples from T. infestans and Colombiana strain-infected mice as T. cruzi I.     

Effects of antiserum to Trypanosoma cruzi on the uptake and rate of killing of vector-borne, metacyclic forms of the parasite by macrophages

Kierszenbaum F., Lima M. F. and Wirth J. J. 1985. Effects of antiserum to Trypanosoma cruzi on the uptake and rate of killing of vector-borne, metacyclic forms of the parasite by macrophages. International Journal for Parasitology15: 409–413. The contribution of phagocytic function to host defense against infection with metacyclic forms of Trypanosoma cruzi isolated from insect vectors was investigated in mice passively transferred with anti-T. cruzi serum. The protective effect resulting from the passive transfers was significantly reduced by administration of either silica or cobra venom factor (CVF). A more pronounced curtailment of the protective effect was seen when both silica and CVF were administered to the mice. This effect was greater than that calculated by adding the effects produced by silica and CVF alone. In in vitro experiments, presence of anti-T. cruzi antibodies enhanced the capacity of mouse macrophages to take up the metacyclic organisms and increased the proportion of macrophages associating with the parasites. Increased macrophage-parasite association was also seen when either the flagellates or the macrophages were preincubated with the antiserum. Antibody-treated metacyclic forms of T. cruzi were more rapidly cleared by untreated macrophages than parasites pretreated with normal mouse serum. These results support a role for macrophages in host defense against the form of T. cruzi responsible for natural infections and emphasize the role played by anti-T. cruzi antibodies. The combined effect of the silica and CVF treatments suggests that C activity may contribute to the protective action of antibodies through its opsonic properties, though a concomitant role for C-dependent immune lysis cannot be ruled out. These results highlight the protective role of antibodymediated mechanisms against infection with the form of T. cruzi responsible for natural infections.     

Trypanosoma lewisi: ultrastructural observations of surface antigen movement induced by antibody.

Cellular antigens of Trypanosoma lewisi have been located with ferritin-conjugated antibody (FCA) at the ultrastructural level. Incubation of live T. lewisi with antibodies from infected rats and ferritin-labeled rabbit anti-rat γ-globulin induced aggregation of surface antigens in the flagellar pockets, the desmosomal regions, and the flagellar membranes of parasites. Aggregation of surface antigens was not observed when the trypanosomes were incubated with γ-globulin and FCA at low temperatures (0–4 C). Sections of trypanosomes incubated at 37 C for 15 or 30 min after incubation at 0 C with FCA showed internalization of FCA in membrane-bound vesicles. Studying the movement and aggregation of these parasites' surface antigens may give information about the molecular dynamics of the plasma membrane and provide insights into the trypanosomes' antigenic modulation and the hosts' immunological responses.     

Trypanosoma lewisi: production of exoantigens during infection in the rat

Exoantigens are produced by Trypanosoma lewisi during infections in the rat. They were detected in rat serum and plasma by gel-diffusion techniques with hyperimmune rat sera and with rabbit antiserum to washed, living trypanosomes. Their parasite origin is indicated by their presence in trypanosome homogenates, which also contain bound antigens, the continued reactivity of rabbit antisera after absorption with normal rat serum, and the reactions of identity obtained with rat and rabbit antisera. Moreover, by immunoelectrophoresis, the exontigens are revealed as new components in infected rat serum with a mobility slightly anodal to the origin. The results also show that the exoantigens are continuously released in vivo and that the trypanosomes avidly bind non-antibody rat serum proteins to their surface. Unlike the complete qualitative changes in exoantigens that accompany antigenic variation of pathogenic species of trypanosomes, at least one exoantigen remains unchanged when antigenic variation occurs with T. lewisi although additional exoantigens may appear and disappear. The relation of the exoantigens to the known ablastic and trypanocidal antibodies is difficult to determine since these antibodies and the exoantigens occur simultaneously in the blood during and after the infection. Although it cannot yet be ruled out that the exoantigens elicit the formation of these antibodies, a review of all the available evidence suggests that the exoantigens of T. lewisi may not be immunogenic during a natural course of infection. Possibly they are hemolysins with a nutritive function.     

Beneficial effects of acetylsalicylic acid (aspirin) on the actions of extracellular vesicles shed by Trypanosoma cruzi in macrophages

Trypomastigote forms of Trypanosoma cruzi, the causative agent of Chagas disease, shed extracellular vesicles (EVs) that promote the susceptibility of host cells to infection. During T. cruzi infection, the immune response of the host is important for controlling parasitism, which is necessary for survival. Macrophages produce inflammatory mediators, such as eicosanoids and nitric oxide (NO), with trypanocidal effects that control the parasite load in the early stages of the disease. In this study, we evaluated the contribution of host cyclooxygenase (COX) to the actions of EVs shed by T. cruzi strain Y (EVs-Y) in infected macrophages. RAW 264.7 macrophages exposed to EVs-Y and then infected with trypomastigote forms of T. cruzi produced less NO, and an increased number of trypomastigote forms were internalized in the cell compared to the controls, indicating that the effects exerted by EVs-Y favor the parasite. Interestingly, when macrophages were pretreated with acetylsalicylic acid, a dual COX inhibitor, before exposure to EVs-Y and subsequent infection with trypomastigote forms, there was an increase in NO production and a decrease in trypomastigote uptake compared to the controls. These results suggest that EVs-Y modulates the macrophage response in favor of T. cruzi and indicate a role for COX in the effects of EVs.     

Interferon-Gamma Promotes Infection of Astrocytes by Trypanosoma cruzi

The inflammatory cytokine interferon-gamma (IFNγ) is crucial for immunity against intracellular pathogens such as the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease (CD). IFNγ is a pleiotropic cytokine which regulates activation of immune and non-immune cells; however, the effect of IFNγ in the central nervous system (CNS) and astrocytes during CD is unknown. Here we show that parasite persists in the CNS of C3H/He mice chronically infected with the Colombian T. cruzi strain despite the increased expression of IFNγ mRNA. Furthermore, most of the T. cruzi-bearing cells were astrocytes located near IFNγ⁺ cells. Surprisingly, in vitro experiments revealed that pretreatment with IFNγ promoted the infection of astrocytes by T. cruzi increasing uptake and proliferation of intracellular forms, despite inducing increased production of nitric oxide (NO). Importantly, the effect of IFNγ on T. cruzi uptake and growth is completely blocked by the anti-tumor necrosis factor (TNF) antibody Infliximab and partially blocked by the inhibitor of nitric oxide synthesis L-NAME. These data support that IFNγ fuels astrocyte infection by T. cruzi and critically implicate IFNγ-stimulated T. cruzi-infected astrocytes as sources of TNF and NO, which may contribute to parasite persistence and CNS pathology in CD.     

Evaluation of some chemotherapeutics against Endamoeba histolytica in cultures with Trypanosoma cruzi

Ten antibiotics and amebacidal agents have been re-studied for their “amebacidal” end points. These determinations were obtained by use of E. histolytica plus T. cruzi and were compared with the end points previously obtained by others utilizing “open” and “sealed” culture technics of E. histolytica plus organism ‘t.’. The end point ranges corresponded closely with those obtained with the “sealed culture” technic. The cultures of E. histolytica growing in association with T. cruzi alone provide an opportunity for study of direct amebacidal action in vitro, without the complication that associated bacterial flora has introduced in previous methods.     

Trypanosoma lewisi: electron microscopy of the infected spleen

The spleens of Lewis rats, both normal and infected with Trypanosoma lewisi were examined by electron microscopy. Special attention was directed to clusters of splenic cells which occur in the course of the infection. The reticular cells first showed alterations of their structure by the second day of infection, with considerable surface membrane activity. By the fourth day and thereafter various cells were found gathered around the reticular cells. These cell clusters mainly contained lymphocytes, plasma cells, and erythropoietic elements in many stages of differentiation. It was not unusual that several cell types were found adjacent to the same central reticular cell. These arrays, similar in geometry to the erythropoietic island of the bone marrow, became more predominantly “plasma cell” islands as the infection progressed. Parasites were recognizable within the reticular cells, and were noted to be in regions where the cellular membranes of adjacent cells demonstrated vesiculations resembling rhopheocytosis. A further observation was the pinching off of neighboring plasma cell cytoplasm into the reticular cells.     

Mannose-Binding Lectin Regulates Host Resistance and Pathology during Experimental Infection with Trypanosoma cruzi

Mannose-binding lectin (MBL) is a humoral pattern-recognition molecule important for host defense. Although recent genetic studies suggest an involvement of MBL/MASP2-associated pathways in Chagas’ disease, it is currently unknown whether MBL plays a role in host resistance to the intracellular protozoan Trypanosoma cruzi, the causative agent of Chagas’ disease. In this study we employed MBL^−/− mice to assess the role of MBL in resistance to experimental infection with T. cruzi. T. cruzi infection enhanced tissue expression of MBL both at the mRNA and protein level. Similarly, symptomatic acute Chagas’ disease patients displayed increased serum concentrations of MBL compared to patients with indeterminate, asymptomatic forms of the disease. Furthermore, increased parasite loads in the blood and/or tissue were observed in MBL^−/− mice compared to WT controls. This was associated with reduced systemic levels of IL-12/23p40 in MBL^−/− mice. Importantly, MBL^−/− mice infected with a cardiotropic strain of T. cruzi displayed increased myocarditis and cardiac fibrosis compared to WT controls. The latter was accompanied by elevated hydroxyproline content and mRNA levels of collagen-1 and -6 in the heart. These observations point to a previously unappreciated role for MBL in regulating host resistance and cardiac inflammation during infection with a major human pathogen.     

Trypanosoma lewisi and T. cruzi: Effect of infection on gestation in the rat

Termination of pregnancy occurred in the rat after infection with Trypanosoma lewisi early in gestation. Rats were inoculated on Day 2 of pregnancy and the uteri were excised and examined on Days 10 and 14 of gestation. There were no detectable differences in size in the fetuses of infected and control females at Day 10, but by Day 14 young of infected females were being reabsorbed, and their weight was markedly less than that of control young.A bioassay of estrogens and progestogens based on the decidual cell response indicated that there was a sufficient hormone level to maintain pregnancy beyond Day 10, so the mechanism of action by which fetal death was produced did not appear to be hormone depletion. The crucial changes in fetal weight occurred between Days 12 and 14 of gestation in rats infected with T. lewisi on Day 2 of gestation.Trypanosoma cruzi caused little or no pathologic change in the pregnant laboratory rat throghout the period of gestation and infected females gave birth to apparently normal young.     

Tumor necrosis factor alpha induced by Trypanosoma cruzi infection mediates inflammation and cell death in the liver of infected mice

Trypanosoma cruzi (T. cruzi) infected C57BL/6 mice developed a progressive fatal disease due to an imbalance in the profile of circulating related compounds accompanying infection like tumor necrosis factor alpha (TNFα). TNFα has been proposed as an important effector molecule in apoptosis. In this work, we evaluate inflammation and the proteins involved in apoptotic process in liver of infected mice and the role of TNFα. C57BL6/mice were infected subcutaneously with 100 viable trypomastigotes of Tulahuén strain of T cruzi. One set of these animals were treated with 375 μg of antihuman TNFα blocking antibody. Animals were sacrificed at 14 days post-infection (p.i).The analyses of Bcl-2 family proteins revealed an increase of the pro-apoptotic proteins Bax and tBid in T. cruzi-infected mice. Compared with control animals, cytochrome c release was increased. Apoptosis was also induced in infected mice. Anti-TNFα treatment decreases hepatic apoptosis. Our results suggest that T. cruzi infection induces programmed cell death in the host liver by increase of TNFα production, associated with TNF-R1 over-expression, that set in motion the Bid cleavage and mitochondrial translocation, Bax mitochondrial translocation, cytochrome c release, and ultimately apoptosis induction.