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 (G1n, 0.56 U; Sn, 0.14 U; G2n, 0.16 U; M, 0.06 U; C, 0.08 U; G1k, 0.65 U; Sk, 0.10 U; G2k, 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.

Phylogenetic, morphological and behavioural analyses support host switching of Trypanosoma (Herpetosoma) lewisi from domestic rats to primates.

We characterized four Brazilian trypanosomes isolated from domestic rats and three from captive non-human primates that were morphologically similar to T. lewisi, a considered non-pathogenic species restricted to rodents and transmitted by fleas, despite its potential pathogenicity for infants. These isolates were identified as T. lewisi by barcoding using V7V8 SSU rDNA sequences. In inferred phylogenetic trees, all isolates clustered tightly with reference T. lewisi and T. lewisi-like trypanosomes from Europe, Asia and Africa and despite their high sequence conservation formed a homogeneous clade separate from other species of the subgenus T. (Herpetosoma). With the aim of clearly resolving the relationships between the Brazilian isolates from domestic rats and primates, we compared sequences from more polymorphic ITS rDNA. Results corroborated that isolates from Brazilian rats and monkeys were indeed of the same species and quite close to T. lewisi isolates of humans and rats from different geographical regions. Morphology of the monkey isolates and their behaviour in culture and in experimentally infected rats were also compatible with T. lewisi. However, infection with T. lewisi is rare among monkeys. We have examined more than 200 free-ranging and 160 captive monkeys and found only three infected individuals among the monkeys held in captivity. The findings of this work suggest that proximity of monkeys and infected rats and their exposure to infected fleas may be responsible for the host switching of T. lewisi from their natural rodent species to primates. This and previous studies reporting T. lewisi in humans suggest that this trypanosome can cause sporadic and opportunistic flea-borne infection in primates.

Activity of D-carnitine and its derivatives on Trypanosoma infections in rats and mice.

Little progress has been made in the treatment of African trypanosomiasis over the past decades. L-carnitine has a major role in glycolysis-based energy supply of blood trypanosomes for it stimulates constant ATP production. To investigate whether administration of the isomer D-carnitine could exert a competitive inhibition on the metabolic pathway of the L-form, possibly resulting in parasite replication inhibition, several formulations of this compound were tested on Trypanosoma lewisi and T. brucei rhodesiense in rodent models. High oral dosages of D-cornitine inner salt and proprionyl-D-carnitine were not toxic to animals and induced about 50% parasite growth inhibition in reversible, i.e. competitive, fashion. A putative mechanism could be an interference in pyruvate kinase activity and hence ATP production. Considering both, lack of toxicity and inhibitory activity, D-carnitine may have a role in the treatment of African trypanosomiasis, in association with available trypanocidal drugs.

Comparative positions of kinetoplasts in Trypanosoma musculi and Trypanosoma lewisi during development in vitro.

The development of Trypanosoma musculi and Trypanosoma lewisi were studied in vitro in the presence of adherent splenic cells. Both parasites developed only when attached by their flagellar tips to adherent splenic cells. During the proliferation of T. musculi, the kinetoplast migrated towards the nucleus, and once in the vicinity of the nucleus, the nuclear division was triggered. The kinetoplast of T. lewisi did not migrate towards the nucleus, but remained at its original location. The nucleus and kinetoplast divided at the same time in both parasites, and parasites started dividing from their flagellar ends and T. musculi and T. lewisi daughter cells were formed within 48 h. The unavailability of the adherent splenic cells in vitro led the parasites to transform into round/oval nonviable forms.

A medium for the continuous cultivation of bloodstream forms of Trypanosoma lewisi at 37 C.

Trypanosoma lewisi has been maintained continuously at 37 C for more than 2 yr in Iscove's modified Dulbecco's medium with 10% fetal calf serum and a feeder layer of rat fibroblasts. In this medium the continuously reproducing hematozoic culture forms resemble bloodstream forms of T. lewisi in that they appear morphologically similar in Giemsa-stained preparations examined by light microscopy and have a surface coat that is absent in culture forms grown at ambient temperatures, when examined by electron microscopy. To determine whether these hematozoic culture forms also are similar functionally to bloodstream forms, comparative tests of the 2 forms were made of infectivity for the natural rat host, growth in vitro in the described culture medium, sensitivity to inhibition of reproduction by the rat antibody ablastin, and agglutinability by the 2 trypanocidal antibodies produced during a natural course of infection in the rat. Initially, differences between the 2 forms were minor, but after 16 mo in vitro greater differences began to emerge. Most marked was a reduction in infectivity by 22 mo, although sensitivity to ablastin, the single most important characteristic of bloodstream forms of T. lewisi, was still appreciable at this time. Nevertheless, despite this limitation, the culture system described supports hematozoic culture forms of T. lewisi for a considerably longer time than has been reported thus far.

Trypanosoma lewisi: stage-specific surface antigens and exoantigens recognized by monoclonal antibodies.

The stage-specific expression of surface antigens by Trypanosoma lewisi was investigated using monoclonal antibodies directed against this parasite. Hybridomas secreting monoclonal antibodies were produced by the fusion of SP2/0-Ag 14 mouse plasmacytomas with spleen cells from rats infected previously with the Taliaferro strain of T. lewisi. Additivity enzyme-linked immunosorbent assays and indirect immunofluorescent antibody tests indicated the determinant recognized by monoclonal antibody TL40.3 (IgM) was different from those recognized by monoclonal antibodies TL40.1 (IgA), TL40.2 (IgM), and TL40.6 (IgG2 alpha). Monoclonal antibody TL40.3 agglutinated trypanosomes collected 3 days after parasite inoculation while monoclonal antibodies TL40.1, TL40.2, and TL40.6 agglutinated trypanosomes collected 6 days after inoculation. Since agglutinin titers against trypanosomes from irradiated (700 rad from a 60Co source) and nonirradiated rats were similar, expression of the antigens recognized by the monoclonal antibodies appeared to be independent of the immunological state of the host and the morphology of the parasite. The reproduction of T. lewisi in in vitro trypanostatic assays was inhibited only when the monoclonal antibodies were present in concentrations greater than or equal to those needed to agglutinate the trypanosomes. Monoclonal antibodies TL40.1 and TL40.3, but not TL40.2 and TL40.6, agglutinated erythrocytes collected later in the infection from irradiated, infected rats. None of the monoclonal antibodies agglutinated erythrocytes from nonirradiated, infected rats, from irradiated, noninfected rats or from nonirradiated, noninfected rats. This suggests that immunocompetent rats may make blocking antibodies against the exoantigens recognized by monoclonal antibodies TL40.1 and TL40.3.

Metabolic activity of Trypanosoma lewisi cultured in vitro in the presence of normal or ablastinic rat serum.

Trypanosoma lewisi, in cultures supplemented with normal rat serum, synthesized the majority of new RNA during the first 13 hr of the culture, whereas DNA synthesis occurred from the 8th hr onwards and amino acids were incorporated into macromolecules uniformly throughout the 24 hr culture period. Thymidine was taken up by the parasite only between the 7th and 14th hr of culture, unlike uracil and amino acids which were taken up as required. Ablastin, a trypanostatic factor in the serum of infected rats, maximally inhibited DNA synthesis if it was added to the cultures before the 5th hr, partially inhibited synthesis if added between the 5th and 11th hr, but if added after this had no inhibitory effect. Ablastin only partially inhibited RNA synthesis and was without effect on amino acid utilisation.

Polyamine oxidase-mediated trypanosome killing: the role of hydrogen peroxide and aldehydes.

Trypanosoma lewisi and T. musculi were lysed when incubated with bovine serum in the presence of either spermine or spermidine. Similar results were obtained when a fraction from bovine serum containing polyamine oxidase (PAO) activity or a commercially available purified beef plasma PAO were used in lieu of bovine serum. Trypanosomes treated with cytotoxic concentrations of PAO-spermine failed to establish infection in rats. These results are similar to those from our previous studies with African trypanosomes. We now extend the properties of PAO by showing that human retroplacental serum (RPS) containing PAO activity was also capable of mediating trypanosome killing. This is of significance because the macrophage PAO resembles the human RPS PAO. In addition, our preliminary studies, in which an attempt was made to characterize the factors responsible for cytotoxicity, suggested that a number of products of the PAO-polyamine reaction display trypanocidal properties. These included hydrogen peroxide (H2O2), the aldehyde acrolein, and possibly aminoaldehydes. No evidence was obtained that the oxygen intermediates, superoxide and hydroxyl radicals, play a role in the PAO-mediated trypanosome killing. Ammonia, an additional product of PAO-polyamine reaction, was not trypanocidal. Furthermore, the data suggested that less than 30 min exposure to the reaction mixture (and possibly to aminoaldehydes) was adequate to cause irreversible damage to trypanosomes.

A semi-automated microassay method for the measurement of ablastin, a division inhibitory antibody.

Division of Trypanosoma lewisi in the rat is terminated by ablastin, a factor(s) in the serum of rats acquired during infection which is believed to be specific antibody. In this study a semi-automated microassay technique for the measurement of ablastin is described. The system involved the use of [3H]TdR as a marker for trypanosome division and a multiple-well cell harvester to harvest trypanosomes from wells of microtitre plates. The method is versatile, quantitative and easy to perform, permitting the simultaneous measurement of numerous samples for this division-inhibitory activity.

Trypanostatic activity of rat IgG purified from the surface coat of Trypanosoma lewisi.

Host IgG is a component of the surface coat of Trypanosoma lewisi; it is specifically acquired during infection in the rat, concomitant with a rise in titer of trypanostatic (ablastic) activity of host serum. Host IgG was eluted from trypomastigotes at 7 to 9 days postinfection with a high salt-low pH buffer. Surface coats and trypanosome ultrastructure were not notably altered by the elution procedure, as determined by electron microscopy. Rat IgG was removed and purified from the trypanosome eluates on an immunoadsorbent column made with the IgG fraction of anti-rat IgG serum coupled to Sepharose beads. Concentrated column eluates, by comparison with a standard, were shown to be rat IgG by immunoelectrophoresis and SDS polyacrylamide gel electrophoresis. As a control, IgG from normal rat serum was purified by the same technique. IgG-negative trypanosomes harvested from immunosuppressed rats bound IgG purified from surface coats of trypanosomes, but not IgG purified from normal rat serum, as demonstrated by subsequent labelling with FITC-conjugated, rabbit anti-rat IgG. The IgG purified from surface coats inhibited the reproduction of T. lewisi in an in vitro assay, but purified, normal IgG did not. These data show that antigen-specific host IgG, adsorbed to the surface of T. lewisi, is ablastic antibody.

Basis of the specificity of rodent trypanosomes for their natural hosts.

The strong natural insusceptibility of mice to infection with the rat-specific Trypanosoma lewisi was investigated in a variety of mouse strains. Parasite elimination by all strains was similar involving a lag phase of about 7 h of constant parasitemia followed by rapid clearance (half-times of 55 and 130 min in different strains). In vitro cultures were utilized to determine whether mouse cells and serum were inherently deficient in supporting T. lewisi or, alternatively, toxic for the parasite; neither was found to be the case. In attempts to prolong T. lewisi survival and growth, mice were treated with various preparations, including T. lewisi sonicates, rabbit antiserum against mouse macrophages, silica dust, normal rat and rabbit sera and crude immunoglobulin fractions, and combinations of silica dust and serum or immunoglobulin fractions. The most striking effects were obtained with combinations of silica dust and serum, resulting in extensive T. lewisi growth and death of a portion of the infected mice. These results, together with microscopic observations, suggested that the principal mechanism responsible for murine resistance to heterologous trypanosomes is a type of antibody-dependent, cell-mediated immunity involving granulocytes and (probably) platelets.

A method for the assay of ablastin in the serum of rats infected with Trypanosoma lewisi.

This paper describes a simple quantitative assay for albastin, a factor present in the serum from rats infected with Trypanosoma lewisi, which prevents the division of the parasite. The assay measures in vitro the inhibition of the incorporation of 3H-TdR into the DNA of T. lewisi in the presence of serum from infected animals. Utilising this method, one can measure the titre of albastin in a particular serum sample and the time and duration of its appearance in the circulation of infected rats.

Haemoflagellates: commercially available liquid media for rapid cultivation.

The successful cultivation of a variety of haemoflagellates in three different liquid media is reported. These media include medium 199, Grace's insect tissue-culture medium and Schneider's drosophila medium, each in combination with 30% (v/v) foetal calf serum. These media were used to cultivate Old and New World species of visceral and cutaneous human Leishmania, as well as Leishmania species isolated from sandflies, rodents, and reptiles. Four strains of Trypanosoma cruzi, an isolate of T. R-angeli and and an isolate of T. lewisi have also been cultivated in these media. One or more of these media have been used to cultivate 121 strains of haemoflagellates, including at least 14 different species (11 Leishmania and 3 Trypanosoma) and many geographic isolates or strains. The Leishmania include L. braziliensis, L. peruviana, L. mexicana, L. tropica, L. donovani, L. chagasi, L. enriettii, L. hertigi, L. hoogstraali, L. adleri, and L. agamae. Using the Schneider's based medium, we have obtained primary isolates of both cutaneous and visceral Leishmania of man and of experimentally infected laboratory rodents and canines. Freeze-dried preparations of the Schneider's based medium that were reconsituted with distilled water after 24 months of storage at ambient temperature have proven to be suitable cultivation media. This feature makes the media valuable field tools. The various species of human Leishmania cultivated in these media have in our experience demonstrated no differences in growth rate, viability after liquid nitrogen preservation, or infectivity for laboratory animals and tissue-culture cells compared with promastigotes derived from blood-agar cultivation.