Infection of white rat peritoneal macrophages with Toxoplasma gondii, (Coccidia: Sarcocystidae) after Trypanosoma lewisi (Kinetoplastida: Trypanosomatidae) infection

Peritoneal macrophages from Wistar rats, inoculated and non-inoculated with 10(6) T. lewisi trypomastigotes, were cultured and infected with 10(6) T. gondii tachyzoites. Multiplication rates of this parasite were studied after 1, 24 and 48 h of infection but there were not significant differences between the number of parasites found inside of macrophages coming, either from T. lewisi infected or non infected rats. On the other hand, in vivo studies of Toxoplasma multiplication inside peritoneal macrophages, showed that there is an increase of parasite number in cells from T. lewisi infected rats, as compared with those macrophages from non infected rats. This effect was statistically significant and was more evident after four days of infection. Therefore, it has been demonstrated that in vivo, but not in vitro T. lewisi infections, causes an important decrease of the natural resistance to T. gondii of the white rats, which is manifested by the major invasion and multiplication of the parasite inside of peritoneal macrophages.     

Rat liver glucose-6-phosphate dehydrogenase isozymes: influence of infection with Trypanosoma

Glucose-6-phosphate dehydrogenase (G6PD) changes were studied in livers of rats inoculated with Trypanosoma lewisi, Trypanosoma rhodesiense, Trypanosoma congolense and Trypanosoma brucei. Marked increases in G6PD were directly related to the degree of parasitemia. No essential differences in G6PD levels were seen in animals inoculated with physiological saline when compared with uninoculated controls. Elevation of G6PD was observed only from day 10 to 20 in rats inoculated with T. lewisi. After day 20, the G6PD levels were not statistically significant from those of uninoculated controls. Liver G6PD levels were increased as early as day 3 post-inoculation and continued up to the time of death in rats inoculated with T. brucei, T. rhodesiense and T. congolense.     

Iron metabolism in Trypanosoma lewisi infection: serum iron and serum iron-binding capacity.

Progressive changes in iron levels, total iron binding capacity and hematocrit values in sera of rats infected with Trypanosoma lewisi are described. The host dietary group were: (1) complete or full complement; (2) iron-deficient, and (3) pair-fed or calorically restricted. The hematocrit values of T. lewisi-infected rats given the various diets were not significantly different from those of the controls. The decrease in total iron binding capacity (TIBC) of rats inoculated with T. lewisi and fed complete and pair-fed diets ranged up to 15% over uninfected controls. TIBC levels in rats fed an iron-deficient diet and inoculated with T. lewisi ranged up to 32% over uninfected controls. TIBC levels of deficient infected rats were significantly different from the controls from day 90 to infection to the end of the observation period. Serum iron (SI) values of non-infected rats regardless of dietary regimen showed significantly higher values than T. lewisi-infected animals between days 95 and 120. The average SI value, for this period, in adequately fed control rats was 204 +/- 7 microgram/100 ml as compared to 172 +/- 5 microgram/100 for trypanosome-infected rats. SI levels of rats on a pair-fed diet and infected with T. lewisi decreased to 17% over uninfected controls. SI levels of animals on an iron-deficient diet and infected with T. lewisi decreased up to 76% over uninfected controls.     

室内条件下莱氏猛叩甲对松墨天牛和黄粉虫的捕食能力

【目的】本研究旨在明确莱氏猛叩甲Tetrigus lewisi老熟幼虫分别对黄粉虫Tenebrio molitor和松墨天牛Monochamus alternatus末龄幼虫和蛹的捕食量的差异,评估莱氏猛叩甲的捕食能力,为松墨天牛M.alteratus的有效治理找到潜在的高效生物防治因子。【方法】于2014年11月在浙江富阳昌东村马鞍山砍下松墨天牛虫害木,通过林间野外调查,明确松墨天牛和莱氏猛叩甲的发生情况;将野外采集到的莱氏猛叩甲幼虫带回室内,分别饲喂黄粉虫和松墨天牛的末龄幼虫和蛹,每天记录捕食量,确定莱氏猛叩甲的捕食能力。【结果】野外调查中,我们在松墨天牛幼虫蛀食的坑道中发现了莱氏猛叩甲幼虫和松墨天牛幼虫被取食后的残体;在采集到1 094头松墨天牛幼虫的危害木内,共采得莱氏猛叩甲末龄幼虫36头,约为松墨天牛幼虫数量的3%。室内捕食实验表明,5 d内莱氏猛叩甲捕食黄粉虫幼虫总量为3.2头,平均每天捕食约0.6头,而捕食松墨天牛幼虫总量为8.0头,平均每天捕食约1.6头,莱氏猛叩甲对松墨天牛的总捕食量显著高于对黄粉虫幼虫的总捕食量(P0.0001);4 d内莱氏猛叩甲捕食黄粉虫蛹总量为5.6头,平均每天捕食约1.4头,而捕食松墨天牛蛹总量为7.8头,平均每天捕食约2.0头,莱氏猛叩甲对松墨天牛蛹的捕食量显著高于黄粉虫蛹(P=0.028)。回归分析还表明,莱氏猛叩甲幼虫的捕食量不受叩甲个体大小的影响。【结论】莱氏猛叩甲普遍分布在松墨天牛发生区,同时发现松墨天牛被取食后的残体,表明莱氏猛叩甲是松墨天牛的捕食性天敌。莱氏猛叩甲均能捕食黄粉虫和松墨天牛的幼虫和蛹;相比于捕食黄粉虫,莱氏猛叩甲对松墨天牛的幼虫和蛹具有更好的捕食效率,具有很好的生物防治前景,是未来松墨天牛治理潜在高效生物防治因子。     

Effect of Trypanosoma lewisi (Kinetoplastida: Trypanosomatidae) on the infection of white rats with Toxoplasma gondii (Eucoccidia: Sarcocystidae) oocysts

White rats were inoculated with 10(6) trypomastigotes of Trypanosoma lewisi, simultaneously or two days before and after inoculation with 10(5) oocysts of T. gondii. A greater number of cysts was found in the brain of the animals having concomitant inoculations, as compared with rats inoculated with either one of the two parasites. An apparent immunosuppressive effect is likely. Since both organisms can be found in rats, it is possible that infections with T. lewisi, could make this rodent another intermediate host for Toxoplasma infections.     

Trypanosoma lewisi-induced immunosuppression: the effects on alveolar macrophage activities against Cryptococcus neoformans

The immunosuppressive effect of Trypanosoma lewisi infection on alveolar macrophage (AM) activities against Cryptococcus neoformans was studied in an animal model. Two groups of rats were treated with T. lewisi and killed after 4 (4d-rats) and 7 days (7d-rats), respectively. A third group not given T. lewisi, served as control. AM were challenged in vitro with C. neoformans. Phagocytosis was assessed with a fluorescence method. Superoxide anion production was evaluated with the nitroblue tetrazolium (NBT) test. The survival of cryptococci was estimated by counting colony-forming units. The numbers of detached AM from culture plates were determined using a Bürker chamber. The NBT response, adhesion to plate surface and killing activity, but not the phagocytosis of AM from 4d-rats were significantly impaired compared to control or 7d-rats. Thus, T. lewisi causes transitory immunosuppressive effects on AM activities. This rapid T. lewisi immunosuppression model may be useful to study new approaches to anticryptococcal therapy.     

Trypanosoma lewisi Infections in Normal Rats and in Rats Treated with Dexamethasone

SYNOPSIS. Administration of dexamethasone to rats infected with Trypanosoma lewisi resulted in the development of exceedingly large populations of trypanosomes which were fatal to their hosts. The elevated levels of parasitemia in treated rats early in infections were thought not to be a result of an increased reproductive rate. However, trypanosomes in treated rats 2 days postinfection did have a higher coefficient of variation in total length and a greater percentage of dividing forms than those observed from infected rats which were not given the drug. The course of infection may be markedly altered not only in intensity but also in length by this corticosteroid. It is suggested that dexamethasone administered at the levels recorded to rats infected with T. lewisi inhibits the production of ablastin and trypanocidal antibodies.     

Suppressor and protector factors derived from Trypanosoma lewisi.

Trypanosoma lewisi is a specific protozoan blood parasite of rats. Normal rats infected 2 h after treatment with plasma from day 8 irradiated (8.5 Gy) infected rats had significantly higher parasitaemia; in contrast, animals infected 7 days post-plasma treatment were significantly protected. Trypanolytic and ablastic antibodies could be demonstrated in the serum of normal rats treated with the plasma; the trypanolytic antibodies were stage-specific. Suppression of normal rat splenocyte responses to Con A and lipopolysaccharide (LPS) stimulation were also observed in the presence of different protein concentrations of whole lysate from epimastigote forms. The suppression by mitogen Con A was ablated by the addition of exogenous IL 2, or by washing cells incubated with the lysate prior to mitogen stimulation. These results indicate that immunoregulatory factors are present in the plasma of rats infected with T. lewisi, and the effect of these factors can be demonstrated in vitro with whole parasite lysate. The restoration of normal splenocyte responses to Con A by addition of exogenous IL 2 or by washing cells suggests that the suppressor factor(s) act(s) on the T cells by inhibiting their proliferation and IL 2 production, and the continued presence of these products is essential in the maintenance of suppression.     

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.     

Elicitation of the reproduction-inhibiting antibody ablastin by serum exoantigens of Trypanosoma lewisi

Serum exoantigens of Trypanosoma lewisi were collected 5 days after infection from immunocompetent (untreated) rats and rats immunosuppressed by treatment with either hydrocortisone acetate or dexamethasone. Normal rats were then immunized with pooled, whole exoantigen-containing serum from 1 of these 3 sources plus alum as an adjuvant, and the immune sera produced were tested individually. All contained agglutinating (trypanocidal) antibodies to both antigenic variants of T. lewisi, but only about two-thirds showed precipitating activity with exoantigens in gels. More importantly, however, when these antisera were thoroughly adsorbed with living trypanosomes (from immunocompetent hosts) to remove agglutinating antibody only and then tested for ablastic activity in vitro, all showed significant (P less than 0.01) reproduction-inhibiting activity, comparable to that shown by ablastic serum collected from rats that experienced a natural infection. Antisera from control rats similarly immunized with normal rat serum were negative in all antibody tests. The exoantigens of T. lewisi are, therefore, a complex mixture of immunogens that are related to the known immune responses to the parasite and can elicit the formation of ablastic antibody with the same biological properties as that produced during a natural infection.     

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.     

Flow cytometric analysis of immunoglobulin and complement component C3 on the surface of Trypanosoma lewisi

We have reinvestigated whether surface immunoglobulin (sIg) on Trypanosoma lewisi is antibody directed toward parasite antigen by using flow cytometry to analyze parasites stained with fluoresceinated F(ab')2 fragments of antibodies to rat IgG and IgM. We have confirmed that IgG antibody to the parasites is present both in the serum of rats and on the surface of parasites between the fourth and twentieth days of infection, that the amount of sIg per cell increases as the infection progresses, and that considerably more IgG is present on parasites harvested from intact rats than on those from rats that had been immunosuppressed by whole body gamma-irradiation. In addition sIgM was detected on trypanosomes from intact, but not on parasites from irradiated rats. We have also made two observations suggesting that not all sIg is specific antibody made in response to T. lewisi. First, a low but significant amount of sIgG was detected on parasites throughout infection in irradiated rats; no sIgM was detected on these parasite. Second, when parasites harvested from immunosuppressed rats were incubated in normal rat serum, the amount of both sIgG and sIgM detected by flow immunofluorescence increased. Parasites harvested from intact animals bound IgM but not IgG from normal rat serum. These results suggest either that natural antibody to the trypanosomes is present in the serum of uninfected rats or that some rat immunoglobulins bind to structures on the trypanosome surface in ways that do not depend on usual antigen-antibody interactions. Finally, flow immunofluorescence was also used to detect complement component C3 on the surface of both intact and trypsinized bloodstream forms harvested from intact or immunosuppressed rats. The amount of sC3 per cell did not increase until late in the infection and consequently did not correlate with the increase of sIgG. Therefore, T. lewisi avoids destruction by the immune system although immune effector molecules, IgG, IgM, and C3, are on its surface.     

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.     

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

Abstract. 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.     

Surface immunoglobulins, a possible mechanism for the persistence of Trypanosoma lewisi in the circulation of rats.

The results presented below show that the dividing and adult forms of T. lewisi share common antigens and indicate that the persistence of adult forms in the circulation of rats immune to reinfection is not due to a change in surface antigens as has been postulated. Using a rabbit anti-rat immunoglobulin serum, the presence of rat immunoglobulins on the surface of adult trypanosomes could be demonstrated. These immunoglobulins were not complement-fixing or opsonic. It is suggested that these immunoglobulins are responsible for the persistence of the adult forms in the circulation of the rat.     

Changes in Antigenicity of Trypanosoma lewisi During the Course of Infection in Rats*

SYNOPSIS. In rats infected with T. lewisi, 2 morphologically distinct forms of the trypanosome occur. By means of the immunodiffusion technic several antigenic differences between the 2 forms were observed. The time course in the antigenic and morphologic changes is somewhat different. The antigenic change from young to adult forms is gradual. It starts 12 days after infection and is complete in 14 days, 3 to 5 days after the morphologic appearance of the trypanosomes is that of adult forms. In addition, there appear to be 2 separate stages of young forms when they are analyzed for antigenic composition.     

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: pyruvate and transketolase activity in normal and thiamine deficient rats

Transketolase and pyruvate changes were studied in rats infected with Trypanosoma lewisi and fed complete, thiamine-deficient and pair-fed control diets. Regardless of the dietary group, marked increases in pyruvate levels were observed in the infected animals. There were no significant differences in erythrocyte transketolase activity of rats given a full complement diet. Significant decreases, however, were observed in the transketolase activity of pair-fed and thiamine deficient rats. The greater decreases occurred in the infected animals.     

Correlation of oligomycin-sensitive ATPase activity in trypanosomes with their content of an antigen to primary biliary cirrhosis

The amounts of an antigen to primary biliary cirrhosis (PBC) which occur in subcellular fractions of Trypanosoma rhodesiense and T. lewisi correlate positively with the oligomycin-sensitive (OS) ATPase activity of these fractions. This result is consistent with the mitochondrial ATPase association of the antigen in mammalian and other cells. Higher levels of OS-ATPase and of PBC antigen in T. lewisi accord with a more extensive mitochondrial development in this species.     

Effect of Holothurin on Trypanosoma lewisi Infections in Rats

SYNOPSIS. Crude holothurin from the Bahamian sea cucumber Actinopyga agassiz , inoculated intraperitoneally into rats with Trypanosoma lewisi infection, altered the host-parasite relationship. The effect was measured by a study of parasite population during infections. Rats treated with holothurin prior to and simultaneously with an infection of trypanosomes had lower parasitemias than controls. A higher level of parasitemia was observed in treated after infection with trypanosomes. Both dosage and timing appeared to have been important parameters of the observed effect. The reticuloendothelial system is suspected to play a role in these findings.