Trypanocidal Antibodies Research Articles

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.

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.

Isolation of protective antigens from Trypanosoma lewisi by using trypanostatic (ablastic) immunoglobulin G from the surface coat.

Antigens were purified from extracts of Trypanosoma lewisi on immunoadsorbent columns of trypanostatic immunoglobulin G eluted from parasite surface coats at 8 days postinfection. Eight absorbed proteins, with molecular weights between 15,000 and 70,000, were identified. These surface coat antigens (SCAgs) were then used to immunize rats. After immunization, sera were assayed in vitro for levels of circulating trypanostatic and trypanocidal antibodies. Approximately half of the immune sera had higher levels of trypanostatic antibody, compared with control sera; no trypanocidal antibodies (agglutinins) were detected in any of the sera. The rats were then challenged intraperitonally, and the parasitemias and division rates of the parasites were monitored. Parasitemias of all immunized rats were significantly (P less than 0.01) lower and of shorter duration than those of the controls. Division rates of trypanosomes were also significantly (P less than 0.01) lower in all immunized rats at all times before total cessation of division compared with control rats. A clear dose-response effect was observed, with greater amounts of SCAg eliciting higher levels of protection. Purified SCAgs were also more effective immunogens than were the crude trypanosome extracts from which they had been purified, and in which other proteins in addition to the SCAgs were present. These data provide conclusive evidence that the immunoglobulin G in the surface coats of T. lewisi, adsorbed during the course of infection, is specific antibody, in that it can be used to isolate parasite antigens that elicit a trypanostatic response in rats immunized with them.

Interference with anti-trypanosome immune responses in rabbits infected with cyclically-transmitted Trypanosoma congolense.

Rabbits were infected with two clones of antigenically distinct stocks of Trypanosoma congolense transmitted through Glossina morsitans. Local skin reaction development and the appearance of neutralizing antibodies were followed in animals infected with one or other of the trypanosome stocks, with both stocks simultaneously or with both stocks consecutively. There was little difference in local skin reaction development on rabbits infected with a single stock or with both stocks simultaneously but, in rabbits exposed to a heterologous stock 14 or 21 days after a primary infection reactions were reduced in size or completely absent. Neutralizing antibodies against metacyclic-derived trypanosomes were detected 21 days after infection in animals infected with a single trypanosome stock and, in rabbits infected with both stocks simultaneously, antibodies against each stock were also detected 21 to 28 days after infection. In rabbits challenged 14 or 21 days after primary infection the appearance of trypanocidal antibodies against the stock used for challenge was delayed from 28 to 49 days.

Interference with anti‐trypanosome immune responses in rabbits infected with cyclically‐transmitted Trypanosoma congolense

Summary Rabbits were infected with two clones of antigenically distinct stocks of Trypanosoma congolense transmitted through Glossina morsitans. Local skin reaction development and the appearance of neutralizing antibodies were followed in animals infected with one or other of the trypanosome stocks, with both stocks simultaneously or with both stocks consecutively. There was little difference in local skin reaction development on rabbits infected with a single stock or with both stocks simultaneously but, in rabbits exposed to a heterologous stock 14 or 21 days after a primary infection reactions were reduced in size or completely absent. Neutralizing antibodies against metacyclic‐derived trypano‐somes were detected 21 days after infection in animals infected with a single trypanosome stock and, in rabbits infected with both stocks simultaneously, antibodies against each stock were also detected 21 to 28 days after infection. In rabbits challenged 14 or 21 days after primary infection the appearance of trypanocidal antibodies against the stock used for challenge was delayed from 28 to 49 days.

Babesia bovis: Attachment of Infected Erythrocytes to Heparin-Sepharose Columns

lewisi infections. Although there were some differences, the hyperimmune antiserum showed several lines of identity with antisera collected at various times during a typical primary infection. Antisera collected 21 and 28 days postinfection shared two precipitin lines with the hyperimmune rat antisera, and antisera collected from 49 to 250 days postinfection produced from three to four common precipitin lines. Why these antibodies are not usually detected in the serum of rats with a typical primary infection is unknown. The presence of blocking antibodies in the sera of infected rats has been suggested by Long and Dusanic (1978, loc. cit.). If blocking antibodies are present, then exoantigens might be released as antigen-antibody complexes in the untreated rats. The ability of hyperimmune antisera to precipitate exoantigens from the immunocompetent rats may be the result of increased avidity or the presence of antibody i ections. Although there were some to the antigen-antibody complexes. In the immunosuppressed host, these exoantigens might be released as free antigens, which would account for their increased reactivity. Ferrante and Jenkin (1977, Austral. J. Exp. Biol. Med. Sci. 55: 275-289) detected nonopsonic, noncomplement fixing, "blocking" antibodies on the surface of T. lewisi and suggested that these immunoglobulins played a role in protecting the parasites from trypanocidal antibodies. Giannini and D'Alesandro (1979, Exp. Parasitol. 47: 342-355) suggested that the bound immunoglobulin is the reproduction inhibiting antibody ablastin. The exact nature and function of antibodies bound to T. lewisi remains to be determined. This work was supported in part by Grant AI-11369 from the U.S. National Institutes of Health and National Science Foundation Grants PCM 76-11922 and PCM 78-23939. the antigen-antibody complexes. In the im-

Trypanosoma lewisi: Avidity and adsorbability of ablastin, the rat antibody inhibiting parasite reproduction

The relation of naturally acquired host IgG in the surface coat of bloodstream forms of Trypanosoma lewisi to ablastin was studied to determine whether, contrary to a long-held conclusion, the antibody is avid and adsorbable. It was found by immunofluorescence and agglutination tests with monospecific antisera to rat IgG that bloodstream forms collected from immunosuppressed hosts, in contrast to those from immunocompetent hosts, have little or no detectable surface IgO. Specificity of adsorption was also demonstrated in other immunofluorescence experiments in which bloodstream forms from immunosuppressed hosts adsorbed IgG from immune serum with ablastic activity only (previously adsorbed with trypanosomes from immunocompetent hosts to remove the trypanocidal antibodies), but did not adsorb IgG from normal rat serum. To determine whether this specific adsorption of IgG by the parasite could be correlated with a reduction in ablastic activity, immune sera were adsorbed with bloodstream forms from immunosuppressed hosts at packed cell/serum ratios of either 1.2 or 2.0, and the adsorbed sera were then tested for ablastic activity in vitro. With both cell/serum ratios, ablastic activity was reduced by 50%. In comparison, similar adsorptions of immune sera with trypanosomes from immunocompetent hosts resulted in reductions of ablastic activity of only about 9 and 27% with the low and high cell/serum ratios, respectively. It is concluded that the failure to effect significant adsorption of ablastin in earlier studies resulted from the use of ablastinsensitized trypanosomes from immunocompetent hosts.

Genetic resistance to Trypanosoma congolense infections in mice.

The mechanisms of genetic resistance or "trypanotolerance" to infection with Trypanosoma congolense were investigated in two strains of mice. One strain C57BL, is outstandingly resistant to most stabilates of T. congolense and can survive for over 80 days, whereas CFLP, in common with most other strains, generally succumbs in less than 20 days. Evaluation of several pathophysiological and immunological parameters showed that after infection both strains initially developed similar levels of parasitemia, anemia, biochemical derangement, and immunosuppression. The most outstanding difference was after day 8 postinfection, when the susceptible strain (CFLP) sustained high levels of parasitemia (10(9) trypanosomes per ml) until death 2 to 4 days later, whereas the resistant strain (C57BL) showed a marked decrease to less than 10(6) trypanosomes per ml by day 10 postinfection. Clear evidence that this was associated with the presence of trypanocidal antibody in the resistant mice was provided by the results of an infectivity neutralization test on serum collected from each strain at 10 days postinfection. Chronically infected C57BL mice showed declining waves of parasitemia and a slow restoration of most hematological and biochemical indexes to near normal levels by 80 days postinfection, although at this stage they remained immunosuppressed.

Trypanosoma lewisi: Accumulation of antigen-specific host IgG as a component of the surface coat during the course of infection in the rat

Abstract Naturally acquired host IgG, adsorbed to the surface of Trypanosoma lewisi during the course of infection in the rat, was labeled with fluorescein-conjugated rabbit IgG, or Fab fragments of this IgG, directed against rat IgG. The intensity of fluorescent labeling increases with time, concomitant with the increase in anti- T. lewisi activity of host plasma. Trypanosomes harvested from immunosuppressed hosts lack detectable surface IgG. Trypanosomes having little or no detectable surface IgG (harvested from immunosuppressed hosts or early in the infection from immunocompetent hosts) can adsorb IgG from serum with ablastic activity only (obtained from other infected rats between the first and second crises and adsorbed to remove trypanocidal antibodies), but not from normal serum. Therefore, the absence of detectable surface IgG on such cells is not caused by the parasites' inability to adsorb host IgG, but rather results from the immune state of the host. Hence surface IgG on T. lewisi is specific antibody. Host albumin is nonspecifically adsorbed, in contrast to IgG. Trypanosomes from immuno-suppressed and immunocompetent rats were positive and visually indistinguishable from each other when labeled with anti-rat albumin, and were equally agglutinable with anti-rat albumin serum.

A culture and biochemical assay system for Trypanosoma lewisi

Trypanosoma lewisi was cultivated as forms which appeared to be physiologically similar to those found in vivo. The medium consisted of 1.0 g peptone, 1.0 g glucose, 10 ml rat serum, 10,000 units penicillin G, 10,000 μg streptomycin and 90 ml Hank's Balanced Salt Solution. It was supplemented with 8.0 × 10 8 rat erythrocytes per milliliter. In the complete medium trypanosomes multiplied for 48–72 hr. Cultured forms were lethal to newborn rats and infective to adults. Adsorbed early immune serum inhibited the growth of the trypanosomes in vitro and the percentage of reproductives declined from 66 to 45%. The cultured trypanosomes were also susceptible to both trypanocidal antibodies.

Immunosuppression and ablastin

Ablastin formed by animals in response to infections by rodent trypanosomes possesses the characteristics of an antibody. Partial resistance to Trypanosoma lewisi is demonstrable in animals previously injected with live Trypanosoma musculi. Antisera from T. musculi infected mice do not inhibit reproduction by T. lewisi bloodstream forms in vitro as efficiently as homologous antisera collected at similar times during infections, indicating a degree of specificity. Ablastin activity in antisera is not altered by treatment with 2-mercaptoethanol or by heatng at 60 °C for 3 hr. Sephadex G-200 gel filtration of early and late antisera from T. lewisi infected rats and assays with bloodstream forms cultured at 37 °C detect ablastin activity in the second major fraction eluted from the columns. Ablastin appears to be an antibody of the immunoglobulin G species. Immunosuppressant procedures utilized in studies of the host responses to rodent trypanosomes are reviewed and include: chemical agents, irradiation, splenectomy, reticuloendothelia blockade and thymectomy, and treatment with antilymphocyte and antithymocyte sera. Evaluation of the results of the application of these procedures to rodent parasite systems indicates ablastin is an antibody and supports the concept that the inhibition of trypanosome reproduction is separate and distinct from the first trypanocidal event responsible for the decreasing parasitemias observed during the infections. Recent studies concur and suggest that the first crisis in the infections is mediated by the combined actions of a thymus-dependent ablastin and a thymus-independent trypanocidal antibody.

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.

Effects of Host Fighting Behavior on the Course of Infection of Trypanosoma Duttoni in Mice

In one experiment, male albino mice were inoculated with Trypanosoma duttoni, and were allowed to begin fighting on the same day; the fighting animals developed significantly lower parasitemias than the isolated controls throughout the reproductive phase of the infection. In two other experiments, mice were allowed to fight for a period of 10 days prior to inoculation; the fighting animal exhibited higher parasitemias than the controls, with the subordinate mice developing slightly, but not significantly, higher parasitemias than the dominant animals. Since all differences noted among controls, dominants and subordinates were during the reproductive phase of the infection, and since no differences were observed in the length of infection among these groups, it was concluded that fighting altered the ablastic, but not the trypanocidal antibody response. This alteration is believed to have resulted from the action of adrenal cortical hormones on antibody—producing cells. Sustained elevation of plasma corticosterone levels during 10 days of fighting in the latter experiments may have been responsible for the different results obtained with the two experimental designs.

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.

Virulent Trypanosoma lewisi Infections in Cortisone-Treated Rats

Administration of four doses of 50 to 100 mg cortisone/kg body wt to rats 2 days prior to, and 2 days postinoculation with ca. 1 X 106 Trypanosoma lewisi causes a prolonged parasite reproductive phase, higher parasitemias, and production of virulent infections. The enhanced virulence is most likely due to an impairment of the host immune response. Reinfection of rats recovered from T. lewisi infections was not possible in spite of protracted cortisone treatment. Trypanosoma lewisi in the rat is a classic example of a nonpathogenic hemoflagellate infection. During the course of infection three phases are discernible in the peripheral circulation: (1) a day or two after inoculation the parasites appear in the blood and undergo active reproduction for a period of 3 to 4 days. At this time the number of dividing forms is high, and there is considerable variability in size and shape; (2) after the 5th or 6th day a number crisis occurs during which most of the parasites are destroyed; the survivors do not divide, but persist in the blood for an additional period, are of uniform size, and are referred to as adults; and (3) a second number crisis terminates the infection after 3 to 4 weeks, and results in complete protective immunity of the rat against reinfection. The immunologic events which underlie these manifestations have been ascribed to the production of a reproduction-inhibiting antibody, called ablastin, which inhibits the reproduction of the parasites without injuring them and without destruction of motility or viability, and the formation of two trypanocidal antibodies, which result in the two number crises (Taliaferro, 1924, 1932). Other workers suggest that only two antibodies are involved (Chandler, 1958; Ormerod, 1963). Modifications of the course of infection have been attempted with varying success. The most successful demonstration of enhanced virulence and prolongation of the parasitic reproductive phase was accomplished by keeping rats on a pantothenate-deficient diet (Becker et al., 1943, 1947) or by oral administration of sodium salicylate (Becker and Received for publication 29 July 1966. * Supported by a grant from the NIH, Institute of Allergy and Infectious Diseases (AI 05226). Gallagher, 1947; Saul and Becker, 1949). The present work suggests that cortisone treatment of the host so modifies its immune response that the infection becomes virulent. MATERIALS AND METHODS Male rats weighing 120 to 175 g were obtained from Berkeley Pacific. The colony was apparently free of Bartonella infections. The strain of Trypanosoma lewisi was obtained from Dr. P. D'Alesandro, of the Rockefeller University. Cortisone was an aqueous suspension of 25 mg/ml hydrocortisone acetate for intramuscular use (Robinson Laboratory). Blood for hematocrits and thin films was obtained by snipping off the tip of the tail. Daily blood films were prepared and stained in Giemsa. Hematocrits were taken using microhematocrit capillary tubes (Clay-Adams). The numbers of trypanosomes per mm' of blood were counted by the method of D'Alesandro (1959) and modified by the lematocrit values. Reproductive activity of the trypanosomes was determined using the method of D'Alesandro (1959), but included broad as well as dividing and small forms. The experiments were divided into a series of 7: Group A: Four animals given 0.5 ml of 0.85% NaCl intramuscularly in the hind limbs 2 days before and 2 days after inoculation. Group B: Four animals given 200 mg cortisone/kg body wt administered as four intramuscular injections with the injection schedule as in A. Group C: Five animals given 300 mg cortisone/kg body wt and administered in a manner described above. Group D: Four animals given 400 mg cortisone/kg body wt with injection schedule similar to groups A, B, and C. Groups A through D were inoculated intraperitoneally with ca. 1 X 10 trypanosomes, derived from animals infected intraperitoneally 5 to 7 days previously. Approximately 50% of the trypanosomes were dividing forms. The amount of antibody present in this inoculum was probably quite low since parasites were taken before the first crisis and infected blood was diluted approximately 10-fold in 0.85% NaCl.

In vitro studies of ablastin, the reproduction-inhibiting antibody to Trypanosoma lewisi.

SYNOPSIS. Previously, the reproduction‐inhibiting effects of ablastin could only be shown in vivo. The present report describes techniques for the in vitro demonstration and titration of this antibody. With a medium composed of Hanks' balanced salt solution, rat serum, lactalbumin hydrolysate, yeast extract and rat blood lysate, blood stream forms of Trypanosoma lewisi can be grown for approximately 24 hours at 37d̀C. Starting with the medium containing normal rat serum and inoculated with adult (inhibited) trypanosomes from infected rat blood, 50% or more of the parasites are in various stages of division after incubation overnight. Under similar conditions with ablastic rat serum, the parasites do not reproduce but remain as adults. If the medium is inoculated with reproducing trypanosomes from the blood, parasites in the presence of normal serum continue to reproduce, whereas those exposed to ablastin are almost completely converted to adult, non‐reproducing forms. Similar results are obtained when the immune sera used are first adsorbed with living parasites to remove all trypanocidal antibodies. Ablastic serum inactivated at 56d̀C for 20 minutes does not lose its inhibitory activity indicating that ablastin is not complement dependent, and parasites grown on media at room temperature are not affected by the antibody suggesting that basic antigenic differences exist between blood stream forms at 37d̀C and culture forms at room temperature. Studies of the conversion of blood stream forms to culture forms indicate that the critical temperature range for the conversion lies between 28d̀ and 30d̀C. The significance of these results is discussed, and possible applications of the techniques described to studies of the mechanism of ablastic action are considered.

A study of cytoplasmic inclusions in Trypanosoma lewisi and their relationship to the formation of antibody.

SUMMARY: Developmental forms of Trypanosoma lewisi continue to increase in number when the ‘crisis’ (normally occurring at the 10th day) is inhibited; the inclusions which they contain also increase in number, density and refraetile power. Suspensions of killed developmental forms containing multiple inclusions have greater protective power against a challenge infection than have similar suspensions of adult forms which contain a single inclusion. It is suggested that the multiple inclusions contain an antigenic factor which stimulates the production of a trypanocidal antibody and reacts with it to produce the crisis, but the single inclusions of the adult forms which survive the crisis have different antigenic properties.

Concerning Salicylate Inhibition of Ablastic Activity in Trypanosoma lewisi Infection

The immune reaction in Trypanosoma lewzisi infection in the rat has been described as operating in two ways (Taliaferro, 1932). An antireproductive antibody, ablastin, is formed first. This antibody inhibits the reproduction of the parasites. A second antibody, the trypanocidal antibody, is then formed which destroys the parasites. The question of the existence, formation, nature and operation of ablastin has received a great deal of attention by many investigators. Taliaferro (1924, 1925, 1926, 1932) reported that ablastin was a protein in the euglobulin fraction of serum, that it was passively transferable, that it would not unite with trypanosomes in vitro, and that it neither killed the parasites nor affected their motility or vitality. Its only manifestation was that of in vivo suppression of the reproduction of the parasite. Becker and co-workers (1943, 1947) reported that pantothenic acid deficiency affected the antireproductive activity of ablastin so that reproduction continued and the infection became pathogenic. Becker and Gallagher (1947) found that treatment of infected rats with sodium salicylate had a similar effect on ablastic activity. Blood sugar studies by Schern (1928) and Poindexter (1933) indicated an inverse relation between the amount of blood sugar and the numbers of trypanosomes in pathogenic trypanosomiases. Tubangui and Yutuc (1931) found that hypoglycemia occurred only shortly before death. Linton (1929) found that in the non-pathogenic T. lewisi there was no change in blood sugar levels in the rat during the course of the infection. Minoru (1940) reported changes in blood sugar levels in rabbits as a result of sodium salicylate administration. In low amounts there was a hypoglycemia. In greater amounts there was a hyperglycemia. Brown (1915) and Taliaferro (1926) suggested that the difference between pathogenic and non-pathogenic trypanosomiases might be based on the absence or presence of antireproductive activity. In this study an attempt was made to examine the interrelationships among antireproductive activity, pathogenicity, blood glucose levels, blood pantothenic acid levels, blood protein levels, as associated with sodium salicylate treatment. This was done in order to learn something of the nature and operation of ablastin and the way in which salicylate interferes with ablastic activity.

Studies on the Mechanism of Host Resistance in Experimental Leptospirosis Icterohemorrhagica

Studies on the Mechanism of Host Resistance in Experimental Leptospirosis Icterohemorrhagica

Some Factors in the Defense Mechanism against Reinfection with Trypanosoma lewisi

Early in experimental studies on Trypanosoma lewisi it was discovered that rats having spontane ously recovered from an infection are refractory to reinfection (Kanthack, Durham and Blanford1). The immunity thus acquired lasts, with an occasional exception, for life of rat. The numerous immunological studies with T. lewisi have been chiefly centered on determining factors involved in recovery of rats from an initial infection (Taliaferro,2 Regendanz and Kikuth,3 and others). Our knowledge of defense mechanism against reinfection is at present meager and largely based on in vitro studies. Only a few observations have been made directly on parasite during course of reinfection. Laveran and Mesnil4 noted that when recovered or hyperimmunized rats are inoculated intraperitoneally with living T. lewisi parasites seldom appear in circulating blood. They be lieved that phagocytosis was chief mechanism involved in both active and passive immunity. Agglutination of parasites was observed by Laveran and Mesnil, but they did not consider it an important event in defense mechanism since parasites were apparently uninjured by reaction. Recent workers have generally failed to observe typical phagocytosis of trypanosomes. Taliaferro5 has noted trypanosomes attached to leucocytes under conditions similar to experiments of Laveran and Mesnil and when inactivated immuna rat serum was mixed with washed T. lewisi and washed guinea pig leucocytes. It would appear that, at present time, phagocytosis is not con sidered to be important or usual manner of destruction and disposal of T. lewisi in either initial infection or in reinfection. Taliaferro5 injected intravenously large quantities of washed adult trypanosomes into recovered rats? but otherwise normal?and also into recovered rats splenectomized during course of their initial infection. In first experiments, 27 rats were inoculated from 6 to 325 days after end of initial infection. There was no apparent correlation between length of time from recovery to second inoculation and duration of reinfec tion. No reproduction by parasites was noted in 8 rats whose reinfections lasted from 2 to 3 days. Dividing forms occurred in 6 rats whose reinfections lasted 2 to 13 days and, for others, trypano somes remained in blood too short a time to determine reproductive activity. In many of rats recovery was apparently rapid, since the parasites were swept from blood in 5 to 15 minutes. To what extent reproduction developed and whether a numerical increase of trypanosomes occurred is not stated. In splenectomized series, 10 rats were reinfected from 31 to 353 days after end of initial infection. The reinfec tions lasted from 1 to 40 days and in 2 rats slight variation of trypanosomes but no division was noted. It was concluded that, in most cases, trypanocidal antibody is so strong in recovered rats that it is largely effective before ablastin (repro duction-inhibiting antibody) has time to play any particular r?le. The nature of trypanocidal antibody has not been determined, but according to Taliaferro,6 it is a lytic antibody which may act as an opsonin in vivo. In vivo studies, thus far, have failed to indicate specific character of this antibody. From a review of literature it would appear that in most attempts to establish reinfection experimental animal was injected intraperitoneally with a dilute suspension of infective blood. Since trypanosomes injected into peritoneal cavity must traverse one or more lymph nodes before they can enter blood stream, possibility was early considered that in spontaneous recovery lymph nodes might become sensitized and thus produc? an effective barrier against parasites entering circulation. In present study, experiments designed to gain information on this question clearly demon strated that trypanosomes pass without hindrance