subsp. brucei have devised a technique whereby trypanosomes mice. The validity of the technique was also tested by using suitable control inocula.

Transcription

1 INFECTION AND IMMUNITY, May 1982, p /82/ $02.00/0 Vol. 36, No. 2 Early Course of Infection in Susceptible and Resistant Strains of Mice, Using [3H]Uridine-Labeled Trypanosoma brucei subsp. brucei LAWRENCE W. ANDERSON* AND KEITH L. BANKS Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington Received 16 October 1981/Accepted 30 December 1981 A radioisotopic technique utilizing [3H]uridine was developed to determine the tissue distribution of intravenously inoculated Trypanosoma brucei subsp. brucei in susceptible (Swiss-Webster) and resistant (deer) strains of mice. The reliability of the technique was tested by using unincorporated [3H]uridine and heat-killed (labeled) trypanosomes in parallel experiments. The inoculations with viable (labeled) organisms showed that during the initial 24 h T. brucei subsp. brucei was cleared from the bloodstream of deer mice to a significantly greater extent than in Swiss-Webster mice. In addition, significantly more radioactivity was found in the liver, spleen, and kidneys of the deer mice. Autoradiographs of trypanosomes in selected tissues supported the distribution observed with labeled organisms. All of the above experiments involved the highly virulent, monomorphic bloodstream form of T. brucei subsp. brucei. Similar experiments with less virulent tissue culture-adapted trypanosomes showed that these organisms were readily cleared from the bloodstream, even in highly susceptible Swiss-Webster mice. The results suggest that avoidance of phagocytosis may be an important virulence factor of T. brucei subsp. brucei and contributes to the variation observed in species and strain susceptibility to trypanosomiasis. The capacity of African trypanosomes to produce disease varies markedly depending upon the trypanosome isolate (8), the species of host (8, 14), and the host strain (12, 18, 25). For example, the course of Trypanosoma brucei subsp. brucei infection in C3H mice is more rapid than corresponding infections in C57BL (3) or deer mice (21). The reasons for this wide variation in disease course are not well defined, but several factors could be involved. First, variation in the magnitude and type of immune response to trypanosomal surface antigens could influence the degree of parasitemia. Indeed, the balance of protective immunity versus immunosuppression has already been shown to influence the course of murine trypanosomiasis (26). A second potential factor is the variety of tissue environments found in susceptible hosts, which either favor or limit trypanosome growth. For example, there is widespread evidence that trypanosomes localize in definite organs, and this organ distribution varies between hosts (14, 21, 22, 28). Finally, the destruction of trypanosomes by phagocytes or soluble blood components may differ between hosts and therefore alter the course of the disease (24). To better understand the factors involved in the pathogenesis of trypanosomiasis in mice, we 525 have devised a technique whereby trypanosomes can be labeled with isotope and tracked in vivo. Using this technique, we have studied the distribution of trypanosomes of low and high virulence in susceptible and resistant strains of mice. The validity of the technique was also tested by using suitable control inocula. MATERLS AND METHODS Mice. Female Swiss-Webster mice were obtained from Lab Animal Resources (Pullman, Wash.) at 6 to 10 weeks of age. Deer mice (Peromyscus maniculatus, male and female) were obtained from our own colony and used for study at 8 to 10 weeks of age. Trypanosomes. T. brucei subsp. brucei (-I-/ EA- TRO/110) was kindly provided by Jack Moulton, Davis, Calif. This stock has been passaged in Swiss- Webster mice for 3 years at Washington State University. In vitro-grown T. brucei subsp. brucei was established by the technique of Hirumi et al. (9), except that goat choroid plexus cells were used as the feeder-cell layer. Trypanosomes were grown in RPMI 1640 containing 20%o fetal bovine serum (Flow Laboratories, Rockville, Md.), 1% nonessential amino acids, 20 mm N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 100 U of penicillin per ml, 100,ug of streptomycin per ml, and 2 mm L-glutamine. Labeling of the trypanosomes. Whole blood was obtained from infected mice 1 to 7 days postinfection by cardiac puncture, diluted in 4:6 or 5:5 phosphate-

2 526 ANDERSON AND BANKS INFECT. IMMUN. saline-glucose, and passed through a DEAE-cellulose column as described by Lanham and Godfrey (13). A total of 60 x 106 to 100 x 106 trypanosomes isolated in this manner were then added to Dulbecco modified Eagle medium (GIBCO Laboratories, Grand Island, N.Y.) containing 10%o fetal bovine serum and 50,uCi of [3H]uridine (5,6-3H, 39.5 Ci/mmol; New England Nuclear Corp., Boston, Mass.) per ml. The trypanosome suspension was incubated at 37 C for 3 h with agitation of the parasites at 1-h intervals. The organisms were spun at 729 x g for 15 min, and the labeling medium was removed. The pellet was subsequently washed with 40 ml of Dulbecco modified Eagle medium containing 10% fetal bovine serum and then 40 ml of serum-free Dulbecco modified Eagle medium. Two washes were necessary to remove unincorporated [3H]uridine, and although 10%o serum protected trypanosome viability, it was deemed undesirable as part of the inoculum based on its antigenic properties. The final pellet was suspended in 2 to 3 ml of Hanks balanced salts solution, and 0.1 ml was added to scintillation cocktail in a glass scintillation vial and counted in a Beckman LS 9000 scintillation counter. A similar volume of the final wash was also counted. If the organisms were adequately labeled and the wash was low in radioactivity (less than 400 cpm), the trypanosomes were counted in a hemocytometer and inoculated (0.2 to 0.3 ml) intravenously into the lateral tail vein of mice. Usually, between 2 x 106 to 5 x 106 organisms were inoculated. In control experiments: (i) trypanosomes were labeled and then killed by heat treatment (55 to 60 C for 15 min) and injected intravenously, and (ii) unincorporated [3H]uridine was similarly inoculated at 10,000 to 20,000 cpm/mouse. In comparative studies with Swiss-Webster and deer mice, the same labeled population of trypanosomes was inoculated into both species. Tissue digestion. At set intervals postinoculation the mice were euthanasized with ether. Fifty microliters of blood was obtained from the tail vein before euthanasia or from the internal vasculature during dissection. Similar levels of radioactivity were found in the blood regardless of the source of the sample. The entire liver, lungs, brain, kidneys, spleen, and heart were removed and weighed. Tissue pieces 250 mg or less were added to 2 ml of Protosol (New England Nuclear) in scintillation vials and heated at 55 C overnight. The digested tissues were then cooled to room temperature, 0.25 ml of H202 was added to each vial, and the vials were reheated to 55 C for 0.5 h. Each vial then received 10 ml of Aquasol (New England Nuclear) or 10 ml of Dimilume (Packard Instrument Co., Downers Grove, Ill.) and was placed in the dark for 7 to 10 days before counting. Control tissues from uninoculated mice were prepared in a similar fashion. All vials were subsequently counted in a Beckman LS 9000 scintillation counter with random coincidence monitor and H- number (quench) calculation incorporated into the counting program. Blood volumes. Blood volumes of Swiss-Webster and deer mice were determined by using standard 5tCr-RBC and 1251-bovine serum albumin techniques (7) Ȧutoradiography. Labeled trypanosomes in blood smears from mice infected with T. brucei subsr. brucei were fixed in absolute methanol and dipped in NTB2 emulsion (Eastman Kodak Co., Rochester, N.Y.) by standard autoradiographic techniques. After 2 to 3 weeks the slides were developed, stained with Giemsa, and observed by light microscopy. In addition, Formalin-fixed tissue pieces of spleen and lung were sectioned after paraffin embedding and coated with emulsion, developed, and stained as above. Virulence. The in vivo form of T. brucei subsp. brucei was inoculated intraperitoneally into Swiss- Webster and deer mice at 5 x 105 trypanosomes per mouse. Tissue culture-adapted T. brucei subsp. brucei was inoculated by identical means into Swiss-Webster mice only. Labeled T. brucei subsp. brucei was compared (as to virulence) with unlabeled organisms by intravenous inoculation of 106 trypanosomes into Swiss-Webster mice. All infections were monitored as to the length of host survival and the mean survival period determined. Statistics. The data were analyzed statistically by Student's t test. RESULTS Labeling technique. Preliminary experiments utilizing [ Cr]- and [1251]5-iodo-2'-deoxyuridine proved unsuccessful due to the instability or toxicity (or both) of these compounds for labeling trypanosomes. Subsequent experiments showed that [3H]uridine could be incorporated into T. brucei subsp. brucei and remain associated with the organisms for a longer period of time than radioisotopes which labeled trypanosomal proteins (e.g., 3H-amino acids). T. brucei subsp. brucei incorporated between 10,000 and 20,000 dpm per 106 organisms during the 3-h labeling period. Autoradiographic analysis of the labeled trypanosome population demonstrated that all trypanosomes were labeled to approximately the same extent. Four of four Swiss-Webster mice died 3 days after inoculation with 106 T. brucei subsp. brucei regardless of whether the organisms were labeled or unlabeled. Hence, [3H]uridine was a suitable label for T. brucei subsp. brucei, having the important qualities of high specific activity, low toxicity, and good stability Ṫissue distribution of trypanosomes in Swiss- Webster mice. The distribution of T. brucei subsp. brucei in Swiss-Webster mice was determined by intravenous inoculation of labeled trypanosomes. For the sake of comparison, unincorporated [3H]uridine and heat-killed T. brucei subsp. brucei labeled before heating were also inoculated into Swiss-Webster mice. The distribution of radioactivity from all three sets of inoculations was determined by analysis of seven major organs and tissues at 0.5 and 24 h postinfection. Autoradiography of blood and lung tissue showed labeled trypanosomes at 0.5 and 9 h postinfection. All trypanosomes observed were labeled to some extent. The distribution of label from inoculation of viable T. brucei subsp. brucei differed substantially from that of control inocula (Table 1). At 0.5 h post-

3 VOL. 36, 1982 TISSUE DISTRIBUTION OF T. BRUCEI SUBSP. BRUCEI 527 TABLE 1. Tissue distribution of [3H]uridine-labeled T. brucei subsp. brucei and control inocula in Swiss- Webster micea % of inoculum per organb Time postinjection Organ T. brucei subsp. brucei Unincorporated Live Killed [3Hluridine 0.5 Bloodc 89.2 ± ± ± 0.0 Spleen 2.3 ± ± ± 0.0 Kidneys 0.5 ± ± ± 0.8 Liver 0.7 ± ± ± 4.1 Heart 0.2 ± ± ± 0.2 Lungs 5.1 ± ± ± 0.2 Brain 0.4 ± ± ± 0.1 Totald Blood 36.8 ± ± ± 0.0 Spleen 4.0 ± ± ± 0.2 Kidneys 1.0 ± ± ± 0.3 Liver 4.5 ± ± ± 1.5 Heart 0.7 ± ± ± 0.3 Lungs 0.9 ± ± 0.2 Brain 1.0 ± ± ± 0.0 Totald Aquasol. a All samples were counted ir b Percentage of inoculum per organ was based upon the disintegrations per minute in each organ minus the disintegrations per minute due to blood volume divided by the total inoculum per mouse. All values are listed with their standard deviation and represent the mean of three to five mice. C Blood disintegrations per minute were deternined from a 5S0j.l sample, and the total disintegrations per minute calculated from blood volume data (per mouse). d Percentage of label (inoculum) recovered per time point. inoculation, 89o of the label associated with live T. brucei subsp. brucei remained in the blood, and 5% was found in the lungs. In contrast, very little of the radioactivity associated with heatkilled trypanosomes remained in the blood, whereas significantly higher levels were found in the liver and kidneys. Similarly, unincorporated [3H]uridine accumulated in the liver and kidneys, though the amount of label present was somewhat lower than that in mice receiving killed trypanosomes. The accumulation of label in the other organs examined was unremarkable regardless of the inoculum used. At 24 h postinoculation the recovery of label associated with viable T. brucei subsp. brucei had decreased to 48.9o, as compared to 98.4% at 0.5 h postinoculation. Nevertheless, most of the label recovered was found in the blood with small increases in radioactivity in all other tissues except the lungs. The total recovery of inoculum (radioactivity) from the control inocula was approximately the same at 24 h as at 0.5 h (Table 1). The distribution of label from the control inoculations was also not substantially different at 24 h than at 0.5 h post-inoculation. It is important to bear in mind that the recovery figures cited above represent only the label found in the seven tissues listed in Table 1. Data not included above indicate that radioactivity from inoculation of unincorporated [3H]uridine could be found in a wide variety of other tissues. Overall, the data support the contention that [3H]uridine associated with viable trypanosomes remains bound, and the distribution of radioactivity from such inoculations is not due to free or released label. Comparative tissue distribution of T. brucei subsp. brucei in susceptible and resistant mice. Two murine hosts were compared to determine the influence of host factors on the early events of trypanosome infection. The data from these experiments showed that there was a marked difference in the course of infection in Swiss- Webster and deer mice with our stabilate of T. brucei subsp. brucei. Three of three Swiss- Webster mice died within 3 days of infection, whereas an identical number of deer mice survived for an average of 90 days when infected with the same population of trypanosomes. When [3H]uridine-labeled organisms were inoculated intravenously into both murine strains, the tissue distribution was similar at 0.5 h, but

4 528 ANDERSON AND BANKS markedly different by 24 h (Table 2). Deer mice contained significantly less label in the blood (P < 0.01) and significantly more label in the spleen (P < 0.01), liver (P < 0.05), and kidneys (P < 0.05) than did Swiss-Webster mice. There were no differences between the two hosts in label accumulation in the lungs and only a small difference in heart and brain activity, which may be related to the increased clearance of trypanosomes in the deer mice (i.e., see brain and heart values of heat-killed trypanosomes, Table 1). Thus trypanosomes were cleared from the bloodstream much quicker in mice resistant to rapidly lethal trypanosomiasis. Comparative tissue distribution of mouse- and tissue culture-passaged T. brucei subsp. brucei. Virulence studies also demonstrated that T. brucei subsp. brucei adapted to tissue culture caused the death of Swiss-Webster mice at approximately 25 days post-inoculation, about 8 times slower than mouse-passaged trypanosomes. These organisms had been passaged in tissue culture for 22 weeks. Intravenous inoculation of labeled tissue culture-passaged T. brucei subsp. brucei showed that the trypanosomes were removed from the bloodstream rapidly and TABLE 2. Tissue distribution of [3H]uridine-labeled T. brucei subsp. brucei in Swiss-Webster and deer mice' Time % of inoculum per organb postinjection Organ Swiss-Webster Deer (h) 0.5 Bloodc 81.2 ± ± 1.4 Spleen 3.7 ± ± 1.0 Kidneys 1.0 ± ± 1.3 Liver 7.9 ± ± 5.1 Heart 0.1 ± ± 0.2 Lungs 4.1 ± ± 1.6 Brain 0.3 ± ± 0.1 Totald Blood 43.2 ± ± 6.7 Spleen 2.4 ± ± 1.6 Kidneys 1.1 ± ± 1.3 Liver 6.1 ± ± 6.0 Heart 0.1 ± ± 0.3 Lungs 1.5 ± ± 0.4 Brain 0.8 ± ± 0.1 Totald a All samples were counted in Dimilume. All values are the mean of three to four mice. b See footnote b of Table 1. c See footnote c of Table 1. d See footnote d of Table 1. INFECT. IMMUN. the label accumulated in the liver, spleen, and kidneys (Table 3). Comparison with results with mouse-passaged T. brucei subsp. brucei showed significantly less label in the bloodstream (P < 0.05) at 0.5 and 24 h and significantly more label in liver (P < 0.05) and kidneys (P < 0.05) at 0.5 h when tissue culture-passaged T. brucei subsp. brucei were used. Little difference was noted in spleen radioactivity with these two inocula. DISCUSSION The technique outlined in this paper has several advantages over other published techniques (10). These include: (i) the labeling of trypanosomal nucleic acids instead of surface proteins, which are continuously shed; (ii) a higher specific activity such that a much smaller inoculum can be used; and (iii) the fact that autoradiography can be used to verify trypanosome distribution. The primary disadvantages are the necessity of digesting whole tissues and the subsequent presence of tissue chemiluminescence. This latter problem was substantially reduced through the use of Dimilume, a commercially available scintillation cocktail which retards chemiluminescence. Nonetheless, the use of the tritium labeling system requires that a substantial effort be put into quench and chemiluminescence monitoring and subsequent correction of the data. Thus, gamma labels such as 75Se are likely preferable for short-term studies. However, those observations which require longer time periods to develop, such as the marked differences in tissue distribution of trypanosomes in deer and Swiss-Webster mice, can only be determined by using more stably labeled trypanosomes (such as the organisms described in this paper). The reliability of the technique as an indicator of trypanosome location was evidenced by several findings. First, labeled trypanosomes were detected by autoradiography in blood and lungs. In addition, the distribution of unincorporated [3H]uridine and label associated with killed trypanosomes was markedly dissimilar from the distribution of label associated with viable trypanosomes. Although after inoculation it is difficult to determine whether the radioactivity is associated with live or dead trypanosomes, the data in Table 1 suggest that dead trypanosomes are removed by the liver. This interpretation is supported by reports showing the clearance of bloodstream trypanosomes within the liver of immune mice (15). In summary, the data support the use of this technique for determining the fate of T. brucei subsp. brucei in mice. One of the initial purposes in developing the labeling technique described above was to determine whether the tissue trophism displayed by trypanosomes in various species (14, 28) was

5 VOL. 36, 1982 TISSUE DISTRIBUTION OF T. BRUCEI SUBSP. BRUCEI 529 TABLE 3. Comparison of mouse- and tissue culture-passaged T. brucei subsp. brucei in Swiss- Webster mice' % of inoculum per organ' Time with T. brucei subsp. brucei postinjection Organ from: (h) Mouse Tissue bloodc culture 0.5 Bloodd 81.2 ± ± 22.9 Spleen 3.7 ± ± 1.1 Kidneys 1.0 ± ± 1.2 Liver 7.9 ± ± 5.8 Heart 0.1 ± ± 0.3 Lungs 4.1 ± ± 11.9 Brain 0.3 ± ± 0.3 Totale Blood 43.2 ± ± 9.3 Spleen 2.4 ± ± 0.5 Kidneys 1.1 ± ± 0.6 Liver 6.1 ± ± 0.4 Heart 0.1 ± ± 0.1 Lungs 1.5 ± ± 0.7 Brain 0.8 ± ± 0.2 Totale a All samples were counted in Dimilume. Values represent the mean of three to five mice. b See footnote b of Table 1. c Repeated from Table 2 for ease of comparison with tissue culture data. d See footnote c of Table 1. e See footnote d of Table 1. detectable early in the infection. The means by which early localization might occur could be related to specific trypanosomal behavior such as adherence to cell membranes (2) or response to tissue substances chemotactic to trypanosomes (Banks et al., manuscript in preparation). The infection of Swiss-Webster mice with our stabilate of T. brucei subsp. brucei did not provide substantial information on tissue trophism probably due to the rapidity of the infective process. However, it was noted in both Swiss-Webster and deer mice that the trypanosomes were retained in the lungs very early in the infection (0.5 h post-inoculation). Killed T. brucei subsp. brucei were not similarly retained. Nonetheless, by 24 h the level of radioactivity in the lungs had dropped considerably, and it is our conclusion that this brief accumulation is due to nonspecific trapping of the trypanosomes in the capillary beds of the lungs after intravenous inoculation. Infection of deer mice results in chronic disease with damage to specific organs (21). Yet experiments with labeled T. brucei subsp. brucei showed that only a small number of organisms could be found in sites commonly reported to be infiltrated by trypanosomes (i.e., heart and brain). These results suggest that the localization of trypanosomes within the heart and brain does not occur during the very early stages of the infection. Further studies will be required to determine the factors that account for tissue localization of this parasite. Data from the labeling experiments indicate that the rapidity with which T. brucei subsp. brucei was cleared from the bloodstream was correlated with the length of host survival. Thus, within 24 h of infection deer mice cleared trypanosomes more completely than did Swiss- Webster mice; tissue culture trypanosomes were cleared more efficiently in Swiss-Webster mice than were comparable numbers of mouse-passaged organisms. The increased clearance of tissue culture-passaged T. brucei subsp. brucei may have been due to the presence of some nonbloodstream forms which have been previously shown to be readily engulfed by tissue macrophages, even in the absence of immune serum (29). Alternatively, bovine and goat proteins adherent to the trypanosome surface due to extensive culture, combined with natural antibodies in the mice, could have led to the quicker removal of these organisms from the bloodstream (5). Experiments are currently under way to determine whether either explanation is correct. As label was cleared from the bloodstream it accumulated in the liver just as label associated with killed trypanosomes was shown to do. Thus, these findings are consistent with the observations of others that the liver is the principle site for removal of trypanosomes from the circulation (15). Although we as yet do not have definitive proof that phagocytosis is actively taking place, the system described herein could be applied to this question (e.g., autoradiography of the liver). Certainly, it is clear that the early removal of bloodstream trypanosomes can influence the course of the subsequent infection. African trypanosomes are often observed within lymphoid tissue (6, 27) and produce a suppression (1, 11, 16, 17, 19, 23) or an activation (or both) of the lymphocytes of the spleen, lymph nodes, and peripheral blood. The spleens of both Swiss-Webster and deer mice were found to accumulate label associated with viable trypanosomes, indicating that spleen cells at an early stage of infection are exposed to soluble (20) and membranous (4) trypanosome components with immunosuppressive characteristics. In agreement with this finding, within 24 h of infection a pronounced splenomegaly was observed in both species. These studies have examined the fate of circu-

6 530 ANDERSON AND BANKS lating trypanosomes in nonimmune hosts. The data suggest that trypanosomes are removed by the liver, most likely by the phagocytic Kupffer cells, and the more readily this defense mechanism is employed the longer the host will survive. Other mechanisms related to be immune response of the mice are undoubtedly also involved in the course of the infection (26). Although T. brucei subsp. brucei is known to accumulate in muscle, brain, and lymphoid tissue in mice (14, 21), little concentration was observed in the initial 24 h, except in the spleen. This accumulation may account for the profound influence these organisms have on spleen cell function. The fate of circulating trypanosomes in other phases of trypanosome infection, as detectable by the technique developed here, will provide additional clues to trypanosome-host interaction. ACKNOWLEDGMENTS This work was supported by Public Health Service grant Al from the National Institutes of Health, Biomedical Research Support Grant RR , Rockefeller Foundation grant GACOH-8013, and funds provided by the State of Washington Initiative Measure no We acknowledge the technical assistance of Linda Norton and the helpful comments of P. Klevjer-Anderson. LITERATURE CITED 1. Assoku, R. K. G., C. A. Hazlett, and I. Tizard Immunosuppression in African trypanosomiasis. Int. Arch. Allergy Appl. Immunol. 59: Banks, K. L Binding of Trypanosoma congolense to the walls of small blood vessels. J. Protozool. 25: Clayton, C. E Trypanosoma brucei: influence of host strain and parasitic antigenic type on infections in mice. Exp. Parasitol. 44: Clayton, C. E., D. L. Sacks, B. M. Ogilvie, and B. A. Askonas Membrane fractions of trypanosomes mimic the immunosuppressive and mitogenic effects of living parasites on the host. 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