Effects of melatonin on thymic and oxidative stress dysfunctions during Trypanosoma cruzi infection.

Although the exact etiology of Chagas disease is not completely elucidated, thymic atrophy and oxidative stress are believed to be important contributors to the pathogenesis during acute Trypanosoma cruzi (T. cruzi) infection. We hypothesized that exogenous melatonin, administered by gavage (5 mg/kg, p.o., gavage) to young (5 weeks old) and middle-aged (18 months old) male Wistar rats, would modulate thymic oxidative damage and reverse the age-related thymus regression during T. cruzi acute infection. Increased levels of superoxide anion (O2- ) were detected in the thymus of infected animals, and treatment with melatonin reverted this response. We found reduced TBARS levels as well as a significant increase in superoxide dismutase (SOD) activity in the thymus of all middle-aged melatonin-treated animals, infected or not with T. cruzi. Furthermore, melatonin increased the thymic expression of SOD1 and SOD2 in middle-aged control animals. Nox2 expression was not affected by melatonin treatment in young or middle-aged animals. Melatonin reverted the age-related thymic regression as revealed by the increase in thymus weight, total number of thymocytes, and reduction in age-related accumulation of double-negative thymocytes. This is the first report to directly examine the effects of melatonin treatment on the thymic antioxidant/oxidant status and thymic changes during T. cruzi infection. Our results revealed new antioxidant features that turn melatonin a potentially useful compound for the treatment of Chagas disease, a condition in which an excessive oxidative damage occurs.

Lethal and nonlethal murine malarial infections differentially affect apoptosis, proliferation, and CD8 expression on thymic T cells.

Although thymic atrophy and apoptosis of the double-positive (DP) T cells have been reported in murine malaria, comparative studies investigating the effect of lethal and nonlethal Plasmodium infections on the thymus are lacking. We assessed the effects of P. yoelii lethal (17XL) and nonlethal (17XNL) infections on thymic T cells. Both strains affected the thymus. 17XL infection induced DP T-cell apoptosis and a selective decrease in surface CD8 expression on developing thymocytes. By contrast, more severe but reversible effects were observed during 17XNL infection. DP T cells underwent apoptosis, and proliferation of both DN and DP cells was affected around peak parasitemia. A transient increase in surface CD8 expression on thymic T cells was also observed. Adult thymic organ culture revealed that soluble serum factors, but not IFN-γ or TNF-α, contributed to the observed effects. Thus, lethal and nonlethal malarial infections led to multiple disparate effects on thymus. These parasite-induced thymic changes are expected to impact the naïve T-cell repertoire and the subsequent control of the immune response against the parasite. Further investigations are required to elucidate the mechanism responsible for these disparate effects, especially the reversible involution of the thymus in case of nonlethal infection.

Thymic stromal lymphopoietin-dependent basophils promote Th2 cytokine responses following intestinal helminth infection.

CD4(+) Th2 cytokine responses promote the development of allergic inflammation and are critical for immunity to parasitic helminth infection. Recent studies highlighted that basophils can promote Th2 cytokine-mediated inflammation and that phenotypic and functional heterogeneity exists between classical IL-3-elicited basophils and thymic stromal lymphopoietin (TSLP)-elicited basophils. However, whether distinct basophil populations develop after helminth infection and their relative contributions to anti-helminth immune responses remain to be defined. After Trichinella spiralis infection of mice, we show that basophil responses are rapidly induced in multiple tissue compartments, including intestinal-draining lymph nodes. Trichinella-induced basophil responses were IL-3-IL-3R independent but critically dependent on TSLP-TSLPR interactions. Selective depletion of basophils after Trichinella infection impaired infection-induced CD4(+) Th2 cytokine responses, suggesting that TSLP-dependent basophils augment Th2 cytokine responses after helminth infection. The identification and functional classification of TSLP-dependent basophils in a helminth infection model, coupled with their recently described role in promoting atopic dermatitis, suggests that these cells may be a critical population in promoting Th2 cytokine-associated inflammation in a variety of inflammatory or infectious settings. Collectively, these data suggest that the TSLP-basophil pathway may represent a new target in the design of therapeutic intervention strategies to promote or limit Th2 cytokine-dependent immunity and inflammation.

[Effect of Toxoplasma gondii infection on thymus cells in rats].

OBJECTIVE: To study the pathological damage of thymus and thymus cell apoptosis of male rats infected with Toxoplasma gondii. METHODS: Fifty Wistar male rats (7-8-week-old) were randomly divided into infection group (40) and control group (10). Rats in infection group were infected with 5 x 10(4) tachyzoites by intraperitoneal injection, while those in control group received same volume of PBS. On the 3rd, 6th, 9th and 12th day post infection, ten rats from infection group and two from control group were sacrificed, the thymus glands were removed. The thymus tissue sections were stained with hematoxylin and eosin (HE) for observation on histopathological changes. Single thymus cell suspensions were prepared. Cell cycle analysis was performed by flow cytometry, and proliferation index was calculated. Thymus frozen sections were stained with Hoechst 33258, and morphologic changes in apoptotic nuclei were observed under fluorescence microscope. Expression of Bcl-2 and Bax proteins were determined by using immunohistochemistry. RESULTS: Microscopic examination showed that pathological changes occurred in thymus grand on the 3rd day after infection. The space between connective tissue capsules was widened, cells in cortex and medulla cells were sparse, and more phagocytes and extravasated blood were found in thymus. On the 6th day post infection the thymus damage was aggravated, and no significant improvement was seen on day 12. On the 3rd, 6th, 9th and 12th day after infection, thymocyte proliferation index was (11.15 +/- 0.99)%, (6.17 +/- 1.02)%, (5.45 +/- 0.96)% and (6.63 +/- 1.52)%, respectively, and each of them was significantly lower than that of the control [(13.81 +/- 1.18)%] (P < 0.01). On the 3rd day after infection, the number of apoptotic cells increased, significantly increased on day 6, and there was no much difference in the number of apoptotic cells between day 6 and day 12. The immunohistochemistry results showed that on the 3rd, 6th, 9th and 12th day post-infection, the gray scale value of Bax positive cells was 88.21 +/- 4.74, 64.69 +/- 6.82, 83.62 +/- 5.79, and 101.09 +/- 6.72, respectively, and each of them was significantly lower than that of the control (128.69 +/- 8.95) (P < 0.01), while there was no significant change in the Bcl-2 protein level (P > 0.05). CONCLUSION: T. gondii causes severe pathological damage in host thymus tissue with a decrease in the proliferation index, an increase in the number of apoptotic cells, and high expression of Bax protein.

Prolonged antigen presentation is required for optimal CD8+ T cell responses against malaria liver stage parasites.

Immunization with irradiated sporozoites is currently the most effective vaccination strategy against liver stages of malaria parasites, yet the mechanisms underpinning the success of this approach are unknown. Here we show that the complete development of protective CD8+ T cell responses requires prolonged antigen presentation. Using TCR transgenic cells specific for the malaria circumsporozoite protein, a leading vaccine candidate, we found that sporozoite antigen persists for over 8 weeks after immunization--a remarkable finding since irradiated sporozoites are incapable of replication and do not differentiate beyond early liver stages. Persisting antigen was detected in lymphoid organs and depends on the presence of CD11c+ cells. Prolonged antigen presentation enhanced the magnitude of the CD8+ T cell response in a number of ways. Firstly, reducing the time primed CD8+ T cells were exposed to antigen in vivo severely reduced the final size of the developing memory population. Secondly, fully developed memory cells expanded in previously immunized mice but not when transferred to naïve animals. Finally, persisting antigen was able to prime naïve cells, including recent thymic emigrants, to become functional effector cells capable of eliminating parasites in the liver. Together these data show that the optimal development of protective CD8+ T cell immunity against malaria liver stages is dependent upon the prolonged presentation of sporozoite-derived antigen.

Thymic alterations induced by Plasmodium berghei: expression of matrix metalloproteinases and their tissue inhibitors.

The thymus plays a crucial role in the generation of T-cells, and so our laboratory has been interested in the study of the intrathymic events that occur during infection diseases and may cause disruption in its functions. Previously, we showed that thymus from experimentally Plasmodium berghei-infected mice present histological alterations with high levels of apoptosis, changes in cell migration-related molecules, and premature egress of immature thymocytes to periphery. In addition, parasites were found inside the thymus. In this work we investigated alterations in the expression pattern and activity of matrix metalloproteinases MMP-2 and -9, and their tissue inhibitors, TIMP-1 and TIMP-2. Our results show enhanced expression and widespread distribution of these molecules in thymus from infected animals. Also, the presence of active MMP-2 was detected. These data are suggestive of MMPs and TIMPs importance in the earlier observed changes in the extracellular matrix during thymic alterations after plasmodium infection.

A human IL10 BAC transgene reveals tissue-specific control of IL-10 expression and alters disease outcome.

Interleukin (IL)-10 is an immunoregulatory cytokine that is produced by diverse cell populations. Studies in mice suggest that the cellular source of IL-10 is a key determinant in various disease pathologies, yet little is known regarding the control of tissue-specific human IL-10 expression. To assess cell type-specific human IL-10 regulation, we created a human IL-10 transgenic mouse with a bacterial artificial chromosome (hIL10BAC) in which the IL10 gene is positioned centrally. Since human IL-10 is biologically active in the mouse, we could examine the in vivo capacity of tissue-specific human IL-10 expression to recapitulate IL-10-dependent phenotypes by reconstituting Il10(-/-) mice (Il10(-/-)/hIL10BAC). In response to LPS, Il10(-/-)/hIL10BAC mice proficiently regulate IL-10-target genes and normalize sensitivity to LPS toxicity via faithful human IL-10 expression from macrophages and dendritic cells. However, in the Leishmania donovani model of pathogen persistence, Il10(-/-)/hIL10BAC mice did not develop the characteristic IL-10(+)IFN-gamma(+)CD4 T cell subset thought to mediate persistence and, like Il10(-/-) mice, cleared the parasites. Furthermore, the IL-10-promoting cytokine IL-27 failed to regulate transgenic human IL-10 production in CD4(+) T cells in vitro which together suggests that the hIL10BAC encodes for weak T cell-specific IL-10 expression. Thus, the hIL10BAC mouse is a model of human gene structure and function revealing tissue-specific regulatory requirements for IL-10 expression which impacts disease outcomes.

Morphological alterations of Thymus during the early murine leishmaniasis.

Leishmaniasis is a neglected and endemic disease that affects poorest population mainly in developing countries. Thymus provides an essential complex environment for T cell maturation and differentiation during leishmania infection. The aim of this study was to investigate the pathological alterations of the Thymus during early Leishmania amazonensis murine infection. BALB/c mice were infected with 105 amastigotes for 24 h, 3 days, 7 days, 15 days or 30 days. At different times of infection, the relative weight of the Thymus was obtained, and the Thymus cellularity was determined by counting total cells of one thymic lobe. The thymic lobe was, alternatively, processed for standard Haematoxylin and Eosin protocol. Our results suggest thymic alteration during the early days of BALB/c mice infection with L. amazonensis. The thymic hypertrophy was accompanied by histological alterations in Thymus architecture with thickening cortex at 3 days p.i. and loss of an evident delimitation between the cortex and medulla at 7 days p.i. when compared to the control mice. That is the first time that Thymus hypertrophy was observed during the early leishmaniasis. However, how it may contribute to infection susceptibility requires further investigation.

Changes in cell migration-related molecules expressed by thymic microenvironment during experimental Plasmodium berghei infection: consequences on thymocyte development.

We previously showed alterations in the thymus during experimental infection with Plasmodium berghei. Such alterations comprised histological changes, with loss of cortical-medullary limits, and the intrathymic presence of parasites. As the combination of chemokines, adhesion molecules and extracellular matrix (ECM) is critical to appropriate thymocyte development, we analysed the thymic expression of ECM ligands and receptors, as well as chemokines and their respective receptors during the experimental P. berghei infection. Increased expression of ECM components was observed in thymi from infected mice. In contrast, down-regulated surface expression of fibronectin and laminin receptors was observed in thymocytes from these animals. Moreover, in thymi from infected mice there was increased CXCL12 and CXCR4, and a decreased expression of CCL25 and CCR9. An altered thymocyte migration towards ECM elements and chemokines was seen when the thymi from infected mice were analysed. Evaluation of ex vivo migration patterns of CD4/CD8-defined thymocyte subpopulations revealed that double-negative (DN), and CD4(+) and CD8(+) single-positive (SP) cells from P. berghei-infected mice have higher migratory responses compared with controls. Interestingly, increased numbers of DN and SP subpopulations were found in the spleens of infected mice. Overall, we show that the thymic atrophy observed in P. berghei-infected mice is accompanied by thymic microenvironmental changes that comprise altered expression of thymocyte migration-related molecules of the ECM and chemokine protein families, which in turn can alter the thymocyte migration pattern. These thymic disturbances may have consequences for the control of the immune response against this protozoan.

[The reaction of the bursa and thymus eosinophils in the herring gull after the experimental infection with gull-tapeworm].

Using the methods of light microscopy, eosinophil topography, quantitative and qualitative changes (degranulation level of and a cationic protein content) were studied in the thymus and bursa of 36 herring gull nestlings Larus argentatus mongolicus (Suskin, 1925) 2 weeks after experimental infestation with gull-tapeworm Diphyllobothrium dendriticum (Nitsch, 1824). Eosinophils in the thymus were located in trabecules, mainly close to the blood vessels, thymic (Hassall's) corpuscles and also directly inside them, while in the bursa they were found within the internodular space. As compared with the control bird counts, relative eosinophil count in the birds with an average invasion intensity was increased 3.8 times in the thymus and 2.5 times in the bursa. In birds with high invasion intensity, these counts were increased 4 times in the thymus and 1.2 times in the bursa.

Detection of amastigotes and histopathological alterations in the thymus of Leishmania infantum-infected dogs.

INTRODUCTION: In canine visceral leishmaniasis (CVL), lymphopenia, and the disorganization of lymphoid organs such as spleen and lymph nodes have been demonstrated. However, the involvement of thymus in CVL has not been evaluated so far. Herein, we investigated whether the thymus can be colonized by Leishmania infantum in naturally infected dogs. METHODS: Thymus were obtained from 16 of 58 dogs and samples of this organ were submitted to immunohistochemistry for laminin and fibronectin detection, histopathology, in situ hybridization and polymerase chain reaction (PCR) targeting the gene ITS-1 for Leishmania and sequenced. Samples of spleen, skin and popliteal lymph nodes were collected and submitted to immunohistochemistry and parasitological culture followed by multilocus enzyme electrophoresis. RESULTS: L. infantum was identified in all dogs. DNA and amastigote forms of Leishmania were detected in the thymus from 16 dogs by PCR and in eight by immunohistochemistry. Besides thymus, parasites were detected in spleen, lymph nodes, and skin. A granulomatous or pyogranulomatous thymitis was observed in eight dogs associated to intact amastigotes forms of this parasite. Fibronectin deposition in thymus was higher in dogs with more clinical signs. CONCLUSIONS: These results demonstrate that the thymus of dogs can be parasitized by L. infantum, which may generate inflammatory reactions leading to alterations in thymic microarchitecture.

Impaired Thymic Output Can Be Related to the Low Immune Reconstitution and T Cell Repertoire Disturbances in Relapsing Visceral Leishmaniasis Associated HIV/AIDS Patients.

Background: Visceral leishmaniasis/HIV-co-infected patients (VL/HIV) accounts for around 8% of VL reported cases in Brazil. Relapses of Leishmania infection after anti-leishmanial treatment constitute a great challenge in the clinical practice because of the disease severity and drug resistance. We have shown that non-relapsing-VL/HIV (NR-) evolved with increase of CD4+ T-cell counts and reduction of activated CD4+ and CD8+ T cells after anti-leishmanial treatment. This immune profile was not observed in relapsing-VL/HIV patients (R-), indicating a more severe immunological compromising degree. Elevated activation status may be related to a deficient immune reconstitution and could help to explain the frequent relapses in VL/HIV co-infection. Our aim was to evaluate if this gain of T cells was related to changes in the peripheral TCRVß repertoire and inflammatory status, as well as the possible thymus involvement in the replenishment of these newly formed T lymphocytes. Methods: VL/HIV patients, grouped into non-relapsing (NR- = 6) and relapsing (R- = 12) were evaluated from the active phase up to 12 months post-treatment (mpt). HIV-infected patients (non-VL) and healthy subjects (HS) were included. The TCRVß repertoire was evaluated ex vivo by flow cytometry, whereas the plasmatic cytokine levels were assessed by Luminex assay. To evaluate the thymic output, DNA was extracted from PBMCs for TCR rearrangement excision circles (TREC) quantification by qPCR. Results: VL/HIV cases presented an altered mobilization profile (expansions or retractions) of the TCRVß families when compared to HS independent of the follow-up phase (p < 0.05). TCRVß repertoire on CD4+ T-cells was more homogeneous in the NR-VL/HIV cases, but heterogeneous on CD8+ T-cells, since different Vß-families were mobilized. NR-VL/HIV had the inflammatory pattern reduced after 6 mpt. Importantly, VL/HIV patients showed number of TREC copies lower than controls during all follow-up. An increase of recent thymic emigrants was observed in NR-VL/HIV individuals at 10 mpt compared to R- patients (p < 0.01), who maintained lower TREC contents than the HIV controls. Conclusions: VL/HIV patients that maintain the thymic function, thus generating new T-cells, seem able to replenish the T lymphocyte compartment with effector cells, then enabling parasite control.

Thymic Microenvironment Is Modified by Malnutrition and Leishmania infantum Infection.

Detrimental effects of malnutrition on immune responses to pathogens have long been recognized and it is considered a main risk factor for various infectious diseases, including visceral leishmaniasis (VL). Thymus is a target of both malnutrition and infection, but its role in the immune response to Leishmania infantum in malnourished individuals is barely studied. Because we previously observed thymic atrophy and significant reduction in cellularity and chemokine levels in malnourished mice infected with L. infantum, we postulated that the thymic microenvironment is severely compromised in those animals. To test this, we analyzed the microarchitecture of the organ and measured the protein abundance in its interstitial space in malnourished BALB/c mice infected or not with L. infantum. Malnourished-infected animals exhibited a significant reduction of the thymic cortex:medulla ratio and altered abundance of proteins secreted in the thymic interstitial fluid. Eighty-one percent of identified proteins are secreted by exosomes and malnourished-infected mice showed significant decrease in exosomal proteins, suggesting that exosomal carrier system, and therefore intrathymic communication, is dysregulated in those animals. Malnourished-infected mice also exhibited a significant increase in the abundance of proteins involved in lipid metabolism and tricarboxylic acid cycle, suggestive of a non-proliferative microenvironment. Accordingly, flow cytometry analysis revealed decreased proliferation of single positive and double positive T cells in those animals. Together, the reduced cortical area, decreased proliferation, and altered protein abundance suggest a dysfunctional thymic microenvironment where T cell migration, proliferation, and maturation are compromised, contributing for the thymic atrophy observed in malnourished animals. All these alterations could affect the control of the local and systemic infection, resulting in an impaired response to L. infantum infection.

Thymic alterations in Plasmodium berghei-infected mice.

The primary function of the thymus is to develop immature T-cells into cells that further in the periphery will be able to carry out immune functions. The Literature has shown that thymus can be a target for many pathogens and severe structural alterations take place in this organ during infectious diseases. Here, we investigated if thymus is also a target organ during experimental malaria infection by analyzing the presence of parasites inside the organ and histological alterations in thymuses from Plasmodium berghei NK65-infected BALB/c. After 14 days of infection, parasites were found inside the thymus that presented a profound atrophy with total loss of its architecture. We propose that the presence of parasites in the thymus induces histological modifications that alter the microenvironment, impairing by consequence the successful T cell development. Additional studies are currently being developed in our laboratory to verify if such thymic alterations can influence the systemic immune response to the parasite.

Oral Outbreak of Chagas Disease in Santa Catarina, Brazil: Experimental Evaluation of a Patient's Strain.

Chagas disease is a worldwide public health problem. Although the vectorial transmission of Chagas disease has been controlled in Brazil there are other ways of transmission, such as the ingestion of T. cruzi contaminated food, which ensures the continuation of this zoonosis. Here, we demonstrate the influence of the inoculation route on the establishment and development of the SC2005 T. cruzi strain infection in mice. Groups of Swiss mice were infected intragastrically (IG) or intraperitoneally (IP) with the T. cruzi SC2005 strain derived from an outbreak of oral Chagas disease. The results revealed that 100% of IP infected mice showed parasitemia, while just 36% of IG infected showed the presence of the parasite in blood. The parasitemia peaks were later and less intense in the IG infected mice. Mortality of the IP infected animals was more intense and earlier when compared to the IG infected mice. In the IP infected mice leucopenia occurred in the early infection followed by leucocytosis, correlating positively with the increase of the parasites. However, in the IG infected mice only an increase in monocytes was observed, which was positively correlated with the increase of the parasites. Histopathological analyses revealed a myotropic pattern of the SC2005 strain with the presence of inflammatory infiltrates and parasites in different organs of the animals infected by both routes as well as fibrosis foci and collagen redistribution. The flow cytometric analysis demonstrated a fluctuation of the T lymphocyte population in the blood, spleen and mesenteric lymph nodes of the infected animals. T. cruzi DNA associated with the presence of inflammatory infiltrates was detected by PCR in the esophagus, stomach and intestine of all infected mice. These findings are important for the understanding of the pathogenesis of T. cruzi infection by both inoculation routes.

Thymocyte depletion during acute Trypanosoma cruzi infection in C57BL/6 mice is partly reverted by lipopolysaccharide pretreatment.

Infection with Trypanosoma cruzi in C57BL/6 mice leads to a progressive fatal disease accompanied by thymocyte depletion, which is not related with a higher parasite burden but with increased serum levels of tumour necrosis factor alpha (TNF- alpha). Because this situation may result from an excessive inflammatory syndrome, mice were now given anti-TNF-alpha mAbs throughout their acute infection, or subjected to a LPS desensitization protocol before parasite challenge. Treatment with anti-TNF-alpha mAbs failed to ameliorate thymocyte depletion but shortened survival time and increased parasite load. Pretreatment with LPS (desensitization followed by a sublethal LPS dose) prolonged survival time with a trend to reduce parasitemias and TNF-alpha serum concentrations. Given that pentoxifylline (PTx) interferes with in vitro LPS tolerance, experiments by administering PTx in combination with the tolerance-inducing LPS doses were also performed. Such schedule significantly reduced mortality, TNF-alpha and IL-6 serum concentrations, and CD4+ CD8+ thymocyte loss. LPS pretreatment allowed a better infection control and protected from the accompanying tissue damage.

Effects of ultraviolet-B exposure on the resistance to Listeria monocytogenes in the rat.

A rat infection model using the bacterial pathogen Listeria monocytogenes was employed to analyze the immunosuppressive activity of UVB radiation. Rats were exposed to suberythemal doses of UVB radiation for 5 or 7 consecutive days, using Kromayer or FS40 lamps respectively. Subsequently, the rats were infected subcutaneously or intravenously with Listeria. Exposure to UVB resulted in an increased number of bacteria in the spleen 4 days after infection. Listeria-specific lymphocyte proliferation assays as well as delayed-type hypersensitivity reactions demonstrated that T cell-mediated immunity to Listeria was impaired by UVB as measured 4 and 8 days after infection. In addition, UVB exposure decreased phagocytotic activity of peripheral blood macrophages. This study demonstrated that suberythemal doses of UVB radiation caused a delay in the clearance of Listeria bacteria from the spleen of the rats and that this was probably caused by impaired nonspecific phagocytosis of Listeria by macrophages in addition to an impaired activity of Listeria-specific T cells.

Cryptosporidiosis of the bursa of Fabricius of chickens.

Light-microscope and electron-microscope studies of a coccidial organism found in the bursa of Fabricius from 3 chickens clearly established the parasite as belonging to the family Cryptosporiidae. Hyperplasia and heterophil infiltration were associated with the presence of organisms attached to the microvillus border of epithelial cells lining the plicae of the bursa of Fabricius. Although there were no clinical signs or gross lesions common to the 3 cases described, all had similar histologic lesions in the epithelium lining the bursa of Fabricius.

Early schizonts of Leucocytozoon caulleryi.

Sporozoites of Leucocytozoon caulleryi were inoculated intravenously into 26-day-old specific pathogen-free chickens. Six days after inoculation, early schizonts were detected in lung, spleen, and thymus. They existed separately and were spherical or ovoid, 30-35 microns in diameter, and had well defined walls.

Tissue damage in cattle infected with Theileria annulata accompanied by metastasis of cytokine-producing, schizont-infected mononuclear phagocytes.

The distribution of schizont-infected cells in six calves undergoing acute, lethal sporozoite-induced infections with Theileria annulata was examined, the calves being killed in the early, middle or late stages of disease. A combination of histological and immunocytochemical techniques showed that schizont-infected cells became disseminated rapidly through the lymphoid tissues from the prescapular lymph node draining the site of inoculation to distant lymph nodes (e.g., precrural, mesenteric and mediastinal) and to the spleen and thymus. The parasitized cells also spread rapidly into non-lymphoid organs, being found in the liver, kidney, lung, abomasum, adrenal glands and pituitary gland by day 7, in the brain by day 12 and in the heart by day 14 after infection. As infection progressed, the schizonts differentiated into merozoites. By the late stages of disease, the cells containing merozoites greatly out-numbered schizont-infected cells. The parasitized mononuclear cells were labelled by antibodies to bovine interferon-alpha1 and tumour necrosis factor-alpha and, during the later stages of the disease, contained erythrocytes parasitized by piroplasms. The results suggested that the parasitized mononuclear cells themselves played a role in the development of clinical disease and in tissue damage. These findings provide new evidence that tropical theileriosis can no longer be viewed as a lymphoproliferative disease resulting from the uncontrolled multiplication and metastasis of lymphoid cells infected with T. annulata schizonts, but is caused by a parasite that lives in, and is disseminated by, cytokine-secreting, proliferating mononuclear phagocytes.