Intestinal Transit Time During Infection with Eimeria nieschulzi in Rats*

SYNOPSIS. Experiments were designed to test whether or not intestinal transit time increases significantly during severe coccidiosis in the rat. Intraduodenal catheters were surgically implanted into 25 rats. Six to 12 days after surgery 11 rats were inoculated orally with 10⁴ sporulated oocysts of Eimeria nieschulzi Dieben, and 11 were inoculated with 10⁶ oocysts; 3 rats were retained as uninfected controls. At 2, 4, 8, 9, and 16 days postinoculation (PI) Na₂⁵¹CrO₄ was injected through the catheter into the duodenum of fasted rats and allowed to progress through the small bowel for 15 min, at which time the rats were killed. The distribution of ⁵¹Cr in the gut was plotted as a function of gut length. The leading edge of radioactivity traversed 70% of the gut length in controls, and ～ 50–60% in parasitized rats on days 2, 4, 8, and 9 PI. Also, a reflux of gut contents, as evidenced by radioactivity in the stomach, occurred early (PI days 2 & 4) in rats infected with 10⁴ oocysts and throughout patency in rats infected with 10⁶ oocysts. A 2nd study was undertaken to determine if chemically induced suppression of gut transit time during early infection would enhance infectivity as measured by increased parasite fecundity. Nine rats were injected subcutaneously with an antidiarrheal agent, Loperamide®, known to slow small bowel motility significantly. Another group of 9 control rats was injected with the ethanol-propylene glycol solvent. Ten min after injection, all rats were inoculated per os with 10⁴E. nieschulzi oocysts. The daily number of oocysts discharged/rat was followed from PI days 5–11. Patency began for all rats on PI day 7. The total number of oocysts discharged by the drugged rats as compared with controls was not significantly different.     

The protective capacities of fractionated immune thoracic duct lymphocytes against Nippostrongylus brasiliensis

Thoracic duct lymphocytes (TDL) obtained from rats either on the tenth day of a primary infection (Day 10 TDL) or 1 or 5 weeks after a tertiary infection (hyperimmune TDL) with Nippostrongylus brasiliensis were fractionated into cells lacking (sIg^?) or bearing (sIg⁺) surface immunoglobulin by a rosetting procedure. The abilities of unfractionated TDL, of the two subpopulations, and of the reconstituted cells to confer protection against the parasite were examined. The effector cells which cause worm expulsion were found only in (sIg^?) cells from Day 10 TDL and also predominantly in (sIg^?) cells from hyperimmune TDL. However, a small but significant degree of protection was conferred by (sIg⁺) cells from hyperimmune TDL. These results suggest that the mechanisms involved in worm expulsion are regulated by (sIg^?) cells but that (sIg⁺) cells from hyperimmune rats can also contribute to the mechanisms of worm expulsion.     

Eimeria nieschulzi and Trichinella spiralis: Analysis of concurrent infection in the rat

The effects of concurrent primary infection of the rat with Eimeria nieschulzi and Trichinella spiralis on the number of oocysts of E. nieschulzi shed by the host and on the number, distribution, and fecundity of adult T. spiralis were analyzed. When rats were initially infected with E. nieschulzi followed 9 days later by infection with T. spiralis there occurred a significant decrease in the total numbers of adult worms in the small intestine, a significant shift in the position of these worms along the length of the small gut, a decrease in the fecundity of adult female worms, and a decrease in muscle parasitism when compared with rats infected with T. spiralis alone. When rats were initially infected with T. spiralis, followed 9 days later by infection with E. nieschulzi, there occurred a significant decrease in the numbers of oocysts shed over 24 hr on Days 7, 9, and 11 postinfection below that seen with rats infected only with Eimeria. These changes are discussed in terms of the enteropathophysiologic lesions and enteric inflammation known to occur during infections with these two parasites.     

Fate of H2-activated T lymphocytes in syngeneic hosts. I. Fate in lymphoid tissues and intestines traced with 3H-thymidine, 125I-deoxyuridine and 51chromium.

Information was sought on the fate of T cells activated to H2 determinants in vivo. The cells were obtained from thoracic duct lymph of irradiated F₁ mice injected with parental strain T cells. The fate of the lymph-borne cells—nearly all of which were donor-cell-derived, host-reactive T blasts (T.TDL)—was studied by labelling the cells with either ³HTdR, ¹²⁵IUdR or ⁵¹Cr and transferring them to syngeneic mice.A large proportion of T.TDL (20%) homed to the intestines on transfer. In the small intestine 40% of the cells were located in Peyer's patches; this was lower than with normal TDL (>70%) but higher than with a population of B (θ-negative) blasts (<10%). Some T.TDL were situated within the surface epithelium of the gut. Studies with ⁵¹Cr-labelled cells suggested that a proportion of these cells entered the gut lumen.T.TDL also homed to the large intestine but only when derived from a small inoculum of T cells. T.TDL derived from a large dose of T cells homed preferentially to the small intestine; in this respect they resembled B blasts.Homing to the intestines seemed a general property of T cells activated to transplantation antigens. It was observed irrespective of whether the T.TDL were activated against H2 determinants, M-locus determinants or H2-plus M-locus determinants.Most T.TDL died in the lymphoid tissues within 1–2 weeks of transfer. This conclusion was derived from comparative studies of (a) autoradiographs prepared from recipients of ³HTdR-labelled T.TDL and TDL and (b) the migratory properties of labelled cells harvested from recipients of ⁵¹Cr-labelled T.TDL, normal TDL and irradiated TDL. Rapid clearance of radioactivity from recipients of T.TDL labelled with ¹²⁵IUdR was consistent with this conclusion. Adequate control experiments with this isotope were not possible, however, because attempts to label long-lived lymphocytes (TDL) with ¹²⁵IUdR were unsuccessful.Studies with a variety of cells labelled with ¹²⁵IUdR indicated that a proportion of the label was excreted via the stomach. In certain situations, e.g., in mice with tied renal vessels, extremely high counts (>40% of the injected counts) appeared in the stomach contents.     

The Effect of Eimeria nieschulzi Infection on Leukocyte Levels in the Rat*

SYNOPSIS. Rats inoculated with 10,000 sporulated oocysts of Eimeria nieschulzi had significantly higher total leukocyte counts on postinoculation days (PI) 1, 5, 6 and 7 when compared to control rats. Relative and absolute neutrophil counts increased concomitantly with a decrease in the relative lymphocyte levels in E. nieschulzi-infected rats on PI day 7. Absolute and relative neutrophil counts in infected rats on PI days 7 and 8 were closely correlated with the host's total oocyst discharge. The E. nieschulzi infection had no significant effect on the relative or absolute levels of monocytes or eosinophils. The described changes in leukocyte levels were not paralleled by a significant change in the erythrocyte count.     

Adoptive transfer of the intestinal mast cell response in rats infected with Nippostrongylus brasiliensis.

The intestinal mast cells (IMC) were examined in normal and adoptively immunized rats infected with Nippostrongylus brasiliensis. An increase in the numbers of IMC was observed in infected recipients of thoracic duct lymphocytes (TDL) obtained from donor rats which had themselves been infected 10 days previously (Day 10 TDL). The increase in the number of IMC in the mucosa was related to the number of Day 10 TDL transferred into infected recipients. When TDL were fractionated into populations of cells either bearing (sIg⁺) or lacking (sIg^?) surface immunoglobulin, only sIg^? cells were able to confer the IMC response. Antigenic stimulation was necessary for the differentiation of intestinal mast cells. There was a marked difference between different strains of rats with regard to worm burden and intestinal mast cell kinetics although the increase in intestinal mast cells was always closely related to the final stage of the rapid phase of worm expulsion. These results are compatible with the concept that intestinal mast cells are derived from T cells and suggest that sIg⁺ cells do not influence IMC differentiation. Alternatively, the possibility that the transferred TDL regulate the differentiation of cells of host origin into IMC cannot be excluded.     

Host and Parasite Interactions During Single and Concurrent Infections with Eimeria nieschulzi and E. separata in the Rat1

SYNOPSIS. Some effects of 2 species of Eimeria in the rat were studied in single and concurrent infections. Groups included singly infected E. nieschulzi and E. separata controls (15 rats each) and 4 groups of 5 rats each infected with E. separata (shorter life cycle) at different days of an established E. nieschulzi infection. Experiments were conducted using inoculations of approximately 3,000, 5,000, and 7,000 sporulated oocysts of each species. Hosts showed consistently poor weight gain on day 3 postinoculation (PI) in E. separata infections and usually showed weight loss on days 7 and/or 8 PI in E. nieschulzi infections. Spleen weights indicated that the hosts responded immunologically to both parasites. Small intestine weights increased during E. nieschulzi infections as did cecum-colon weights during E. separata infections. In severe infections both species seemed to cause pathologic change in tissues at a distance from the site of endogenous development. Fecal collections were made at 24-hr intervals from the time of 1st inoculation till the end of patency. Patency usually began on day 7 PI for E. nieschulzi and on day 4 PI for E. separata with oocyst discharge peaking on days 8 and 5, respectively. In 2 instances, a biphasic pattern of oocyst discharge was found for E. separata during concurrent infections. The occurrence of interspecific interactions was indicated by an increased discharge of oocysts of E. separata, including those with the ability to sporulate, in doubly-infected animals as compared with those in singly-infected controls. It was suggested that the increased oocyst discharges may have resulted from additional production of merozoites during schizogony. Concurrent infections weakened the host more than single infections and this seemingly allowed more multiplication of the parasites.     

Eimeria nieschulzi: glucose absorption in infected rats

Liquid scintillation spectrophotometry was employed to determine absorption of ³H-glucose by rats infected with the coccidium Eimeria nieschulzi. In vivo studies showed increased uptake of label into small intestinal tissue and hepatic portal plasma at 3 days postinoculation and decreased absorption at 8 days postinfection compared to uninfected control animals. Observations of tissues incubated in labeled glucose in vitro confirm in vivo findings of increased uptake early in infection and malabsorption coinciding with the observation of clinical disease symptoms.     

Fate of H2-activated T lymphocytes in syngeneic hosts. III. Differentiation into long-lived recirculating memory cells.

Memory to H2 determinants was studied with an adoptive transfer system using a population of H2-activated blast T cells (T.TDL) obtained from thoracic duct lymph of irradiated F₁ hybrid mice injected with parental strain T cells. CBA T.TDL activated either to DBA/2 or C57BL determinants were transferred to syngeneic “B” mice. Thoracic duct lymphocytes (TDL) were obtained from the recipients 4–6 weeks later and tested for their capacity to produce (a) a graft-versus-host (GVH) reaction, (b) a mixed lymphocyte reaction (MLR) (measured by an in vivo technique) and (c) allograft rejection (suppression of the growth of allogeneic tumour cells in vivo). Control experiments involved testing the function of TDL obtained from “B” mice preinjected with TDL or no cells.TDL from “B” mice injected with TDL (passaged TDL) gave strong MLR and GVH reactions to both DBA/2 and C57BL determinants. Passaged T.TDL activated to C57BL antigens gave intermediate MLR and GVH reactions to the specific (C57BL) determinants but only very low responses to third-party (DBA/2) determinants; reciprocal results were obtained with passaged T.TDL activated to DBA/2 determinants. TDL from “B” mice given no cells failed to respond to either set of determinants.Since the responses by the passaged T.TDL did not exceed those by passaged TDL there was no evidence that adoptive transfer of T.TDL had conferred to the recipients a state of memory to either MLR or GVH determinants. Adoptive transfer did, however, lead to qualitative changes in the properties of T.TDL since, before transfer, they were unable to evoke GVH reactions or produce an MLR of normal kinetics.Passaged T.TDL were far superior to passaged TDL at suppressing the growth of allogeneic tumour cells. The protection was specific since protection against DBA/2 tumour cells was, cell for cell, 5–10 fold more effective with passaged T.TDL activated to DBA/2 determinants than with cells activated to C57BL determinants. No protection was observed with cells treated with anti-θ serum. The protective cells appeared to be precursors of effector cells rather than effector cells per se since they failed to lyse the tumour cells in vitro. These data suggest therefore that the descendants of T.TDL which survived after transfer to “B” mice were highly enriched in long-lived recirculating T lymphocytes reactive to determinants expressed by specific tumour allografts.     

Vitamin B6 deficiency and the lymphoid system: I. Effects on cellular immunity and in vitro incorporation of 3H-uridine by small lymphocytes

Thoracic duct lymphocytes from vitamin B₆-deficient rats were found to have a reduced capacity to respond to foreign lymphoid cells in the mixed lymphocyte reaction (MLR), to produce normal lymphocyte transfer reactions, and to incorporate ³H-uridine in vitro. These findings indicate that specific nutritional deficiencies may impair cellular immunity and that this impairment can be monitored by the MLR. It is suggested that the reduction in MLR activity and in ³H-uridine uptake by TDL cells reflected either a shift in the proportions of T and B cells in the TDL and/or an impairment in the capacity of such cells to function in the MLR and in the in vitro test for ³H-uridine incorporation.     

In vivo and in vitro studies of the effects of immune rat serum on Fasciola hepatica

Significant protection against infection with 10 or 30 metacercariae of Fasciola hepatica was conferred on naive rats by the passive transfer of serum derived from rats which had been exposed to primary and challenge infections with 5 or 10 and 30 or 20 metacercariae respectively. Immune serum did not have a pronounced effect on the mortality of metacercariae in vitro. However, its presence was associated with the formation of a precipitate on the tegument of each metacercaria and in the culture medium. The precipitate contained rat antibody and other components, presumably parasite antigens, which elicited the formation of antibody when the precipitate was injected into rats. Viability of metacercariae cultured in immune and normal sera as well as freshly excysted specimens was tested in rats by intraperitoneal infection. Metacercariae cultured in immune serum did not develop. By comparison with the viability of freshly excysted metacercariae, that of some metacercariae cultured in normal serum was impaired; this was attributed to inadequacies in the culture technique. A relationship between precipitate formation in vitro and impaired viability of metacercariae in vivo has yet to be established.     

Local and Systemic Effects on Inflammation during Eimeria nieschulzi Infection1

Rats infected with Eimeria nieschulzi, a coccidium that inhabits intestinal epithelium, have a lower basal inflammatory state in their intestinal mucosa eight days postinoculation as reflected by a drop in mucosal peroxidase activity and a decrease in the number of granulocytes in the lamina propria. The reduction of systemic inflammation in infected rats was assessed from a reduction in the formation of granulation tissue around a sterile cotton string implanted under the abdominal skin of the hosts. This reduced inflammatory response, both locally and systemically, occurs during the development of gamonts by the parasite and the release of oocysts from the host. These results plus the presence of normal or slightly elevated numbers of granulocytes in peripheral blood lead to the conclusion that the parasite does not affect hematopoiesis but interferes with some phase in the directed migration of leukocytes to specific sites.     

Acetylcholinesterase levels in Angiostrongylus cantonensis in relation to the immune response in rats

Beaver J. P. and Dobson C. 1978. Acetylcholinesterase levels in Angiostrongylus cantonensis in relation to the immune response in rats. International Journal for Parasitology8: 9–13. Angiostrongylus cantonensis larvae and adult nematodes synthesize three acetylcholinesterase (AChE) isozymes. The K_m of this isozyme complex changes with the development and migrations of the parasite in the rat host. The levels of parasite AChE changed as the development of A. cantonensis progressed; increasing quantities of AChE were found in young adult A. cantonensis from the brain of rats. After migration to the pulmonary arteries, the quantity of AChE in the parasite was reduced and continued to decline in the aging parasite. Anti-A. cantonensis antibody inhibited parasite AChE activity; this inhibition of the parasite AChE activity changed at stages during development of the parasite which suggested variation in parasite AChE isozyme levels. Haemagglutinating anti-A. cantonensis antibody appeared in the serum of infected rats when the parasites commenced to lay eggs and increased in titre thereafter until 103 days after infection.     

Nucleic Acid Precursor Incorporation by Eimeria nieschulzi (Protozoa: Apicomplexa) and Jejunal Villus Epithelium*

Autoradiography was used to investigate incorporation of tritiated adenine, adenosine, guanosine and thymidine by Eimeria nieschulzi and rat jejunal villus epithelial cells. At 2 1/2 days postinoculation, parasitized and control tissues were incubated for 20 min in oxygenated Tyrode's solution (37 C, pH 7.5) containing 30 μCi/ml of each nucleic acid precursor. Treatment of tissues with ribonuclease revealed that E. nieschulzi incorporated label from [³H]adenine primarily into RNA while that from [³H]adenosine and [³H]guanosine was present mainly in DNA. Label from [³H]thymidine was not utilized by parasites. Host villus epithelial cells incorporated label from [³H]purines primarily into RNA. Labeled cytoplasmic RNA was significantly increased in parasitized cells after incubation in [³H]adenine. Tritiated nuclear RNA and cytoplasmic RNA were significantly decreased in parasitized cells after incubation in [³H]adenosine. Incorporation of label from [³H]guanosine was similar for parasitized and control cells. A small quantity of label from each [³H]precursor was incorporated into DNA of villus epithelial cell nuclei.     

B-1 cells upregulate CD8 T lymphocytes and increase proinflammatory cytokines serum levels in oral encephalitozoonosis

Microsporidia are intracellular pathogens that cause severe disease in immunocompromised humans and animals. We recently demonstrated that XID mice are more susceptible to Encephalitozoon cuniculi infection by intraperitoneal route, evidencing the role of B-1 cells in resistance against infection. The present study investigated the resistance and susceptibility against E. cuniculi oral infection, including the role of B-1 cells. BALB/c and BALB/c XID (B-1 cells deficient) mice were orally infected with E. cuniculi spores. No clinical symptoms were observed in infected animals; histopathology showed lymphoplasmocytic enteritis with degeneration of the apexes of the villi in all infected groups. Higher parasite burden was observed in infected BALB/c XID mice. In the spleen and peritoneum, all infected mice showed a decrease of lymphocytes, including CD8⁺ T cells, mostly in infected BALB/c XID mice. Adoptive transfer of B-1 cells (XID + B-1) was associated with a lower parasite burden. Pro-inflammatory cytokines (IFN-γ, TNF-α and IL-6) increased mostly in infected XID + B1 mice. Together, the present results showed that BALB/c XID mice infected by the oral route were more susceptible to encephalitozoonosis than BALB/c mice, demonstrating the B-1 cells importance in the control of the immune response against oral E. cuniculi infection.     

EmTIP,a T-Cell Immunomodulatory Protein Secreted by the Tapeworm Echinococcus multilocularis Is Important for Early Metacestode Development

Background

Alveolar echinococcosis (AE), caused by the metacestode of the tapeworm Echinococcus multilocularis, is a lethal zoonosis associated with host immunomodulation. T helper cells are instrumental to control the disease in the host. Whereas Th1 cells can restrict parasite proliferation, Th2 immune responses are associated with parasite proliferation. Although the early phase of host colonization by E. multilocularis is dominated by a potentially parasitocidal Th1 immune response, the molecular basis of this response is unknown.

Principal Findings

We describe EmTIP, an E. multilocularis homologue of the human T-cell immunomodulatory protein, TIP. By immunohistochemistry we show EmTIP localization to the intercellular space within parasite larvae. Immunoprecipitation and Western blot experiments revealed the presence of EmTIP in the excretory/secretory (E/S) products of parasite primary cell cultures, representing the early developing metacestode, but not in those of mature metacestode vesicles. Using an in vitro T-cell stimulation assay, we found that primary cell E/S products promoted interferon (IFN)-γ release by murine CD4+ T-cells, whereas metacestode E/S products did not. IFN-γ release by T-cells exposed to parasite products was abrogated by an anti-EmTIP antibody. When recombinantly expressed, EmTIP promoted IFN-γ release by CD4+ T-cells in vitro. After incubation with anti-EmTIP antibody, primary cells showed an impaired ability to proliferate and to form metacestode vesicles in vitro.

Conclusions

We provide for the first time a possible explanation for the early Th1 response observed during E. multilocularis infections. Our data indicate that parasite primary cells release a T-cell immunomodulatory protein, EmTIP, capable of promoting IFN-γ release by CD4+ T-cells, which is probably driving or supporting the onset of the early Th1 response during AE. The impairment of primary cell proliferation and the inhibition of metacestode vesicle formation by anti-EmTIP antibodies suggest that this factor fulfills an important role in early E. multilocularis development within the intermediate host.     

Contribution of the Ly49E Natural Killer Receptor in the Immune Response to Plasmodium berghei Infection and Control of Hepatic Parasite Development

Natural killer (NK) cells have different roles in the host response against Plasmodium-induced malaria depending on the stage of infection. Liver NK cells have a protective role during the initial hepatic stage of infection by production of the T_H1-type cytokines IFN-γ and TNF-α. In the subsequent erythrocytic stage of infection, NK cells also induce protection through Th1-type cytokines but, in addition, may also promote development of cerebral malaria via CXCR3-induction on CD8⁺ T cells resulting in migration of these cells to the brain. We have recently shown that the regulatory Ly49E NK receptor is expressed on liver NK cells in particular. The main objective of this study was therefore to examine the role of Ly49E expression in the immune response upon Plasmodium berghei ANKA infection, for which we compared wild type (WT) to Ly49E knockout (KO) mice. We show that the parasitemia was higher at the early stage, i.e. at days 6–7 of Plasmodium berghei ANKA infection in Ly49E KO mice, which correlated with lower induction of CD69, IFN-γ and TNF-α in DX5⁻ liver NK cells at day 5 post-infection. At later stages, these differences faded. There was also no difference in the kinetics and the percentage of cerebral malaria development and in lymphocyte CXCR3 expression in WT versus Ly49E KO mice. Collectively, we show that the immune response against Plasmodium berghei ANKA infection is not drastically affected in Ly49E KO mice. Although NK cells play a crucial role in Plasmodium infection and Ly49E is highly expressed on liver NK cells, the Ly49E NK receptor only has a temporarily role in the immune control of this parasite.     

Loss of natural resistance to schistosome in T cell deficient rat

Schistosomiasis is among the major neglected tropical diseases and effective prevention by boosting the immune system is still not available. T cells are key cellular components governing adaptive immune response to various infections. While common laboratory mice, such as C57BL/6, are highly susceptible to schistosomiasis, the SD rats are extremely resistant. However, whether adaptive immunity is necessary for such natural resistance to schistosomiasis in rats remains to be determined. Therefore, it is necessary to establish genetic model deficient in T cells and adaptive immunity on the resistant SD background, and to characterize liver pathology during schistosomiasis. In this study we compared experimental schistosomiasis in highly susceptible C57BL/6 (B6) mice and in resistant SD rats, using cercariae of Schistosoma japonicum. We observed a marked T cell expansion in the spleen of infected B6 mice, but not resistant SD rats. Interestingly, CD3e^−/− B6 mice in which T cells are completely absent, the infectious burden of adult worms was significantly higher than that in WT mice, suggesting an anti-parasitic role for T cells in B6 mice during schistosome infection. In further experiments, we established Lck deficient SD rats by using CRISPR/Cas9 in which T cell development was completely abolished. Strikingly, we found that such Lck deficiency in SD rats severely impaired their natural resistance to schistosome infection, and fostered parasite growth. Together with an additional genetic model deficient in T cells, the CD3e^−/− SD rats, we confirmed the absence of T cell resulted in loss of natural resistance to schistosome infection, but also mitigated liver immunopathology. Our further experiments showed that regulatory T cell differentiation in infected SD rats was significantly decreased during schistosomiasis, in contrast to significant increase of regulatory T cells in infected B6 mice. These data suggest that T cell mediated immune tolerance facilitates persistent infection in mice but not in SD rats. The demonstration of an important role for T cells in natural resistance of SD rats to schistosomiasis provides experimental evidences supporting the rationale to boost T cell responses in humans to prevent and treat schistosomiasis.     

Cellular events in protein-tolerant inbred rats. III. Antigen-reactive B cells in rats tolerant to human serum albumin.

Rats tolerant to human serum albumin (HSA) were injected with selected lymphocyte populations and challenged with HSA plus adjuvant to test for loss of tolerance. Thoracic duct lymphocytes (TDL) from normal or immune rats, either untreated or depleted of Ig-bearing cells or HSA-binding cells by affinity chromatography were all equally effective in restoring the HSA antibody response in previously tolerant recipients. In contrast, recirculating B cells (TDL from B rats) were not effective. The results indicated that unresponsiveness to HSA was a lesion of the T- but not the B-cell compartment. However, antibody affinity failed to mature to a high level in tolerant rats that were restored with T cells, and the response of transferred primed B cells into unresponsive recipients was inhibited, suggesting that the tolerant state was not merely due to a T-cell deletion.