Eimeria tenella, an intracellular protozoan parasite infecting the epithelial cells of the ceca of chickens, causes severe diarrhea and bleeding that can lead its host to death. It is of interest that E. tenella first penetrate into the mucosal intraepithelial lymphocytes (IEL) before they parasitize crypt or villous epithelial cells. This in vitro study was undertaken to know whether the penetration of E. tenella into such a lymphoid cell is a beneficial step for the parasite survival and development. Three sequential experiments were performed. First, the in vitro established bovine kidney cell line, MDBK cells, were evaluated for use as host cells for E. tenella, through morphological observation. Second, the degree of parasite development and multiplication in MDBK cells was quantitatively assayed using radioisotope-labelled uracil (3H-uracil). Third, the E. tenella sporozoites viability was assayed after preincubation of them with chicken spleen cells. E. tenella oocysts obtained from the ceca of the infected chickens were used for the source of the sporozoites. Spleen cells (E) obtained from normal chickens (FP strain) were preincubated with the sporozoites (T) at the E:T ratio of 100:1, 50:1 or 25:1 for 4 or 12 hours, and then the mixture was inoculated into the MDBK cell monolayer. Morphologically the infected MDBK cells revealed active schizogonic cycle of E. tenella in 3-4 days, which was characterized by the appearance of trophozoites, and immature and mature schizonts containing merozoites. The 3H-uracil uptake by E. tenella increased gradually in the MDBK cells, which made a plateau after 48-60 hours, and decreased thereafter. The uptake amount of 3H-uracil depended not only upon the inoculum size of the sporozoites but also on the degree of time delay (preincubation; sporozoites only) from excystation to inoculation into MDBK cells. The 3H-uracil uptake became lower as the preincubation time was prolonged. In comparison, after preincubation of sporozoites with spleen cells for 4 or 12 hours, the 3H-uracil uptake was significantly increased compared with that of control group. From the results, it was inferred that, although the penetration of E. tenella sporozoites into the lymphoid cells such as IEL is not an essential step, it should be at least a beneficial one for the survival and development of sporozoites in the chicken intestine.
Cattle- ; Cell-Line ; Cells,-Cultured ; Chickens- ; English-Abstract ; *Eimeria-growth-and-development ; *Kidney-parasitology ; *Lymphocytes-parasitology ; *Spleen-cytology

To study the basic pathological changes of small intestine in metagonimiasis, light- and electron microscopic studies were made, using a total of 21 cats which were experimentally infected with metacercariae of Metagonimus yokogawai. The metacercariae were obtained from naturally infected sweetfish (Plecoglossus altivelis) by digestion technique. The cats were divided in control, light-infection(10,000 metacercariae infected) and heavy-infection(50,000 metacercariae infected) groups. Cats were killed at the 5th, 10th, 15th day, and 4th, 8th and 10th week after the infection. And the small intestine was prepared for the study. Pathological studies comprised gross examination, worm distribution pattern, light microscopic examination and both transmission and scanning electron microscopic examinations. The results obtained were summarized as follows. Gross morphologic changes were the most marked during the first 2 weeks after infection. The gross abnormalities were severer in the heavily infected animals. The changes were dryness and listlessness of serosal surface due to dehydration, mushy and/or watery intestinal content, effacement of transverse nodes and enlargement of mesenteric lymph folds and Peyer's patches. After 4 weeks of infection, these changes became less marked showing a tendency to return to normal. The sectioned flukes were distributed from duodenum to proximal ileum. However, individual variation was marked in distribution. In the heavy-infection group, the locality of parasitism tended to extend more distally. The locality of M. yokogawai in the intervillous space was mostly in the lower-most portion of intervillous space, where they compressed and eroded epithelial cells probably due to mechanical damage to the structure. Very rarely the worms were found in lumen of Lierberkuehn's crypt, and reaching, in two occasions, into proprial lymphoid tissue. Light-microscopically the lesion was restricted in mucosa: Early mucosal changes were shortening, blunting, fusion, and thickening of the villi, crypt hypertrophy with consequent decrease of villus/crypt ratio, as well as stromal changes of edema, capilliary ectasia and marked inflammatory cell infiltration of lymphocytes and plasma cells. Goblet cells were markedly reduced in number as with depletion of its cytoplasmic content. In the later stages of infection, mucosa restored its normal configuration in spite of persistent parasitism of the worms. At the infection stage of 5-15 days, there was significant shortening of the microvillous height with varible destruction of glycocalyx in electron microscopic examination. With lapse of infection time, microvilli became to restore the normal pattern. With these morphological changes, it appears that diarrhea in experimental metagonimiasis would be related to the decrease of absorptive surface of the small intestine particularly in the early phase of infection. The significant changes seen in villi and microvilli might be due to massive intrusion or invasion of Metagonimus worms into the crypts, causing direct mechanical and possible host-immune response to the small bowel mucosa.
parasitology-helminth-trematoda ; metagonimiasis ; Metagonimus yokogawai ; pathology ; cat-intestine ; edema ; lymphocytes ; plasma cells ; goblet cell

Several studies have shown the mechanisms and importance of immune responses against Toxoplasma gondii infection and the notable role of cholinesterases in inflammatory reactions. However, the association between those factors has not yet been investigated. Therefore, the aim of this study was to evaluate the acetylcholinesterase (AChE) activity in blood and lymphocytes and the activity of butyrylcholinesterase (BChE) in serum of rats experimentally infected with T. gondii during the acute phase of infection. For that, an in vivo study was performed with evaluations of AChE and BChE activities on days 5 and 10 post-infection (PI). The activity of AChE in blood was increased on day 5 PI, while in lymphocytes its activity was enhanced on days 5 and 10 PI (P<0.05). No significant difference was observed between groups regarding to the activity of BChE in serum. A positive (P<0.01) correlation was observed between AChE activity and number of lymphocytes. The role of AChE as an inflammatory marker is well known in different pathologies; thus, our results lead to the hypothesis that AChE has an important role in modulation of early immune responses against T. gondii infection.
Acetylcholinesterase/blood/*metabolism ; Animals ; Butyrylcholinesterase/blood/*metabolism ; Humans ; Lymphocytes/enzymology/parasitology ; Male ; Rats ; Rats, Wistar ; Toxoplasma/*physiology ; Toxoplasmosis/*enzymology/genetics/parasitology

This study was carried out to investigate fifteen cases of acute lethal infection of calves (< or = 4 months of age) by the protozoan parasite Theileria (T.) annulata in the south of Portugal. Calves developed multifocal to coalescent nodular skin lesions, similar to multicentric malignant lymphoma. Infestation with ticks (genus Hyalomma) was intense. Theileria was seen in blood and lymph node smears, and T. annulata infection was confirmed by isolation of schizont-transformed cells and sequencing of hypervariable region 4 of the 18S rRNA gene. At necropsy, hemorrhagic nodules or nodules with a hemorrhagic halo were seen, particularly in the skin, subcutaneous tissue, skeletal and cardiac muscles, pharynx, trachea and intestinal serosa. Histologically, nodules were formed by large, round, lymphoblastoid neoplastic-like cells. Immunohistochemistry (IHC) identified these cells as mostly CD3 positive T lymphocytes and MAC387 positive macrophages. A marker for B lymphocytes (CD79alphacy) labeled very few cells. T. annulata infected cells in these nodules were also identified by IHC through the use of two monoclonal antibodies (1C7 and 1C12) which are diagnostic for the parasite. It was concluded that the pathological changes observed in the different organs and tissues were caused by proliferation of schizont-infected macrophages, which subsequently stimulate a severe uncontrolled proliferation of uninfected T lymphocytes.
Animals ; Base Sequence ; Cattle ; Cattle Diseases/epidemiology/*parasitology/pathology ; Cell Growth Processes/physiology ; DNA, Protozoan/chemistry/genetics ; Disease Outbreaks/*veterinary ; Female ; Immunohistochemistry/veterinary ; Lymphocytes/parasitology/*pathology ; Male ; Molecular Sequence Data ; Polymerase Chain Reaction/veterinary ; Portugal/epidemiology ; RNA, Ribosomal, 18S/chemistry/genetics ; Sequence Analysis, DNA ; Skin Diseases/epidemiology/parasitology/pathology/*veterinary ; Theileria annulata/*isolation & purification ; Theileriasis/epidemiology/parasitology/*pathology

Theileria annulata is a tick-borne intracellular protozoan parasite that causes tropical theileriosis, a fatal bovine lymphoproliferative disease. The parasite predominantly invades bovine B lymphocytes and macrophages and induces host cell transformation by a mechanism that is not fully comprehended. Analysis of signaling pathways by quantitative real-time PCR (qPCR) could be a highly efficient means to understand this transformation mechanism. However, accurate analysis of qPCR data relies on selection of appropriate reference genes for normalization, yet few papers on T. annulata contain evidence of reference gene validation. We therefore used the geNorm and NormFinder programs to evaluate the stability of 5 candidate reference genes; 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ACTB (β-actin), PRKG1 (protein kinase cGMP-dependent, type I) and TATA box binding protein (TBP). The results showed that 18S rRNA was the reference gene most stably expressed in bovine PBMCs transformed and non-transformed with T. annulata, followed by GAPDH and TBP. While 18S rRNA and GAPDH were the best combination, these 2 genes were chosen as references to study signaling pathways involved in the transformation mechanism of T. annulata.
Animals ; B-Lymphocytes/parasitology ; Cattle ; Cell Line ; Cells/*parasitology ; Cells, Cultured ; Gene Expression Profiling ; Host-Parasite Interactions/*genetics ; Real-Time Polymerase Chain Reaction/*veterinary ; Reproducibility of Results ; Signal Transduction/*genetics ; Theileria annulata/physiology ; Theileriasis/*physiopathology

The enteric protozoan parasite Entamoeba histolytica is the causative agent of human amebiasis. During infection, adherence of E. histolytica through Gal/GalNAc lectin on the surface of the amoeba can induce caspase-3-dependent or -independent host cell death. Phosphorylinositol 3-kinase (PI3K) and protein kinase C (PKC) in E. histolytica play an important function in the adhesion, killing, or phagocytosis of target cells. In this study, we examined the role of amoebic PI3K and PKC in amoeba-induced apoptotic cell death in Jurkat T cells. When Jurkat T cells were incubated with E. histolytica trophozoites, phosphatidylserine (PS) externalization and DNA fragmentation in Jurkat cells were markedly increased compared to those of cells incubated with medium alone. However, when amoebae were pretreated with a PI3K inhibitor, wortmannin before being incubated with E. histolytica, E. histolytica-induced PS externalization and DNA fragmentation in Jurkat cells were significantly reduced compared to results for amoebae pretreated with DMSO. In addition, pretreatment of amoebae with a PKC inhibitor, staurosporine strongly inhibited Jurkat T cell death. However, E. histolytica-induced cleavage of caspase-3, -6, and -7 were not inhibited by pretreatment of amoebae with wortmannin or staurosporin. In addition, we found that amoebic PI3K and PKC have an important role on amoeba adhesion to host compartment. These results suggest that amebic PI3K and PKC activation may play an important role in caspase-independent cell death in Entamoeba-induced apoptosis.
*Apoptosis ; Caspases/metabolism ; Entamoeba histolytica/*enzymology/*growth & development ; Humans ; Hydrolysis ; Jurkat Cells ; Phosphatidylinositol 3-Kinases/*metabolism ; Protein Kinase C/*metabolism ; T-Lymphocytes/*parasitology/*physiology

Different functions have been attributed to CD4+CD25+Foxp3+ regulatory T-cells (Tregs) during malaria infection. Herein, we describe the disparity in Treg response and pro- and anti-inflammatory cytokines during infection with Plasmodium berghei ANKA between young (3-week-old) and middle-aged (8-month-old) C57BL/6 mice. Young mice were susceptible to cerebral malaria (CM), while the middle-aged mice were resistant to CM and succumbed to hyperparasitemia and severe anemia. The levels of pro-inflammatory cytokines, such as TNF-alpha, in young CM-susceptible mice were markedly higher than in middle-aged CM-resistant mice. An increased absolute number of Tregs 3-5 days post-inoculation, co-occurring with elevated IL-10 levels, was observed in middle-aged CM-resistant mice but not in young CM-susceptible mice. Our findings suggest that Treg proliferation might be associated with the suppression of excessive pro-inflammatory Th1 response during early malaria infection, leading to resistance to CM in the middle-aged mice, possibly in an IL-10-dependent manner.
Aging/*immunology ; Animals ; Cytokines/genetics/metabolism ; Female ; Gene Expression Regulation ; Malaria/*immunology/*parasitology ; Mice ; Plasmodium berghei/*classification ; T-Lymphocytes, Regulatory/classification/*physiology

The present study surveyed the prevalence of natural infection of the sheep esphagus muscle with sarcocysts of Sarcocystis ovicanis and examined induction of protective immunity using UV-attenuated sporocysts. The overall prevalence of natural infection of the sheep was 95%. Infectivity of the collected sarcocysts was confirmed by shedding of sporulated oocysts after feeding infected esophageal tissues to dogs. To induce protective immunity, lambs were immunized 3 times (once a week) with 1.5 x 10(4) sporocysts exposed to UV-light for 30 min (UV-30 group) or 60 (UV-60 group) min and then challenged with 1.5 x 10(4) normal sporocysts at the 3rd week post the 1st vaccination. These lambs showed high survival and less clinical signs of sarcocystosis than normal infected lambs. The attenuated sporocysts produced abnormal cysts; small in size and detached from the muscle fiber. These abnormalities were more obvious in UV-60 group than UV-30 group. Also, the IFN-gamma level and lymphocyte percentage were increased while the total leukocyte count was decreased in the UV-60 group compared with other groups. The high level of IFN-gamma may be an evidence for the induction of Th1 responses which may have protective effect against a challenge infection.
Animals ; Dogs ; Esophagus/parasitology ; Feces/parasitology ; Interferon-gamma/secretion ; Lymphocytes/immunology ; Oocysts/*immunology ; Peptide Fragments/secretion ; Prevalence ; Protozoan Vaccines/immunology ; Sarcocystis/cytology/*immunology/*radiation effects ; Sarcocystosis/epidemiology/immunology/prevention & control/*veterinary ; Severity of Illness Index ; Sheep/immunology/parasitology ; Sheep Diseases/immunology/*prevention & control ; Survival Analysis ; *Ultraviolet Rays ; Vaccines, Attenuated/immunology

We report two cases of hepatocellular carcinoma with prominent lymphocytic infiltration, which has been described as a subtype of hepatocellular carcinoma with good prognosis. One case showed lymphoid follicles and dense lymphocytic infiltrates within the tumor and its periphery, and the other case showed marked lymphocytic infiltration in the cancerous tissue. Piecemeal necrosis of cancer cells and atypical reactive changes were evident. The two cases were seronegative for hepatitis B surface antigen, antibody to hepatitis C virus, and Epstein-Barr virus DNA. One of the cases showed Clonorchis infestation. The prognostic significance of lymphocytic stroma in hepatocellular carcinoma requires further investigation.
Animals ; Carcinoma, Hepatocellular/*diagnosis/pathology ; Clonorchiasis/diagnosis/parasitology ; Clonorchis sinensis ; Diagnosis, Differential ; Female ; Humans ; Liver Neoplasms/*diagnosis/pathology ; Lymphocytes, Tumor-Infiltrating/*pathology ; Male ; Middle Aged ; Tomography, X-Ray Computed

In order to determine the role of Peyer's patch lymphocytes (PPL) in self-clearing of Cryptosporidium parvum infection in murine models, changes in PPL subsets, their cytokine expression, and in vitro IgG1 and IgA secretions by PPL were observed in primary- and challenge-infected C57BL/6 mice. In primary-infected mice, the percentages of CD4+ T cells, CD8+ T cells, sIgA+ B cells, IL-2+ T cells, and IFN-gamma+ T cells among the PPL, increased significantly (P < 0.05) on day 10 post-infection (PI). Secretion of IgG1 and IgA in vitro by PPL also increased on day 10 PI. However, all these responses, with the exception of IgG1 and IgA secretions, decreased in challenge-infected mice on day 7 post-challenge (= day 13 PI); their IgG1 and IgA levels were higher (P > 0.05) than those in primaryinfected mice. The results suggest that murine PPL play an important role in self-clearing of primary C. parvum infections through proliferation of CD4+, CD8+, IL-2+, and IFN-gamma+ T cells, and IgG1 and IgA-secreting B cells. In challenge infections, the role of T cells is reduced whereas that of B cells secreting IgA appeared to be continuously important.
Animals ; Antibodies, Protozoan/analysis/metabolism ; Cattle ; Cryptosporidiosis/*immunology/parasitology ; Cryptosporidium parvum/*immunology ; Feces/parasitology ; Female ; Humans ; Immunoglobulin A/analysis/biosynthesis ; Immunoglobulin G/analysis/biosynthesis ; Interferon-gamma/analysis/biosynthesis ; Interleukin-2/analysis/biosynthesis ; Lymphocytes/*immunology ; Male ; Mice ; Mice, Inbred C57BL ; Peyer's Patches/cytology/*immunology ; Specific Pathogen-Free Organisms