Antigenic Similarity Between the Coccidian Parasites Toxoplasma gondii and Hammondia hammondi1

The antigens that are present in the coccidian parasites Toxoplasma gondii and Hammondia hammondi were demonstrated and defined by using SDS-PAGE and immunoenzymatic techniques with ¹²⁵I-labeled and unlabeled antigens of T. gondii and sera of mice infected orally or intraperitoneally with H. hammondi. All cell surface antigens of T. gondii that were labeled with ¹²⁵I were recognized by antibodies in the sera of the mice infected with H. hammondi except the antigen of approximate molecular weight of 21.5 Kd. This suggests that this antigen is specific for T. gondii. Various antigens in the T. gondii-lysed antigen preparations were recognized by antibodies to H. hammondi. The number of recognized antigens increased as the infection of the mice with H. hammondi progressed. Oral infection with H. hammondi appeared to induce the formation of antibodies that recognized more T. gondii antigens than infection by intraperitoneal inoculation.     

A Comparison of Cross Protection Between BCG,Hammondia hammondi,Besnoitia jellisoni and Toxoplasma gondii in Hamsters*

SYNOPSIS. The effect of pretreatment with BCG strain of Mycobacterium tuberculosis or Hammondia hammondi 21 days before challenge with lethal doses of Toxoplasma gondii and Besnoitia jellisoni was studied in hamsters. The results indicated that the intracardial administration of BCG provided no protection against either T. gondii or B. jellisoni. The hamsters immunized with H. hammondi survived challenge with 10⁴ lethal doses of T. gondii but only 1 lethal dose with B. jellisoni, indicating strong cross protection between H. hammondi and T. gondii and only a marginal one between H. hammondi and B. jellisoni.     

Besnoitia darlingi (Apicomplexa,Sarcocystidae, Toxoplasmatinae): Transmission between Opossums and Cats1

ABSTRACT. Opossums (Didelphis marsupialis), act as intermediate hosts for Besnoitia darlingi and could be infected orally with sporozoites (oocysts) and bradyzoites (tissue cysts), or intraperitoneally (i.p.) with tachyzoites. Infections could presumably be transmitted through cannibalism. Cats (Felis catus), the definitive host, could be infected only with bradyzoites but not sporozoites. Oocysts shed by cats measure about 12 × 12 μm, resemble similarly sized oocysts of Toxoplasma gondii and Hammondia hammondi, and must be differentiated by the appearance of tissue cysts after experimental infection of intermediate hosts. Cats did not form tissue cysts of B. darlingi. Tachyzoites from the related B. jellisoni could be used in the Sabin-Feldman dye test to determine the development of antibody to B. darlingi in opossums after infection.     

Development of Ox-Coyote Cycle of Sarcocystis cruzi1

The ox-coyote cycle of Sarcocystis cruzi was studied by killing 38 calves between 4 and 153 days postinoculation (DPI) with 55 × 10³-5 × 10⁸ sporocysts from the intestines of coyotes. At 4 DPI, a zoite was found within the lumen of a mesenteric lymph node artery. At 7 DPI, zoites were found in mononuclear cells and in endothelial cells in mesenteric arteries. First generation meronts (41.0 × 17.5 μm in diameter) occurred 7–26 DPI in mesenteric lymph nodes. At 19–46 DPI, second generation meronts occurred in kidneys, muscles, and other tissues: renal meronts were 19.6 × 11.0 μm, and intramuscular meronts were 25.0 × 11.1 μm. Merozoites were found in the peripheral blood 17 DPI and later at 24–46 DPI. They divided by endodyogeny in mononuclear cells. Sarcocysts were seen first in the heart at 45 DPI and contained one or two metrocytes. At 55 DPI, sarcocysts containing only metrocytes were found in striated muscles, heart, and in smooth muscles of the urinary bladder, rumen, omasum, abomasum, and small intestine. At 67, 87, 112, and 153 DPI, sarcocysts were found only in striated muscles and in the heart. At 67 DPI, sarcocysts were up to 360 μm long. They contained only metrocytes and were not infective to the dog. At 86 DPI, sarcocysts contained mostly bradyzoites, a few metrocytes, and were infective to a coyote. The thin-walled sarcocysts grew to a maximum length of 800 μm and contained bradyzoites that were 10.9 × 3.0 μm. At 90 DPI, two mature sarcocysts were found in 2 of 73 sections of brain and spinal cord; hundreds of sarcocysts were present in sections of tongue and heart of this calf. Gametogony occurred in the small intestine of the coyote. Macro-and microgamonts were found in goblet cells of the small intestines of coyotes 6 h after the ingestion of infected meat. Microgamonts were few and contained 3–11 slender gametes. Oocysts were seen at 12 h and sporulation was completed 9 DPI. The prepatent period in the coyote was 8 days. The ox-coyote cycle is compared with ox-dog cycle.     

Dysregulation of focal adhesion kinase upon Toxoplasma gondii infection facilitates parasite translocation across polarised primary brain endothelial cell monolayers

The apicomplexan parasite Toxoplasma gondii invades tissues and traverses non‐permissive biological barriers in infected humans and other vertebrates. Following ingestion, the parasite penetrates the intestinal wall and disseminates to immune‐privileged sites such as the brain parenchyma, after crossing the blood–brain barrier. In the present study, we have established a protocol for high‐purification of primary mouse brain endothelial cells to generate stably polarised monolayers that allowed assessment of cellular barrier traversal by T. gondii. We report that T. gondii tachyzoites translocate across polarised monolayers of mouse brain endothelial cells and human intestinal Caco2 cells without significantly perturbing barrier impermeability and with minimal change in transcellular electrical resistance. In contrast, challenge with parasite lysate or LPS increased barrier permeability by destabilising intercellular tight junctions (TJs) and accentuated transmigration of T. gondii. Conversely, reduced phosphorylation of the TJ‐regulator focal adhesion kinase (FAK) was observed dose‐dependently upon challenge of monolayers with live T. gondii but not with parasite lysate or LPS. Pharmacological inhibition of FAK phosphorylation reversibly altered barrier integrity and facilitated T. gondii translocation. Finally, gene silencing of FAK by shRNA facilitated transmigration of T. gondii across epithelial and endothelial monolayers. Jointly, the data demonstrate that T. gondii infection transiently alters the TJ stability through FAK dysregulation to facilitate transmigration. This work identifies the implication of the TJ regulator FAK in the transmigration of T. gondii across polarised cellular monolayers and provides novel insights in how microbes overcome the restrictiveness of biological barriers.     

An Experimental Toxoplasma gondii Dose Response Challenge Model to Study Therapeutic or Vaccine Efficacy in Cats

High numbers of Toxoplasma gondii oocysts in the environment are a risk factor to humans. The environmental contamination might be reduced by vaccinating the definitive host, cats. An experimental challenge model is necessary to quantitatively assess the efficacy of a vaccine or drug treatment. Previous studies have indicated that bradyzoites are highly infectious for cats. To infect cats, tissue cysts were isolated from the brains of mice infected with oocysts of T. gondii M4 strain, and bradyzoites were released by pepsin digestion. Free bradyzoites were counted and graded doses (1000, 100, 50, 10), and 250 intact tissue cysts were inoculated orally into three cats each. Oocysts shed by these five groups of cats were collected from faeces by flotation techniques, counted microscopically and estimated by real time PCR. Additionally, the number of T. gondii in heart, tongue and brains were estimated, and serology for anti T. gondii antibodies was performed. A Beta-Poisson dose-response model was used to estimate the infectivity of single bradyzoites and linear regression was used to determine the relation between inoculated dose and numbers of oocyst shed. We found that real time PCR was more sensitive than microscopic detection of oocysts, and oocysts were detected by PCR in faeces of cats fed 10 bradyzoites but by microscopic examination. Real time PCR may only detect fragments of T. gondii DNA without the presence of oocysts in low doses. Prevalence of tissue cysts of T. gondii in tongue, heart and brains, and anti T. gondii antibody concentrations were all found to depend on the inoculated bradyzoite dose. The combination of the experimental challenge model and the dose response analysis provides a suitable reference for quantifying the potential reduction in human health risk due to a treatment of domestic cats by vaccination or by therapeutic drug application.     

Elucidating pathways of Toxoplasma gondii invasion in the gastrointestinal tract: involvement of the tight junction protein occludin

Toxoplasma gondii is an obligate intracellular parasite infecting one third of the world's population. The small intestine is the parasite's primary route of infection, although the pathway of epithelium transmigration remains unclear. Using an in vitro invasion assay and live imaging we showed that T. gondii (RH) tachyzoites infect and transmigrate between adjacent intestinal epithelial cells in polarized monolayers without altering barrier integrity, despite eliciting the production of specific inflammatory mediators and chemokines. During invasion, T. gondii co-localized with occludin. Reducing the levels of endogenous cellular occludin with specific small interfering RNAs significantly reduced the ability of T. gondii to penetrate between and infect epithelial cells. Furthermore, an in vitro invasion and binding assays using recombinant occludin fragments established the capacity of the parasite to bind occludin and in particular to the extracellular loops of the protein. These findings provide evidence for occludin playing a role in the invasion of T. gondii in small intestinal epithelial cells.     

Toxoplasma gondii: an improved rat model of congenital infection

The objective of this study was to refine the rat model of congenital toxoplasmosis. In Fischer rats we found that visualization of spermatozoa in vaginal exudates and the detection of at least 6 g body weight increase between days 9 and 12 of pregnancy, allowed the diagnosis and timing of pregnancy with 60% specificity and 84% sensitivity. A dose of 10⁴Toxoplasma gondii bradyzoites or 10²T. gondii oocysts of the Prugniaud strain resulted in more than 50% of congenital infection of the rat litters. Transmission of T. gondii via lactation was not detected in rats inoculated with either bradyzoites or oocysts. Bioassays of 51 neonates born from mothers inoculated with bradyzoites (in tissue cysts) and 29 neonates from mothers inoculated with oocysts demonstrated that both liver and lungs can be used for the diagnosis of congenital transmission in this model.     

Functional analysis of Toxoplasma lactate dehydrogenases suggests critical roles of lactate fermentation for parasite growth in vivo

Glycolysis was thought to be the major pathway of energy supply in both fast‐replicating tachyzoites and slowly growing bradyzoites of Toxoplasma gondii. However, its biological significance has not been clearly verified. The genome of T. gondii encodes two lactate dehydrogenases (LDHs), which are differentially expressed in tachyzoites and bradyzoites. In this study, we knocked out the two LDH genes individually and in combination and found that neither gene was required for tachyzoite growth in vitro under standard growth conditions. However, during infection in mice, Δldh1 and Δldh1 Δldh2 mutants were unable to propagate and displayed significant virulence attenuation and cyst formation defects. LDH2 only played minor roles in these processes. To further elucidate the mechanisms underlying the critical requirement of LDH in vivo, we found that Δldh1 Δldh2 mutants replicated significantly more slowly than wild‐type parasites when cultured under conditions with physiological levels of oxygen (3%). In addition, Δldh1 Δldh2 mutants were more susceptible to the oxidative phosphorylation inhibitor oligomycin A. Together these results suggest that lactate fermentation is critical for parasite growth under physiological conditions, likely because energy production from oxidative phosphorylation is insufficient when oxygen is limited and lactate fermentation becomes a key supplementation.     

Histochemical distribution of digestive enzymes in intestine of goldline, Sarpa salpa L. 1758

Histochemical localization of non‐specific esterase, alkaline and acid phosphatase in the intestine of free‐living goldline (Sarpa salpa L. 1758) was investigated. Fish were caught in the vicinity of the town of Zadar (Adriatic Sea, Croatia), and samples of three parts of the intestine proper (anterior, middle and posterior) as well as the rectum were used for presentation of non‐specific esterases, alkaline phosphatase and acid phosphatase. Non‐specific esterase activity was found in the cytoplasm and brush border of enterocytes in all investigated intestinal segments and the rectum. The activity was stronger in the middle and posterior part of the intestine but weaker in the anterior segment of the intestine as well as in the rectum. Intestinal alkaline phosphatase was detected in the brush border and supranuclear cytoplasm of enterocytes of all investigated intestinal segments. Enzymatic activity gradually decreased in a posterior direction. Acid phosphatase activity was observed as a fine granular reaction product in the supranuclear region of enterocytes and was almost equal in all investigated intestinal segments as well as in the rectum. The possible role of enzymes in intracellular digestion and transport is discussed.     

Eimeria apsheronica in the goat: endogenous development and host cellular response

Most first generation schizonts of Eimeria apsheronica developed in the jejunum; others were distributed throughout the small intestine and occasionally in the caecum. Some were also found in the mesenteric lymph nodes, which were oedematous and haemorrhagic. In the intestine, haemorrhage and congestion were seen before parasites were detected, and continued throughout all later stages. Schizonts occurred in the lamina propria and occasionally in the submucosa, where they sometimes caused a cellular inflammatory response. Schizonts were first seen at 8 days post-infection (DPI); they had poorly defined nuclei and were enclosed in a capsule-like wall. At 16 DPI, many had matured, had a mean size of 125 x 82 microns, and were filled with numerous spindle-shaped merozoites, which were in ranks and loops. At 18 and 20 DPI, when small white lesions (1-3 mm in diameter) were observed in the jejunum and elsewhere in the small intestine, a second generation of schizonts, macrogametes, microgametocytes and maturing oocysts were seen, in the epithelial cells of the small intestine and caecum. Their mean sizes, respectively, were: 26.2 x 18.9, 24.7 x 18.5, 30.2 x 21.7 and 26.6 x 19.3 microns. Macrogametes contained basophilic central and eosinophilic peripheral granules. The sexual stages were associated with a generalized cellular inflammatory response.     

Pre-Erythrocytic Development and Associated Host Responses to Haemoproteus meleagridis (Haemosporina: Haemoproteidae) in Experimentally Infected Domestic Turkeys12

ABSTRACT. Two generations of pre-erythrocytic schizogony occurred in skeletal and cardiac muscle of domestic turkeys infected with sporozoites of Haemoproteus meleagridis. First generation schizonts reached maturity approximately five days post-inoculation (DPI) and developed in capillary endothelial cells and myofibroblasts. The schizonts ranged from 12 to 20 μm in diameter and produced long (5–6 μm), slender merozoites. Early second generation schizonts were first detected in capillary endothelial cells between 5 and 8 DPI. They were cylindrical and ranged in size from 5 to 8 μm in diameter and up to 28 μm in length. Second generation schizonts which reached maturity by 17 DPI were surrounded by a thick, hyaline wall and were packed with numerous spherical merozoites less than 1 μm in diameter. Mature megaloschizonts were fusiform, ranged from 30 to 113 μm in diameter, and extended as much as 465 μm along the long axis of muscle fibers. Merozoites developed as buds from cytomeres that formed between 8 and 14 DPI. Infected turkeys developed a moderate to severe myositis within 5 DPI and were lame in one or both legs. The myositis was associated with the necrosis of scattered groups of muscle fibers. Muscle fibers surrounding mature megaloschizonts were swollen and hyaline. Megaloschizonts were surrounded occasionally by fibroblasts and infiltrates of mononuclear cells. The morphology and site of development of mature megaloschizonts of Haemoproteus meleagridis are contrasted with those of other avian haemosporidians.     

Fatal Toxoplasmosis and Enteroepithelial Stages of Toxoplasma gondii in a Pallas Cat (Felis manul)1

Toxoplasma gondii was found in tissues of a six-year-old female Pallas cat (Felis manul) from the Milwaukee County Zoo. Toxoplasma gondii meronts (types D and E), gamonts, and oocysts were present in the epithelium of the small intestine. Numerous unsporulated oocysts were present in the intestinal lumen. The cat died of acute, overwhelming toxoplasmosis. Necrotic enteritis, multifocal necrotizing granulomatous hepatitis, and pneumonia were the prominent lesions.     

Histochemical distribution of four types of enzymes and mucous cells in the gastrointestinal tract of reared half‐smooth tongue sole Cynoglossus semilaevis

The histochemical distribution of acid phosphatase (ACP), alkaline phosphatase (ALP), non‐specific esterase (NSE), peroxidase (POD) and mucous‐cell types was evaluated in the gastrointestinal tract of the half‐smooth tongue sole Cynoglossus semilaevis. The enzymes were detected in the entire stretch of the gastrointestinal tract. ACP activity was found in the supranuclear region of enterocytes and the lamina propria of the intestine, as well as the cytoplasm of epithelial cells of the stomach. The staining intensity of ACP in the anterior and posterior intestines was stronger than in the stomach. ALP activity was detected in the striated border of enterocytes and muscularis of the whole intestine, lamina propria and supranuclear cytoplasm of the enterocytes in the anterior intestine, as well as in the blood vessels of the stomach. The staining intensity for ALP in the anterior intestine was stronger than in the posterior segment and the latter was stronger than in the stomach. NSE activity was detected in the cytoplasm of the epithelial cells in the entire gastrointestinal tract, with the anterior intestine showing stronger intensity than the stomach. POD activity was located in the blood cells of the lamina propria of the gastrointestinal tract and the levels in the stomach were similar to the anterior and posterior intestines. Alcian blue (pH 2·5) periodic acid Schiff (AB‐PAS) histochemical results revealed three types of mucous cells in the gastrointestinal tract. Type I cells (PAS+AB‐) were observed among the gastric mucosa columnar cells in the stomach and enterocytes in the basal region of the villi and in the middle and top regions of the intestinal villi. Type II cells (PAS‐AB+) and type III cells (PAS+AB+) were not detected in the stomach but were distributed ubiquitously among enterocytes in the middle and top regions of the intestinal villi.     

A study of morphology and histology of the alimentary tract of Glyptosternum maculatum (Sisoridae,Siluriformes)

Xiong, D., Zhang, L., Yu, H., Xie, C., Kong, Y., Zeng, Y., Huo, B. and Liu, Z. 2011. A study of morphology and histology of the alimentary tract of Glyptosternum maculatum (Sisoridae, Siluriformes). —Acta Zoologica (Stockholm) 92 : 161–169. The structure of alimentary tract has been studied in a cold water fish Glyptosternum maculatum, an endemic teleost species of notable economic importance and with high potential for controlled rearing of the species in Tibet, by light and electron microscope. Glyptosternum maculatum has short oesophagus, large caecal‐type stomach and short intestine, and the digestive tract with four layers: mucosa, submucosa, muscularis and serosa. Taste buds were found in the epithelium of lips, buccopharynx and oesophagus. The stratified epithelium of buccopharynx and oesophagus was located with numerous goblet cells. The U‐shaped stomach has three parts, corresponding to mammalian cardiac, fundus and pyloric portion, lined with a single‐layered columnar epithelium, and tubular gastric glands are present in cardiac and fundic portion, but absent in pyloric portion. No pyloric caeca was detected. The intestine is separated from the stomach by a loop valve. The intestine epithelium is composed of simple columnar cells with a distinct microvillus brush border and many goblet cells. Meanwhile, the intestinal coefficient was 0.898. At the ultrastuctural level, three type cells (mucous, glandular and endocrine cell) were found in the stomach, and glandular cell with a great amount of pepsinogen granules. The enterocytes of the intestinal mucosa display abundant endoplasmic reticulum, mitochondria and well‐developed microvilli. Congxin Xie, College of Fisheries, Huazhong Agricultural University, Wuhan, Hubei 430070, China. E‐mail: xiecongxin@mail.hzau.edu.cn or dreamsail_2005@yahoo.com.cn     

Toxoplasma gondii: Identification and characterization of bradyzoite-specific deoxyribose phosphate aldolase-like gene (TgDPA)

Toxoplasma gondii undergoes stage conversion from tachyzoites to bradyzoites in intermediate hosts. There have been many reports on bradyzoite-specific genes which are thought to be involved in stage conversion. Here, we described a novel T. gondii deoxyribose phosphate aldolase-like gene (TgDPA) expressing predominantly in bradyzoites. The TgDPA gene encodes 286 amino acids having a predicted molecular weight of 31 kDa. Sequence analysis revealed that TgDPA had a deoxyribose phosphate aldolase (DeoC) domain with about 30% homology with its Escherichia coli counterpart. RT- and quantitative PCR analyses showed that the TgDPA gene was more expressed in bradyzoites and that its expression gradually increased during in vitro tachyzoite-to-bradyzoite stage conversion. A polyclonal antibody against recombinant TgDPA protein was raised in rabbits, and immunofluorescent analysis demonstrated that TgDPA was expressed in bradyzoites in vivo and in vitro. These findings indicate that the TgDPA gene is a new bradyzoite-specific marker and might play a role in bradyzoites.     

Serologic evidence that ascaris and toxoplasma infections impact inflammatory responses to Helicobacter pylori in Colombians

Background: Helicobacter pylori‐infected children from coastal Tumaco, Colombia, have more parasitism, and adults have lower gastric cancer risk compared with high‐altitude Pasto/Tuquerres residents. Because helminth and Toxoplasma gondii infections alter helicobacter gastritis in rodent models, we determined whether seropositivity to Ascaris lumbricoides or T. gondii was associated with Th2‐IgG1 or Th1‐IgG2 responses to H. pylori. Methods: Sera (240) from the two populations were evaluated for A. lumbricoides and T. gondii seropositivity and results correlated with IgE and IgG isotype responses to H. pylori. Results: Most Tumaco children and adults were seropositive for A. lumbricoides (89%, 66%), T. gondii (59%, 98%), or both (45%, 66%). In contrast, seropositivity among Pasto/Tuquerres children was much lower (9%A. lumbricoides, 11%T. gondii, and 2% dual positive) but increased in adults (58%A. lumbricoides, 82%T. gondii, and 41% dual positive). A. lumbricoides seropositivity correlated with elevated IgE and anti‐inflammatory Th2‐IgG1 responses to H. pylori, while T. gondiigondii seropositivity was linked to elevated IgE, pro‐inflammatory Th1‐IgG2, IgG3, and IgG4 responses to H. pylori. Individuals with high T. gondii titers had reduced Th1‐IgG2, IgG3, and IgG4 responses to H. pylori. Conclusions: Results support regional differences for childhood parasitism and indicate A. lumbricoides and T. gondii infections may impact inflammatory responses to H. pylori and partially explain differences in gastric cancer risk in Colombia.