Immunomagnetic separation of Toxoplasma gondii and Hammondia spp. tissue cysts generated in cell culture.

Toxoplasma gondii is commonly transmitted among animals and humans by ingestion of infected animal tissues or by consumption of food and water contaminated with environmentally-resistant oocysts excreted by cats. Tissue cysts and oocysts have different walls, whose structures and compositions are poorly known. Herein, we describe an immunomagnetic separation (IMS) method that was successfully used for purification of T. gondii tissue cysts generated in cell culture. We used an IgG monoclonal antibody (mAb) that reacts against antigens in tissue cyst walls. Many in vitro produced cysts were obtained by this IMS; >2,000 T. gondii cysts were isolated from a single culture flask of 25 cm2. Tissue cysts from two Hammondia spp., H. hammondi, and H. heydorni, produced in cell culture were also separated using this method. As a reference, purification of tissue cysts by Percoll gradients was used. Percoll was able to separate T. gondii tissue cysts produced in mice but was not suitable for purifying T. gondii tissue cysts produced in vitro. The IMS described here should favor proteomic studies involving tissue cysts of T. gondii.

Cystoisospora belli, liver disease and hypothesis on the life cycle.

Cystoisospora belli causes chronic diarrhoea, acalculous cholecystitis, cholangiopathy and disseminated cystoisosporosis in patients with AIDS. Clinical manifestations and histological stages during C. belli infection in a patient with AIDS and liver disease were described. It was possible to identify sporozoite-like structures in the villus epithelium of the duodenum, close to the vascularization that underlies the basal membrane and unizoite tissue cysts near to the vascularization in the lamina propria. Unizoite tissue cysts were found inside of sinusoids in the liver communicating with the central vein and with a bile canaliculus and portal spaces. Based on these findings a hypothesis on C. belli life cycle could consider that the route of migration of unizoite tissue cysts up the liver is via the portal blood. The unizoite tissue cysts located in hepatic portal vein could migrated via sinusoid to central vein and general circulation through the venous system. The unizoite tissue cysts could also return via bile canaliculus to bile duct to portal triad. This hypothesis allows to understand the presence of unizoite stages in blood, the pathway by which the bile ducts become infected and unizoites in the liver being able to behave like hypnozoites that favour relapses and treatment failures.

First record of an infection by tissue cyst-forming coccidia in wild vizcachas (Lagostomus maximus, Rodentia) of Argentina.

Endoparasites of the Sarcocystidae family share the ability to form tissue cysts in their intermediate hosts, ultimately leading to pathogenesis in the definitive hosts that include various mammals, reptiles and birds. In our research on the endocrinology of the female vizcachas (Lagostomus maximus), we have found abnormal cystic structures in the ovaries of some individuals. So far, no cases of infection by tissue cyst-forming parasites have been reported in this species. To evaluate whether this autochthonous wild rodent is an intermediate host of an undescribed endoparasite, histological sections from various organs were examined. Pinhead-sized tissue cysts were found in the ovaries, mammary glands, uterus, pituitary, brain, adrenals and spleen, of both pregnant and non-pregnant females. The presence of cysts in the adult brain and embryonic tissue is indicative of the ability of the parasite to cross both the blood-brain and placental barriers. The infected brains exhibited a lower cyst density than that seen in other organs. Regardless of their location in superficial or deep tissue, the cysts were surrounded by a layer of connective tissue. Histologically, the cyst wall consisted of an outer layer of fibroblasts and collagen fibers, and an inner, granular-looking layer composed of host nucleated cells surrounding thousands of spindle-shaped bradyzoites. Outside the cysts, the host cellular structures showed normal appearance. The remarkable morphological similarities between the cysts studied here with those reported in naturally infected rabbits from an area neighboring the one inhabited by the vizcachas point to Besnoitia sp. as a plausible candidate. More studies will be necessary to confirm the identity of the parasite. Nevertheless, this is the first report of L. maximus as an intermediate host for a tissue cyst-forming coccidia.

Development of Cystoisospora felis in Cell Culture and in vitro Formation of Monozoic Tissue Cysts.

Cystoisospora felis is a coccidian parasite commonly found in feces of domestic cats. Infection in cats occurs by ingestion of sporulated oocysts or consumption of rodents infected by the parasite. Scarce information is available about extraintestinal stages of C. felis in naturally infected intermediate hosts, as well as in cell culture. The aim of the current work was to investigate the development of C. felis in Vero cells (African green monkey kidney) and MDCK cells (Madin-Darby canine kidney). Cell monolayers were inoculated with mechanically released sporozoites of C. felis, and parasite growth was daily examined using light microscopy. After cell invasion, only parasitophorous vacuoles containing a single zoite were observed. Five days post-inoculation with sporozoites, unstained cell monolayers were evaluated by differential interference contrast (DIC), and also by Romanovsky stain using conventional light microscopy. Single zoites, each surrounded by a cyst wall, were observed by both methods. Multiplication by endodyogeny did not occur in any cell monolayer. Treatment of encysted parasites with HCl-pepsin for 15 min led to dissolution of the cyst wall and release of intact and motile zoites. To our knowledge, this is the first demonstration of in vitro production of monozoic tissue cysts of C. felis. As kittens commonly shed C. felis in their feces, oocysts are easily available for in vitro production of monozoic tissue cysts of the parasite. Development of C. felis in cell culture may be employed as a model on tissue cyst formation of Cystoisospora spp. and closely related coccidia.

Presence of Toxoplasma gondii in Pork Intended for Human Consumption in Tropical Southern Mexico.

BACKGROUND: Toxoplasmosis is caused by the protozoon Toxoplasma gondii, which is one of the most widespread parasites that infect animals and humans worldwide. One of the main routes of infection for humans is through the consumption of infected meat containing bradyzoites in tissue cysts. Pork is one of the foremost meat types associated with outbreaks of acute toxoplasmosis in humans. MATERIALS AND METHODS: Sixty blood samples were collected from finished pigs at slaughter and their sera was evaluated by an indirect-IgG ELISA. Matched muscle samples were obtained from the tongue and loin. Whole blood and tissue samples were evaluated to search for T. gondii DNA using a nested-polymerase chain reaction. RESULTS: Seroprevalence of T. gondii was 96.6% (58/60) of sampled pigs. Meanwhile, T. gondii DNA was present in 23.21% of tongue tissue samples (13/56), 7% of loin tissues (4/57), and 0% in blood samples (0/44), respectively. Two pigs were serologically indeterminate. CONCLUSION: This is the first report of the presence of T. gondii DNA in tissue samples obtained from finalized pigs. Results from the present study suggest a high exposure to T. gondii in pigs intended for human consumption from the tropical region of Mexico. Thus, the consumption of some undercooked pork meat meals typical from the southern region of Mexico could represent a significant risk for acquiring infection for the human population.

Phenotypic and genotypic characterization of two Toxoplasma gondii isolates in free-range chickens from Uberlândia, Brazil.

The aim of this study was to determine the seroprevalence of Toxoplasma gondii infection in free-range chickens from Uberlândia, Minas Gerais state, Brazil, and characterize the genotypic and phenotypic features of two isolates of this parasite, considering the importance of these hosts in the epidemiology of toxoplasmosis. Serum samples from 108 free-range chickens were obtained from ten different districts, and submitted to the modified agglutination test (MAT) for the presence of anti-T. gondii antibodies, and brain and heart tissue samples from infected chickens were processed for mouse bioassay. An overall seroprevalence of 71·3% was found and antibody titres ranged from 16 to 4096. After confirmation of seropositivity by mouse bioassay, the determination of the T. gondii genotypes of two isolates was performed by PCR-RFLP, using primers for the following markers: SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, new SAG2, Apico and CS3. These T. gondii isolates, designated TgChBrUD1and TgChBrUD2, were obtained from heart samples of free-range chickens. The TgChBrUD1 isolate belonged to ToxoDB PCR-RFLP genotype 11 and the TgChBrUD2 isolate belonged to ToxoDB PCR-RFLP genotype 6. Both isolates demonstrated high virulence in a rodent model, with the TgChBrUD1 isolate able to induce brain cysts, in accord with its pattern of multiplication rates in human fibroblast culture. Taken together, these results reveal high prevalence of T. gondii infection in free-range chickens throughout Uberlândia, indicating an important degree of oocyst environmental contamination and the existence of considerable risk for T. gondii transmission to humans by consumption of free-range chicken as a food source.