Comparative infectivity of Toxoplasma gondii bradyzoites in rats and mice

Infectivity of Toxoplasma gondii bradyzoites was compared in outbred female Sprague Dawley rats and outbred Swiss Webster mice. Rats inoculated subcutaneously with 1-10 bradyzoites of the 2 strains of T. gondii (VEG and GT-1) developed persistent infection, whereas an infective dose by the oral route was 10-1,000 bradyzoites. The infectivity of bradyzoites of the VEG and the GT-1 strains of T. gondii in rats by the subcutaneous route was comparable to that in mice.     

Antigenic similarity between Hammondia hammondi and Toxoplasma gondii tachyzoites

Hammondia hammondi is an obligate heteroxenous intestinal coccidian of cats, sharing many characteristics with Toxoplasma gondii. The tachyzoite stage antigens of T. gondii and H. hammondi were studied by immunofluorescence assays (IFA) and western blotting (WB) techniques to demonstrate antigenic similarities. Five monoclonal antibodies (MAbs), anti-T. gondii antigens, P22, P23, P30, P35, and P43, and mice polyclonal anti-H. hammondi serum were investigated. Antigens of H. hammondi were recognized by anti-P30 MAb both in IFA and in WB and by anti-P22 and anti-P35 MAbs only in IFA. Polyclonal anti-H. hammondi serum revealed many common antigens between the 2 parasites (30, 32, 35, 66, and 90 kDa). The differences of host parasite relationship between these 2 coccidians lead us to suggest that many of these antigens with similar molecular weights are not the same, but homologous, molecules or that they are not the only factors involved in these differences.     

Bradyzoite-induced murine toxoplasmosis: stage conversion, pathogenesis, and tissue cyst formation in mice fed bradyzoites of different strains of Toxoplasma gondii

The development of Toxoplasma gondii was studied in mice fed bradyzoites. At one hour after oral inoculation (HAI), bradyzoites were found in cells of the surface epithelium and the lamina propria of the small intestine, primarily the ileum. Division into two tachyzoites was first observed at 18 HAI in the intestine. At 24 HAI, organisms were also seen in mesenteric lymph nodes. Organisms were first detected in the brain at six days after oral inoculation with bradyzoites (DAI) but not consistently until 10 DAI. Immunohistochemical staining with bradyzoite specific (BAG-5 antigen) anti-serum showed that bradyzoites retained their BAG-5 reactivity even after the first division into two tachyzoites in the intestine at 18 HAI. BAG-5 positive organisms were not seen 2-5 DAI. BAG-5 antigens reappeared in T. gondii at 6 DAI. Whole mice and individual tissues of mice fed bradyzoites were bioassayed in cats and mice for the presence of bradyzoites. Feces of cats fed murine tissues were examined for oocyst shedding for short prepatent periods. Bradyzoites were present in the intestines of mice up to 12 HAI but not at 18 HAI, and tachyzoites and not bradyzoites disseminated to other tissues from the intestine. Bradyzoites were again detected 6 DAI. Using the mouse bioassay, T. gondii was first detected in peripheral blood at 24 HAI and more consistently at 48 HAI. Using a pepsin-digestion procedure and mouse bioassay, organisms were demonstrated in many tissues of mice 15 and 49 DAI.     

A cell culture system for study of the development of Toxoplasma gondii bradyzoites

Toxoplasma gondii is a ubiquitous apicomplexan parasite and a major opportunistic pathogen under AIDS-induced conditions, where it causes encephalitis when the bradyzoite (cyst) stage is reactivated. A bradyzoite-specific Mab, 74.1.8, reacting with a 28 kDa antigen, was used to study bradyzoite development in vitro by immuno-electron microscopy and immunofluorescence in human fibroblasts infected with ME49 strain T. gondii. Bradyzoites were detected in tissue culture within 3 days of infection. Free floating cyst-like structures were also identified. Western blotting demonstrated the expression of bradyzoite antigens in these free-floating cysts as well as in the monolayer. Bradyzoite development was increased by using media adjusted to pH 6.8 or 8.2. The addition of gamma-interferon at day 3 of culture while decreasing the total number of cysts formed prevented tachyzoite overgrowth and enabled study of in vitro bradyzoites for up to 25 days. The addition of IL-6 increased the number of cysts released into the medium and increased the number of cysts formed at pH 7.2. Confirmation of bradyzoite development in vitro was provided by electron microscopy. It is possible that the induction of an acute phase response in the host cell may be important for bradyzoite differentiation. This system should allow further studies on the effect of various agents on the development of bradyzoites.     

A method to obtain large quantities of Toxoplasma gondii tachyzoites with extreme purity

In order to extract genetic material from the intracellular parasite Toxoplasma gondii, large numbers of organisms free of host cells are necessary. A method is described in which the RH strain of T. gondii was cultivated in nonadherent U937 cells. The purification was done by countercurrent elutriation. The resulting population of 10(10) T. gondii contained only 0.17% host cell material. The method is simple, rapid, and minimizes the use of animals.     

Toxoplasma gondii: metabolism of intracellular tachyzoites is affected by host cell ATP production

PURPOSE: To evaluate different-caliber biopsy cutting needles in terms of the benefits and potential risk of bleeding in a swine model. MATERIALS AND METHODS: A total of 190 sequential liver biopsy specimens were obtained in 11 Yorkshire pigs (weight, 50-70 lb [22.5-31.5 kg]) by using 14-, 18-, and 20-gauge cutting needles. For each biopsy procedure, blood loss was determined by weighing sponges used to absorb bleeding, and sample-tissue DNA content was measured with spectrofluorometry. Analysis of variance was used to compare results. RESULTS: The larger the caliber of needle, the greater the absolute blood loss (for 14-gauge, 1.69 g; for 18-gauge, 0.74 g; for 20-gauge, 0.32 g) and DNA content per sample (for 14 gauge, 40.38 microg; for 18-gauge, 12.18 microg; for 20-gauge, 5.86 microg). The ratio of blood loss to amount of DNA recovered did not differ among the different-caliber needles. To obtain the same amount of diagnostic tissue, more passes were needed with the smaller-caliber needles. CONCLUSION: Use of larger-caliber needles is more efficient despite the greater amount of blood loss, because more tissue can be recovered and because fewer passes are necessary, which reduces the chances of complications.     

An immunochemical study of the antigens from Toxoplasma gondii tachyzoites obtained in different cultivation systems

Ureteral obstruction in a renal allograft, due to a variety of etiologies, is both a challenging diagnostic and therapeutic disorder. Since ureteral obstruction in a renal transplant recipient usually presents as azotemia, it must also be distinguished from acute rejection. Although ultrasound is non-invasive and readily available, the most definitive diagnostic tool is percutaneous nephrostomy tube placement with antegrade nephrostogram. A variety of therapeutic approaches are available to treat ureteral obstruction in a renal allograft. These procedures can be open (e.g., repeat ureteroneocystotomy) or utilize an endourological approach (e.g., transluminal ureteral dilatation). From an experimental standpoint, recent data in rodent models of experimental hydronephrosis demonstrate similar pathobiologic events in both the obstructed kidney and an allograft undergoing the chronic rejection process. To this end, investigation needs to be conducted to assess whether partial, unrecognized ureteral obstruction in an allograft hastens the development of chronic rejection. This would further underscore the importance of ureteral obstruction as a cause for not only acute azotemia in an allograft, but also chronic deterioration in renal transplant function.     

Detection of Toxoplasma gondii in 94 placentae from infected women by polymerase chain reaction, in vivo, and in vitro cultures

BACKGROUND: Several epidemiological studies have implicated hypercholesterolemia and hypertriglyceridaemia as a dietary risk factor in the etiology of vascular disease. To date, there are virtually no blood lipid data available for Negroid Black African Seventh-Day Adventist vegetarians. This study was undertaken to gain a preliminary and better understanding of the relationships between BP, blood lipids, and diets in adults at the Seventh-Day Adventist Seminary of West Africa, Ilisan-Remo, Nigeria. METHODS: Three randomly selected groups of the Nigerian populace with different dietary habits were investigated. The Seventh-Day Adventist Seminary of West Africa was the study area. Anthropometric measurements, blood pressure, serum cholesterol, triglycerides, and serum glucose were estimated using standard methods. FINDINGS: The vegetarians (VEGs) had significantly lower body weight 75.0 +/- 1.9 kg than the semi-vegetarians (SEMI-VEGs) 77.3 +/- 1.8 kg (p < 0.05). There was no significant difference between the blood pressure (BP) of the three groups studied, although the VEGs exhibited lower systolic BP. The VEGs had significantly lower serum total cholesterol and triglycerides (p < 0.05), than non-vegetarians (NON-VEGs). The SEMI-VEGs had blood triglycerides values in between NON-VEGs and VEGs levels but these were not significant. There were no differences in blood glucose in the three groups. CONCLUSIONS: The vegetarian diet as well as the African natural diet are associated with lower levels of important cardiovascular disease risk factors. The significantly lower cardiovascular disease risk factors in vegetarian African Adventists could be a protective measure against the development of premature IHD and CVD incidence.     

Characterization of Toxoplasma gondii from the feces of naturally infected cats

Feces of 1,000 cats from a humane shelter in Columbus, Ohio, were examined microscopically for oocysts of Toxoplasma gondii and by inoculation into mice. From the first 541 cats examined, oocysts of Toxoplasma were found in the feces of seven cats but in none of the remaining 459 cats. Results of the dye test in these seven cats showed titers of antibody of less than 1:2 in four cats, and of 1:8, 1:6, and 1:32 in the remaining three cats. The pathogenicity and infectivity of oocysts and cysts of all seven strains were compared in mice after oral and intraperitoneal inoculations. Oocysts and cysts were more pathogenic when administered by the oral route than by the intraperitoneal route. The cysts were less pathogenic than the oocysts. Excellent cross-immunity between six of these seven feline strains and the M-7741 strain was deomonstrated in cats by the fact that oocysts were not shed in feces of cats challenged with cysts of homologous or heterologous strains.     

Effects of 2',3'-dideoxyinosine on Toxoplasma gondii cysts in mice

The activity against Toxoplasma gondii of 2',3' dideoxyinosine (ddI), an anti-human immunodeficiency virus drug, was examined in an in vitro and in vivo study. Cell cultures infected with a strain known to cause chronic infections were used to show the dose-dependent effect of this drug compared with spiramycin and sulfadiazine. When a dose of 4 microg/ml was used, no infected THP-1 cells or parasites were found after 60 h of incubation. An electron-microscopic study confirmed that after 12 h at 1 microg/ml, the few parasites observed were severely altered. The treatment of chronically infected mice 3 months postinfection showed that a 30-day treatment with 2 mg of ddI/ml induced a significant reduction in the number of T. gondii cysts in the cerebral tissue. These cysts were not viable, as confirmed by immunofluorescence and reinfection experiments. These experiments suggest a possible role for ddI in the treatment of toxoplasmosis, and this possibility deserves further investigation.     

Rosette-forming cells during immune response to Toxoplasma gondii in mice

The rosette-forming cell (RFC) response of mice immunized with varying doses of Toxoplasma gondii was studied by immunocytoadherence (ICA). The specificity of ICA in the present system was tested by passive sensitization with hyperimmune serum in vivo and in vitro. A slight increase in RFC was observed with the latter. Prior treatment of spleen cells from immunized animals with rabbit anti-mouse immunoglobulin resulted in total inhibition of ICA. During the primary and the secondary response after 10 days, the number of RFC rose rapidly to reach the peak on the 3rd day. With secondary immunization 30 days later, the peak shifted to the 2nd day. Mice infected with a lower dose of Toxoplasma had a greater number of RFC during the secondary response after 10 days than with a larger dose.     

Detection by enzyme immunosorbent assay of Toxoplasma gondii IgG antibodies in dried blood spots on PKU-filter paper from newborns

OBJECTIVE: To assess the relative efficacy of a pacing esophageal stethoscope and intermittent boluses (40 mg) of gallamine in correcting sinus bradycardia (SB) during coronary artery surgery. DESIGN: The study was prospective, randomized, and controlled. SETTING: A community hospital. PARTICIPANTS: Fifty patients scheduled for elective coronary artery surgery. INTERVENTIONS: The patients were randomly allocated to receive treatment for an SB (less than 60 BPM) with either transesophageal atrial pacing (TAP) or gallamine. MEASUREMENTS AND MAIN RESULTS: Heart rate, blood pressure, and systemic hemodynamics were measured. The electrocardiogram was monitored for rate, rhythm, and conduction abnormalities. Twenty-four of the 25 TAP patients could be paced at a rate of 70 BPM after SB. Cardiac index increased from 1.90 to 2.56 L/min/m2. In the gallamine group, heart rate was increased from 50 to 66 BPM, but cardiac index only increased to 2.2 L/min/m2, and 2 patients developed nodal rhythms. Eight of these patients had peak heart rates over 80 BPM, and two were over 90 BPM. CONCLUSIONS: The ability to reliably and precisely control heart rate was superior with TAP compared with intermittent bolus dosing with gallamine.     

Invasiveness of 2 selected strains of Toxoplasma gondii with respect to mice, rats and rabbits

In Experiment I, groups of 22-and 140+-day rats were trained in acquisition and extinction of 1-way avoidance with a CS that consisted of the opening of a guilotine door 5 sec before US onset or a combination of door-opening plus a tonal signal that remained on until the occurrence of the motor response. Under both CS conditions, avoidance acquistion was similar at each age level. The extinction date indicated comparable performance for the young subjects but differential performance and greater resistance to extinction for the adult subjects. Adults trained with the door-opening CS persisted in responding for an entire series of 100 extinction trials, whereas the adults trained with the compound CS extinguished well within the 100-trial, whereas the adults trained with the compound CS extinguished well within the 100-trial limit. A 2nd experiment included 10 pre-exposures of the simple or compound CS's prior to avoidance training. Although the pups were insensitive to pre-exposure effects, the adults that were pre-exposed and trained with identical CS's showed evidence of pre-exposure effects. Results of both experiments were interpreted as indicative of differential cue saliency between ages.     

Kinetics study and characterisation of target excreted/secreted antigens of immunoglobulin G, M, A and E antibodies from mice infected with different strains of Toxoplasma gondii

A kinetics study and characterisation of target excreted/secreted antigens of immunoglobulin (Ig) G, M, A and E antibodies were realised by Western blotting with immune sera of mice inoculated with three strains of Toxoplasma gondii: RH, C56 and S3. IgG antibodies of the immune sera recognised the major proteins of the three excreted/secreted antigen preparations with molecular masses of 30, 45, 63 and 77 kDa. IgM antibodies recognised proteins revealed by IgG antibodies but with variable intensities; some proteins were revealed during a short period. IgA antibodies did not recognise the 35-kDa antigen or the antigens inferior to 28 kDa. The RH excreted/secreted antigens were revealed with the highest intensity. The IgE antibodies were briefly detected in trace amounts during period from the 20th to the 35th day. The RH strain with its excreted/secreted antigens had the best antigenicity and is a good model for immunoprotection studies.     

A dichotomous role for nitric oxide during acute Toxoplasma gondii infection in mice

Production of nitric oxide by macrophages is believed to be an important microbicidal mechanism for a variety of intracellular pathogens, including Toxoplasma gondii. Mice with a targeted disruption of the inducible nitric oxide synthase gene (iNOS) were infected orally with T. gondii tissue cysts. Time to death was prolonged compared with parental controls. Histologic analysis of tissue from infected mice showed scattered small foci of inflammation with parasites in various tissues of iNOS-/- mice, whereas tissue from the parental C57BL/6 mice had more extensive tissue inflammation with few visible parasites. In particular, extensive ulceration and necrosis of distal small intestine and fatty degeneration of the liver was seen in the parental mice at day 7 postinfection, as compared with the iNOS-/- mice where these tissues appeared normal. Serum interferon gamma and tumor necrosis factor alpha levels postinfection were equally elevated in both mouse strains. Treatment of the parental mice with a NO synthase inhibitor, aminoguanidine, prevented early death in these mice as well as the hepatic degeneration and small bowel necrosis seen in acutely infected control parentals. These findings indicate that NO production during acute infection with T. gondii can kill intracellular parasites but can be detrimental, even lethal, to the host.     

Acute and chronic phases of Toxoplasma gondii infection in mice modulate the host immune responses

Murine antibody responses to soluble proteins are generally restricted to the immunoglobulin G1 (IgG1) isotype. When mice were infected with Toxoplasma gondii Beverley and concomitantly immunized with a soluble unrelated protein antigen, a modification in the isotypic distribution of antibodies directed against this nonparasite antigen was observed, with a preferential production of IgG2a. Interestingly, when mice were immunized with a soluble protein antigen during the chronic phase (day 40) of infection with T. gondii Beverley, a similar modification in the isotypic distribution of antiprotein antibodies was observed.     

Serologic prevalence of Toxoplasma gondii in chickens in Madras, India

Serum samples from 185 chickens (Gallus gallus) collected from the various slaughter markets in and around Madras City, India were examined for antibodies to Toxoplasma gondii using the modified agglutination test incorporating mercaptoethanol. Antibodies (> or = 1:25) to T. gondii were found in 39.5% of sera. Antibody titers of individual sera (% in parentheses) were 1:25 (8.1%), 1:50 (10.8%), 1:100 (6.5%), 1:200(2.7%), 1:400 (4.3%), 1:800 (5.9%) 1:1,600 (0.5%), and 1:3,200 (0.5%).     

Ultrastructure of the oocysts, sporocysts, and sporozoites of Toxoplasma gondii

In this study we have investigated the subcellular localization in transfected COS-1 cells of the two major forms of the hepatitis C virus core protein: the immature protein of 191 residues (p21) and its proteolytically cleaved product of 173 residues (p19). In this study, and unlike previous investigations, we have been able to distinguish separately the localization of p21 from p19. This was achieved by the addition of a C-terminal HSV Tag to the p21 full coding sequence, and exploiting the fact that it is subsequently lost in the p19 product. In order to obtain an accurate localization of both p21 and p19 we used a mouse anti-HSV Tag MAb together with a human anti-core MAb (B12.F8) to perform double immunofluorescence studies. The results have shown that p21 is always localized around the nuclear envelope. On the other hand, p19 can be found diffused in the cytoplasm to different degrees. These in vivo results reinforce the proposed links between the regulated processing of the hepatitis C virus core protein and the possibility that this may contribute towards the regulation of its diverse biological functions.