Host cells of Toxoplasma gondii encystation in infected primary culture from mouse brain

In order to identify brain cell types that serve as host cells of Toxoplasma gondii encystation primary cultures from murine brain were infected and stained for neural and parasite stage-specific markers. In mixed culture inoculated with T. gondii tachyzoites, MAP2⁺ neurons, GFAP⁺ astrocytes, F4/80⁺ microglia, and O1⁺ oligodendrocytes proved to be infected as detected by parallel labeling of SAG1. At 4 days following infection with bradyzoites, cysts developed in neuronal, astroglial, and microglial host cells as clarified using bradyzoite-specific antibody 4F8. Additional staining of SAG1 revealed that astrocytes in bradyzoite-infected brain cell culture can also harbor tachyzoite-containing vacuoles. Stage conversion was observed shortly after inoculation and was accompanied by an increase in parasite proliferation. However, tachyzoites became rare in prolonged culture. By contrast, the numbers of cysts and of the bradyzoites isolated multiplied during long-term culture. These findings demonstrate that both glial and neuronal host cells allow T. gondii encystation in the absence of T cell-derived cytokines and imply that a brain-internal spreading of bradyzoites may sustain chronic infection. Received: 25 March 1997 / Accepted: 16 April 1997     

Histopathology of the reproductive system of male sheep experimentally infected with Toxoplasma gondii

The aim of this study was to investigate the histopathological changes in reproductive system (testicles, epididymis, seminal vesicles, and prostate) of small male ruminants after Toxoplasma gondii infection. Eight sheep were inoculated with T. gondii: group I, four sheep (2.0 × 10⁵ P-strain oocysts); group II, four sheep (1.0 × 10⁶ RH-strain tachyzoites); and group III, two uninfected sheep maintained as control. Infection with T. gondii was confirmed by seroconversion (indirect fluorescent antibody test-IgG) in all the infected animals beginning on post-inoculation day (PID) 7. On PID 70, all the animals were euthanized and tissue samples (testicles, epididymis, seminal vesicles, and prostate) were collected and processed for histological analysis. The main changes detected were a focal mononuclear interstitial inflammatory infiltrate in the prostate and seminal vesicles; diffuse testicular degeneration associated with calcification foci and a multifocal mononuclear interstitial inflammatory infiltrate; and a mononuclear interstitial infiltrate and focal necrotic areas of the muscle fibers surrounding the seminal vesicles. The histopathological findings of this work, along with the detection of T. gondii in the examined parenchyma tissues (immunohistochemistry) and the results obtained by other authors examining different tissues, suggest that histological changes diagnosed in the reproductive system of rams infected with T. gondii are strongly suggestive of toxoplasmatic infection.     

Transplacental transmission of Toxoplasma gondii in reinfected pregnant female canines

Twelve pregnant female canines, naturally infected with Toxoplasma gondii, were reinfected with T. gondii: three (GI) received tachyzoites subcutaneously (1.0 × 107), three (GII) were orally inoculated with oocysts (1.5 × 104), and six (GIII) were kept as a nonreinfected control group. All the reinfected female canines (GI and GII) miscarried or presented fetal death, while only one GIII female presented a stillborn in a litter of four pups (P < 0.01). Fever, lymphoadenopathy, miscarriage, and fetal death were the main clinical alterations observed. The highest serological titers detected through the indirect fluorescence antibody test (IFAT) were 1,024 (GI) and 4,096 (GII). In group III, the titers ranged between 64 and 256. By bioassays in mice, T. gondii was isolated in 17 organs of the reinfected adult canines, in 11 of the control group, and in 20 of the neonates. Positive immunostaining of cysts and/or tachyzoites were observed in 26 canine tissues (14 from GI and GII and ten from GIII). The agent was detected by immunohistochemistry in the encephalon of a neonate and in the spinal cord of a stillborn, thus, confirming that T. gondii infected canine fetuses, provoking miscarriages, even in bitches that presented primoinfection.     

Gerbils (Meriones unguiculatus) are highly susceptible to oral infection with Neospora caninum oocysts

Laboratory-reared gerbils (Meriones unguiculatus) were found to be highly susceptible to oral infection with Neospora caninum (NC-Liv strain) oocysts. Gerbils fed ∼1000 oocysts became sick or died at 6–13 days post feeding of oocysts (PFO). N. caninum was isolated in cell culture and from γ-interferon-knockout mice inoculated with homogenates of mesenteric lymph nodes of gerbils examined as early as 1 day PFO. Numerous N. caninum tachyzoites were found in ulcerative lesions in the intestines of gerbils examined at 7–9 days PFO. In a gerbil fed 10 oocysts, N. caninum tachyzoites were found in lesions in the brain. Gerbils fed 10 oocysts developed antibodies to N. caninum by 18 days PFO as determined by the Neospora agglutination test (titers ≥1:500). All gerbils remained negative for antibodies to Toxoplasma gondii as determined by the Toxoplasma agglutination test. Received: 10 September 1999 / Accepted: 10 September 1999     

Optimization of <Emphasis Type="Italic">Toxoplasma gondii</Emphasis> DNA extraction from amniotic fluid using NucliSENS easyMAG and comparison with QIAamp DNA minikit

Antenatal diagnosis of congenital toxoplasmosis relies on PCR in amniotic fluid. Because parasitic load is often low, DNA extraction must be optimized. Manual methods remain widespread although automated methods appear more effective. This study aimed at optimizing an automated method and at comparing it with a widespread manual method: QIAamp DNA minikit. To optimize NucliSens easyMAG, we evaluated the addition of proteinase K pre-treatment and the increase of the amount of silica particles used for the extraction. The optimized method was then compared to QIAamp DNA minikit on samples containing less than 25 tachyzoites/ml. NucliSens easyMAG DNA yield was improved after proteinase K pre-treatment (p < 0.01), but not with a higher silica particle input. The optimized method yielded more positive PCRs than the manual method, especially for samples containing 5 tachyzoites/ml or less (71% vs 26%, p < 10^-4). The DNA amount in samples found positive by PCR was higher after optimized automated extraction than after manual extraction (p < 10^-4). Proteinase K pre-treatment should be added to extract DNA from amniotic fluid using NucliSens easyMAG. Using this optimized automated method rather than manual methods would improve the sensitivity of Toxoplasma PCR and simplify the daily workflow.     

Improved methods for examination of Toxoplasma gondii cytoskeleton at ultrastructural level

To evaluate an improved method for identifying the presence of the structural elements of the cytoskeleton of Toxoplasma gondii and their influence on invasion of the parasite in host cells, copper grids coated with plastic film were used for adhesion of whole parasites. Tachyzoites were incubated with 0.5% Triton X-100 in PHEM buffer containing protease inhibitors, post-fixed in 1% glutaraldehyde, stained with uranyl acetate and submitted to critical point drying. In order to analyze the presence of the structural elements of the cytoskeleton, immunolocalization was carried out using colloidal gold. Invasion of the parasite was examined on cell culture after treatment of tachyzoites with cytochalasin B (CB). In order to observe this effect, an immunocytochemical assay using alkaline phosphatase was carried out. A very well conserved extraction of the cytoskeleton elements of T. gondii, such as conoid and microtubules, as well as the rhoptries, was observed. By immunolocalization with colloidal gold, it was possible to detect the actin in its globular form. In the assay of invasion of the parasite on the host cell, after treatment of the T. gondii tachyzoites with CB, the invasion process was totally inhibited. Received: 18 April 2000 / Accepted: 19 August 2000     

In vivo and in vitro activity of roxithromycin againstToxoplasma gondii in mice

Roxithromycin protected mice against a lethal infection with the extremely virulent RH strain and the CS6 strain ofToxoplasma gondii. Therapy initiated 2 h after infection with 2×10³ or 2×10⁴ RH or C56 tachyzoites protected more than 80% of the mice, compared with 0% of untreated controls (p < 0.001). Paradoxically,Toxoplasma gondii was isolated more frequently (> 80%)in treated mice surviving infection with the less virulent strain (CS6) than those surviving infection with the more virulent RH strain (< 25%). Furthermore, in vitro studies revealed that roxithromycin at dosages up to 250 g/ml had no effect on the proliferation ofToxoplasma gondii in murine peritoneal macrophage cultures.     

The effect of prolactin (PRL) on the growth of Toxoplasma gondii tachyzoites in vitro

During the development and effector phases of the anti-Toxoplasma response, the immunological system of a host is involved in several complex interactions with the endocrine system, and prolactin (PRL) is one of the most important hormones involved in immunoregulation. In this work, the influence of the recombinant human prolactin (rhPRL) on the viability, penetration, and intensity of intracellular proliferation of Toxoplasma gondii BK strain in vitro was evaluated. Using one murine (L929) and two human cell lines (Hs27 and HeLa), no toxic effect of the rhPRL on host cells was found (by determining cellular viability using MTT assay). A similar lack of rhPRL cytotoxic activity was found in the case of the extracellular tachyzoites of T. gondii BK. Replication of parasites in the presence of rhPRL was analyzed first by simultaneous addition of the hormone and the parasites into a microculture of the host cells (treatment during infection). No statistically significant changes in the intensity of parasite proliferation in all used host cells were found for a wide range of the hormone concentrations. However, pre-incubation of the tachyzoites with rhPRL resulted in a significant reduction (up to 36.15%) in the replication abilities of the parasite. Further experiments revealed that in fact, the inhibition of replication was caused by a limited capacity of the parasites to penetrate host’s cells as demonstrated by the reduced number of infected cells.     

Effect of hyperprolactinaemia on <Emphasis Type="Italic">Toxoplasma gondii</Emphasis> prevalence in humans

Recent studies have shown that hormones could induce anti-parasitic functions of the host immune system; thus, the aim of the present study was to estimate the seroprevalence of Toxoplasma gondii antibodies by an enzyme-linked immunosorbent assay in a Polish population of women and men with hyperprolactinaemia (n = 234) and hypoprolactinaemia (n = 41) and in a control group (n = 281) with the physiological level of prolactin (PRL). Women with hyperprolactinaemia revealed lower seroprevalence than those with normal PRL level (33.90% and 45.58%, respectively; p = 0.025). Detailed analysis of the results showed that twofold, threefold, fourfold and fivefold increase of the PRL concentration above the normal was correlated to the decrease of the T. gondii seroprevalence, but only in the group of women with a very high PRL level (>86 ng/ml) seroprevalence (12.50%) was significantly lower (p = 0.0004) than in the control subjects. These results confirm previously described suggestions on the relationship between hyperprolactinaemia and parasitic infection frequency. We postulate that a high level of PRL may be one of the important factors preventing T. gondii infection in women.     

In vitro sensitivity of Blastocystis hominis to garlic, ginger, white cumin, and black pepper used in diet

To determine the growth pattern and in vitro susceptibility of Blastocystis hominis to metronidazole (MTZ), garlic, ginger, white cumin, and black pepper. Stool specimens were collected from 16 irritable bowel syndrome (IBS) and 10 controls between July–November 2010. Stool microscopy and culture for B. hominis was performed. Drug susceptibility assays was done using 0.01 and 0.1 mg/ml of MTZ, garlic, ginger, white cumin, and black pepper. Effect was assessed on B. hominis culture after 48 h. Stool DNA was extracted using stool DNA extraction kit (Qiagen) and polymerase chain reaction (PCR) done using subtype-specific sequence-tagged-site primers. B. hominis genotype 3 and coinfection of 1 and 3 tended to grow well in culture compared to isolated type 1 infection. Exposed to MTZ at a concentration of 0.01 mg/ml, 38% (6/16) B. hominis from IBS did not grow in culture compared to 100% (10/10) of B. hominis from control (p = 0.001). When they were exposed to MTZ at 0.1 mg/ml, 56% (9/16) B. hominis from IBS did not grow in cultures compared to 100% (10/10) from control (p = 0.01). Forty-four percent (7/16) B. hominis from IBS did not grow in culture compared to 100% (10/10) B. hominis from control when exposed to garlic at a concentration of 0.01 mg/ml (p = 0.003) and following exposure to garlic at 0.1 mg/ml, 38% (6/16) B. hominis from IBS did not grow in cultures compared to 100% (10/10) from control (p = 0.001). B. hominis isolates from IBS had a cell count of 6,625 at a MTZ concentration of 0.01 mg/ml that reduced to 1,250 as MTZ concentration was increased to 0.1 mg/ml (p = 0.08). B. hominis from IBS with a mean cell count of 3 × 10⁵ at baseline decreased to 1 × 10⁴ when exposed to garlic at 0.01 mg/ml (p < 0.001) and to 1 × 10³ (p < 0.001) when garlic was 0.1 mg/ml. B. hominis from IBS cell count decreased to 1 × 10⁵ when exposed to white cumin at 0.01 mg/ml (p = 0.01) and to 1 × 10⁵ (p < 0.001) when white cumin was 0.1 mg/ml. Exposed to black pepper at 0.1 mg/ml, cell count of B. hominis from IBS decreased to 1 × 10⁵ (p = 0.01). B. hominis from IBS decreased to 1.3 × 10⁵ exposed to ginger at 0.01 mg/ml (p = 0.001). B. hominis isolates were mostly genotypes 3, type 1 and 3 coinfection, and non-typeable B. hominis isolates. B. hominis isolates from IBS mostly genotype 1 demonstrated an increased sensitivity to garlic at 0.01 mg/ml with a B. hominis cell count of 3,714 compared to 6,142 when exposed to 0.01 mg/ml of MTZ. However, this sensitivity did not increase as garlic concentration was increased to 0.1 mg/ml, for B. hominis cell count was 6,000 compared to 1,428 as MTZ was increased to 0.1 mg/ml.     

A study of virulence parameters for Toxoplasma gondii infections in mice

With the aim of establishing assessments of the virulence of Toxoplasma gondii isolates for mice, we investigated weight loss, serum levels of haptoglobin, and serum levels of tumor necrosis factor-alpha (TNF-α) as alternative parameters to mouse mortality. Groups of BALB/c mice were inoculated i.p. with increasing parasite doses (5 × 10¹, 10⁴, and 10⁶) of isolate 119 and with a low dose (5 × 10¹) of isolates GT1 and NED. The inoculation dose was inversely correlated with the interval to the onset of weight loss (starting at days 9, 7, and 5, respectively) and the onset of increase in serum haptoglobin (starting at about days 8, 6, and 4, respectively). The GT1 strain (inducing 100% mortality at day 9) also gave the fastest response in terms of weight loss (onset by day 8) and increase in serum haptoglobin (onset by day 6), which occurred 1–2 days before these parameters were affected in the other low-dose groups. The low-dose NED and 119 inoculations were distinguished by continued weight loss, which lasted until day 20 in the former group (max. average weight loss 2.9 g as compared with 0.4 g). Received: 31 August 1997 / Accepted: 15 October 1997     

Seroprevalence of and risk factors for Toxoplasma gondii antibodies among asymptomatic blood donors in Egypt

A cross-sectional study was conducted to evaluate the seroprevalence of and risk factors for Toxoplasma gondii antibodies in 260 blood donors seen at blood banks in Mansoura University Hospital, Egypt. Blood donors were interviewed about sociodemographic characteristics and risk factors for T. gondii infection. A blood sample was taken to document their T. gondii antibody status using enzyme-linked immunosorbent assay. Overall, 155 (59.6%) of 260 blood donors were positive for anti-T. gondii IgG antibodies. Multivariate logistic regression analysis showed a significant association between T. gondii seropositivity and eating meat by-products (luncheon/shawerma) (adjusted odds ratio [OR] 80.82 [95% CI 18.62–350.81], P < 0.0001) or being non-educated (adjusted OR 32.25 [95% CI 7.46–139.44], P < 0.0001). These findings highlight that T. gondii is prevalent among blood donors in Egypt.     

Toxoplasma gondii: Uptake of fetuin and identification of a 15-kDa fetuin-binding protein

Lectin-binding studies demonstrated the presence of a 68-kDa glycoprotein in tachyzoites ofToxoplasma gondii harvested from P388D1 macrophage cell cultures but not in tachyzoites maintained in peritoneal cavities of NMRI mice. This protein was identified as the embryonic protein fetuin that regularly is contained in fetal calf serum, a component of cell-culture media. Uptake of fetuin byT. gondii was demonstrated by intracellular localization of this protein. As shown by latex agglutination and immunofluorescence, no specific binding of fetuin to the parasite's surface was detected. Using affinity chromatography of fetuin-agarose, it was demonstrated that fetuin bound specifically to a 15-kDa antigen of tachyzoites. As revealed by inhibition studies with sialic acid and the lectinSambucus nigra agglutinin, the 15-kDa protein probably recognized glycan structures of fetuin.     

Seroprevalence of Neospora caninum and Toxoplasma gondii in camels (Camelus dromedarius) in Mashhad, Iran

One hundred twenty camels were blood-sampled and used to evaluate serological screening for Neospora caninum and Toxoplasma gondii infection by indirect fluorescent antibody test (IFAT) in Mashhad, Iran, during years 2004–2005. Of the 120 camels, antibodies to N. caninum were found in three in titers of 1:20 and in four in titers of 1:40 using whole N. caninum tachyzoites as IFAT slide (VMRD Inc., Pullman, WA 99163, USA). Antibodies to T. gondii were found in three camels in titers 1:20 and in two camels in titers 1:40 using whole T. gondii tachyzoites as IFAT slide (BIOGENE, Iran).     

Theileria-infected cell line from an African buffalo (Syncerus caffer)

Mononuclear cells were isolated from the peripheral blood of a buffalo infected with a Theileria sp. using density gradient centrifugation, and the cells were put into culture flasks covered by a monolayer of bovine endothelial cells. Twenty days after culture initiation, cells containing macroschizonts were detected in Giemsa-stained smears. The first subculture was carried out on day 45 of culture propagation. Subsequently, infected cells were subcultured twice a week, and each time 1 to 2 × 10⁶ per milliliter cells were harvested. DNA was extracted from culture material and a partial polymerase chain reaction amplification of the 18S ribosomal RNA (rRNA) gene was carried out using Theileria genus-specific primers. Sequence data and phylogenetic analysis using the 18S rRNA gene indicated a close relationship to Theileria sp. buffalo, previously described in literature. Here, the first successful attempt to establish a macroschizont-infected lymphoblastoid cell line of Theileria sp. (buffalo) from an African buffalo is described.     

Transient Extremity Ischemia Augments CD34+ Progenitor Cell Availability

Peripheral blood is an easily accessed source for stem cell production; however, the number of cells produced is relatively low. We hypothesized that ischemic preconditioning may serve as a safe method to increase the number of CD34+ cells that can be harvested and cultured in a short period. This study was conducted to test this hypothesis by examining the safety and efficacy of brief, transient ischemia of the lower limbs to augment the number of cells that can be produced from blood of healthy volunteers. Following induction of ischemia, blood samples were withdrawn at baseline, 30 min, 12 h and 24 h. The number of progenitor cells was determined by flow cytometry after the harvested cells were cultured for 5 days. We also analyzed the blood samples to determine IL-8 and VEGF concentrations. No serious adverse events were observed. The total number of cells increased from 0.46 ± 0.1 × 10⁶ cells/ml in the pretreatment blood samples to 0.7 ± 0.1 × 10⁶ cells/ml in blood taken 12 h after the conclusion of transient ischemia, p = 0.0029. The number of CD34+ cells increased from 4.23 ± 0.8 × 10⁴ cells/ml in the pretreatment samples to 7.17 ± 1.34 × 10⁴ cells/ml in blood taken 12 h after ischemia, p = 0.0001. The harvested stem cells maintained their ability to construct tubular structures. The augmentation in the number of CD34+ cells was positively correlated with the increase of IL-8, but not with VEGF concentrations. Ischemic preconditioning is a safe and effective technique to increase the availability of stem cells for therapeutic purposes.     

Mutation of the herpes simplex virus 1 KOS UL45 gene reveals dose dependent effects on central nervous system growth

Summary.  The herpes simplex virus type 1 (HSV-1) UL45 gene encodes an 18 kDa virion envelope protein whose function remains unknown. Previous studies using a UL45 null mutant, UL45Δ, demonstrated that deletion of the UL45 gene altered plaque size in Vero and HeLa cells, but was not essential for replication in these cell types. The goal of this study was to determine if mutation of the UL45 gene influenced virus growth in the CNS. Two UL45 mutants, as well as a repaired revertant virus, were constructed and tested for their ability to cause encephalitis and replicate in the CNS. The UL45 mutants were not lethal when 1 × 10³ pfu were injected intracerebrally into Balb/c mice. In contrast, at inocula greater than 1 × 10³, the UL45 mutants were lethal. In vivo growth curves derived from mice inoculated intracerebrally with 1 × 10³ pfu of virus revealed that the mutants grew poorly compared to wild type or revertant viruses. These results suggest that the 18 kDa UL45 gene product is required for efficient growth in the central nervous system at low doses. We propose that the UL45 gene may play an important role under the conditions of a naturally acquired infection. Received June 23, 2001 Accepted November 1, 2001     

Toxoplasma gondii from liquid nitrogen for continuous cell culture: methods to maximise efficient retrieval

This study aims to increase the efficiency of continuous growth of Toxoplasma gondii in HeLa cells from tachyzoite stocks frozen in liquid nitrogen. Freezing and retrieval of tachyzoites for continuous cell culture requires more stringent protocols than those published for animal culture. The freezing and retrieval conditions are optimised so that a quality harvest (> or = 1 x 10(6) tachyzoites/mL, > or = 90% viability) can be produced using T. gondii recovered from liquid nitrogen as fast and reliably as possible. Retrieval success rate increased from 36% to 100%. An improved freezing procedure using chilled reagents and freshly harvested parasites, and adoption of an effective recovery protocol with retrieval of 3 x 10(7) tachyzoites into 75 cm2 flasks, change of maintenance media after six hours and subsequent blind passage all contributed to this success. The result is faster and more dependable production of T. gondii for diagnostic and experimental use.     

Comparative evaluation of the sensitivity of LAMP, PCR and in vitro culture methods for the diagnosis of equine piroplasmosis

The sensitivity of LAMP, PCR and in vitro culture methods for the detection of Theileria equi and Babesia caballi was evaluated using tenfold serially diluted culture parasites. On day 1 post-culture, both T. equi and B. caballi parasites could only be observed at 1% parasite dilution from the in vitro culture method, whereas LAMP could detect up to 1 × 10⁻³% of both T. equi and B. caballi parasite dilutions, whilst PCR could detect 1 × 10⁻³% T. equi and 1 × 10⁻¹% B. caballi parasite dilutions. On day 7 post-culture, the detection limit for T. equi and B. caballi in the in vitro culture increased up to 1 × 10⁻⁶%, whereas LAMP detection limit increased to 1 × 10⁻¹⁰% for both parasites, whilst the PCR detection limit increased to 1 × 10⁻¹⁰% and 1 × 10⁻⁶% for T. equi and B. caballi, respectively. Furthermore, LAMP and PCR amplified the T. equi DNA extracted from the organs of an experimentally infected horse. This study further validates LAMP as an alternative molecular diagnostic tool, which can be used in the diagnosis of early infections of equine piroplasmosis and together with PCR can also be used as supplementary methods during post-mortems.     

Detection of <Emphasis Type="Italic">Toxoplasma gondii</Emphasis> in water by an immunomagnetic separation method targeting the sporocysts

An immunomagnetic separation (IMS) method was developed to detect Toxoplasma gondii in fresh waters by using the monoclonal antibody 4B6 targeting the sporocyst wall of T. gondii, Hammondia hammondi, Hammondia heydorni, and Neospora caninum. Water concentrates obtained by filtering 10- to 20-l samples samples were spiked with Toxoplasma oocysts, sonicated to release the sporocysts, and analyzed by IMS-4B6. Mean sporocyst recoveries were 74.5 ± 5.3% in drinking water, 30.6 ± 2.4 and 37.1 ± 3.2% in surface waters, and 81.6 ± 2.1% in IMS buffer. Then, this IMS method was integrated in a multistep procedure (i.e., filtration, IMS, immunofluorescence and autofluorescence) to detect Toxoplasma in unspiked and spiked water samples (10–30 l) of various qualities. Sporocyst recoveries ranged from 14.4 to 44.7% in drinking water samples spiked with 1–10 oocysts/l, and from 17.8 to 32.5% in surface water samples spiked with 10 oocysts/l. Sporocysts were not detected in 25 unspiked water samples. A sporocyst-like structure was seen in one of these unspiked samples, but its coccidian nature could not be proved by three polymerase chain reaction (PCR) methods targeting sequences of coccidian small and large subunit rRNA genes and Toxoplasma repetitive elements. In conclusion, IMS-4B6 is relevant for the detection of Toxoplasma in water generating small concentrates (<1 ml). Due to 4B6 cross-reactions, a PCR would be useful to further characterize coccidian sporocysts found microscopically.