Genetic evidence that an endosymbiont-derived endoplasmic reticulum-associated protein degradation (ERAD) system functions in import of apicoplast proteins.

Most apicomplexan parasites harbor a relict chloroplast, the apicoplast, that is critical for their survival. Whereas the apicoplast maintains a small genome, the bulk of its proteins are nuclear encoded and imported into the organelle. Several models have been proposed to explain how proteins might cross the four membranes that surround the apicoplast; however, experimental data discriminating these models are largely missing. Here we present genetic evidence that apicoplast protein import depends on elements derived from the ER-associated protein degradation (ERAD) system of the endosymbiont. We identified two sets of ERAD components in Toxoplasma gondii, one associated with the ER and cytoplasm and one localized to the membranes of the apicoplast. We engineered a conditional null mutant in apicoplast Der1, the putative pore of the apicoplast ERAD complex, and found that loss of Der1(Ap) results in loss of apicoplast protein import and subsequent death of the parasite.

Basic heel prick test: inclusion of screening, diagnosis and criteria for early confirmation of congenital infection by Toxoplasma gondii.

Toxoplasma gondii can cross the placental barrier, causing fetal infection with potentially severe sequelae. The aim of this study was to evaluate whether the serological screening for toxoplasmosis should be included in the basic neonatal heel prick test in order to establish criteria for the confirmation and/or exclusion of the diagnosis of congenital infection in newborns treated at three public health units in the metropolitan region of Goiania, Goias State, Brazil. Blood samples were collected on filter paper from newborns and later, peripheral blood samples from the mothers and their respective children were obtained to confirm or exclude the diagnosis of suspected congenital infection, by means of an enzyme-linked immunosorbent assay (IgM and IgG) and a polymerase chain reaction assay. From a total of 1,159 blood samples collected on filter paper, 43.92% were reactive to IgG and 0.17% to anti-T. gondii IgM and IgG. One hundred and twenty-seven paired samples (mother and child) were collected following consensual protocols for peripheral blood collection. Results obtained from the filter paper and peripheral blood of the newborns were 90.55% concordant. A comparison of the mother and child blood test results showed agreement regarding the detection of IgG in 90.48% of the samples. The parasite DNA was detected in the peripheral blood of one child. In view of the results obtained in this study, the inclusion of the serological screening for toxoplasmosis in the newborn heel prick test proved to be effective for the early detection of congenital T. gondii infection.

Apicomplexan autophagy and modulation of autophagy in parasite-infected host cells.

Apicomplexan parasites are responsible for a number of important human pathologies. Obviously, as Eukaryotes they share a number of cellular features and pathways with their respective host cells. One of them is autophagy, a process involved in the degradation of the cell's own components. These intracellular parasites nonetheless seem to present a number of original features compared to their very evolutionarily distant host cells. In mammals and other metazoans, autophagy has been identified as an important contributor to the defence against microbial pathogens. Thus, host autophagy also likely plays a key role in the control of apicomplexan parasites, although its potential manipulation and subversion by intracellular parasites creates a complex interplay in the regulation of host and parasite autophagy. In this mini-review, we summarise current knowledge on autophagy in both parasites and their host cells, in the context of infection by three Apicomplexa: Plasmodium, Toxoplasma, and Theileria.

Gram-negative bacteria aggravate murine small intestinal Th1-type immunopathology following oral infection with Toxoplasma gondii.

Oral infection of susceptible mice with Toxoplasma gondii results in Th1-type immunopathology in the ileum. We investigated gut flora changes during ileitis and determined contributions of gut bacteria to intestinal inflammation. Analysis of the intestinal microflora revealed that ileitis was accompanied by increasing bacterial load, decreasing species diversity, and bacterial translocation. Gram-negative bacteria identified as Escherichia coli and Bacteroides/Prevotella spp. accumulated in inflamed ileum at high concentrations. Prophylactic or therapeutic administration of ciprofloxacin and/or metronidazole ameliorated ileal immunopathology and reduced intestinal NO and IFN-gamma levels. Most strikingly, gnotobiotic mice in which cultivable gut bacteria were removed by quintuple antibiotic treatment did not develop ileitis after Toxoplasma gondii infection. A reduction in total numbers of lymphocytes was observed in the lamina propria of specific pathogen-free (SPF), but not gnotobiotic, mice upon development of ileitis. Relative numbers of CD4(+) T cells did not differ in naive vs infected gnotobiotic or SPF mice, but infected SPF mice showed a significant increase in the frequencies of activated CD4(+) T cells compared with gnotobiotic mice. Furthermore, recolonization with total gut flora, E. coli, or Bacteroides/Prevotella spp., but not Lactobacillus johnsonii, induced immunopathology in gnotobiotic mice. Animals recolonized with E. coli and/or total gut flora, but not L. johnsonii, showed elevated ileal NO and/or IFN-gamma levels. In conclusion, Gram-negative bacteria, i.e., E. coli, aggravate pathogen-induced intestinal Th1-type immunopathology. Thus, pathogen-induced acute ileitis may prove useful to study bacteria-host interactions in small intestinal inflammation and to test novel therapies based on modulation of gut flora.

Neosporosis y toxoplasmosis en la liebre europea (Lepus europaeus) en la provincia de La Pampa (Argentina) / Infectious diseases and reproductive status in european hare (Lepus europaeus) in La Pampa province (Argentina)

El objetivo de este trabajo fue determinar la prevalencia a Neospora caninum (Nc) y Toxoplasma gondii (Tg) y conocer el periodo reproductivo de la liebre europea (Lepus europaeus) en la provincia de La Pampa (Argentina). Para ello se muestrearon 106 liebres procedentes de acopiadores de la provincia de La Pampa. Los sueros fueron analizados por un ensayo inmunoenzimático de competición para determinar anticuerpos a Nc y para Tg se realizó una prueba de hemaglutinación indirecta. Se encontraron 66 hembras, de las cuales 13 estaban preñadas, y 40 machos (p=0,01). Sobre un total de 44 sueros analizados para Nc, 5 (11,4%) dieron positivo. De los 106 sueros estudiados para Tg ninguno fue positivo.

The objective of this study was to determine the prevalence of these diseases and to know the reproductive season of the European hare (Lepus europaeus) in the Province of La Pampa (Argentina). To that aim, 106 hares coming from different places from La Pampa province were sampled. .Nc antibody diagnosis was performed by means of a competitive enzimoimmunoassay (ELISA-c), and an hemo-agglutination indirect assay (HAI) for Tg were carried out to know the disease status. From the total of hares sampled, 40 were male, 66 female (p=0.01) and 13 (19.7%) of them were detected as pregnant. Nc sero-prevalence was 11.4% over a total of 44. From 106 sera analyzed by HAI no one was positive for Tg.

A plastid segregation defect in the protozoan parasite Toxoplasma gondii.

Apicomplexan parasites--including the causative agents of malaria (Plasmodium sp.) and toxoplasmosis (Toxoplasma gondii)--harbor a secondary endosymbiotic plastid, acquired by lateral genetic transfer from a eukaryotic alga. The apicoplast has attracted considerable attention, both as an evolutionary novelty and as a potential target for chemotherapy. We report a recombinant fusion (between a nuclear-encoded apicoplast protein, the green fluorescent protein and a rhoptry protein) that targets to the apicoplast but grossly alters its morphology, preventing organellar segregation during parasite division. Apicoplast-deficient parasites replicate normally in the first infectious cycle and can be isolated by fluorescence-activated cell sorting, but die in the subsequent host cell, confirming the 'delayed death' phenotype previously described pharmacologically, and validating the apicoplast as essential for parasite viability.

Método sencillo para determinar la presencia de Toxoplasma gondii (Eucoccidia: Sarcocystidae) en carne / Simplifie method to determinated presence of Toxoplasma gondii in meat

Presence of T. gondii in meat from several sources was determined by traditional methods and by a new simplified method in wich animals are fed meat directly without any previous treatment with artificial gastric fluid. Tissues are ground and the animals ingest them naturally. Determination of Toxoplasma in lungs or brain (as well as by specific antibody test), showed no statistically significant differences between both methods.