Whole genome sequencing of a natural recombinant Toxoplasma gondii strain reveals chromosome sorting and local allelic variants

Background  

Toxoplasma gondii is a zoonotic parasite of global importance. In common with many protozoan parasites it has the capacity for sexual recombination, but current evidence suggests this is rarely employed. The global population structure is dominated by a small number of clonal genotypes, which exhibit biallelic variation and limited intralineage divergence. Little is known of the genotypes present in Africa despite the importance of AIDS-associated toxoplasmosis.     

Immunogenicity and Efficacy of Single Antigen Gp63, Polytope and PolytopeHSP70 DNA Vaccines against Visceral Leishmaniasis in Experimental Mouse Model

Polytope approach of genetic immunization is a promising strategy for the prevention of infectious disease as it is capable of generating effective cell mediated immunity by delivering the T cell epitopes assembled in series. Leishmaniasis is a significant world wide health problem for which no vaccine exists. In this study we have compared immunogenicity and efficacy of three types of DNA vaccines: single antigen Gp63 (Gp63/pcDNA), polytope (Poly/pcDNA) and Polytope fused with hsp70 (Poly/hsp/pcDNA) against visceral leishmaniasis in susceptible BALB/c mice. Mice vaccinated with these plasmids generated strong Th1 immune response as seen by dominating IFN-γ over IL-10 cytokine. Interestingly, cytotoxic responses generated by polytope DNA plasmid fused with hsp70 of Leishmania donovani were significantly higher when compared to polytope and single antigen Gp63 vaccine. Challenge studies revealed that the parasite load in liver and spleen was significantly lower with Poly/hsp/pcDNA vaccination compared to other vaccines. Therefore, our study indicates that polytope DNA vaccine is a feasible, practical and effective approach for visceral leishmaniasis.     

Multiplex PCR assay for rapid identification of three endangered snake species of India

Species identification has been the core issue in all approaches of conservation of endangered wild life. In this regard molecular techniques for species authentication have proved indispensable. A novel multiplex PCR assay for the identification of three Indian snake species Python morulus, Ptyas mucosus, and Naja naja is successfully demonstrated using 16S rRNA gene. Three reverse primers and a common forward primer were designed to generate three different size species-specific PCR fragments. Absence of any PCR amplification in non-target species proves the specificity of the primers. These four primers were combined in a multiplex assay to enable identification of three snake species in a single reaction. The assay described here shows its utility in identifying unknown snake specimen and in case of samples yielding low quality DNA. This multiplex PCR technique using novel primers is an unprecedented approach offered for forensic identification of exhibits originating from three Indian snake species. It is expected that this endeavor will help strengthening conservation efforts for these species.     

Molecular identification of crocodile species using novel primers for forensic analysis

All crocodilians are under varying degrees of threat due to over exploitation and these species have been listed in Appendix I or II of CITES. The lack of molecular techniques for the identification of confiscated samples makes it difficult to enforce the law. Conclusive forensic identification of species requires a complete gene sequence which is difficult in case of degraded samples. We have developed two novel sets of primers to amplify two partial cytochrome b gene sequences of six crocodile species i.e. Crocodylus palustris, Crocodylus porosus, Crocodylus siamensis, Crocodylus niloticus, Gavialis gangeticus and Caiman crocodilus. These partial sequences were edited to give a complete cyt b gene sequence, which can be used as an effective tool for forensic authentication of crocodile species. A phylogeny of crocodile species was reconstructed using these sequences. The described primers hold great promise in forensic identification of crocodile species, which can aid in the effective enforcement of law and conservation of these ancient species.     

Effects of high-pressure processing on Toxoplasma gondii tissue cysts in ground pork

Ingestion of Toxoplasma gondii tissue cysts can result in severe disease in immunocompromised individuals and pregnant women. Treatment of meat and meat products to eliminate viable T. gondii tissue cysts would provide a means to protect consumers. In this study, we examined the effects of high-pressure processing (HPP) on ground pork containing viable tissue cysts of the VEG strain of T. gondii. Ground pork containing tissue cysts was exposed to 400, 300, 200, 100, or 0 MPa treatment for 30, 60, or 90 sec in a commercial HPP unit. The HPP-treated ground pork was subjected to acid-pepsin digestion and bioassayed in mice. The results of the mouse bioassay revealed that none of the mice inoculated with tissue cysts exposed to 400 or 300 MPa became infected, whereas all mice inoculated with tissue cysts exposed to 200, 100, or 0 MPa became infected with T. gondii regardless of exposure time. Results indicate that HPP treatment of ground pork with 300 MPa of pressure will render tissue cysts of T. gondii nonviable and make pork safe for human consumption.     

Seroprevalence of Toxoplasma gondii antibodies in humans from rural Western Amazon, Brazil

Antibodies to Toxoplasma gondii were assayed in sera of 266 humans from 71 farms located at Rond?nia State, Western Amazon, Brazil, by the modified agglutination test (MAT) and the indirect immunofluorescent antibody test (IFAT). Antibodies were found in 195 humans (73.3%), with MAT titers of 1:25 in 11, 1:50 in 11, 1:100 in 16, 1:200 in 27, 1:400 in 38, 1:800 in 37, 1:1,600 in 22, and 1:3,200 or higher in 33. From the 71 farms visited, 69 had seropositive humans. Prevalence of anti-T. gondii antibodies increased with age of the people (P < 0.05), and no difference was observed in the occurrence by gender (P > 0.05). A sanitary questionnaire was applied in each farm, and statistical association between the serologic status and several variables were analyzed. Home-grown vegetable consumption and origin of drinking water (well or river) were the independent variables that displayed significant association (P = 0.002 and 0.02, respectively). Higher values of occurrence were found in people with consumption of home-grown vegetables (76.1%) and people that drink well water (75.4%) compared with people that did not consume this type of food (61.9%) and drink river water (55.2%). By IFAT (> or = 1:16), 194 of 266 (73%) humans were seropositive and there was a good correlation between MAT and IFAT.     

Serologic survey for Toxoplasma gondii and Neospora caninum in the common brushtail possum (Trichosurus vulpecula) from urban Sydney, Australia

The common brushtail possum (Trichosurus vulpecula) has well adapted to increasing urbanization, resulting in greater interaction with humans and their domestic pets. Wildlife species in urban areas face a higher risk of exposure to zoonotic pathogens and may be affected by parasites hosted by cats (Toxoplasma gondii) or dogs (Neospora caninum), yet it is unknown to what extent urban T. vulpecula are exposed to these parasites. Antibodies to T. gondii and N. caninum were assayed in sera of 142 adult possums from the city of Sydney, Australia. Using the modified agglutination test, antibodies to T. gondii were found in 9 (6.3%) of the 142 animals in titers of 1:25 (4), 1:50 (1), 1:100 (1), 1:800 (1), 1:3,200 (1), 1:6,400 (1), and 1:12,800 (1). Of some T. vulpecula multiple sera samples within a 2-yr frame could be collected, but seropositive animals in general were not recaptured after initial seroconversion. One possum had a high T. gondii titer on 2 consecutive bleedings, 14 mo apart, and seropositive possums appeared normal when captured. Sex seemed not to have an affect on antibody prevalence, but age and location may play a role. Antibodies to N. caninum were not detected in 1:25 dilution of sera in the N. caninum agglutination test, indicating that T. vulpecula may not have been exposed to this parasite. This is the first serological survey for T. gondii and N. caninum infections in urban T. vulpecula.     

A differential drug screen for compounds that select against antibiotic resistance

Antibiotics increase the frequency of resistant bacteria by providing them a competitive advantage over sensitive strains. Here, we develop a versatile assay for differential chemical inhibition of competing microbial strains, and use it to identify compounds that preferentially inhibit tetracycline-resistant relative to sensitive bacteria, thus "inverting" selection for resistance. Our assay distinguishes compounds selecting directly against specific resistance mechanisms and compounds whose selection against resistance is based on their physiological interaction with tetracycline and is more general with respect to resistance mechanism. A pilot screen indicates that both types of selection-inverting compounds are secreted by soil microbes, suggesting that nature has evolved a repertoire of chemicals that counteracts antibiotic resistance. Finally, we show that our assay can more generally permit simple, direct screening for drugs based on their differential activity against different strains or targets.