High prevalence and abundant atypical genotypes of Toxoplasma gondii isolated from lambs destined for human consumption in the USA

Little information is available on the presence of viable Toxoplasma gondii in tissues of lambs worldwide. The prevalence of T. gondii was determined in 383 lambs (<1 year old) from Maryland, Virginia and West Virginia, USA. Hearts of 383 lambs were obtained from a slaughter house on the day of killing. Blood removed from each heart was tested for antibodies to T. gondii by using the modified agglutination test (MAT). Sera were first screened using 1:25, 1:50, 1: 100 and 1:200 dilutions, and hearts were selected for bioassay for T. gondii. Antibodies (MAT, 1:25 or higher) to T. gondii were found in 104 (27.1%) of 383 lambs. Hearts of 68 seropositive lambs were used for isolation of viable T. gondii by bioassay in cats, mice or both. For bioassays in cats, the entire myocardium or 500g was chopped and fed to cats, one cat per heart and faeces of the recipient cats were examined for shedding of T. gondii oocysts. For bioassays in mice, 50g of the myocardium was digested in an acid pepsin solution and the digest inoculated into mice; the recipient mice were examined for T. gondii infection. In total, 53 isolates of T. gondii were obtained from 68 seropositive lambs. Genotyping of the 53 T. gondii isolates using 10 PCR-restriction fragment length polymorphism markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico) revealed 57 strains with 15 genotypes. Four lambs had infections with two T. gondii genotypes. Twenty-six (45.6%) strains belong to the clonal Type II lineage (these strains can be further divided into two groups based on alleles at locus Apico). Eight (15.7%) strains belong to the Type III lineage. The remaining 22 strains were divided into 11 atypical genotypes. These results indicate high parasite prevalence and high genetic diversity of T. gondii in lambs, which has important implications in public health. We believe this is the first in-depth genetic analysis of T. gondii isolates from sheep in the USA.     

Sporulation and Survival of Toxoplasma gondii Oocysts in Seawater

ABSTRACT. We have been collaborating since 1992 in studies on southern sea otters ( Enhdyra lutris nereis ) as part of a program to define factors, which may be responsible for limiting the growth of the southern sea otter population. We previously demonstrated Toxoplasma gondii in sea otiers. We postulated that cat feces containing oocysts could be entering the marine environment through storm run-off or through municipal sewage since cat feces are often disposed down toilets by cat owners. The present study examined the sporulation of T. gondii oocysts in seawater and the survival of sporulated oocysts in seawater. Unsporulated oocysts were placed in 1.5 ppt artificial seawater, 32 ppt artificial seawater or 2% sulfuric acid (positive control) at 24 C in an incubator. Samples were examined daily for 3 days and development monitored by counting 100 oocysts from each sample. From 75 to 80% of the oocysts were sporulated by 3 days post-inoculation under all treatment conditions. Groups of 2 mice were fed 10,000 oocysts each from each of the 3 treatment groups. All inoculated mice developed toxoplasmosis indicating that oocysts were capable of sporulating in seawater. Survival of sporulated oocysts was examined by placing sporulated T. gondii oocysts in 15 ppt seawater at room temperature 22–24 C (RT) or in a refrigerator kept at 4 C. Mice fed oocysts that had been stored at 4C or RT for 6 months became infected. These results indicate that T. gondii oocysts can sporulate and remain viable in seawater for several months.     

Impact of rRNA methylations on ribosome recycling and fidelity of initiation in Escherichia coli

Ribosomal RNA (rRNA) contains a number of modified nucleosides in functionally important regions including the intersubunit bridge regions. As the activity of ribosome recycling factor (RRF) in separating the large and the small subunits of the ribosome involves disruption of intersubunit bridges, we investigated the impact of rRNA methylations on ribosome recycling. We show that deficiency of rRNA methylations, especially at positions 1518 and 1519 of 16S rRNA near the interface with the 50S subunit and in the vicinity of the IF3 binding site, adversely affects the efficiency of RRF-mediated ribosome recycling. In addition, we show that a compromise in the RRF activity affords increased initiation with a mutant tRNA^fMet wherein the three consecutive G-C base pairs (₂₉GGG₃₁:₃₉CCC₄₁), a highly conserved feature of the initiator tRNAs, were mutated to those found in the elongator tRNA^Met (₂₉UCA₃₁:₃₉ψGA₄₁). This observation has allowed us to uncover a new role of RRF as a factor that contributes to fidelity of initiator tRNA selection on the ribosome. We discuss these and earlier findings to propose that RRF plays a crucial role during all the steps of protein synthesis.     

Serological survey for antibodies to Encephalitozoon cuniculi in ownerless dogs from urban areas of Brazil and Colombia

There are 3 strains of Encephalitozoon cuniculi that occur in mammals. Strain III is associated with clinical disease in dogs, although some can be asymptomatic carriers and excrete spores in their urine. Several cases of human E. cuniculi infection caused by strain III have been observed in immunocompromised patients, indicating that E. cuniculi should be considered a zoonotic agent. Encephalitozoon cuniculi can cause fatal disease in maternally-infected or young dogs. Clinical signs in these animals included blindness, encephalitis, retarded growth rate, and nephritis. Encephalitozoon cuniculi has also been associated with primary renal failure in adult dogs. The present study used the direct agglutination test (DAT, cut-off 1:50) and the indirect fluorescent antibody test (IFAT, cut-off 1:10) to examine the prevalence of antibodies to E. cuniculi in dogs from Brazil and Colombia. Using the DAG, 31 (27.4%) of 113 dogs from Brazil and 47 (18.5%) of 254 dogs from Colombia were seropositive. Nine (14.3%) of 63 dogs from Brazil and 18 (35.3%) of the 51 dogs from Colombia were seropositive by indirect immunofluorescent antibody test. These results indicate that dogs from Brazil and Colombia are exposed to E. cuniculi.     

Concentrating Toxoplasma gondii and Cyclospora cayetanensis from surface water and drinking water by continuous separation channel centrifugation

Aims:  To evaluate the effectiveness of continuous separation channel centrifugation for concentrating Toxoplasma gondii and Cyclospora cayetanensis from drinking water and environmental waters.
Methods and Results:  Ready-to-seed vials with known quantities of T. gondii and C. cayetanensis oocysts were prepared by flow cytometry. Oocysts were seeded at densities ranging from 1 to 1000 oocysts l⁻¹ into 10 to 100 l test volumes of finished drinking water, water with manipulated turbidity, and the source waters from nine drinking water utilities. Oocysts were recovered using continuous separation channel centrifugation and counted on membrane filters using epifluorescent microscopy. Recovery efficiencies of both parasites were ≥84% in 10 l volumes of drinking water. In source waters, recoveries ranged from 64% to 100%, with the lowest recoveries in the most turbid waters. Method precision was between 10% and 20% coefficient of variation.
Conclusion:  Toxoplasma gondii and C. cayetanensis are effectively concentrated from various water matrices by continuous separation channel centrifugation.
Significance and Impact of the Study:  Waterborne transmission of T. gondii and C. cayetanensis presents another challenge in producing clean drinking water and protecting public health. Detection of these parasites relies on effectively concentrating oocysts from ambient water, otherwise false negatives may result. Validation data specific to T. gondii and C. cayetanensis concentration methods are limited. Continuous separation channel centrifugation recovers oocysts with high efficiency and precision, the method attributes required to accurately assess the risk of waterborne transmission.     

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.