Molecular identification of Kalicephalus isolated from snakes in Hunan province, subtropical China

@#Parasite classification and identification are central to controlling parasitosis. Traditional methods for identifying parasite species are based on morphological features, but these are time-consuming and inaccurate, especially for cryptic species. The purpose of the present study was to select molecular markers to promote the development of molecular systematic for parasites. The internal transcribed spacers (ITS) of nuclear ribosomal DNA (rDNA) falls in between 18S, 5.8S, and 28S rDNA sequences, including ITS-1 and ITS-2 sequences. Previous studies have demonstrated that rDNA ITS sequences provide useful genetic markers for identifying parasitic nematodes. With the ultimate goal of controlling parasite transmission, we identified Kalicephalus belonging to three species using ITS rDNA genes. The ITS genes (750–797 bp) of 21 Kalicephalus belonging to 3 species were cloned and sequenced. Intra- and interspecific identities were 98.4% and 80%–89%, respectively. The phylogenetic tree reconstructed with the neighbour-joining (NJ) method revealed that congener Kalicephalus form the same branch, which is far apart from other branches of other nematodes. This is consistent with morphological classifications, demonstrating the accuracy of our molecular method. This is the first report stating that ITS genes can be used to classify Kalicephalus, and it lays the foundation for identification, molecular epidemiology, and phylogenetics of Kalicephalus and related parasitic nematodes.

Combinations of three immunological assays for detecting anti-Toxoplasma IgG in the sera of patients infected with Toxoplasma gondii.

Enzyme-linked immunosorbent assay (ELISA), Dot-ELISA and Dot-immunogold silver staining (Dot-IGSS) were simultaneously used to detect the specific IgG against Toxoplasma gondii in 65 patients infected with the protozoa. The positive rates were 86.51%, 92.51% and 98.64%, respectively. When ELISA and Dot-ELISA results were put together, the positive rate increased to 95.38%. When Dot-IGSS results were combined with those of ELISA or Dot-ELISA, the positive rate was raised to 100%. The difference in positive rate between ELISA and Dot-IGSS was significant (x2 = 6.93, p < 0.01), but no statistically significant differences were found between ELISA and Dot-ELISA or between Dot-ELISA and Dot-IGSS. Paired comparison of the reacting intensities of the sera in the 3 assays showed the correlations were highly significant (p < 0.001), with r = 0.608 between Dot-IGSS and Dot-ELISA, r = 0.8194 between Dot-IGSS and ELISA and r = 0.517 between Dot-ELISA and ELISA. Hence combination of different serological assays may increase their sensitivity and specificity for detecting the anti-Toxoplasma antibodies.

Comparative study of Dot-immunogold silver staining and Dot-ELISA for the detection of serum antibodies against Wuchereria bancrofti.

Dot-immunogold silver staining (Dot-IGSS) and Dot-ELISA, using the soluble antigen of Brugia malayi, were employed to detect anti-Wuchereria bancrofti antibodies in 50 cases of Wuchereria bancrofti microfilaremia. The positive rates were 100% and 90% in Dot-IGSS and Dot-ELISA respectively. The average titer in the 45 positive cases was 1:184 (1:10-1:2560) for Dot-IGSS and 1:150 (1:10-1:2560) for Dot-ELISA, with 30 cases showing the same titer in both tests, 13 cases showing higher titer in Dot-IGSS than in Dot-ELISA and 2 cases in the former showing lower titers than in the latter. There was a linear relationship between the titers of antibodies detected by Dot-IGSS and by Dot-ELISA (r = 0.8443). Dot-IGSS, similar to Dot-ELISA, is easy to carry out and the result is easy to read. It is seen that Dot-IGSS is highly sensitive and specific and is practicable for immunodiagnosis and surveillance of filariasis.