Genetic variation and phylogenetic relationship of Hypoderaeum conoideum (Bloch, 1782) Dietz, 1909 (Trematoda: Echinostomatidae) inferred from nuclear and mitochondrial DNA sequences

Objective: To explore genetic variations of Hypoderaeum conoideum collected from domestic ducks from 12 different localities in Thailand and Lao PDR, as well as their phylogenetic relationship with American and European isolates. Methods: The nucleotide sequences of their nuclear ribosomal DNA (ITS), mitochondrial cytochrome c oxidase subunit 1 (CO1), and NADH dehydrogenase subunit 1 (ND1) were used to analyze genetic diversity indices. Results: We found relatively high levels of nucleotide polymorphism in ND1 (4.02%), whereas moderate and low levels were observed in CO1 (2.11%) and ITS (0.96%), respectively. Based on these polymorphisms, the 20 ND1, 12 CO1, and 18 ITS haplotypes were classified, and several common haplotypes were observed in all samples. At least three major lineages, namely American, European and Asian lineages, have been classified by phylogenetic analyses based on ND1 sequences. Conclusions: Our report demonstrates that the ND1 gene is the most suitable genetic marker to explore genetic variation and phylogenetic relationship of Hypoderaeum conoideum. However, a combination of all loci for ND1, CO1 and ITS would be of great value toward further genetic investigation of this endemic worldwide parasite. Thus, comprehensive molecular genetic analyses of Hypoderaeum conoideum from its worldwide distribution is needed to further understanding of the evolutionary and systematic relationships of this parasite.

Genetic variation and phylogenetic relationship of Hypoderaeum conoideum (Bloch, 1782) Dietz, 1909 (Trematoda: Echinostomatidae) inferred from nuclear and mitochondrial DNA sequences

Objective: To explore genetic variations of Hypoderaeum conoideum collected from domestic ducks from 12 different localities in Thailand and Lao PDR, as well as their phylogenetic relationship with American and European isolates. Methods: The nucleotide sequences of their nuclear ribosomal DNA (ITS), mitochondrial cytochrome c oxidase subunit 1 (CO1), and NADH dehydrogenase subunit 1 (ND1) were used to analyze genetic diversity indices. Results: We found relatively high levels of nucleotide polymorphism in ND1 (4.02%), whereas moderate and low levels were observed in CO1 (2.11%) and ITS (0.96%), respectively. Based on these polymorphisms, the 20 ND1, 12 CO1, and 18 ITS haplotypes were classified, and several common haplotypes were observed in all samples. At least three major lineages, namely American, European and Asian lineages, have been classified by phylogenetic analyses based on ND1 sequences. Conclusions: Our report demonstrates that the ND1 gene is the most suitable genetic marker to explore genetic variation and phylogenetic relationship of Hypoderaeum conoideum. However, a combination of all loci for ND1, CO1 and ITS would be of great value toward further genetic investigation of this endemic worldwide parasite. Thus, comprehensive molecular genetic analyses of Hypoderaeum conoideum from its worldwide distribution is needed to further understanding of the evolutionary and systematic relationships of this parasite.

Rapid Detection and Identification of Wuchereria bancrofti, Brugia malayi, B. pahangi, and Dirofilaria immitis in Mosquito Vectors and Blood Samples by High Resolution Melting Real-Time PCR

A simple, rapid, and high-throughput method for detection and identification of Wuchereria bancrofti, Brugia malayi, Brugia pahangi, and Dirofilaria immitis in mosquito vectors and blood samples was developed using a real-time PCR combined with high-resolution melting (HRM) analysis. Amplicons of the 4 filarial species were generated from 5S rRNA and spliced leader sequences by the real-time PCR and their melting temperatures were determined by the HRM method. Melting of amplicons from W. bancrofti, B. malayi, D. immitis, and B. pahangi peaked at 81.5+/-0.2degrees C, 79.0+/-0.3degrees C, 76.8+/-0.1degrees C, and 79.9+/-0.1degrees C, respectively. This assay is relatively cheap since it does not require synthesis of hybridization probes. Its sensitivity and specificity were 100%. It is a rapid and technically simple approach, and an important tool for population surveys as well as molecular xenomonitoring of parasites in vectors.

Zoonotic Echinostome Infections in Free-Grazing Ducks in Thailand

Free-grazing ducks play a major role in the rural economy of Eastern Asia in the form of egg and meat production. In Thailand, the geographical location, tropical climate conditions and wetland areas of the country are suitable for their husbandry. These environmental factors also favor growth, multiplication, development, survival, and spread of duck parasites. In this study, a total of 90 free-grazing ducks from northern, central, and northeastern regions of Thailand were examined for intestinal helminth parasites, with special emphasis on zoonotic echinostomes. Of these, 51 (56.7%) were infected by one or more species of zoonotic echinostomes, Echinostoma revolutum, Echinoparyphium recurvatum, and Hypoderaeum conoideum. Echinostomes found were identified using morphological criteria when possible. ITS2 sequences were used to identify juvenile and incomplete worms. The prevalence of infection was relatively high in each region, namely, north, central, and northeast region was 63.2%, 54.5%, and 55.3%, respectively. The intensity of infection ranged up to 49 worms/infected duck. Free-grazing ducks clearly play an important role in the life cycle maintenance, spread, and transmission of these medically important echinostomes in Thailand.

The Number and Characteristics of Endothelial Progenitor Cells from Peripheral Blood of Type 2 Diabetic Patients

Introduction: Type 2 diabetes, caused by an insulin resistant, is associated with endothelial cell dysfunction and impaired vascular homeostasis, resulting in vascular inflammation and atherosclerosis. Recently, endothelial progenitor cells (EPCs) which originated from the bone marrow are considered to be an important regulator in vascular homeostasis and a surrogate marker for vascular complications. According to this, EPC dysfunction may play an important role in causing the diabetic-associated vascular complications. Objective: To compare number and characteristic of EPCs in Type 2 diabetic patients with those of normal healthy subjects. Methods: The number of EPCs from twenty Type 2 diabetic patients and twenty healthy subjects was studied by using flow cytometry. The isolated EPCs were cultured and characterized by Dil-AcLDL engulfment. The various glucose concentrations were employed in the EPC culture in order to determine the effect of hyperglycemia on the number and viability of the EPCs. Results: The number of EPCs in Type 2 diabetic patients was significantly decreased compared to healthy subjects (5.5 \× 10⁶ \± 0.5 \× 10⁶ vs. 23 \× 10⁶ \± 2.3 x 10⁶; P \< 0.01). There was an inverse correlation between the EPC numbers and the concentrations of plasma glucose (r = -0.045) as well as HbA1c (r = -0.336). The culture of EPCs from diabetic patients took a significantly longer period of time to develop mature EPCs than control (30.8 \± 3.9 days vs. 22.4 \± 2.7 days, P \< 0.01). In addition, the number of cultured EPCs was significantly reduced when the glucose concentration in culture medium was greater than 16.5 mmol/l (P \< 0.05). These results demonstrated that glucose has a negative effect on the number and viability of EPCs in a dose-dependent fashion. Conclusion Hyperglycemic condition in Type 2 diabetes has a negative effect on the number and viability of circulating EPCs in a dose-dependent fashion.

The characteristic of mesenchymal stem cells from peripheral blood and mobilized peripheral blood in comparison to bone marrow derived mesenchymal stem cells

Objective: To isolate and characterize mesenchymal stem cells (MSCs) from peripheral blood and G-CSF mobilized peripheral blood. Methods: Mononuclear cells (MNCs) were isolated from peripheral blood, G-CSF mobilized peripheral blood and bone marrow using gradient centrifugation. The numbers of MSCs in these three sources were quantified using flow cytometry. The isolated MNCs were then cultured to generate MSCs. The MSCs generated from those three sources were studied in term of the MSC marker (CD73, CD90, CD105 and CD106) expression, the ability to generate colony (CFU-F) in culture and the ability to differentiate toward osteocyte and adipocyte-lineages. Results: The percentage of cells that expressed CD90 in fresh MNC populations isolated from bone marrow (BM-MNCs), peripheral blood (PB-MNCs) and mobilized peripheral blood (MPB-MNCs) is 1.94, 2.1 and 0.05 respectively, while the expression of CD73 in BM-MNCs, PB-MNCs and MPB-MNCs is 5.2, 15.2 and 7.8 respectively. The percentage of cells that expressed CD105 in BM-MNCs, PB-MNCs and MPB-MNCs is 5.3, 4.1 and 2.75, respectively while the expression of CD106 in those three populations is 2.82, 2.36 and 4.5 respectively. The ability of BM-MNCs, PB-MNCs and MPB-MNCs to generate colony in culture (CFU-F) is 67, 30 and 48 colonies per 10⁶ plating MNCs, respectively. After culture for three passages, more than 64% of BM-MNCs, PB-MNCs and MPB-MNCs homogeneously expressed CD73, CD90 and CD105. In contrast, the expression of CD45 (marker of hematopoietic cells) in those populations is negative. In addition, the bone marrow-derived MSCs also have an ability to differentiate toward osteocyte and adipocyte-lineages. Conclusion: We have successfully isolated and characterized MSCs from both peripheral blood and G-CSF mobilized peripheral blood. Those MSCs expressed several MSC markers, including CD73, CD90, CD105 and CD106, and able to generate colonies in culture in a manner similar to those of BM-MSCs. Our results suggest that these PB-MSCs and MPB-MSCs might be used as an alternative source for the clinical treatment in the future.

Characterization of mesenchymal stem cells from umbilical cord and Wharton’s jelly in comparison to bone marrow derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) are capable of differentiating into different mesenchymal lineages, including adipose tissue, bone and cartilage. For clinical use, adult bone marrow is the most common source of MSCs. However, the frequency and differentiating capacity of MSCs in adult bone marrow decrease with age of donors. Different post-natal tissues have been studied for the presence of MSCs. However, there is no report about the characteristic of MSCs isolated from those sources in comparison to MSCs isolated from bone marrow. This study comprehensively compared several characteristics of MSCs isolated from umbilical cord, Wharton’s jelly and bone marrow in aspects of cell morphology, immunophenotype and growth kinetics as well as the differentiation capacity to be adipogenic and osteogenic lineages. We successfully isolated MSCs from umbilical cord and Wharton’s jelly, as well as bone marrow. Our results indicated that MSCs isolated from those three sources have fibroblast-like morphology and exhibited similar cell surface markers including CD73, CD90, and CD105. Umbilical cord and Wharton’s jelly derived MSCs cultured in NH OsteoDiff Medium and NH AdipoDiff Medium showed the ability to differentiate to be osteocytes and adipocytes in a comparable degree to bone marrow derived MSCs. Moreover, the proliferation capacity of MSCs isolated from umbilical cord and Wharton’s jelly at passage 2 to 4 was significantly higher than bone marrow derived MSCs (p \< 0.05). The results obtained from this study indicated that MSCs isolated from umbilical cord and Wharton’s jelly had similar characteristic in comparison to bone marrow derived MSCs. Therefore, post-natal tissues, especially umbilical cord and Wharton’s jelly, are attractive alternative sources of MSCs for future clinical application.