Development and standardization of the Dot Blot test for serological diagnosis of bovine leptospirosis

Leptospirosis a public health problem and an endemic zoonosis in Brazil, is diagnosed by serological methods. Therefore, low-cost and easy to execute methodologies with good/high sensitivity, such as Dot Blot, are an important diagnostic tool. The aim of this study was to standardize and validation the dot-blot technique for the serological diagnosis of bovine leptospirosis. Several concentrations of antigens applied to nitrocellulose membranes, and different dilutions of conjugated bovine serum were evaluated to develop and standardize the test. The best distinction/contrast between positive and negative samples was observed for 1μL antigen (0.11μg/μL outer membrane protein of the Hardjo serovar (OMPH) and 0.08 μg/μL outer membrane protein of the Wolffi serovar (OMPW)), 1:500 and 1:10000 bovine serum dilution and conjugate, respectively. The Dot Blot test efficiency was 71.87% and kappa index, 0.46 (p<0.0001). The other parameters measured were: sensitivity 91.89%; specificity 59.32%; positive predictive value 58.62%; and, negative predictive value 59.32%. In addition to high sensitivity, other advantages of the Dot Blot technique have been identified, such as practicality, low cost since it does not require sophisticated devices and the fact that the Hardjo and Wolffi OMP also reacted with serovars from other pathogenic serogroups. The results provided positive expectations for the use of Dot Blot as support in the diagnosis of bovine leptospirosis, especially if used as a screening test, stimulating further research for the future development of kits for diagnostic purposes.

Western Blot Analysis to Detect Cross-reaction in Toxocara vitulorum Protein with Anti-Mecistocirrus digitatus Serum

Worm infections are found in livestock and can be transmitted to humans. Toxocara vitulorum is a worm species which commonly infected people. Cross-reaction among worms can generate false positive to establish helminthiasis diagnosis through antibody inspection. This study aimed to determine specific proteins that caused cross-reaction between Toxocara vitulorum antigen and anti-M. digitatus serum by using the western blot technique. In the present study, the whole worms extracted of T. vitulorum and M. digitatus have been used to make polyclonal antibodies from M. digitatus with Wistar rats as hosts. The cross-reaction between whole worm extract of T. vitulorum protein and anti-M. digitatus serum obtained 12 protein bands that each relative molecular mass (Mr) valued of 176, 124, 92, 68, 59, 47, 31, 29, 26, 16, 12, and 10 kDa. Cross-reaction occurred between T. vitulorum protein and anti-M. digitatus.

Cross Reaction of Haemonchus contortus Protein with Toxocara vitulorum Anti-L2 Serum Using Western Blot Technique

In the adult stage, Haemonchus contortus worms infect the abomasum host causing anemia and even death in animals. However, identifying the H. contortus protein can be used as a reference for the diagnosis of diseases. The diagnosis is performed by serological cross-reaction between H. contortus protein and anti-L2 Toxocara vitulorum (T. vitulorum) serum using the western blot technique. The main purpose of the current research was to identify the cross-reaction between H. contortus proteins and anti-L2 T. vitulorum serum using the western blot technique. T. vitulorum worms were collected from the intestine of cattle and H. contortus worms were collected from the abomasum of goats. The first step was making antibodies by oral infection of rats with infective eggs (L2) of T. vitulorum. The blood was taken 21 days after infection. Then, the blood was centrifuged at 1500 rpm for 10 minutes to get the serum. The second step was making homogenates from the whole worm extract of H. contortus. After crushing the worms, it was centrifuged at 5000 rpm for 15 minutes and the supernatant was taken. The supernatant was then analyzed using Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) with coomassie brilliant blue staining. The third step was the analysis of H. contortus protein with serum anti-L2 T. vitulorum using the western blot technique. From the H. contortus homogenates analysis using SDS-PAGE, 16 protein bands were obtained. The cross-reactions were 141.3, 81.3, 64. 6, 51.3, 46.8, and 38 kDa. The data from cross-reactions suggested that the H. contortus protein cannot be used as a diagnostic material. It is serologically Haemonchosis because it caused false positives with diagnostic Toxocariasis.

MicroRNA-520c-3p Modulates Doxorubicin-Chemosensitivity in HepG2 Cells

Background: Doxorubicin (DOX) is the most common drugs used in cancer therapy, including Hepatocellular Carcinoma (HCC). Drug resistance, is one of chemotherapy’s significant problems. Emerging studies have shown that microRNAs (miRNAs) could participate in regulating this mechanism. Nevertheless, the impact of miRNAs on HCC chemoresistance is still enigmatic. Objective: Investigating the role of miR-520c-3p in enhancement of anti-tumor effect of DOX against HepG2 cells. Methods: Expression profile for liver related miRNAs (384 miRNAs) has been analyzed on HepG2 cells treated with DOX using qRT-PCR. miR-520c-3p, the most deregulated miRNA, was selected for combination treatment with DOX. Expression level for LEF1, CDK2, CDH1, VIM, Mcl-1 and TP53 was evaluated in miR-520c-3p transfected cells. Cell viability, colony formation, wound healing as well as apoptosis assays have been demonstrated. Furthermore, Mcl-1 protein level was measured using western blot technique. Results: The present data indicated that miR-520c-3p overexpression could render HepG2 cells chemo-sensitive to DOX through enhancing its suppressive effects on proliferation, migration, and induction of apoptosis. The suppressive effect of miR-520c-3p involved altering the expression levels of some key regulators of cell cycle, proliferation, migration and apoptosis including LEF1, CDK2, CDH1, VIM, Mcl-1 and TP53. Interestingly, Mcl-1 was found to be one of the potential targets of miR-520c-3p, and its protein expression level was down-regulated upon miR-520c-3p overexpression. Conclusion: Our data referred to the tumor suppressor function of miR-520c-3p that could modulate chemosensitivity of HepG2 cells toward DOX treatment, providing a promising therapeutic strategy in HCC.

A new ELISA and western blot technique based on recombinant TES antigen and/or larval antigen for the detection of toxocariasis in humans

Serological antibody detection by enzyme-linked immunosorbent assay (ELISA)- and immunoblot-based methods constitutes the best indicator of human Toxocara infection. Nevertheless, the availability of serological tests, particularly western blots (WB), evaluated for sensitivity and specificity is limited. Therefore, an Anti-Toxocara-ELISA immunoglobulin g (IgG) prototype (Proto-ELISA) and an Anti-Toxocara-Westernblot (IgG) prototype (Proto-WB) were evaluated by testing 541 human sera pre-determined for Toxocara infection by an established in-house Anti-Toxocara-ELISA (IH-ELISA). To evaluate sensitivity and specificity of the newly developed ELISA and WB prototypes, results were compared to IH-ELISA and a commercial WB (Com-WB). Compared to the IH-ELISA, a sensitivity of 93.1% (229/246) and a specificity of 94.6% (279/295) of the Proto-ELISA with a Cohen's κ of 0.88 were obtained. The sensitivity of the Proto-WB was 76.7% (240/313) and specificity was 99.6% (227/228) with a Cohen's κ of 0.73 compared to those of Com-WB. A comparison to the IH-ELISA revealed 91.5% (225/246) sensitivity and 94.6% (279/295) specificity of the Proto-WB with a Cohen's κ of 0.86. Cross-reactivity was observed for some samples positive for Ascaris and Trichinella spp. in the Proto-ELISA, Proto-WB and Com-WB. Overall, the evaluated ELISA and WB prototypes showed high sensitivity and specificity, indicating high reliability of these newly developed tests.