Search

Dermatophytes are the group of keratinophilic fungi that cause superficial cutaneous infection, which traditionally belong to the genera Trichophyton, Microsporum, and Epidermophyton. Dermatophyte infection is not only a threat to the health of small animals, but also an important zoonotic and public health issue because of the potential transmission from animals to humans. Rabbit dermatophytosis is often clinically identified; however, limited information was found in Asia. The aims of this study are to investigate the prevalence and to evaluate the risk factors of dermatophytosis in pet rabbits in Northern Taiwan. Between March 2016 and October 2018, dander samples of pet rabbits were collected for fungal infection examination by Woods lamp, microscopic examination (KOH preparation), fungal culture, and PCR assay (molecular identification). Z test and Fishers exact test were performed to evaluate the potential risk factors, and logistic regression analysis was then performed to build the model of risk factors related to dermatophyte infection. Of the collected 250 dander samples of pet rabbits, 29 (11.6%) samples were positive for dermatophytes by molecular identification. In those samples, 28 samples were identified as the T. mentagrophytes complex and 1 sample was identified as M. canis. Based on the results of the Firths bias reduction logistic analyses, animal source (rabbits purchased from pet shops) and number of rearing rabbits (three rabbits or more) were shown as the main risks for dermatophyte infection in the pet rabbits in Taiwan. The results of the present study elucidate the prevalence of rabbit dermatophyte infection, pathogens, and risk factors in Taiwan, and provide an important reference for the prevention and control of rabbit dermatophytosis.

Abstract Background Apoptin, a nonstructural protein encoded by the VP3 gene of chicken anemia virus (CAV), has been shown to not only induce apoptosis when introduced into the precursors of chicken thymocytes, but has been found to specifically kill human cancer cells, tumor cell and transformed cells without affecting the proliferation of normal cells. This tumor-specific apoptotic characteristic of the protein potentially may allow the development of a protein drug that has applications in tumor therapy. However, several major problems, which include poor expression and poor protein solubility, have hampered the production of apoptin in bacteria. Results Significantly increased expression of recombinant full-length apoptin that originated from chicken anemia virus was demonstrated using an E. coli expression system. The CAV VP3 gene was fused with a synthetic sequence containing a trans-acting activator of transcription (TAT) protein transduction domain (PTD). The resulting construct was cloned into various different expression vectors and these were then expressed in various E. coli strains. The expression of the TAT-Apoptin in E. coli was significantly increased when TAT-Apoptin was fused with GST-tag rather than a His-tag. When the various rare amino acid codons of apoptin were optimized, the expression level of the GST-TAT-Apoptinopt in E. coli BL21(DE3) was significantly further increased. The highest protein expression level obtained was 8.33 g/L per liter of bacterial culture after induction with 0.1 mM IPTG for 4 h at 25 °C. Moreover, approximately 90% of the expressed GST-TAT-Apoptinopt under these conditions was soluble. After purification by GST affinity chromatography, the purified recombinant TAT-Apoptinopt protein was used to evaluate the recombinant protein’s apoptotic activity on tumor cells. The results demonstrated that the E. coli-expressed GST-TAT-apoptinopt showed apoptotic activity and was able to induce human premyelocytic leukemia HL-60 cells to enter apoptosis. Conclusions On expression in E. coli, purified recombinant TAT-Apoptinopt that has been fused to a GST tag and had its codons optimized, was found to have great potential. This protein may in the future allow the development of a therapeutic protein that is able to specifically kill tumor cells.

Abstract Background VP2 of chicken anemia virus (CAV) is a dual-specificity phosphatase required for virus infection, assembly and replication. The functions of the nuclear localization signal (NLS) and nuclear export signal (NES) of VP2 in the cell, however, are poorly understood. Our study identified the presence of a NLS in VP2 and showed that the protein interacted significantly with mini-chromosome maintenance protein 3 (MCM3) in the cell. Results An arginine-lysine rich NLS could be predicted by software and spanned from amino acids 133 to 138 of VP2. The critical amino acids residues between positions 136 and 138, and either residue 133 or 134 are important for nuclear import in mammalian cells based on systematic mutagenesis. A NES is also predicted in VP2; however the results suggest that no functional NES is present and that this protein is CRM1 independent. It was also shown that VP2 is a chromatin binding protein and, notably, using a co-immunoprecipitation assay, it was found that VP2 association with MCM3 and that this interaction does not require DSP activity. Conclusions VP2 contains a NLS that span from amino acids 133 to 138. VP2 is a CRM1 independent protein during nuclear export and associates with MCM3 in cells.

Abstract Background Chicken anemia virus (CAV), the causative agent chicken anemia, is the only member of the genus Gyrovirus of the Circoviridae family. CAV is an immune suppressive virus and causes anemia, lymph organ atrophy and immunodeficiency. The production and biochemical characterization of VP1 protein and its use in a subunit vaccine or as part of a diagnostic kit would be useful to CAV infection prevention. Results Significantly increased expression of the recombinant full-length VP1 capsid protein from chicken anemia virus was demonstrated using an E. coli expression system. The VP1 gene was cloned into various different expression vectors and then these were expressed in a number of different E. coli strains. The expression of CAV VP1 in E. coli was significantly increased when VP1 was fused with GST protein rather than a His-tag. By optimizing the various rare amino acid codons within the N-terminus of the VP1 protein, the expression level of the VP1 protein in E. coli BL21(DE3)-pLysS was further increased significantly. The highest protein expression level obtained was 17.5 g/L per liter of bacterial culture after induction with 0.1 mM IPTG for 2 h. After purification by GST affinity chromatography, the purified full-length VP1 protein produced in this way was demonstrated to have good antigenicity and was able to be recognized by CAV-positive chicken serum in an ELISA assay. Conclusions Purified recombinant VP1 protein with the gene's codons optimized in the N-terminal region has potential as chimeric protein that, when expressed in E. coli, may be useful in the future for the development of subunit vaccines and diagnostic tests.

Background: Pigeon circovirus (PiCV) is considered to be a viral agent central to the development of young pigeon disease syndrome (YPDS). The Cap protein, a structural protein encoded by the cap (or C1) gene of PiCV, has been shown to be responsible for not only capsid assembly, but also has been used as antigen for detecting antibody when the host is infected with PiCV. The antigenic characteristics of the Cap protein potentially may allow the development of a detection kit that could be applied to control PiCV infection. However, poor expression and poor protein solubility have hampered the production of recombinant Cap protein in the bacteria. This study was undertaken to develop the optimal expression of recombinant full-length Cap protein of PiCV using an E. coli expression system., Results: The PiCV cap gene was cloned and fused with different fusion partners including a His-tag, a GST-tag (glutathioine-S-transferase tag) and a Trx-His-tag (thioredoxin-His tag). The resulting constructs were then expressed after transformation into a number of different E. coli strains; these then had their protein expression evaluated. The expression of the recombinant Cap protein in E. coli was significantly increased when Cap protein was fused with either a GST-tag or a Trx-His tag rather than a His-tag. After various rare amino acid codons presented in the Cap protein were optimized to give the sequence rCapopt, the expression level of the GST-rCapopt in E. coli BL21(DE3) was further increased to a significant degree. The highest protein expression level of GST-rCapopt obtained was 394.27 ± 26.1 mg/L per liter using the E. coli strain BL21(DE3)-pLysS. Moreover, approximately 74.5% of the expressed GST-rCapopt was in soluble form, which is higher than the soluble Trx-His-rCapopt expressed using the BL21(DE3)-pLysS strain. After purification using a GST affinity column combined with ion-exchange chromatography, the purified recombinant GST-rCapopt protein was found to have good antigenic activity when tested against PiCV-infected pigeon sera., Conclusions: These findings shows that the E. coli-expressed full-length PiCV Cap protein has great potential in terms of large-scaled production and this should allow in the future the development of a serodiagnostic kit that is able to clinically detect PiCV infection in pigeons.

Background: Chicken anemia virus (CAV) is an important viral pathogen that causes anemia and severe immunodeficiency syndrome in chickens worldwide. Generally, CAV infection occurs via vertical transmission in young chicks that are less than two weeks old, which are very susceptible to the disease. Therefore, epidemiological investigations of CAV infection and/or the evaluation of the immunization status of chickens is necessary for disease control. Up to the present, systematically assessing viral protein antigenicity and/or determining the immunorelevant domain(s) of viral proteins during serological testing for CAV infection has never been performed. The expression, production and antigenic characterization of CAV viral proteins such as VP1, VP2 and VP3, and their use in the development of diagnostic kit would be useful for CAV infection prevention., Results: Three CAV viral proteins VP1, VP2 and VP3 was separately cloned and expressed in recombinant E. coli. The purified recombinant CAV VP1, VP2 and VP3 proteins were then used as antigens in order to evaluate their reactivity against chicken sera using indirect ELISA. The results indicated that VP2 and VP3 show good immunoreactivity with CAV-positive chicken sera, whereas VP1 was found to show less immunoreactivity than VP2 and VP3. To carry out the further antigenic characterization of the immunorelevant domains of the VP2 and VP3 proteins, five recombinant VP2 subunit proteins (VP2-435N, VP2-396N, VP2-345N, VP2-171C and VP2-318C) and three recombinant VP3 subunit proteins (VP3-123N, VP3-246M, VP3-366C), spanning the defined regions of VP2 and VP3 were separately produced by an E. coli expression system. These peptides were then used as antigens in indirect ELISAs against chicken sera. The results of these ELISAs using truncated recombinant VP2 and VP3 subunit proteins as coating antigen showed that VP2-345N, VP2-396N and VP3-246M gave good immunoreactivity with CAV-positive chicken sera compared to the other subunit proteins. Moreover, the VP2-396N and VP2-345 based ELISAs had better sensitivity (97.5%) and excellent specificity (100%) during serodiagnosis testing using a mean plus three standard deviations cut-off. The VP3-246M based ELISA showed a sensitivity of 85% and a specificity of 100% at the same cut-off value., Conclusions: This is the first report to systematically assess the antigenic characteristics of CAV viral proteins for sero-diagnosis purposes. Purified recombinant VP2-396N and VP2-345N subunit proteins, which span defined regions of VP2, were demonstrated to have good antigenicity and higher sensitivities than VP3-246M and were able to recognize CAV-positive chicken serum using an ELISA assay. The defined antigenicity potential of these chimeric subunit proteins produced by expression in E. coli seem to have potential and could be useful in the future for the development of the CAV diagnostic tests based on a subunit protein ELISA system.

The giant freshwater prawn Macrobrachium rosenbergii is commercially cultured throughout the world including Taiwan. From 1992 to 1995, Taiwanese production decreased by approximately 50% due to disease. The yeast Metschnikowia bicuspidata is considered to be one of the major causes of white muscle disease, but the molecular mechanism of its pathogenesis is not known. Using RNA differential display (DD) with muscle and hepatopancreatic tissue, we identified a 324 nucleotide (nt) message specifically expressed by M. rosenbergii infected with M. bicuspidata but not in the controls. A ribonuclease protection assay (RPA) confirmed expression in both tissues. RPA data also revealed an additional 230 bp mRNA message that was not identified by DD. Using RNA ligase-mediated rapid amplification of 5' cDNA ends (5'-RACE), we successfully isolated a 1357 bp full-length gene (c57) that showed 92 and 87% sequence identity to the actin gene of the Kuruma shrimp Marsupenaeus japonicus (also called Penaeus japonicus) (GenBank accession number AB055975) and the beta-actin gene of the white shrimp Litopenaeus vannamei (also called Penaeus vannamei) (GenBank accession number AF300705), respectively. The deduced amino acid sequence of c57 showed 83 % sequence similarity to M. japonicus and L. vannamei actin proteins. Based on this high homology, we suggest that upregulation of actin expression in the muscle and hepatopancreas is part of the shrimp response to M. bicuspidata infection. Increased expression may be related to repair of tissues damaged by yeast infection.