Circulating hypervirulent Marek's disease viruses in vaccinated chicken flocks in Taiwan by genetic analysis of meq oncogene.

Marek's disease (MD) is an important neoplastic disease caused by serotype 1 Marek's disease virus (MDV-1), which results in severe economic losses worldwide. Despite vaccination practices that have controlled the MD epidemic, current increasing MD-suspected cases indicate the persistent viral infections circulating among vaccinated chicken farms in many countries. However, the lack of available information about phylogeny and molecular characterization of circulating MDV-1 field strains in Taiwan reveals a potential risk in MD outbreaks. This study investigated the genetic characteristics of 18 MDV-1 strains obtained from 17 vaccinated chicken flocks in Taiwan between 2018 and 2020. Based on the sequences of the meq oncogene, the phylogenetic analysis demonstrated that the circulating Taiwanese MDV-1 field strains were predominantly in a single cluster that showed high similarity with strains from countries of the East Asian region. Because the strains were obtained from CVI988/Rispens vaccinated chicken flocks and the molecular characteristics of the Meq oncoprotein showed features like vvMDV and vv+MDV strains, the circulating Taiwanese MDV-1 field strains may have higher virulence compared with vvMDV pathotype. In conclusion, the data presented demonstrates the circulation of hypervirulent MDV-1 strains in Taiwan and highlights the importance of routine surveillance and precaution strategies in response to the emergence of enhanced virulent MDV-1.

Authentication of olive oil in commercial products using specific, sensitive, and rapid loop-mediated isothermal amplification.

Olive oil is an important and popularly used plant oil in the daily diet or chemical industry. Due to its biological benefits on human health and higher selling prices, adulteration of olive oil for commercial fraud by other plant oils is becoming a serious issue. In this study, a specific, sensitive and rapid loop-mediated isothermal amplification (LAMP) was first developed for the detection of Olea europaea DNA for olive oil authentication. The oleosin gene was used for the primer design of the LAMP assay. After primer validation, the results showed that the LAMP primers were specific and rapid to isothermally authenticate the oleosin gene of Olea europaea within 1 h at 62 °C and had no cross-reaction with other DNA of plant oils. The sensitivity of LAMP was 1 ng of genomic DNA in olive oil, and only 1% olive oil in the sample was requisite during DNA amplification. Additionally, positive detection by LAMP in all the collected commercial olive oil products was practically performed but not in PCR assays. In conclusion, herein, the established LAMP assay with specificity could not only be capable for rapid identification but also applicable for olive oil authentication for precluding adulteration in plant oil products. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-023-05726-y.

Prevalence and Risk Factors of Zoonotic Dermatophyte Infection in Pet Rabbits in Northern Taiwan.

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 Wood's lamp, microscopic examination (KOH preparation), fungal culture, and PCR assay (molecular identification). Z test and Fisher's 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 Firth's 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.

Investigation of Transovarial Transmission of Bartonella henselae in Rhipicephalus sanguineus sensu lato Ticks Using Artificial Feeding.

Bartonella henselae is a slow-growing, Gram-negative bacterium that causes cat scratch disease in humans. A transstadial transmission of the bacteria from larvae to nymphs of Rhipicephalus sanguineus sensu lato (s.l.) ticks, suspected to be a potential vector of the bacteria, has been previously demonstrated. The present study aims to investigate transovarial transmission of B. henselae from R. sanguineus s.l. adults to their instars. Adult ticks (25 males and 25 females) were fed through an artificial feeding system on B. henselae-infected goat blood for 14 days, and 300 larvae derived from the experimentally B. henselae-infected females were fed on noninfected goat blood for 7 days. Nested PCR and culture were used to detect and isolate B. henselae in ticks and blood samples. Bartonella henselae DNA was detected in midguts, salivary glands, and carcasses of the semi-engorged adults and pooled tick feces (during feeding and post-feeding periods). After the oviposition period, B. henselae DNA was detected in salivary glands of females (33.3%), but not in pooled eggs or larvae derived from the infected females. However, B. henselae DNA was detected by nested PCR from the blood sample during larval feeding, while no viable B. henselae was isolated by culture. According to our findings, following infected blood meal, B. henselae could remain in the tick midguts, move to other tissues including salivary glands, and then be shed through tick feces with limited persistency. The presence of bacterial DNA in the blood during larval feeding shows the possibility of transovarial transmission of B. henselae in R. sanguineus s.l. ticks.

Specific, sensitive and rapid Curcuma longa turmeric powder authentication in commercial food using loop-mediated isothermal nucleic acid amplification.

Turmeric (Curcuma longa) is a rhizomatous plant of the ginger family Zingiberaceae that is usually dried and ground into powder for use as a seasoning. Because turmeric has become increasingly popular in the functional food market, adulteration of C. longa by other turmeric species is becoming an increasingly significant problem. In this study, loop-mediated isothermal amplification (LAMP) was developed for the detection of C. longa DNA for turmeric authentication. ITS2-26S rDNA was used for the LAMP primer designation. The results demonstrated that the specific primers exhibited high specificity, authenticated C. longa DNA within 30 min at 65 °C isothermally and had no cross-reaction with other adulterants. LAMP was sensitive to 0.1 ng of turmeric C. longa DNA, and only 0.01% of C. longa turmeric powder in the sample was required for DNA amplification. The sensitivity of LAMP was 10-fold higher than that of PCR (0.1%) from a previous report. Moreover, all the collected commercial turmeric products were positively detected by LAMP and RtF-LAMP (real-time fluorescence LAMP). The developed LAMP assay not only had higher specificity and rapidity than that of other methods but could also be applied to authenticate turmeric to prevent adulteration in food products.

Utilizing Odor-Adsorbed Filter Papers for Detection Canine Training and Off-Site Fire Ant Indications.

The red imported fire ant (RIFA, Solenopsis invicta) is an exotic aggressive pest that is notorious for its ability to seriously harm humans and animals, cause economic loss to agriculture, and damage ecosystems. This is the first study to validate the capability of filter paper adsorption as a feasible odor bearer of RIFAs and evaluate its use in detection dog training. Two live RIFA-experienced detection dogs achieved a mean 92% positive indication rate (PIR) on RIFA-scented papers with a relatively low false response rate (0.8%). The similar accuracies in recognizing live RIFAs (96%) and scented papers (92%) suggest that a filter paper is an effective odor reservoir. After training with live RIFA and scented filter papers, both RIFA-experienced and inexperienced detection dogs successfully indicated filter papers that were scented with at least 10 RIFAs for 4 h with a high PIR (>93%) and low false response rate (2%). Detection dogs correctly recognized the filter papers scented by 10 RIFAs for 24 h with a 97.6% PIR. Even for scented samples stored at -20 °C and 4 °C for 13 weeks, the positive indication rates (PIRs) were as high as 90%. These results suggest that filter paper is an effective RIFA odor bearer, and the scent can be maintained at least 13 weeks for dog identification. After RIFA-scented paper training, detection dogs showed high (>95%) PIRs for both RIFA-scented paper and live RIFAs and also successfully performed field studies. Using filter paper as a RIFA odor bearer is an effective and economical method for detection dog training and RIFA identification.

Transmission of Bartonella henselae within Rhipicephalus sanguineus: Data on the Potential Vector Role of the Tick.

Bartonella henselae is a fastidious intraerythrocytic, gram-negative bacteria that causes cat scratch disease in humans. Ixodes ricinus has been confirmed to be a competent vector of B. henselae, and some indirect evidences from clinical cases and epidemiological studies also suggested that some other tick species, including Rhipicephalus sanguineus, may transmit the bacteria. B. henselae has been detected in R. sanguineus but no experimental investigations have been performed to evaluate the vector competency of this tick species regarding B. henselae transmission. To this end, this work aimed to assess the transstadial transmission of B. henselae between larvae and nymphs of R. sanguineus as well as transmission by nymphs infected at the larval stage. Four hundred B. henselae negative larvae were fed with B. henselae-infected blood by using an artificial membrane feeding system. After five days of feeding, B. henselae was detected by PCR in 57.1% (8/14) of engorged larval pools, 66.7% (4/6) of semi-engorged larval pools, and 66.7% (2/3) of larval feces pools. After molting, B. henselae DNA was also detected in 10% (1/10) of nymph pools, but not in tick feces. After a pre-fed step of nymphs infected at the larval stage on non-infected blood meal, B. henselae was detected by PCR in blood sample from the feeder, but no Bartonella colonies could be obtained from culture. These findings showed that B. henselae could be transstadial transmitted from R. sanguineus larvae to nymphs, and also suggest that these nymphs may retransmitted the bacteria through the saliva during their blood meal. This is the first study that validated the artificial membrane feeding system for maintaining R. sanguineus tick colony. It shows the possibility of transstadial transmission of B. henselae from R. sanguineus larvae to nymphs.

Development of a specific isothermal nucleic acid amplification for the rapid and sensitive detection of shrimp allergens in processed food.

Food allergens that cause anaphylactic reactions have become an important health problem worldwide. Among them, shrimp is a popular seafood in many cuisines. The best way to avoid allergic reactions is to mitigate the intake of food allergens. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for the detection of shrimp DNA. Using LAMP primers, the identification of shrimp DNA by the LAMP assay was specific and rapid (within 30 min). It exhibited no cross-reaction with the DNA of other Crustacea, including crabs and lobster, and at least 0.01% shrimp DNA existed in the test sample. Additionally, the sensitivity of LAMP for detecting shrimp DNA was 100-fold greater than that of conventional PCR. LAMP for the detection of shrimp DNA was reproducible regardless of whether the genomic DNA was extracted from boiled, steamed or roasted shrimp samples. In summary, the LAMP assay established herein not only could be potentially used for diagnosing shrimp DNA but could also be applicable for identifying shrimp allergens in commercial food products in marketplaces.

Specific, sensitive and rapid authentication of donkey-hide gelatine (Colla corii asini) in processed food using an isothermal nucleic acid amplification assay.

Donkey-hide gelatine (DHG) is a well-known, animal-derived traditional Chinese medicine material called Colla corii asini (known in Chinese as "E'jiao"). Because DHG is claimed to have properties that are beneficial to health, its consumption has increased, but its production has decreased. Thus, the incidence of DHG adulteration has become increasingly serious. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for the authentication of DHG. Identification of donkey DNA from DHG was performed specifically and rapidly within one hour by LAMP primers. Moreover, the sensitivity of LAMP in authenticating DHG was 10-3 ng, which revealed a 105-fold higher sensitivity than that of conventional PCR. The relative detection limit was 0.1% DHG in the adulterants, including gelatines of horse, cow, pork, goat, sheep or chicken origins. When genomic DNAs extracted from heat-treated DHG samples, including boiling or autoclaving for 40 min, were used as templates, DHG detection by LAMP was unchanged and reproducible. In conclusion, the LAMP assay established herein could potentially be applied for the authentication of DHG and DHG-related products in herbal or food markets.

Oral Inoculation of Specific-Pathogen-Free Chickens with Chicken Anemia Virus Induces Dose-Dependent Viremia and Transient Anemia.

Chicken infectious anemia caused by chicken anemia virus (CAV) is a very important immunosuppressive disease in chickens. The horizontal spread of CAV in field chickens has been confirmed mainly through oral infection in our published article. Anemia is the main symptom of this disease. Studies by other scientists have shown that infection of CAV in 1-day-old chicks can cause anemia, and the degree of anemia is directly proportional to the dose of infectious virus. However, the pathogenesis of oral inoculation of CAV in older chickens is still not well understood. The purpose of this study was to determine whether 3-weeks-old specific-pathogen-free (SPF) chickens infected with different viral doses in oral route would cause anemia, as well as other signs associated with age-resistance. The experimental design was divided into a high-dose inoculated group (106 1050), low-dose inoculated group (103 TCID50), and non-virus inoculated control group, and 12 birds in each group at the beginning of the trial. The packed cell volumes (PCVs), CAV genome copies in tissues, CAV titer in peripheral blood fractions, and serology were evaluated at 7, 14, and 21 days post-infection (dpi). Virus replication and spread were estimated using quantitative polymerase chain reaction (qPCR) and viral titration in cell culture, respectively. The results showed that the average PCVs value of the high-dose inoculated group was significantly lower than that of the control group at 14 dpi (p < 0.05), and 44.4% (4/9) of the chickens reached the anemia level (PCVs < 27%). At 21 dpi, the average PCV value rebounded but remained lower than the control group without significant differences. In the low-dose inoculated group, all birds did not reach anemia during the entire trial period. Peripheral blood analysis showed that the virus titer in all erythrocyte, granulocyte and mononuclear cell reached the peak at 14 dpi regardless of the high-dose or low-dose inoculated group, and the highest virus titer appeared in the high-dose inoculated group of mononuclear cell. In the low-dose inoculated group, CAV was detected only at 14 dpi in erythrocyte. Taken together, our results indicate that the older birds require a higher dose of infectious CAV to cause anemia after about 14 days of infection, which is related to apoptosis caused by viral infection of erythrocytes. In both inoculated groups, the viral genome copies did not increase in the bone marrow, which indicated that minimal cell susceptibility to CAV was found in older chickens. In the low-dose inoculated group, only mononuclear cells can still be detected with CAV at 21 dpi in seropositive chickens, indicating that the mononuclear cell is the target cell for persistent infection. Therefore, complete elimination of the CAV may still require the aid of a cell-mediated immune response (CMI), although it has previously been reported to be inhibited by CAV infection. Prevention of early exposure to CAV could be possible by improved hygiene procedures.

Identification of nuclear localization signal and nuclear export signal of VP1 from the chicken anemia virus and effects on VP2 shuttling in cells.

BACKGROUND: VP1 of the chicken anemia virus (CAV) is a structural protein that is required for virus encapsulation. VP1 proteins are present both in the nucleus and cytoplasm; however, the functional nuclear localization signal (NLS) and nuclear export signal (NES) of VP1 are still unknown. This study aimed to characterize the NLS and NES motifs of VP1 using bioinformatics methods and multiple-site fragment deletions, and investigate shuttling of VP2 from nucleus to cytoplasm by co-transfection with VP1. METHODS: Two putative NLS motifs were predicted by the WoLF PSORT and NLStradamus programs from the amino acid sequence of VP1. Three NES motifs of VP1 were predicted by the NetNES 1.1 Server and ELM server programs. All mutants were created by multiple-site fragment deletion mutagenesis. VP1 and VP2 were co-expressed in cells using plasmid transfection. RESULTS: A functional NLS motif was identified at amino acid residues 3 to 10 (RRARRPRG) of VP1. Critical amino acids 3 to 10 were significantly involved in nuclear import in cells and were evaluated using systematic deletion mutagenesis. Three NES motifs of VP1 were predicted by the NetNES 1.1 Server and ELM server programs. A functional NES was identified at amino acid residues 375 to 388 (ELDTNFFTLYVAQ). Leptomycin B (LMB) treatment demonstrated that VP1 export from nucleus to cytoplasm occurred through a chromosome region maintenance 1 (CRM1)-dependent pathway. With co-expression of VP1 and VP2 in cells, we observed that VP1 may transport VP2 from nucleus to cytoplasm. CONCLUSION: Our data showed that VP1 of CAV contained functional NLS and NES motifs that modulated nuclear import and export through a CRM1-dependent pathway. Further, VP1 may play a role in the transport of VP2 from nucleus to cytoplasm.

Persistent Infection with Chicken Anemia Virus in 3-Week-Old Chickens Induced by Inoculation of the Virus by the Natural Route.

Naturally acquired chicken anemia virus (CAV) infection in chickens frequently occurs from 3 weeks of age onward after maternally derived antibodies have decayed. The oral inoculation of older chickens with CAV was reported to have negative effects on cell-mediated immune function, and pathological changes were identified. To date, there has been no complete illustration of an immunological and persistent infection. To understand the pathogenesis of persistent CAV infection, an immunological study of CAV-infected 3-week-old specific pathogen-free (SPF) chickens was carried out by different routes of inoculation. The weight, packed cell volumes, and organ samples were obtained at 7, 14, 21, and 28 days postinfection (dpi). Here, we compared hematological, immunological, and sequential pathological evaluations and determined the CAV tissue distribution in different organs. Neither a reduction in weight gain nor anemia was detected in either the inoculated or the control group. The immune-pathological changes were investigated by evaluating the body and thymus weight ratio and specific antibody titer. Delayed recovery of the thymus corresponding to a low antibody response was detected in the orally inoculated group. This is different from what was found in chickens intramuscularly infected with the same dose of CAV. The CAV remaining in a wide range of tissues was examined by viral reisolation into cell culture. The absence of the virus in infected tissues was typically found in the intramuscularly inoculated group. These chickens were immediately induced for a protective antibody response. A few viruses replicating in the thymus were found 21 dpi due to the regression in the antibody titer in the orally inoculated group. Our findings support that a natural infection with CAV may lead to the gradual CAV viral replication in the thymus during inadequate antibody production. The results clearly confirmed that virus-specific antibodies were essential for viral clearance. Under CIA-risk circumstances, administration of the CAV vaccine is important for achieving a sufficient protective immune response.

The rapid and sensitive detection of edible bird's nest (Aerodramus fuciphagus) in processed food by a loop-mediated isothermal amplification (LAMP) assay.

Edible bird's nest (EBN) is a well-known and precious traditional Chinese herbal material (CHM). Because of this, preventing the adulteration of EBN efficiently and precisely is crucial to protect consumers' interests and health. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for the detection of EBN using specifically designed LAMP primers. The results demonstrated that the identification of EBN by LAMP assay was specific and rapid (within 1 h). It had no cross-reaction with EBN adulterants, including white fungus, egg white and pig skin, in different ratios. The relative detection limit was 0.01% EBN in the adulterants. Moreover, the sensitivity of LAMP in authenticating EBN was 10-8 µg, it showed higher sensitivity than that of conventional PCR with 105 fold. When genomic DNAs extracted from boiled or steamed EBN samples were used as templates, LAMP for EBN detection was not affected and was reproducible after heat processing. In conclusion, the LAMP assay established herein could be applicable for authenticating EBN and for identifying commercial EBN products in herbal markets.

Characterization of the DNA binding activity of structural protein VP1 from chicken anaemia virus.

BACKGROUND: Chicken anaemia virus (CAV) is commonly found in poultry. VP1 is the sole structural protein of CAV, which is the major component responsible for capsid assembly. The CAV virion consists of the VP1 protein and a viral genome. However, there is currently no information on the protein-nucleic acid interactions between VP1 and DNA molecules. RESULTS: In this study, the recombinant VP1 protein of CAV was expressed and purified to characterize its DNA binding activity. When VP1 protein was incubated with a DNA molecule, the DNA molecule exhibited retarded migration on an agarose gel. Regardless of whether the sequence of the viral genome was involved in the DNA molecule, DNA retardation was not significantly influenced. This outcome indicated VP1 is a DNA binding protein with no sequence specificity. Various DNA molecules with different conformations, such as circular dsDNA, linear dsDNA, linear ssDNA and circular ssDNA, interacted with VP1 proteins according to the results of a DNA retardation assay. Further quantification of the amount of VP1 protein required for DNA binding, the circular ssDNA demonstrated a high affinity for the VP1 protein. The preferences arranged in the order of affinity for the VP1 protein with DNA are circular ssDNA, linear ssDNA, supercoiled circular dsDNA, open circular DNA and linear dsDNA. CONCLUSIONS: The results of this study demonstrated that the interaction between VP1 and DNA molecules exhibited various binding preferences that were dependent on the structural conformation of DNA. Taken together, the results of this report are the first to demonstrate that VP1 has no sequence-specific DNA binding activity. The particular binding preferences of VP1 might play multiple roles in DNA replication or encapsidation during the viral life cycle.

Development of loop-mediated isothermal amplification (LAMP) assays for the rapid detection of allergic peanut in processed food.

Peanut is a widely and common used in many cuisines around the world. However, peanut is also one of the most important food allergen for causing anaphylactic reaction. To prevent allergic reaction, the best way is to avoid the food allergen or food containing allergic ingredient such as peanut before food consuming. Thus, to efficient and precisely detect the allergic ingredient, peanut or related product, is essential and required for maintain consumer's health or their interest. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for the detection of allergic peanut using specifically designed primer sets. Two sets of the specific LAMP primers respectively targeted the internal transcribed sequence 1 (ITS1) of nuclear ribosomal DNA sequence regions and the ara h1 gene sequence of Arachia hypogeae (peanut) were used to address the application of LAMP for detecting peanut in processed food or diet. The results demonstrated that the identification of peanut using the newly designed primers for ITS 1 sequence is more sensitive rather than primers for sequence of Ara h1 gene when performing LAMP assay. Besides, the sensitivity of LAMP for detecting peanut is also higher than the traditional PCR method. These LAMP primers sets showed high specificity for the identification of the peanut and had no cross-reaction to other species of nut including walnut, hazelnut, almonds, cashew and macadamia nut. Moreover, when minimal 0.1% peanuts were mixed with other nuts ingredients at different ratios, no any cross-reactivity was evident during performing LAMP. Finally, genomic DNAs extracted from boiled and steamed peanut were used as templates; the detection of peanut by LAMP was not affected and reproducible. As to this established LAMP herein, not only can peanut ingredients be detected but commercial foods containing peanut can also be identified. This assay will be useful and potential for the rapid detection of peanut in practical food markets.

VP2 of Chicken Anaemia Virus Interacts with Apoptin for Down-regulation of Apoptosis through De-phosphorylated Threonine 108 on Apoptin.

Chicken anaemia virus (CAV) is an important contagious agent that causes immunosuppressive disease in chickens. CAV Apoptin is a nucleoplasmic shuffling protein that induces apoptosis in chicken lymphoblastoid cells. In the present study, confocal microscopy revealed co-localisation of expressed CAV non-structural protein VP2 with Apoptin in the nucleus of MDCC-MSB1 cells and the nucleoplasmic compartment of CHO-K1 cells. In vitro pull-down and ex vivo biomolecular fluorescent complementation (BiFC) assays further showed that the VP2 protein directly interacts with Apoptin. Transient co-expression of VP2 and Apoptin in MDCC-MSB1 cells significantly decreased the rate of apoptosis compared with that in cells transfected with the Apoptin gene alone. In addition, the phosphorylation status of threonine 108 (Thr108) of Apoptin was found to decrease upon interaction with VP2. Although dephosphorylated Thr108 did not alter the subcellular distribution of Apoptin in the nucleus of MDCC-MSB1 cells, it did suppress apoptosis. These findings provide the first evidence that VP2 directly interacts with Apoptin in the nucleus to down-regulate apoptosis through alterations in the phosphorylation status of the latter. This information will be useful to further elucidate the underlying mechanism of viral replication in the CAV life cycle.

The development of loop-mediated isothermal amplification (LAMP) assays for the rapid authentication of five forbidden vegetables in strict vegetarian diets.

Plant-based food ingredients such as garlic, Chinese leek, Chinese onion, green onion and onion are widely used in many cuisines around the world. However, these ingredients known as the "five forbidden vegetables" (FFVs) are not allowed in some vegetarian diets. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for the detection of FFVs using five respective LAMP primer sets. The specific primers targeted the ITS1-5.8S-ITS2 nuclear ribosomal DNA sequence regions among the five vegetables. The results demonstrated that the identification of FFVs using the newly developed LAMP assay is more sensitive than the traditional PCR method. Using pepper, basil, parsley, chili and ginger as references, established LAMP primer sets showed high specificity for the identification of the FFV species. Moreover, when FFVs were mixed with other plant ingredients at different ratios (100:0, 50:50, 20:80, 10:90, 5:95, 2:98, and 1:99), no cross-reactivity was evident using LAMP. Finally, genomic DNAs extracted from boiled and steamed FFVs in processed foods were used as templates; the performance of the LAMP reaction was not influenced using validated LAMP primers. Not only can FFV ingredients be identified but commercial foods containing FFVs can also be authenticated. This LAMP method will be useful for the authentication of FFVs in practical food markets in the future.

High molecular weight of polysaccharides from Hericium erinaceus against amyloid beta-induced neurotoxicity.

BACKGROUND: Hericium erinaceus (HE) is a well-known mushroom in traditional Chinese food and medicine. HE extracts from the fruiting body and mycelia not only exhibit immunomodulatory, antimutagenic and antitumor activity but also have neuroprotective properties. Here, we purified HE polysaccharides (HEPS), composed of two high molecular weight polysaccharides (1.7 × 10(5) Da and 1.1 × 10(5) Da), and evaluated their protective effects on amyloid beta (Aß)-induced neurotoxicity in rat pheochromocytoma PC12 cells. METHODS: HEPS were prepared and purified using a 95 % ethanol extraction method. The components of HEPS were analyzed and the molecular weights of the polysaccharides were determined using high-pressure liquid chromatography (HPLC). The neuroprotective effects of the polysaccharides were evaluated through a 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay and an MTT assay and by quantifying reactive oxygen species (ROS) and mitochondrial membrane potentials (MMP) of Aß-induced neurotoxicity in cells. RESULT: Our results showed that 250 µg/ml HEPS was harmless and promoted cell viability with 1.2 µM Aß treatment. We observed that the free radical scavenging rate exceeded 90 % when the concentration of HEPS was higher than 1 mg/mL in cells. The HEPS decreased the production of ROS from 80 to 58 % in a dose-dependent manner. Cell pretreatment with 250 µg/mL HEPS significantly reduced Aß-induced high MMPs from 74 to 51 % and 94 to 62 % at 24 and 48 h, respectively. Finally, 250 µg/mL of HEPS prevented Aß-induced cell shrinkage and nuclear degradation of PC12 cells. CONCLUSION: Our results demonstrate that HEPS exhibit antioxidant and neuroprotective effects on Aß-induced neurotoxicity in neurons.

High yield production of pigeon circovirus capsid protein in the E. coli by evaluating the key parameters needed for protein expression.

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.

Expression and characterization of highly antigenic domains of chicken anemia virus viral VP2 and VP3 subunit proteins in a recombinant E. coli for sero-diagnostic applications.

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.