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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.

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.

Mango (Mangifera indica) is one of the most popular tropical fruits around the world. It is also widely and commonly used in many cuisines and products in the food industry. However, the anaphylactic reaction caused by mango has long been reported as a major problem for consumption in recent years. To prevent allergens in mango, the best way is to avoid mango in the diet. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for the detection of mango in food. Four specifically designed LAMP primers targeting the internal transcribed sequence 1 (ITS1) of nuclear ribosomal DNA sequence regions were used to address the LAMP reaction for amplifying mango DNA. The results demonstrated that the detection of mango DNA was specifically validated by the LAMP primer. The sensitivity of LAMP for detecting mango DNA is equivalent to that of the traditional PCR method. The LAMP primer sets showed high specificity for detecting the DNA of mango and had no cross-reactions to other species. Moreover, when mango was mixed with other fruits at different ratios, no cross-reactivity for the detection of mango DNA was manifested during LAMP. Finally, genomic DNAs extracted from different heat-processed mangos were used as templates; the detection of mango DNA by LAMP was not significantly affected and was reproducible. As to this established LAMP herein, mango ingredients can be detected, and commercial foods containing mango can also be identified. This assay will be useful and have potential for the rapid detection of mango DNA in practical food markets.

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 (&gt, 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 (&gt, 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.

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.

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 10(5)-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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13197-020-04319-3) contains supplementary material, which is available to authorized users.

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 &lt, 0.05), and 44.4% (4/9) of the chickens reached the anemia level (PCVs &lt, 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.

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.

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.

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.

Flagellin, a bioactive Toll-like receptor (TLR) 5 ligand, may trigger the innate immunity that in turn is important for subsequent adaptive immune responses. In the present study, the adjuvant effects of the monomeric and polymeric forms of Salmonella flagellin (mFliC and pFliC, respectively) were examined in specific-pathogen free (SPF) chickens immunized intramuscularly (i.m.) or intranasally (i.n.) with formalin-inactivated avian influenza virus (AIV) H5N2 vaccines. Results showed that mFliC cooperating with the 64CpG adjuvant significantly induced influenza-specific antibody titers of plasma IgA in the i.m.-vaccinated animals. The nasal IgA levels in the i.n.-mFliC-coadministrated AIV vaccinated chickens were significantly elevated compared to levels observed in the control group (H5N2 vaccine alone). The pFliC cooperating with the 64CpG adjuvant significantly enhanced cell proliferation of splenocytes in the i.m.-vaccinated animals. TLR3 and TLR5 expressions were activated by flagellin stimulation in vitro and in vivo. These results suggest that flagellin can be used as an adjuvant in an AIV H5N2 vaccine, especially for mucosal immunity.

Chicken anemia virus (CAV) is an anemia agent of breeder and young chicks. This virus is the cause of economic losses across the chicken industry worldwide as a consequence of severe anemia and immunodeficiency among the birds. Two genes of CAV encoding the VP1 and VP2 proteins were cloned and expressed in Escherichia coli BL21 (DE3). A Western blot assay using His-tag antiserum was used to assess the expression level of the CAV viral proteins in E. coli. The results demonstrated that only full-length VP2 can be successfully expressed in E. coli, but not full-length VP1. A serial of N-terminus deletions of the VP1 protein, VP1Nd30, VP1Nd60 and VP1 Nd129, were created using PCR in order to improve VP1 expression. The results demonstrated that all three of these recombinant VP1 mutant proteins can be expressed in E. coli. VP1Nd129 protein demonstrates the highest expression level compared to the other two proteins. The specificity of Nd129-VP1 and VP2 protein were confirmed by mass spectrometry. By comparing the expression level of VP1Nd129 and VP2 protein after the addition of IPTG, the results indicated that the VP1Nd129 protein gave a higher level of protein expression than VP2. The highest yields of VP1Nd129 and VP2 were 26.2 and 15.5 mg/L, respectively, after IPTG induction with 0.1 mM IPTG for 6 h, respectively. The identification of the optimized conditions for production of the CAV viral proteins VP1 and VP2 will allow them to be used in the future as an antigen for the development of vaccines and diagnostic tests.

A multistrain formula (MF) consisted of four selected lactic acid bacteria (LAB) strains was evaluated for its function to ameliorate the growth rate and inflammation of broiler chickens caused by Eimeria tenella parasitizing. Thirty one day broiler chickens were divided into 3 groups with 10 chickens in 2 cages per group. These groups were control, infected and MF groups. In control group, chickens were fed with 0.2 ml PBS and not challenged with E. tenella. In MF group, chickens were gavage fed with 0.2 ml of MF (109 CFU/ml) from day 1 to day 23. On day 14, each chicken in both the infected and MF groups was challenged with 5,000 sporulated E. tenella oocysts. Seven days post infection, it was found that the average body weight gain (ABWG) for chickens in the infected and MF groups were 281.51 ± 61.00 and 295.52 ± 62.16 g per chicken, respectively. Chickens in each group were then sacrificed and their cecal tonsils collected. It was observed that feeding MF could reduce the cecal lesion scores (LS) of the Eimeria infected chickens significantly. In addition, for chickens in the MF group, the levels of cecal gene expression of proinflammatory cytokines, such as interleukin (IL)-1β, IL-6, and interferon (IFN)-γ, were found significantly lower, while anti-inflammation cytokine, ie, IL-10, was significantly higher than those of the chickens in the infected group. This study thus suggests that the MF, which previously has been shown to have antagonistic effect against Salmonella infection and the induced inflammation either in live or in heat killed form, may also be useful for the amelioration of the growth rate and the inflammation of broiler chickens caused by E. tenella infection.

Isolates of Newcastle disease virus (NDV) from chicken cases were obtained from various locations in Taiwan during 2003-2006 and were genotypically analyzed by using reverse transcription polymerase chain reaction (RT-PCR) with primers specific to the viral fusion (F) protein gene (534 bp). Part of the amplified F protein DNA product (nucleotide sequence 47-418) and the deduced amino acid sequences were compared phylogenetically with those from strains previously reported in Taiwan and other geographic regions. Our results showed that all Taiwanese isolates (n=20) collected during 2003-2006, according to the phylogenetic tree, belong to the genotype VIId. In addition, all the six Taiwanese isolates obtained in 2003, carry the motif (112)R-R-Q-K-R(116) and have the amino acid L(23) replaced by F(23) (assigned as Group 1). On the other hand, 12 out of the 14 Taiwanese isolates obtained during 2004-2006 possess the motif (112)R-R-K-K-R(116) and have the amino acid G(74), instead of E(74) (assigned as Group 2). To our best knowledge, this is the first reported VIId isolates that possess the sequences of G(74)/(112)R-R-K-K-R(116) within the F0 protein. Since a high mortality, severe clinical signs, typical postmortem lesions, and a high intra-cerebral pathogenicity index (ICPI) were observed in the NDV-infected chickens, these isolates acquired between 2003 and 2006 are considered as the velogenic type. The Group 2 viruses have become dominant and responsible for the majority of Taiwanese outbreaks during recent years. Based on our phylogenetic analysis, it can be postulated that these isolates were evolved from previously reported local strains, and the Group 2 family emerged the latest in the genotype VIId. The information is fundamental to improving the efficiency of controlling strategies and vaccine development for NDV.

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 num- ber 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.

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.

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.

In this investigation, detection dogs are trained and used in identifying red imported fire ants, Solenopsis invicta Buren, and their nests. The methodology could assist in reducing the frequency and scope of chemical treatments for red imported fire ant management and thus reduce labor costs and chemical use as well as improve control and quarantine efficiency. Three dogs previously trained for customs quarantine were retrained to detect the scents of red imported fire ants. After passing tests involving different numbers of live red imported fire ants and three other ant species--Crematogaster rogenhoferi Mayr, Paratrechina longicornis Latreille, and Pheidole megacephala F.--placed in containers, ajoint field survey for red imported fire ant nests by detection dogs and bait traps was conducted to demonstrate their use as a supplement to conventional detection methods. The most significant findings in this report are (1) with 10 or more red imported fire ants in scent containers, the dogs had >98% chance in tracing the red imported fire ant. Upon the introduction of other ant species, the dogs still achieved on average, a 93% correct red imported fire ant indication rate. Moreover, the dogs demonstrated great competence in pinpointing emerging and smaller red imported fire ant nests in red imported fire ant-infested areas that had been previously confirmed by bait trap stations. (2) Along with the bait trap method, we also discovered that approximately 90% of red imported fire ants foraged within a distance of 14 m away from their nests. The results prove detection dogs to be most effective for red imported fire ant control in areas that have been previously treated with pesticides and therefore containing a low density of remaining red imported fire ant nests. Furthermore, as a complement to other red imported fire ant monitoring methods, this strategy will significantly increase the efficacy of red imported fire ant control in cases of individual mount treatment.

Unmethylated CpG motifs are capable of evoking a range of immunostimulatory effects in vertebrates and have tremendous potential to be used as therapeutic agents and adjuvants. This particular type of CpG motif has been demonstrated to be an excellent immune adjuvant mediated by Toll-like receptor 9 (TLR9) in various mammalian vaccines; however, only a few studies confirm its efficacy in avian vaccines. In the present study, immunomodulatory activities of plasmids with various copy numbers of a CpG motif were evaluated in chickens inoculated with an avian influenza vaccine. Results showed that the plasmid with 64 copies of the CpG motif (64CpG-plasmid) significantly enhanced the mRNA expressions of interferon-gamma (IFN-γ), TLR3 and TLR7 in chicken splenocytes compared to plasmids with lesser copies of the CpG motif in vitro. Chickens inoculated with the H5N2 avian influenza inactivated vaccines (V52) coadministrated with the 64CpG-plasmid (V52-64CpG) showed significant increments of hemagglutination inhibition (HI) titers, peripheral blood mononuclear cell (PBMC) proliferation, and mRNA expressions of IFN-α, IFN-γ, TLR3, TLR7 and TLR21 in splenocytes as compared to those of chickens inoculated with V52 alone, V52 adjuvanted with aluminum gel (V52-gel), or with V52-gel plus vector. Additionally, following challenge with a highly virulent H5N1 strain, a higher survival rate (100%) was observed in chickens inoculated with V52-64CpG as compared to those that received V52-gel (80%) or PBS (0%). The 64CpG-plasmid significantly enhanced chicken immunity in vitro and in vivo; thus it can be a potent adjuvant in an avian influenza vaccine for chickens.

Interleukin-18 (IL-18) can induce interferon-gamma (IFN-gamma) production and promote Th1 immunity, and hence, it modulates immune functions. In the present study, the in vitro and in vivo immunomodulatory activities of full length or mature chicken IL-18 expressed in a prokaryotic expression system (pCHIL18-F and pCHIL18-M, respectively) and chicken IL-18 expressed in a eukaryotic expression system (euCHIL18) were examined. Results showed that pCHIL18-F, pCHIL18-M and euCHIL18 significantly enhanced IFN-gamma mRNA expression in chicken splenocytes, which successfully increased IFN-gamma-induced nitric oxide (NO) synthesis by macrophages. Vaccination with cell-cultured Newcastle disease vaccine (NDTC) co-administrated with pCHIL18-F, pCHIL18-M or euCHIL18 resulted in significant increments of hemagglutination inhibition (HI) titers, cell proliferation of peripheral blood mononuclear cells (PBMC), and ratios of CD8(+) to CD4(+) in chickens compared with inoculation of PBS or NDTC alone. Thus, full length and mature chicken IL-18 expressed using a prokaryotic system and using a eukaryotic system showed equivalent in vitro and in vivo biological activities, and all forms effectively enhanced cell-mediated and humoral immunity, suggesting possible future use as a potential adjuvant in chicken NDTC vaccine production.

Both the sigmaC and sigmaB proteins of avian reovirus (ARV) can induce type- and group-specific neutralizing antibodies, respectively. In this study, the full-length of S1133 sigmaC, 1071-1 sigmaC, S1133 sigmaB, and S1133 sigmaC-sigmaB fusion genes of ARV were cloned into a secreted vector pPICZalphaA and then integrated into the chromosome of Pichia pastoris for induced expression. Western blot assay showed that ARV sigmaC, sigmaB, and sigmaC-sigmaB fusion proteins were expressed and secreted into the medium. Two types of ELISA kits using equal mixtures of 1071-1sigmaC and S1133 sigmaB and S1133 sigmaC-sigmaB fusion proteins as antigens were developed. After a checker board titration for optimal conditions, the cut-off values of positive results for the 1071-1sigmaC/S1133 sigmaB and S1133 sigmaC-sigmaB ELISA kits were 0.24 and 0.12, respectively. Forty-four serum neutralization test-positive and twenty-eight serum neutralization-negative samples from vaccinated and commercial farm chickens were tested by the new ELISA kits and by the conventional ELISA. The new ELISA kits have higher positive rates than the conventional ELISA. The results revealed that the correlation rates for the serum neutralization titer and the absorbance values with the new ELISA kits and the conventional ELISA were 100% and 95.8%, respectively.

Many studies have been undertaken to investigate the mechanisms of skin differentiation. In particular, growth factors and hormones are believed to play important roles in skin proliferation, differentiation and survival. Insulin-like growth factor-1 (IGF-1) has been identified as a survival factor in many tissues including the skin, but the molecular mechanism of IGF-1 in epidermal differentiation is not completely understood. Neonatal mouse skin is useful for studying changes in gene expression, as the mitotic activity of skin cells changes shortly after birth. Using RNA differential display (DD), a 357-nt message that is specifically expressed in the epidermal keratinocytes of IGF-1-injected newborn mice but not in controls, has been identified. Confirmation of expression of this gene by ribonuclease protection assay (RPA) showed that its mRNA expression in the epidermal keratinocytes is induced by IGF-1. Using RNA ligase-mediated rapid amplification of 5′ cDNA ends (RLM-5′-RACE), we have successfully isolated a 3473-bp full-length gene, c98, that has 97% sequence homology to a bcl-2-like gene, bcl-w. The latter has been identified as a proto-oncogene in several murine myeloid cell lines. Amino acid sequence analysis of the c98 showed that it has 97% sequence identity to the bcl-w protein and possesses bcl-2 homology domains (BH) 1, 2 and 3. Immunoblotting data revealed similar increases of c98 protein expression to its mRNA expression in the keratinocytes of IGF-1-injected animals. Weak expression of other bcl-2 family member proteins, bax, bcl-2 and bcl-xL, were also found in the immunoblots. Additionally, IGF-1 was found to be able to protect epidermal keratinocytes from dexamethasone (DEX)-induced apoptosis, based on the findings that after the cells were treated with DEX, DNA laddering was present in the control mice but not in those injected with IGF-1. Further, using a photometric enzyme-linked immunoassay to quantitate keratinocyte death, we found that after addition of DEX, the amounts of cytoplasmic histone-associated DNA fragments were not significantly (P>0.05) different in IGF-1-treated cells compared with untreated control cells during the high mitotic stage of skin epidermis. To assess the role of c98 in these anti-apoptotic processes, we have generated a recombinant plasmid that contains an expression vector and c98 and transfected this plasmid into the keratinocytes from mice without IGF-1-treatment. Expression of the c98 protein was found to completely (P>0.05) block DEX-induced apoptosis after cell transfection. Taken together, our current data demonstrated that IGF-1 plays an anti-apoptotic role in the DEX-induced apoptosis in epidermal keratinocytes and this, at least in part, may be mediated through expression of c98.

The major histocompatibility (B) complex of the chicken contains genes similar to Class I (B-F) and Class II (B-L beta) genes in mammals, as well as a highly-polymorphic gene family (B-G) whose exact function is not known. Specific B-haplotypes are strongly associated with resistance to a number of infectious diseases, and with immune responses to soluble and cellular antigens. In mammals, Class I and Class II molecules control development of the T cell repertoire, including selection of CD4+ and CD8+ T cells. One study of chickens reported that low CD4:CD8 ratio was associated with the B4 haplotype, which shares expressed B-F/B-L genes with the B13 haplotype. In studies reported here, chickens of two haplotypes carried in the Auburn R line, B302 and B305 (which is B13-related), were evaluated for percentages of T cells expressing the CD4, CD8, CD3, TCR1, TCR2 and TCR3 antigens in peripheral blood lymphocytes (PBL), thymus, and spleen. These two haplotypes were chosen for comparison because they differ in resistance to Marek's disease (MD) and are closely-related in B-F and B-L genes by restriction fragment length polymorphism analyses. Homozygous birds of each B haplotype were produced from crosses of (B302 x B305)F1 sires and dams. PBL, thymocytes, and splenocytes from B302 homozygotes had higher CD4:CD8 ratios than B305 homozygotes. However, CD4:CD8 ratio differences could not be attributed to haplotype-controlled differences in V beta usage within CD4/CD8 subsets, as has been described for certain V beta families in mice and humans. These results indicate that thymic selection events involving CD4 and CD8 subsets and TCR V beta usage are controlled by a gene or genes closely-linked to the B-complex, which may or may not be Class I or Class II genes.

Chicken anemia virus (CAV) is an important viral pathogen that causes anemia and severe immunodeficiency syndrome in chickens worldwide. In this study, a potential diagnostic monoclonal antibody against the CAV VP1 protein was developed which can precisely recognize the CAV antigen for diagnostic and virus recovery purposes. The VP1 gene of CAV encoding the N-terminus-deleted VP1 protein, VP1Nd129, was cloned into an Escherichia (E.) coli expression vector. After isopropyl-β-D-thiogalactopyronoside induction, VP1Nd129 protein was shown to be successfully expressed in the E. coli. By performing an enzyme-linked immunoabsorbent assay using two coating antigens, purified VP1Nd129 and CAV-infected liver tissue lysate, E3 monoclonal antibody (mAb) was found to have higher reactivity against VP1 protein than the other positive clones according to the result of limiting dilution method from 64 clones. Using immunohistochemistry, the presence of the VP1-specific mAb, E3, was confirmed using CAV-infected liver and thymus tissues as positive-infected samples. Additionally, CAV particle purification was also performed using an immunoaffinity column containing E3 mAb. The monoclonal E3 mAb developed in this study will not only be very useful for detecting CAV infection and performing histopathology studies of infected chickens, but may also be used to purify CAV particles in the future.

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 × 105 Da and 1.1 × 105 Da), and evaluated their protective effects on amyloid beta (Aβ)-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 Aβ-induced neurotoxicity in cells. Result Our results showed that 250 μg/ml HEPS was harmless and promoted cell viability with 1.2 μM Aβ 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 Aβ-induced high MMPs from 74 to 51 % and 94 to 62 % at 24 and 48 h, respectively. Finally, 250 μg/mL of HEPS prevented Aβ-induced cell shrinkage and nuclear degradation of PC12 cells. Conclusion Our results demonstrate that HEPS exhibit antioxidant and neuroprotective effects on Aβ-induced neurotoxicity in neurons.

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.

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.