Combined use of ELISA and Western blot with recombinant N protein is a powerful tool for the immunodiagnosis of avian infectious bronchitis

Background

The avian infectious bronchitis virus (IBV) remains a significant source of loss in the poultry industry and early diagnosis is required to prevent the disease from spreading. This study examined the combined use of an ELISA and Western blot (WB) to detect antibodies against the nucleocapsid protein (N) of IBV. The coding sequence for N was amplified by RT-PCR and expressed in Escherichia coli. A soluble recombinant N protein (rN) of approximately 50?kDa was obtained. A total of 389 sera were tested against the rN in ELISA and the results were compared with those of the commercial IDEXX IBV Ab test. ELISA-rN achieved a 90.34% sensitivity and 90.16% specificity. WB confirmed all false negative sera in ELISA-rN or IDEXX test as truly positive. The current study indicate that the combined use of rN in ELISA and WB is a powerful tool for the immunodiagnosis of avian infectious bronchitis.

Methods

Constructed recombinant pAE/n expression vectors were used to transform E. coli BL21(DE3) Star competent cells (Invitrogen). The rN of infectious bronchitis virus was purified by affinity chromatography using HisTrap HP 1?mL columns pre-packed with pre-charged Ni Sepharose in the ÄKTAprime Automated Liquid Chromatography system (GE Healthcare). A total of 389 serum samples from chickens were used to develop and evaluate the ELISA-rN test. To standardize the indirect ELISA development, serum dilutions (1:100, 1:200 and 1:400) and different concentrations of purified rN antigen (50, 100 and 200?ng/well) were tested. Positive and negative sera for IBV were used as controls. The results were compared with those obtained from a commercial kit. Serum samples scored as negative with the commercial kit but as positive with the ELISA-rN were further analysed by Western blot analyses using the rN protein as an antigen. The results of the ELISA-rN were compared to the commercial kit results using receiver-operating characteristics curves, area under the curve, and confidence intervals with the software GraphPad Prism version 6.0 for Windows (GraphPad Software, USA).

Results

The expected cDNA fragment of approximately 1240?bp was successfully amplified by PCR using primers designed to select for the coding region of the N protein. The rN was expressed as a soluble protein to avoid the refolding steps and, after purification a yield of 10?mg/L of rN was obtained. The SDS-PAGE results demonstrated the presence of two distinct bands that had a molecular mass of approximately 45 and 50 KDa. Out of 244 sera that scored positive in the commercial ELISA IDEXX IBV Ab Test, 220 were also positive in the ELISA-rN, yielding an ELISA-rN test sensitivity of 90.16%. Out of 145 sera that scored negative in the IDEXX IBV Ab Test, 131 also scored negative in the ELISA-rN, indicating a specificity of 90.34%. Sera that tested negative in the ELISA-rN and positive in the commercial test also reacted with the rN protein in Western blot.

Conclusions

The association between the ELISA and Western blot techniques developed in this study with a subunit of IBV (rN) were able to detect antibodies that the commercial ELISA did not detect suggesting that the ELISA-rN has greater sensitivity.

     

Microenvironmental pH Is a Key Factor for Exosome Traffic in Tumor Cells

Exosomes secreted by normal and cancer cells carry and deliver a variety of molecules. To date, mechanisms referring to tumor exosome trafficking, including release and cell-cell transmission, have not been described. To gain insight into this, exosomes purified from metastatic melanoma cell medium were labeled with a lipid fluorescent probe, R18, and analyzed by spectrofluorometry and confocal microscopy. A low pH condition is a hallmark of tumor malignancy, potentially influencing exosome release and uptake by cancer cells. Using different pH conditions as a modifier of exosome traffic, we showed (i) an increased exosome release and uptake at low pH when compared with a buffered condition and (ii) exosome uptake by melanoma cells occurred by fusion. Membrane biophysical analysis, such as fluidity and lipid composition, indicated a high rigidity and sphingomyelin/ganglioside GM3 (N-acetylneuraminylgalactosylglucosylceramide) content in exosomes released at low pH. This was likely responsible for the increased fusion efficiency. Consistent with these results, pretreatment with proton pump inhibitors led to an inhibition of exosome uptake by melanoma cells. Fusion efficiency of tumor exosomes resulted in being higher in cells of metastatic origin than in those derived from primary tumors or normal cells. Furthermore, we found that caveolin-1, a protein involved in melanoma progression, is highly delivered through exosomes released in an acidic condition. The results of our study provide the evidence that exosomes may be used as a delivery system for paracrine diffusion of tumor malignancy, in turn supporting the importance of both exosomes and tumor pH as key targets for future anti-cancer strategies.     

Diagnostic performance of anti-Zika virus IgM,IgAM and IgG ELISAs during co-circulation of Zika,dengue, and chikungunya viruses in Brazil and Venezuela

BackgroundSerological diagnosis of Zika virus (ZIKV) infection is challenging because of the antibody cross-reactivity among flaviviruses. At the same time, the role of Nucleic Acid Testing (NAT) is limited by the low proportion of symptomatic infections and the low average viral load. Here, we compared the diagnostic performance of commercially available IgM, IgAM, and IgG ELISAs in sequential samples during the ZIKV and chikungunya (CHIKV) epidemics and co-circulation of dengue virus (DENV) in Brazil and Venezuela.Methodology/Principal findingsAcute (day of illness 1–5) and follow-up (day of illness ≥ 6) blood samples were collected from nine hundred and seven symptomatic patients enrolled in a prospective multicenter study between June 2012 and August 2016. Acute samples were tested by RT-PCR for ZIKV, DENV, and CHIKV. Acute and follow-up samples were tested for IgM, IgAM, and IgG antibodies to ZIKV using commercially available ELISAs. Among follow-up samples with a RT-PCR confirmed ZIKV infection, anti-ZIKV IgAM sensitivity was 93.5% (43/46), while IgM and IgG exhibited sensitivities of 30.3% (10/33) and 72% (18/25), respectively. An additional 24% (26/109) of ZIKV infections were detected via IgAM seroconversion in ZIKV/DENV/CHIKV RT-PCR negative patients. The specificity of anti-ZIKV IgM was estimated at 93% and that of IgAM at 85%.Conclusions/SignificanceOur findings exemplify the challenges of the assessment of test performance for ZIKV serological tests in the real-world setting, during co-circulation of DENV, ZIKV, and CHIKV. However, we can also demonstrate that the IgAM immunoassay exhibits superior sensitivity to detect ZIKV RT-PCR confirmed infections compared to IgG and IgM immunoassays. The IgAM assay also proves to be promising for detection of anti-ZIKV seroconversions in sequential samples, both in ZIKV PCR-positive as well as PCR-negative patients, making this a candidate assay for serological monitoring of pregnant women in future ZIKV outbreaks.     

Convergent, modular expression of ebony and tan in the mimetic wing patterns of Heliconius butterflies

The evolution of pigmentation in vertebrates and flies has involved repeated divergence at a small number of genes related to melanin synthesis. Here, we study insect melanin synthesis genes in Heliconius butterflies, a group characterised by its diversity of wing patterns consisting of black (melanin), and yellow and red (ommochrome) pigmented scales. Consistent with their respective biochemical roles in Drosophila melanogaster, ebony is upregulated in non-melanic wing regions destined to be pigmented red whilst tan is upregulated in melanic regions. Wing regions destined to be pigmented yellow, however, are downregulated for both genes. This pattern is conserved across multiple divergent and convergent phenotypes within the Heliconii, suggesting a conserved mechanism for the development of black, red and yellow pattern elements across the genus. Linkage mapping of five melanin biosynthesis genes showed that, in contrast to other organisms, these genes do not control pattern polymorphism. Thus, the pigmentation genes themselves are not the locus of evolutionary change but lie downstream of a wing pattern regulatory factor. The results suggest a modular system in which particular combinations of genes are switched on whenever red, yellow or black pattern elements are favoured by natural selection for diverse and mimetic wing patterns.     

Prediction of the in planta Phakopsora pachyrhizi secretome and potential effector families

Asian soybean rust (ASR), caused by the obligate biotrophic fungus Phakopsora pachyrhizi, can cause losses greater than 80%. Despite its economic importance, there is no soybean cultivar with durable ASR resistance. In addition, the P. pachyrhizi genome is not yet available. However, the availability of other rust genomes, as well as the development of sample enrichment strategies and bioinformatics tools, has improved our knowledge of the ASR secretome and its potential effectors. In this context, we used a combination of laser capture microdissection (LCM), RNAseq and a bioinformatics pipeline to identify a total of 36 350 P. pachyrhizi contigs expressed in planta and a predicted secretome of 851 proteins. Some of the predicted secreted proteins had characteristics of candidate effectors: small size, cysteine rich, do not contain PFAM domains (except those associated with pathogenicity) and strongly expressed in planta. A comparative analysis of the predicted secreted proteins present in Pucciniales species identified new members of soybean rust and new Pucciniales‐ or P. pachyrhizi‐specific families (tribes). Members of some families were strongly up‐regulated during early infection, starting with initial infection through haustorium formation. Effector candidates selected from two of these families were able to suppress immunity in transient assays, and were localized in the plant cytoplasm and nuclei. These experiments support our bioinformatics predictions and show that these families contain members that have functions consistent with P. pachyrhizi effectors.     

Essential Oil of Thymus munbyanus subsp. coloratus from Algeria: Chemotypification and in vitro Biological Activities

Thymus munbyanus subsp. coloratus (Lamiaceae) is a small shrub endemic to Algeria and Morocco where is found in lawns, rockeries and mountainous regions. From a phytochemical point of view this taxon has never been characterized. In this work we have analysed the chemical compositions of the essential oils obtained from inflorescences and vegetative parts by GC/MS. A new chemotype, i.e. borneol‐chemotype, was characterized for the first time in the species. Furthermore, we assessed the biological activities of essential oils, namely the antioxidant, antimicrobial and cytotoxicity on tumor cells that were evaluated by the DPPH, ABTS, and FRAP, disc diffusion, and MTT methods, respectively. Biological assays highlighted a moderate inhibitory effect on Staphylococcus aureus, Escherichia coli and Candida albicans (inhibition zone diameter in the range 9 – 10 mm), and noteworthy cytotoxicity on A375 human melanoma cells (IC₅₀ of 46.95 μg/ml).     

Chemical Composition of the Essential Oils of Three Endemic Species of Anthemis Sect. Hiorthia (DC.) R.Fern. Growing Wild in Sicily and Chemotaxonomic Volatile Markers of the Genus Anthemis L.: An Update

The chemical composition of the essential oils isolated from the aerial parts of Anthemis pignattiorum Guarino, Raimondo & Domina and A. ismelia Lojac . and the aerial parts and flowers of Anthemis cupaniana Tod . ex Nyman , three endemic Sicilian species belonging to the section Hiorthia, was determined by GC‐FID and GC/MS analyses. (Z)‐Muurola‐4(14),5‐diene (27.3%) was recognized as the main constituent of the A. pignattiorum essential oil, together with isospathulenol (10.6%), sabinene (7.7%), and artemisyl acetate (6.8%), while in the oil obtained from the aerial parts of A. ismelia, geranyl propionate (8.8%), bornyl acetate (7.9%), β‐thujone (7.8%), neryl propionate (6.5%), and τ‐muurolol (6.5%) prevailed. α‐Pinene was the main compound of both the aerial part and flower oils of A. cupaniana (18.4 and 13.2%, resp.). Also noteworthy are the considerable amounts of artemisyl acetate (12.7%) and β‐thujone (11.8%) found in the oil from the aerial parts and those of tricosane (9.8%) and sabinene (7.6%) evidenced in the flower oil. Furthermore, an update on the main compounds identified in the essential oils of all the Anthemis taxa studied so far was presented, and cluster analyses were carried out, to compare the essential oils of these taxa.     

Lantibiotics biosynthesis genes and bacteriocinogenic activity of <Emphasis Type="Italic">Lactobacillus</Emphasis> spp. isolated from raw milk and cheese

Lactobacillus species are usually used as starters for the production of fermented products, and some strains are capable of producing antimicrobial substances, such as bacteriocins. Because these characteristics are highly desirable, research are continually being performed for novel Lactobacillus strains with bacteriocinogenic potential for use by food industries. The aim of this study was to characterise the bacteriocinogenic potential and activity of Lactobacillus isolates. From a lactic acid bacteria culture collection obtained from raw milk and cheese, 27 isolates were identified by 16S rDNA as Lactobacillus spp. and selected for the detection of lantibiotics biosynthesis genes, bacteriocin production, antimicrobial spectra, and ideal incubation conditions for bacteriocin production. Based on the obtained results, 21 isolates presented at least one of the three lantibiotics biosynthesis genes (lanB, lanC or lamM), and 23 isolates also produced antimicrobial substances with sensitivity to at least one proteinase, indicating their bacteriocinogenic activity. In general, the isolates had broad inhibitory activity, mainly against Listeria spp. and Staphylococcus spp. strains, and the best antimicrobial performance of the isolates occurred when they were cultivated at 25 °C for 24 or 48 h or at 35 °C for 12 h. The present study identified the bacteriocinogenic potential of Lactobacillus isolates obtained from raw milk and cheese, suggesting their potential use as biopreservatives in foods.     

Distribution patterns of the fish assemblage in the Mamanguape River Estuary,North-eastern Brazil

The Mamanguape River Estuary was studied along a continuum ranging from shallow sandstone reefs adjacent to the river mouth up to the limit of influence of seawater, the upper portions of the estuary. Fish samples were gathered using three types of nets along 17 sample sites, grouped in four regions according to salinity range (reefs and low, middle and upper estuaries), to seasonality (dry and rainy seasons) and to habitat usage (marine visitor, marine estuarine-opportunist, marine estuarine-dependent, estuarine resident and estuarine &; marine). Differences were found in the fish assemblages along the estuarine-reef gradient, with most species (n?=?30) being considered marine, estuarine-opportunists or estuarine-dependent, according to its abundance and distribution. A low number of species (n?=?11) were considered estuarine residents. Some species exhibited significant differences in spatial distribution pattern, in which juveniles and adults predominated in different portions of the estuary, suggesting an ontogenetic migration both in relation to the adjacent reef area and across estuarine regions. Several species were newly recorded in the Mamanguape Estuary: Anchoa spinifer, Halichoeres poeyi, Hyporhamphus roberti, Scomberomorus cavalla, Sphyraena barracuda and Ocgocephalus vesperilio.